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.

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.

Cycloheximide, a potent eukaryotic protein synthesis inhibitor (Sisler and Siegel, 1967) was used in a biochemical and genetical investigation of the beisidiomycete fungus, Coprinus cinereus. An optimised polyuridylic acid dependant cell-free polyphenylalanine synthesising system was developed for Coprinus cinereus, in order to identify the cellular component conferring cycloheximide-resistance in two cycloheximide-resistant mutant strains, CY 8.2 and CY9.23 In both of these strains, resistance to cycloheximide was found to be associated with the cytoplasmic ribosome fraction. It was not possible to Identify the particular cytoplasmic ribosomal subunit which conferred cycloheximide-resistance. Analysis of cytoplasmic ribosomal proteins by two-dimensional gel electrophoresis did not reveaú. any difference between the small subunit proteins of CY 8 and CY 8.2. There were a considerable number of differences between the proteins extracted from the large subunit of CY 8 and CY 8.2, and CY 9 and CY9.23. There wan no conclusive evidence to identify a cytoplasmic ribosomal protein associated with cycloheximide resistance although several candidates were proposed. An analysis using carboxymethyl-cellulose chromotography did not Identify any cytoplasmic ribosomal proteins conferring cycloheximide resistance. CY 8.2 was one of 174 cycloheximide-resistant mutant strains produced by the ultraviolet mutagenesis of cycloheximide-sensitive strains, according to a method modified from North (1982). Cycloheximide- resistance in each mutant strain was considered to be conferred a single gene, which in those strains examined, was recessive in heterozygous cycloheximide-resistant dikaryons and diploids . The cycloheximide-resistance mutation in all strains examined belonged to the cy-2 complementation group (North, 1982) and hence were allelic with the resistance gene carried by CY 9.23. A classification of the cycloheximide-resistant mutants was proposed on the basis of their growth responses to cycloheximide.

D.vulgaris (Woolwich) OMs can be extracted successfully and reproducibly from the cell envelopes by the selective solubilisation of cell membranes by sarkosyl. Three Omps (Omps 1, 2 & 3) occur consistently in the OMs of C-Fe, NC & C+Fe cells. NC C+Fe OMs have, in addition, minor proteins absent from C-Fe OMs. Omp2 band is seen to be enhanced in C-Fe and NC OMs as indicated by PAGE. PAGE-immunoblotting of LPS indicates heterogeneity of these molecules. Immunological analyses of NC, C-Fe & C+Fe cells using specific antisera indicate no differences among these cell-types. Neither are antigenic differences revealed by further analyses of OM Western blots by specific antisera. However, pyrolysis analyses indicate that the cell-types analyses show variations in the cell ability of this technique to bacterial species cultured under different growth conditions indicates its potential as a tool for numerical taxonomy of the sulphate reducers. D.vulgaris (Woolwich) does not produce extra Omps in response to iron deprivation. Instead, it increases the synthesis of 0mp2 and LPS in its OM as reflected in the OM PAGE pattern and increased yields of LPS extracted from C-Fe cultures. HPLC sugar analyses indicate the LPS contain N-glucosamine, possibly rhamnose and an unidentified sugar. A change in HPLC sugar pattern of the C+Fe LPS samples indicates interactions between Fe(II) and LPS. This interaction is substantiated by X-ray microanalysis of the elemental content extracted LPS. The evidence indicates that LPS may be involved in Fe(II) uptake by the cells. LPS also takes part in the adhesion of D.vulgaris (Woolwich) to mild steel surfaces. This is demonstrated by the data from experiments using anti-LPS Fab fragments. The presence of these antibodies reduces the number of cells adhering to mild steel coupons. Fe-radiolabelling of OM components immobilised on nitrocellulose shows that Omps 1, 2 & 3 bind Fe(II). Blocking experiments using copper & magnesium indicate Omp1 to be Fe(II) specific while Omps 2 3 are not. The enhancement of Omp2 observed in C-Fe NC OMs in PAGE analyses indicates Omp2 to be principal cation binder, whose synthesis is increased under iron deprivation.

It has been established that the previous synthetic route to 3-benzyloxy-l, 2-dimethylpyrid-4-one and l,2-dimethyl-3-hydroxypyrid-4-one, under the reported conditions, does not lead to their free bases but to their protonated forms. The structures of those pyridones in both free base and protonated forms have been clarified by spectroscopic techniques and a hydroxypridinium structure is proposed for the salts. The direct synthesis of l,2-dimethlyl-3- hydroxyprid-4-one, has also been achieved by Modifying a reported route. Two new, and general synthetic routes have been investigated in a preliminary way. One route involving the reaction of l-bromobutano-2,3-diono with N?-methylbenzamide, has Ied to the recovery of the starting axide and decomposition of the bromo compound. The second route involves the use of pyridine as the starting material. In this study, 1-ethoxycarbonylpiperidene-3,4- diol was synthesised using a previously reported procedure. The oxidation of this diol with Jones' reagent gave a mixture of products, one of which was identified as the desired product, 1-ethoxycarbonylpiperidene-3,4-dione using G.C.-mass spectrometry techniques. ...

Satisfactory preparations of cell walls from Desulfovibrio vulgaris cells grown in iron rich (C+Fe) and iron poor (C-Fe) media were obtained by partial detergent solubilisation of the cell envelopes. Polyacrylamide gel electrophoresis (PAGE) has shown the presence in the outer membrane (OM) of three major proteins (OMPs l, 2 and 3) and a relatively homogeneous lipopolysaccharide (LPS) containing no ketodeoxyoctonate (KDO). A protein of OMP 1 PAGE mobility is readily removed by the EDTA treatment of cells showing its loose association with the cell surface. Only a portion of OMP l is removed by digestion or acetate extraction indicating the existence of two proteins, OMP la and lb which comigrate in PAGE. 1251 lactoperoxidase labelling of whole cells shows proteins of OMP 1 and 3 mobility to be exposed on the cell surface. OMPs do not show hydrogenase activity and this enzyme is inactive in C-Fe cells. Numerous minor proteins are present in C+Fe OM but are absent from C-Fe OM indicating ’protection’ of the cell wall by iron to detergent solubilisation. In response to iron limitation D.vulgaris Woolwich shows an increase in the proportion of carbohydrate in the cell wall and in the yield of extractable LPS. HPLC studies have shown changes in the LPS sugars with iron limitation indicating an interaction between Fed I) and the LPS. Studies on the release of LPS from Intact cells by an EDTA washing procedure have shown a selective interaction between Fe2+ and LPS In D.vulgaris. Fe(II) appears to have an important role in the stabilisation in vivo of the OM and this selective interaction may play a part in iron uptake. In vitro OM reconstitutions using extracted material have given further support to this selective interaction. The incorporation of acetate extracted OMPs into reconstituted OM has indicated a possible iron transport function for one or more of these proteins. Calculations of Fe2+ :LPS molar binding ratios based on compositional data and these in vivo and in vitro studies show two forms of iron binding to the surface LPS, which may be applicable to the parameters at work in the natural environment of the bacteria.

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.

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 project is concerned with the total synthesis of plant growth regulators related to abscisic acid (ABA). The biological activity of these plant growth regulators, ABA and Xanthoxin, and their derivatives is influenced by the stereochemistry of the double bond system in the side chain, the 2Z,4E-isomers being most potent.