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Firefighters are at elevated risk of cardiovascular disease and myocardial infarction. Overweight and obesity prevalence in this occupational group may be greater than in the general population. This is the first nutrition-based study for UK firefighters, involving 28 fire stations and 575 firefighters based in London. This study was designed to develop population specific tools: body composition reference charts, a validated food frequency questionnaire (FFQ) and a cookery book for use in a multicomponent worksite intervention. The validity of BMI for classification of firefighter adiposity status was investigated. Combined prevalence of male firefighter (n=497) overweight and obesity by BMI was 80%, which contrasted widely with the adiposity indices: percentage body fat (BF%) (63%), waist circumference (WC) (43%) and waist-to-height-ratio (WHtR) (59%). Female firefighters (n=78) exhibited lower combined prevalence of overweight and obesity compared to the male firefighters: BMI (43%), BF% (18%), WC (36%) and WHtR (27%). However, misclassification caused by BMI was widespread for both sexes, showing BMI's specificity to be particularly poor, leading to high rates of false positives. This represents the first study to assess the adiposity of UK female firefighters and the first to comprehensively identify BMI to generate widespread misclassification of UK firefighters. Following this, a novel body composition reference system for UK firefighters was developed. This took the form of centile reference curves illustrating age-related changes in fat mass and skeletal muscle mass of firefighters, offering an improvement upon the limitations of BMI and BF%. Overweight and obesity cut-offs were defined at the 85th and 95th centiles which were chosen due to relative suitability and good agreement (97%; Kappa 0.86, p< 0.001) between the BF% reference curves and the fat-mass index reference curves at these centiles. A novel FFQ was developed via population specific modification of the EPIC-Norfolk FFQ and validated against three 24hr recalls. Correlations between the methods were significant (p< 0.01) for energy (r=0.42), carbohydrate (r=0.42), protein (r=0.42), fat (r=0.35), fibre (r=0.34), saturated fatty acids (r=0.36), monounsaturated fatty acids (r=0.32), polyunsaturated fatty acids (r=0.24, p=0.05), vitamin C (r=0.26), calcium (r=0.45), iron (r=0.38) and sodium (r=0.32). Bland-Altman (BA) analyses indicated good agreement between methods for energy and each nutrient, with an average of 96% of cases falling between the limits of agreement. Cross-quartile analysis identified a low mean rate of misclassification (4.2%). In terms of reproducibility, the mean correlation between repeat administrations was 0.7 (p< 0.01), with >95% of cases falling between the BA limits of agreement. This constitutes the first FFQ validated for UK firefighters. A worksite cookery workshop intervention and accompanying cookery book were developed and tested, demonstrating practical methods of healthy meal preparation along with environmental modification suggestions designed to ameliorate the established obesogenic food environment. This resulted in several key significant (p< 0.01) improvements to the mess, including 8 firefighting watches reinstating smaller plates, 10 watches leaving leftovers in the kitchen, 11 watches incorporating wholegrain products and 8 watches switching to making sauces/soups from scratch. Finally, the efficacy of a fire station-based nutrition intervention was tested in a cluster-controlled trial. This encompassed group education on non-communicable disease risk factors and the health benefits of adopting a Mediterranean diet. Dietary assessment was undertaken utilising the newly developed FFQ, which informed personalised nutrition consultation for each participant. Mixed design ANOVAS demonstrated significant improvements at 4-months post-intervention (p< 0.01) in daily mean intakes of energy (-244 kcal), total fat (-12 g), saturated fatty acids (-5g), sodium (-311 mg), sugars, preserves and snacks (-19 g). This contributed to concomitant mean body composition improvements in fat mass (-2 kg), BF% (-1.7%), WC (-1.7 cm), weight (-1.7 kg), BMI (-0.5 kg/m2), mood (+0.9) and energy level (+1.2) compared with the control group increasing in WC (+1.9 cm). In conclusion, the population specific tools can be utilised in a worksite intervention which led to improvements in nutrient intake and body composition. The external validity of the tools, along with the intervention's high level of feasibility further renders it suitable for rollout on a national level.

This thesis describes colon targeted, fast dissolving delivery systems prepared through electrospraying and electrospinning. An overview of relevant literature on electrospraying and electrospinning is given at the start. Biodegradable polymer polyvinylpyrrolidone (PVP), and polycaprolactone (PCL) were used in the projects to produce nanomaterials using simple and straight forward techniques of electrospinning and electrospraying. In the first project, preparation, and characterization of PCL and PVP nanoparticles, using coaxial electrospraying was carried out. Electrosprayed nanocomposite coaxial nanoparticles with polyvinylpyrrolidone 10000 (PVP10) in the shell and polycaprolactone (PCL) 14000 in the core, were fabricated. Spherical, fiber-free particles were obtained with 5% solutions of polyvinylpyrrolidone. Confirmation of the presence of polycaprolactone inside the polyvinylpyrrolidone particles remained a challenge. Due to time constraints further work on this aspect of the project was abandoned, but a useful outcome was that low molecular weight PVP (av. mw10 kDa) could be used to prepare particles less than 100 nm. For the second project, hemin was selected for preparation of formulations for treatment of anaemia, as current treatments have side effects like gastric irritation and low bioavailability. Electrospun nanofibers and electrosprayed nanoparticles were tailored to enhance cellular uptake of low bioavailability drugs (hemin). DMF and MeOH was used to make hemin soluble. PVP-360 and eudragit L 100 were initially selected to make hemin formulations by electrospinning. A variety of electrospinning conditions were attempted and optimal conditions for producing high quality fibers were found to be 0.5 mL/hr, 14 cm (distance) and 12.22 kV (voltage) for PVP/1% (w/w) hemin fibers. Preliminary dissolution tests showed that PVP/ hemin fibres completely dissolved in PBS within 10 minutes. Whereas eudragit/hemin fibres took 2 to 3 days to dissolve. PVP-360 made hemin dissolve successfully in PBS, so further development was focused on developing PVP formulations. DLS (Dynamic light scattering) gave two or three sizes of populations in biological media. The proportion of aggregates increased as the concentration of hemin increased which might be due to hydrogen bond between PVP and hemin.

Increasing concerns about obesity and related metabolic risk have demanded better measures to identify individuals who are at higher health risk. This is particularly important in minority ethnic groups, especially those with a South Asian (SA) heritage. Variation in body composition between ethnicities is important in identifying risk. For example, South Asians have a characteristic body composition with a higher proportion of body fat, lower skeletal muscle mass (SMM) and a tendency to accumulate body fat (BF) in the abdominal region at equivalent BMI’s compared with those of a Caucasian heritage. The higher percentage of body fat (BF%) in SA children tends to track into adult life and consequently increases the risk of type 2 diabetes and cardiovascular disease. Across infancy, childhood, adolescence and even young adulthood, overweight and obesity are defined using statistically derived cut-off values, typically using centile charts based upon age and gender specific reference data. It remains a challenge to define optimum reference cut-offs for any body composition or anthropometric variable because of the age-related changes in growth and body composition which occurs during the growth and maturation. In view of this continuing challenge, the aim of this study was to extend the work previously undertaken in this research group to develop a full range of growth references for better characterisation of body composition in the UK SA population for the 4-21 years age range. Up until now, there had been a gap in data for adolescents and young adults between 16 to 21 years of age, hence the objective of this study was to generate SA- specific centile curves for anthropometric measurements (height, weight, BMI, waist circumference (WC), leg length and sitting height) and body composition measures (fat mass (FM, BF%), fat-free mass (FFM) and skeletal muscle mass (SMM) between the ages 16 to 21 years. This was a field-based study where anthropometric measurements of 546 participants were collected from volunteers from a SA background living in the UK. Single-frequency bioelectrical impedance analysis technology was used for assessment of body composition. Body composition and anthropometric data were used to construct the gender and ethnic specific centile curves using the LMS method for all variables. Comparison of centile charts with equivalent data for Caucasians was performed. It was found that mean height (172.5 cm for male, 159.3 cm for female) for both genders and mean weight for female (57.8 kg) of the population group was lower (as determined by numerical difference in SD score) compared to the UK 1990 reference. Sitting height increased with increasing age whereas there was no or minimal increase in leg length with increasing age. Participants from the SA ethnic group had (numerically) higher WC and slightly high BMI compared to reference population. At equivalent BMIs, SA tended to have a greater WC. BF% in females increased steadily from 6 to 21 years of age while in males BF% decreased after puberty, most likely reflecting the sex hormone driven changes in body composition during adolescence. FM (kg) increased with increasing age and was similar in both genders. At the 50th centile, SA had a lower SMM compared with Caucasians for all age groups in both genders. In conclusion, this study provides a range of assessment tools in the form of SA specific reference centile charts for children, adolescents and young adults in the UK and are the first set of reference centile charts for anthropometric measurements and body composition in this population group. The findings indicate that the pattern of body composition and anthropometry differs in the SA population compared to their UK Caucasian counterparts. These reference charts will allow health practitioners and researchers to measure and plot child and adolescent growth, development and measures of metabolic risk for this population group, allowing for comparisons with reference data for the same gender and age.

Microvesicles (MVs) are shed to the extracellular environment upon activation by both normal and diseased cells. Increasing evidence has shown that cancer cell-derived MVs carry pathogenic components, such as proteins, messenger RNAs, microRNAs, long non-coding RNAs, DNAs, lipids, and transcriptional factors, which can mediate paracrine signaling in the tumor microenvironment, and that they are thus highly capable of altering the function of the recipient cells. MVs released from tumour cells are thought to play an important role in cancer metastasis. Most carcinomas are derived from epithelial cell lines, which normally adhere together tightly to form the structural foundations of many organs. Epithelial Mesenchymal Transition (EMT) has been linked to malignancy in many carcinomas of epithelial origin. In this study, I look at the role of PC3 (Prostate Cancer cell line) derived MVs in the induction of EMT in PNT2 (normal prostate) cells. Uptake of PKH26-labelled PC3-derived MVs by recipient cells was greater at a low pH of 5.4 and was dependent on MV-based protein and phosphatidylserine interaction with recipient cells. Biochemical and morphological changes, indicative of EMT, (elongation, increased expression of mesenchymal marker Vimentin and reduced expression of epithelial marker, E-cadherin) were observed using fluorescent microscopy and flow cytometry. To further investigate the protein cargo of PC3, PNT2 and the transformed PNT2 (tPNT2) cells and their respective MVs LC-MS/MS analysis was carried out. Around 1290 proteins were identified collectively in all the samples. Pathway analysis tools such as DAVID, PANTHER, KEGG and STRING were used to analyse the protein data. Importantly this study identified key proteins in PC3 MVs that may have contributed to the process of EMT, such as integrins which play a vital role in the biology of invasive carcinoma and setting up a pre-metastatic niche.

Northern Cypriots follow a unique diet and little existing literature has documented this. One component, black olive oil (BOO) is distinct from other olive oils in terms of its taste, appearance and its manufacture. The aim of this research was to assess whether BOO, and other components of the diet can influence outcomes for breast cancer (BC) patients in Northern Cyprus, and to assess attitudes and dietary habits of BC patients during recovery. The research was undertaken in North Cyprus and and investigated the dietary habits of a cohort of 140 BC patients with a view to assessing compliance to the Mediterranean diet and BOO consumption. The patients were assigned to two groups, those actively undergoing treatment for BC or those who had stopped being treated for three years or more. It was found that participants frequently consumed olive oil, which is a basic feature of the Mediterranean diet. An enhanced Mediterranean Diet Score (MDS) was significantly correlated with age, education, physical activity, total energy intake, protein, fat, carbohydrate, fatty acids and cholesterol. Similar to previous research, this study concluded that there is a relationship between improved breast cancer biomarkers and olive oil consumption, indicating that high olive oil consumption may aid recovery. A correlation was seen between monounsaturated fatty acid (MUFA) and oleic acid consumption with significantly reduced CEA BC biomarkers. There was also a statistically significant reduction in CA15-3 CB biomarker associated with the consumption of BOO in the active treatment group, but not with in the treatment stopped group. This study is the first to be conducted on the eating habits of BC patients, with a focus on the dietary awareness in relation to BC and the consumption of BOO, in North Cyprus. Interviews from a cross-section of 14 BC patients were analysed qualitatively using interpretative phenomenological analysis (1PA). Results indicated that participants were confused regarding what dietary advice to follow, and variety of sources was used. Widepsread uses of the internet to gain information as well as local opinion were noted. The views of patients on black olive oil fell into two groups: 1) that black olive oil was preferred because of its distinct taste and smell and 2) that black olive oil was not preferred because of its distinct taste and smell. The results indicate a need for clear professional advice on diet, as an aid to BC recovery. The work ends with nutritional analysis of black olive oil and the effects of BOO polyphenols on BC and hepatoma cells and their growth rate. The level of polyphenols in BOO was found to be much lower than in extra virgin olive oil (EVOO) and they had less anti-oxidant activity. It was however found that BOO polyphenols inhibited growth of breast cancer cells at lower concentrations compared with polyphenols from traditional EVOO. Moreover polyphenols extracted from one source of BOO could selectively inhibit growth of BC cell lines compared with a non-cancerous control cell line. The results of this study has shed light on, and identified the need for, further studies on BOO to understand its relationship to human health.

The aim of this research was to enumerate, isolate, identify and characterize lactic acid bacteria (LAB) from Nono, a Nigerian naturally fermented cow milk product for the selection of multifunctional starter cultures to develop a controlled fermentation process for Nono production. This study focused on screening the diversity of the LAB associated with Nono and characterizing their potential probiotic and functional properties including tolerance to acidic pH and bile salt, antimicrobial activity against indicators of food borne pathogens, and resistance to antimicrobials. The LAB were enumerated and isolated from Nono using MRS, M l7 and MRS + LCysteine agars. These media were selected to harvest a diverse range of LAB associated with Nono. Further, they were identified using conventional phenotypic methods including Gram, catalase and oxidase and the Standard Analytical Profile Index (API 50 CHL) identification system. Genotypic methods including repetitive element sequence-based polymerase chain reaction (rep-PCR) and sequencing of the 16S rRNA, phenylalanyl-tRNA synthase a-subunit (pheS) and RNA polymerase, alpha subunit (rpoA) genes were used to identify the bacteria. The sequences were analysed using the Genbank and Eztaxon databases. Phenotyping revealed a bacterial count at a level of 107 CFU/ml for all samples. A total of 128 LAB were isolated and characterized as Gram positive, catalase and oxidase negative and non-spore forming bacteria. The shape and organization of the isolates were variable: rod, cocci, vibrio, V-shaped and coccobacillus bacteria organized as single, pairs or groups. The repPCR allowed the differentiation of multiple groups within the same species and sequencing of the 16S rRNA, pheS and rpoA genes allowed the identification of various genus and species including Lactobacillus fermentum (40%), Lactobacillus senioris (2%), Lactobacillus delbrueckii (23%), Streptococcus thermophilus (22%) Streptococcus infantarius (10%), Leuconostoc pseudomesenteriodes (2 %) and Enterococcus thailandicus (1%>). Further characterisation of the isolates for probiotic and functional properties focused on seven isolates selected on the basis of differences in their rep-PCR profiles. These include Lactobacillus fermentimi, Lactobacillus senioris, Lactobacillus delbrueckii, Streptococcus thermophilus, Streptococcus infantarius, Leuconostoc pseudomesenteriodes, and Enterococcus thailandicus. The isolates were screened for tolerance to different acidic pH and bile salt concentrations to characterise their resistance to gastric acid and bile. The survival of the isolates at different acidic pH varied according to the isolates and incubation time, Lactobacillus fermentum followed by Lactobacillus senioris survived better at pH 3 and pH 4 for 3 h incubation compared to other test isolates. All the isolates survived high concentration 1.5% and 2% of the bile salt for 3 h incubation. The isolates were further screened for antimicrobial activities against indicators of pathogenic bacteria including Samonella enteritidis serovar Typhimurium variant D TI24, Escherichia coli NCTC 12900, Listeria monocytogenes NCTC 11994, Staphylococcus aureus CMCC 1930 and Bacillus cereus LMG 1356. Inhibitory activity of the test organisms was evaluated using a spot test and also spectrophotometric method by measuring and comparing the optical density (OD) of the indicator bacteria after the 24 h incubation period in both test and control experiment. The test isolates exhibited varying levels of inhibition against common Gram positive and Gram negative foodbome pathogens. Among the seven species of LAB screened for antimicrobial activity, Lactobacillus fermentum not only showed broad antimicrobial activities against the indicators but also exhibited antilisterial activity against Listeria monocytogenes and this is of significant spotlight in the starter culture selection. The susceptibility of the selected LAB to 18 antimicrobials was evaluated by screening the Minimal Inhibitory Concentration (MIC) for each antimicrobial. This was followed by the detection of resistance genes by PCR. The ability of two isolates of LAB to transfer to other bacteria the tet(S), tet{M) genes coding for tetracycline resistance and aad(E) gene coding for streptomycin resistance was investigated by conjugation experiments. The latter experiements revealed a variable antimicrobial susceptibility according to the LAB isolate and the antimicrobial tested. The tet(S), tet(M) were detected in the isolates of Enterococcus thailandicus (52) and Streptococcus infantarius(lO). Additionally, aad{E) was detected in Enterococcus thailandicus (52). The conjugation experiments suggested that the tet(S) gene was transferable in vitro from isolates 52 and 10 to E.faecalis JH2-2 and aad(E) from 52 only to E.faecalis JH2-2. Both tet(S) and aad(E) are located at least on plasmids that have mediated the transfer of the genes to Enterococcus faecalis JH2-2 because positive amplicons were obtained in the donors and transconjugants by amplification of the gene from plasmid DNA samples. This research concluded that various genus, species and sub-species of LAB are involved in the production of Nono. The data obtained in this research are relevant for the selection of multifunctional starter cultures for a control production of Nono in Nigeria.

The aims of the research were to investigate the population and diversity of lactic acid bacteria (LAB) in human breast milk, to assess the probiotic potential of the identified LAB, to examine the antimicrobial resistance profile of the LAB and to investigate the possible relationship between the identified bacterial population and diversity with stage of lactation, number of children and diet. Samples of breast milk were collected from 20 breast feeding mothers. Standard spread plating on MRS agar and MRS agar supplemented with L-cysteine HCl (MRS-cys) was used for the enumeration of the organisms. Phenotypic identification of 108 recovered isolates was carried out. All isolates were Gram positive and oxidase negative, some were catalase positive and others were catalase negative. Further identification was carried out by grouping the isolates using repetitive sequence based PCR (rep-PCR). A total of nineteen groups were generated from the rep-PCR DNA profiles. This was followed by genotypic identification using 16S rRNA gene sequencing. The 16S rRNA gene sequencing identified LAB and non-LAB. Most of the LAB isolates belonged to the Lactobacillus genus, the rest were Leuconostoc, Weissella, Streptococcus and Enterococcus spp. The non-LAB were Staphylococcus epidermidis and Staphylococcus hominis. To examine the potential probiotic characteristics of the isolates, at least one representative isolate from each species of LAB (11 in total) were selected and characterised through a series of experiments. Initially the acid and bile resistance of the selected isolates were studied. All eleven LAB showed good tolerance to low pH and high concentration of bile salt. The antimicrobial activity of the isolates was also assessed. Again all eleven LAB produced antibacterial metabolites that inhibited the growth of all the four indicator bacteria (Bacillus cereus, Escherichia coli, Salmonella Enteritidis and Staphylococcus aureus) in both unbuffered and buffered agar spot tests. The antimicrobial activity of the LAB using Agar well diffusion assay revealed antimicrobial properties of some of the studied LAB. The ability of the eleven selected LAB to produce exopolysaccharide, deconjugate bile salt and reduce cholesterol was investigated. The entire eleven LAB produced exopolysaccharide, deconjugate bile salt and reduce cholesterol. The phenotypic antimicrobial resistance profile of the eleven LAB was assessed. Furthermore, the genetic background of the phenotypic resistance was also investigated. All eleven LAB were sensitive to ciprofloxacin, gatifloxacin and quinupristin/dalfopristin and resistant to daptomycin. Most of the LAB were resistant to erythromycin, gentamicin tetracycline and vancomycin. Antimicrobial resistance genes for erythromycin and tetracycline were confirmed for three LAB.

Extracellular vesicles (EV) are heterogeneous populations of small vesicles released by virtually all cells and are classified depending on their biogenesis and size. Microvesicles (MV) are a subtype of EV and are released under optimal cell conditions and/or upon stimuli. In this study, the influence of erythrocyte derived MV (eMV) on the growth of THP-1 and Jurkat cell lines was observed. HeLa cells and those infected with Human Rhinovirus type 16 derived MV (HRV16iHMV) were used as parallel samples for all experiments. This is because virus infected cell derived EV have been extensively studied and it is understood that viral-MV uses the virus mechanism of entry to communicate with neighbouring cells. Whereas the eMV mechanism is still not fully understood. Therefore, to understand the mechanism of eMV action on the growth of cell lines, their characterisation was explored. Flow cytometry analysis using fluorescence sub-micron particle size reference indicates that the samples acquired during this PhD research meet the 'current requirements' in the field, to be classified as MV according to their sizes (100-1000 nm), and that the amount of release of eMV increases depending on the stimulus used (CaCl2, Normal Human Serum), due to remodelling of the cell membrane. Immunoblotting and flow cytometry surface marker profiling data shows that eMV are composed of key molecules that may allow their survival in the extracellular environment and communication with neighbouring cells. CD235 surface marker confirms their parent cells and the presence of CD47 suggests how eMV may avoid phagocytosis. Surface receptors including CD36, CD58 and CD63 indicate how they may communicate and enter the neighbouring cells through endocytosis. The identification of proteases suggests how eMV may be capable of tissue remodelling and degradation, whereas the presence of miRNAs may indicate how eMV can have an impact on the recipient cell by downregulating gene expression. Fluorescence microscopy shows eMV interacting with the cell membrane and the flow cytometry data suggests that eMV inhibit the growth of THP-1. The mechanism which clarifies this impact is unknown. Nevertheless, this PhD project has identified key biological molecules which constitute biologically active entities and might therefore, help eMV to transfer information and substances to recipient cells displaying their potential as delivery vectors. Thus, data obtained here can positively contribute towards the EV field.

Coptidis rhizoma and Euodiae fructus have been used as herbal medicine in Asian countries including China for more than two thousand years. The bioactivities of the crude extracts and isolated compounds of both herbs have been widely reported including anti-cancer activity. Dual herbal formulae combined with Coptis and Euodia with different ratio have been clinically used to treat multiple digestive system disorders. The aims of this research are to evaluate the anti-breast cancer potential of Coptis-Euodia formulae, and standard alkaloids berberine and evodiamine, associated with their possible mechanisms as anti-cancer agent candidates. Thereby, a hypothesis of "Interaction and synergy between compounds might enhance, reduce, or even revise the bioactivities of medicinal herbs and their formulae compared with bioactive components isolated from them." is to be proved. MTT colorimetric assay, scratch wound-healing assay along with adhesion assay have been used to evaluate anti-breast cancer activities of Coptis-Euodia formulae and standard alkaloids. Cell cycle analysis along with topoisomerase I relaxation assay and topoisomerase II decatanation assay were operated to understand the anti-cancer mechanisms of them. Anti-proliferative activity of Zuo Jin pill (Coptis : Euodia = 6 : 1) against both MDA-MB-231 and MCF-7 human breast cancer cell lines has been proved to be the strongest of the three dual herb combination with IC50 values of 0.16 and 0.19 mg/mL. Coptis-Euodia inhibited the cellular migration and adhesion ability of MDA-MB-231 cells which suggested the anti-metastasis potential. Results indicated that Coptis dominated the anti-cancer activities of Coptis-Euodia formulae with the contribution of berberine. Cell cycle analysis describes one of the anti-cancer mechanisms of Coptis-Euodia extracts. Berberine (5 μM) arrested over 60% of MDA-MB-231 cells at G0/G1 phase with 24 hrs treatment, while evodiamine (10 μM) arrested over 90% of MDA-MB-231 and 70% of MCF-7 cells at G2/M phase after 24 hrs. However, the cell cycle arresting activity of evodiamine did not reflect on the Euodia included herbal extracts. TCM formulae, with the presence of Coptis, arrested MDA-MB-231 cells at G0/G1 phase to a great extent. The effects of Coptis-Euodia extracts on DAN topoisomerase I and II further explained the possible anti-breast cancer mechanism of these herbal medicine formulae. All Coptis-Euodia extracts exhibited topoisomerase poison like activities in a concentration-dependent manner. Berberine has been confirmed to behave as a DNA intercalator at 100 μM while evodiamine did not show topoisomerase inhibition activity at the same concentration. Chapter six attempted to establish fluorescence based assay targeted sodium and calcium ion channels on cell membrane. This study is concluded with a discussion of combination therapy potential of herbal medicine and a novel conception of "Artificial Herbal Medicine" which is expected to improve the understanding of traditional used herbal formulae and the standardization of herbal medicine.

Both MVs and exosomes are isolated by similar methodologies such as differential centrifugation of cell culture supernatant (S/N), filtration, floating on chemical gradients or using antibody labelled magnetic beads. Hence, the isolation techniques, commonly in use, provide low levels of purification and therefore require improvements and an in-depth analysis of observed results. This research has established a protocol for isolation of these vesicles and a means to identify them using known markers and their respective properties. The isolation techniques used and developed in this research include differential centrifugation, sucrose gradient centrifugation, and filtration. MVs and exosomes were later identified and distinguished according to their relative ability to bind FITC and PE conjugated annexin V, anti-LAMP1, anti-CD63 and anti-CD11b antibodies using flow cytometry. Protein gels were run with MVs and exosome samples isolated by different techniques to identify some distinct proteins in MVs/exosomes that may be uniquely present on one or either and hence that could be used as a marker to distinguish MVs from exosomes. To prevent exosomal contamination of MV preparations, in a further improvement of the newly developed reverse filtration technique, gentle disruption of exosome aggregates by gentle water sonication was used and measurements of plasma MV levels made from a population of healthy donors. This found plasma MV levels to range between 0.51 and 2.82 x 105 MVs/ml. The MVs were characterised as phosphatidylserine-positive and ≥0.2 μm in diameter. Most of the variables looked at in this study including freezing at -20ºC, gender and subject age did not alter MV absolute counts significantly. For the first time the effect of fasting on MV levels has been studied. Fasting individuals had a wider spread and appeared to have higher MV levels (2.8x105-5.8x105 MVs/ml). This could be defined as the normal baseline fasting reference range, which is almost 3-fold higher compared to the non-fasting group reference range (0.9-1.5x105) MVs/ml. These results suggest that when analysing total MVs, further investigations should use fasting subjects, as for quantification of other fasting analytes. Prospective markers of (monocyte-derived) MVs, IL-1α, RANTES, G-CSF, CCL-1 and IL-17E are proposed for further investigation. MVs were shown to inhibit phagocytosis of apoptotic bodies more effectively than exosomes. With a view to understanding likely pathways being stimulated during MV release, and to also shortlist possible pharmacological reagents capable of being used to therapeutically inhibit Y27632, calpeptin and methyl-β cyclodextrin were shown to inhibit MV release. MV release from K562 cells was inhibited by Y27632 and calpeptin but less effectively by chlorpromazine or methyl-β-cyclodextrin, MV release from THP-1 acute monocytic leukaemia cells was greater than from peripheral blood monocytes which were in turn shown to express more phosphotidylserine than on exosomes. With a view to using MVs as drug delivery vehicles, THP-1 MVs were found to fuse/hemifuse to K562 erythroleukaemia cells, but not at 4°C or if the MV surface was blocked with annexin V. β-Gly (10 μM) (as well as MVs carrying β-Gly [β-Gly MVs]) was found to inhibit proliferation at least 3-fold compared to untreated control. β-Gly and β-Gly MVs increased the doubling time but neither induced apoptosis of K562 cells (unlike MVs from apoptotic cells). Finally, β-Gly increased the percentage of cells in G0/G1 whilst decreasing the cells in G2/M and appears to induce erythroid differentiation as seen by the increase in the percentage of benzidine-stained cells.

The aim of this project was to examine whether exposure to environmental and physiological stress conditions could affect some functional properties for the selection of probiotic microorganisms. The study was focused on two commercial strains of Bifidobacterium animalis ssp. lactis and two non-commercial Bifidobacterium strains, namely B. breve NCTC 11815 and B. longum NCTC 11818. The effects of exposure to acid, bile, osmotic and oxidative stresses on their antimicrobial activity, biofilm formation capacity and antibiotic susceptibility profiles were assessed. The conditions to generate acid stress in the organisms were chosen as pH 3 for one hour, for both B. animalis ssp. lactis strains, and pH 4 for one hour, for B. breve and B. longum. Conditions for bile stress were 1% (w/v) bile for one hour, for both B. animalis ssp. lactis strains and B. breve, and 0.5% (w/v) bile for one hour, for B. longum. Osmotic stress conditions were 3% (w/v) NaCl for one hour, for both B. animalis ssp. lactis strains and B. breve, and 2% (w/v) NaCl for one hour, for B. longum. Oxidative stress was generated for all organisms by shaking at 200 rpm for two hours. The antimicrobial activities of all four bifidobacteria against pathogenic bacteria, namely Escherichia coli NCTC 12900, Salmonella enterica ser. Typhimurium DT124 and S. enterica ser. Enteritidis PT4, were maintained after exposure to each stress, although there appeared to be lower inhibition after exposure to stress. This varied with strain and type of stress. The antibiotic susceptibility profiles of all four bifidobacteria for five antibiotics, namely tetracycline, erythromycin, ampicillin, chloramphenicol and vancomycin, were unchanged after exposure to each stress. The expression of tetracycline resistance gene tet(W) in one of the B. animalis ssp. lactis strains, designated as strain C, was significantly higher (P ≤ 0.05) after exposure to acid, bile and osmotic stresses, although this did not translate to higher resistance of B. animalis ssp. lactis (C) to tetracycline. Effects of each stress on biofilm formation in the four bifidobacteria varied with the strain. In general, more positive effects of exposure to stress were observed in both B. animalis ssp. lactis strains, while more negative effects of exposure to stress were shown by B. breve and B. longum. The expression of exopolysaccharide-synthesis gene gtf01207 in B. animalis ssp. lactis (C) was significantly higher after exposure to osmotic stress, although it also appeared to be higher after exposure to acid and bile stresses. Studying the effects of exposure to stress on in vitro probiotic selection properties could give a better reflection of what applies in vivo, since microorganisms for probiotic use would be inevitably exposed to stresses. This could give a more accurate insight on the potential to provide health benefit. The results of this study may justify the commercial use of the B. animalis ssp. lactis strains.

Contemporary climate change politics, dominated by neoliberal and ecological modernisation framings, has reached an impasse. This thesis investigates whether a Marxist critique focused on trade unions might offer an alternative approach. It critically analyses the social science literature on climate change and utilises insights from the employment relations literature to interrogate trade union activities. The thesis makes original contributions to both the climate change and employment relations literatures. First, it offers a critique of the dominant climate politics and suggests an alternative framing. Second, it proposes a theorisation of organised workers and trade unions as strategic climate actors, applying Hyman's triangular model of market, society and class to understand union behaviour on climate change. Third, it contains a theoretically informed and empirically grounded investigation of UK trade union policy and practice on climate change at the start of the 21 st century. The main findings are that some socialists, trade unionists and activists in the UK have introduced a working class perspective into climate discourse, including challenges to property relations, climate inequality and through mobilisation. Some trade unionists have foreground the occupational aspects of climate change and climate policy, and made distinctive demands for a radical just transition, climate jobs and socially useful work. Trade union climate representatives ("green reps") in some workplaces have made an independent contribution to climate mitigation and adaptation. The occupation of the Vestas wind turbine manufacturing factory and the resulting climate solidarity illustrate the potential power of workers' climate action. The principal conclusions are that workers have the interest and collective capacity to reduce greenhouse gas emissions, to address the differential impacts of climate and climate policy, and to coalesce other actors to tackle climate change. The recent experience of UK trade unions suggests they have a vital role to play as climate actors and, suitably reconfigured, the capability to lead a renewed climate movement.

The growth profiles of five strains of Cronobacter spp. at low temperature (4 oC to 8 oC) and their thermal resistance to mild heat treatment (48 oC to 50 oC) were investigated with a view to developing a model to predict lag times of individual cells, using a strain with a relatively high thermotolerance and able to grow well at refrigeration temperature (7 oC). The effect of heat stress (49 oC for 7 min) and subsequent recovery temperatures (7 oC to 22 oC) on the individual cellular lag times of one strain of Cronobacter turicensis were analysed using optical density measurements. It was found that the distribution of individual lag times of Cr. turicensis shifted right and became more spread when the recovery temperature decreased, and the distribution was more skewed after heat stress. Assuming the lag of a single cell follows a shifted Gamma distribution with a fixed shape parameter, the effect of recovery temperature on the individual lag times of untreated and sublethally heat stressed cells of Cr. turicensis were modelled. It was found that the shift parameter (Tshift) increased asymptotically as the temperature decreased, while the logarithm of the scale parameter (θ) decreased linearly as recovery temperature increased. To test the validity of the model, growth of low numbers of untreated and heat stressed Cr. turicensis in tryptone soy broth (TSB) and infant first milk was measured experimentally between 7 oC and 22 oC and compared with predictions obtained by Monte Carlo simulations. It was found that in TSB, in most cases, the simulations from both models underestimated the actual growth of individual cells of Cr. turicensis from challenge tests; while in first milk, the untreated model slightly underestimated the actual growth at low temperatures (7 oC and 12 oC). The heat stressed model in first milk was generally in agreement with the data derived from the challenge tests and provides a basis for reliable quantitative microbiological risk assessments for Cronobacter spp. in infant milk. The study has made a contribution to understanding and modelling the responses of untreated and sublethally heat stressed Cr. turicensis at 7 oC, 12 oC and 22 oC at single cell level.

Precisely how Hephaestin (Heph) facilitate iron release from cells is poorly understood. The work in this thesis tried to establish the role of different iron metabolic proteins, Mitoferrin (Mfrn), IRPs and Heph in iron homeostasis. Analysis of 18 tagSNPs in the Mfrn gene was carried out in an Asian-Caucasian population to establish any correlation between the Mfrn tagSNPs, haemoglobin levels and birth weight in the presence of covariates such as sex of the fetus, gestational age and mother's booking weight. Two-way ANCOVA analysis was carried out to check if the covariates have any influence on the dependent variable in the presence of fixed factors. From the ANCOVA analysis of Mfrn tagSNPs it can be concluded that neither the haemoglobin levels nor the birth weight are dependent on the genotype, fetal sex, nor on their interaction. Owing to the significance in identifying the interacting partners of IRPs and Heph to understand more about their role in iron metabolism, protein-protein interaction studies were also carried out. IRPs and Heph genes were successfully cloned with One-Strep tag. Full length clones were sequence confirmed for any variation after PCR. Before carrying out immunoprecipitation to identify the interacting partners, transfection efficiency, viability and the role of magnetic particles on K562 cells was performed by using IRPs and Heph cloned with One-Strep tag. Lipofectamine-LTX plus transfection had more viable cells and higher efficiency compared with magnetic-assisted transfection. Also, this study confirms that magnetic nanoparticles do not have any adverse or significant effect on IRPs during the transfection. An unsuccessful attempt was made to identify the interacting partners of IRPs and Heph by immunoprecipitation. The current thesis work also involved identification of a potential ferroxidase. Ceruloplasmin (Cp) was used as a postive control. Non-denaturing gel eletrophoresis of the K562, MDA-MB-231 and PNT2-C2 cell fractions confirmed the presence of the extra band establishing the ubiquitous nature of the band. Mass spectrometry analysis identified the excised band as Calreticulin (CALR). This is the first report of calreticulin having ferroxidase activity.

Background: Global incidence of obesity and Type 2 diabetes (T2D) has increased rapidly in the last fifty years and even more rapidly in immigrant ethnic minority communities in Western countries. There is evidence of a connection between birth weight and the subsequent risk of obesity and T2D in adulthood, with several T2D susceptibility loci reported to be associated with fetal birth weight, including the leptin receptor gene (LEPR). Aims: To ascertain if maternal candidate loci contribute to fetal birth weight variation in a Gujarati South Asian immigrant population in North London and to investigate three variants in the extracellular region of LEPR to determine their effect on LEPR protein structure and function. Methods: Two commercially-available methods (Sequenom iPLEX and Life Technologies TaqMan) were used to genotype specific polymorphisms in the genes HHEX-IDE, ADCY5 and FTO, while molecular cloning and computer modelling techniques were used to investigate the variants in LEPR. Results: Significant associations were found with lower birth weight and rs 12765131 in the HHEX-IDE locus (p=0.002) and with higher birth weight and markers in FTO, namely rs9939609 (p=0.001), rsl7817449 (p=0.0273) and rs8050136 (p=0.0231). No significant associations were found with fetal birth weight and markers in the ADCY5 gene. Although molecular cloning of three LEPR variants was unsuccessful, in silico modelling of the LEPR protein revealed that the Gln223Arg polymorphism was predicted to affect the surface electrostatic potential of the receptor and create hydrogen bonding with a neighbouring cysteine residue, potentially lowering the binding affinity with leptin. Conclusion: These results demonstrate that maternal variants in HHEX-IDE and FTO are associated with birth weight variation in the Gujarati South Asian population. Furthermore, LEPR polymorphisms have been predicted to affect its overall structure and possibly its leptin signalling function.

The research presented in this thesis encompasses a detailed biophysical and biochemical analysis of extracellular vesicles: in particular microvesicles (MVs). The characterisation utilised novel applications of existing protocols, demonstrated for the first time within this thesis, resulting in an up-to-date and greatly improved series of techniques for the analysis of microvesicles. Application of these techniques revealed two distinct subtypes of MVs that can be collected from the same parent cell types in culture, originating via different pathways of biogenesis. cMVs are constitutively released and sMVs stimulated for release. They express distinct biochemistries leading to the potential to perform different physiological functions. These subtypes exhibit distinct FACs morphologies, distinct protein, non-specific carbohydrate and nucleic acid profiles. The techniques used demonstrated in this thesis also show that the MVs have remarkable differences in size as shown for the first time by TEM, flow cytometry, DLS and Nanosight particle tracking. FT-IR spectroscopy was used for the first time to profile similarities and differences between cell lines by analysis of the fingerprint region (1800cm-1 to 900cm-1). The MV subtypes derived from parent cells were analysed using FT-IR to demonstrate similarity in non-disclosed protein expression. BCA protein analysis, modified schiffs test, spectroscopic and quartz crystal microbalance techniques demonstrated cMVs to be smaller, denser and more slowly released from cells than the sMVs are larger, lighter and released sMVs rapidly in response to a stress stimuli. Analysis of calcium homeostasis has revealed that MVs contain high calcium levels, that are deliverable to cells via MVs as cargo. Furthermore, the MV subtypes express different arrays of deliverable proteins and receptors that were transiently expressible upon the recipient cell.

This thesis examines the relation of systems theory to biosemiotics. To this end, it considers the claim that biosemiotics is an uneven development of systems theory. To do this, this thesis explores theories of 'modelling' and 'information' in biosemiotics from the enhanced point of view of systems theory. It will do so following the example set by Deely's 'archaeology of concepts' (1981). By means of the concept of 'isomorphism as structural similarity' (Bertalanffy 1945) the thesis argues that biosemiotics and cybernetics/systems theory share a systems thinking which is grounded in 'transdisciplinarity', 'history' and 'function'. This thesis also argues that such a common methodological perspective is an instance of historical continuity due primarily to biosemiotics' and systems theory's involvement with Tartu-Moscow semiotics. Subsequently, the thesis argues that biosemiotics' and cybernetics' systems thinking differ in their view of 'information'. It shows how biosemiotics broadly conceives information in terms of Peirce's notion of 'abduction', whereas cybernetics conceives information in terms of deduction. It also shows, as a consequence, how biosemiotics' modelling strategies are identifiable with logic as semiotics, while systems theory's modelling strategy is more closely identifiable with mathematical logic alone. Such a methodological difference is argued to be an 'uneven development' (Althusser 1965). The following thesis reveals much that biosemiotics has left unconsidered: the continuing relevance of systems theory, especially in relation to information and observership, and reveals this through the strength of the principle of abduction. Lastly, the thesis 'Biosemiotics as Systems Theory' provides the grounding for a new form of cultural analysis that supersedes the fallacies of semiology. It does so by proposing the following guidelines for the analysis of culture: substituting 'interpretation' with modelling, dispensing of 'representation' in favour of purely objective reality, and recasting 'motivation' in terms of cybersemiotic constraints.

The aim of this research was to study the identity and survival of probiotic bifidobacteria in fermented dairy products from the UK market throughout their shelf- life and carry out in vitro studies on some of the probiotic criteria and potential functional properties of the isolates including resistance to stomach-intestine conditions, antimicrobial activities, antibiotic resistance/susceptibility, antibiotic resistance genes and their transferability, biofilm formation and production of health- promoting secondary metabolites, such as conjugated linoleic acid (CLA). The initial study for choosing the right selective medium for isolation and enumeration of bifidobacteria included a comparative study on selectivity of well known media transgalactosylated oligosaccharides-mupirocin lithium salt (TOS- MUP) and Bifidobacterium iodoacetate medium 25 (BIM-25). Considering selectivity of the medium, recovery of the bifidobacteria and ease of preparation, it was concluded that TOS-MUP is the medium of choice for determination of total viable counts of bifidobacteria in fermented milks. The result of enumeration study indicated that most of the tested products (22 out of total 24) on TOS-MUP comply with the recommended minimum therapeutic level for probiotics (106 CFU/g) at the time of purchase and at the end of their shelf-life. All presumptive Bifidobacterium isolates were identified as Bifidobacterium animalis subsp. lactis by analysis of partial sequences of the 16S ribisomal RNA gene. Further use of rep-PCR for differentiation of all isolates using two different primers (GTG-5 and BOXA 1 R) did not show any difference among tested isolates as all isolates presented the same band profile. As far as the claims on the label are concerned, most of the products (19 out of 24) failed to name the Bifidobacterium species at all or correctly.