Selected snake and spider venom fractions were screened for in-vitro insulinotropic activity in glucose responsive BRIN-BDll cells, with insulin secretion measured using radioimmunoassay. Significant insulin secretion was noted for 27 snake venom fractions, containing a diverse range of snake toxin families including phospholipases A2, a-neurotoxins, disintegrins, serine proteinases, CRISP (cysteine-rich secretory proteins), metalloproteinases and nucleotidases. The partial N-terminal sequences are reported for 15 snake venom components. Elevated levels of insulin secretion were recorded for 16 fractions from the Grammostola rosea venom and 31 fractions from the Aphonopelma chalcodes venom. The synthetic version of a novel 28 amino acid residue peptide isolated from the Aphonopelma chalcodes produced a significant concentration dependent increase in insulin secretion. A number of theraphotoxins are proposed as constituents of the active Aphonopelma chalcodes fractions. Paliial sequences are presented for 3 unknown Grammostola rosea peptides with insulinotropic activity reported as a novel function of a number of known Grammostola peptides. A microtitre assay was used to assess antimicrobial activity of snake and spider venom against both Gram-positive and Gram-negative bacteria. Snake venom fi.-actions containing an L-amino acid oxidase or metalloproteinase component showed preferential activity against Staphylococcus aureus, while phospholipases A2 were most active against Bacillus subtilis. Activity against Salmonella typhimurium was greatest for fractions containing L- amino acid oxidase. Escherichia coli was least susceptible to the test fractions. Antimicrobial activity for the Aphonopelma chalcodes crude venom was confmed to fractions 26 to 35, containing low mass compounds of mJz 730- 830 alongside a peptide component of mJz 2919, for which the novel sequence is reported. A quantitative study was performed on the selected low molecular mass components isolated ill the Hap/ope/rna lividurn spider venom usmg liquid chromatography / electro spray ionisation mass spectrometry. This study of the bioactive constituents of snake and spider venom serves to enhance the existing body of evidence supporting the study of venomics in pursuit of novel leads for pharmaceutical research and development.

The identification of the chemical acrylamide (AA) in food was followed by intense research which led to the discovery that AA forms in starch-rich foodstuff when roasted, fried and baked, due to the reaction of the amino acid asparagine and reducing sugars. Animal studies have shown the carcinogenic effect of AA leading to tumours in multiple sites and in 1994 the IARC classified AA as a probable human carcinogen. After ingestion AA is metabolised to the reactive epoxide glycidamide (GA) that is able to react with bio-macromolecules such as DNA and haemoglobin (Hb). Three major DNA adducts of GA have been reported of which the N7-(2-carbamoyl-2-hydroxyethyl)guanine (N7-GA-Gua) is the most abundant. The aim of this project is to develop a sensitive method for the detection of the N7-GA-Gua adduct in human leukocyte DNA and urine with two different analytical techniques, namely mass spectrometry, applying LC-MS/MS, and the immunoassay ELISA. A mass spectrometric method was developed utilising online column-switching achieving a LOD of 7 adducts/10[superscript 8] nucleotides and a LOQ of 9 adducts/10[superscript 8] nucleotides for the detection of N7-GA-Gua in human leukocyte DNA. The developed method was applied to analyse the leukocyte DNA of 32 healthy volunteers. In a few samples peaks were detectable, indicating the presence of the N7-GA-Gua adduct but below the LOD and with a high variability. For 10 samples the AA- and GA-Hb adducts were analysed; AA-Hb adducts correlated with AA intake 24 hours prior to donation but there was a non-significant correlation between Hb adducts and N7-GA-Gua adduct levels. It was not feasible to develop a method for the detection of N7-GA-Gua in urine due to the high salt concentration of this matrix and difficult clean-up procedures prior to LC-MS/MS. Two polyclonal antibodies were raised against N7-GA-Gua to develop a competitive ELISA but due to very strong binding with low sensitivity towards the N7-GA-Gua adduct the antibodies were not suitable for use in an ELISA assay. Aiming for a better sensitivity might allow detecting the N7-GA-Gua in human samples.

Botulinum neurotoxins are one of the most potent toxins known to man; they work upon the nervous system blocking the release of chemical neurotransmitters causing flaccid paralysis. Despite their toxicity when administered in low doses they have therapeutic benefits and are used to treat conditions such as muscle spasms and twitches. Due to their extreme toxicity methods of determining the concentration of botulinum neurotoxins in pharmaceutical products must be highly sensitive and accurate. Currently the mouse bioassay is used to monitor the activity of the toxin however this assay is lengthy and has both cost and ethical issues due to the use of live animals. Replacement assays would have a large potential market with a number of users benefiting from them. These include the pharmaceutical industry, clinical environments for point of care sensors in suspected cases of botulism, the food industry for quality control steps and the military for detecting botulinum toxins being used as biowarfare agents. This project details the development of different biosensors for the detection of botulinum neurotoxin all based on measuring the changes to a monolayer of the toxin specific protein SNAP-25. SNAP-25 is selectively cleaved by botulinum neurotoxin types A, C and E; this project focusses on type A as this is used by IPSEN Biopharm in their product Dysport@. In the assays presented in this thesis SNAP-25 is immobilised on gold substrates through its four, naturally occurring, cysteine amino acids which are clustered together in the middle of the protein. The changes to this protein are then monitored electrochemically by cyclic voltammetry or electrochemical impedance spectroscopy and through colourimetry using a UV-visible spectrometer and a microplate reader. These assays have been successful in identifying botulinum neurotoxin with the most sensitive the electrochemical impedance assay, detecting down to 500 ag/ml and the quickest, the microplate reader, only taking seven minutes to perform. The results also show clear differences between the toxin product and the placebo, which contained the excipients RSA and lactose, thus proving that the assays are detecting active botulinum neurotoxin.

Endocrine disruptors (EDs) have been associated with various disorders including disrupted reproductive, metabolic and immune function. We are exposed to EDs via environment and through our diet, thus it is of real importance to monitor its main components, such as milk, for ED contamination, its composition and biological effects. The aim of the thesis is to assess biological activity of various environmental contaminants gaining entry to the food chain as well as to analyze a variety of milk samples for their total estrogenic hormonal load and its chemical composition. The assessment of environmental contaminants has been performed employing estrogenic reporter gene assay (RGA) and revealed number of industrial chemicals, possessing estrogenic activities, some quite substantial, including UV-filters, parabens, phthalates, pesticides and their metabolites. For the analysis of milk samples two assays have been developed and validated according to EU 2002/657/EC criteria i.e. estrogenic RGA and ultra high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) targeted method. Results of a screening of a range of milk samples revealed an extensive milk contamination with both natural and man-made EDs. The chemical contamination did not translate to enhanced estrogenic load as only 3% of samples presented an increased response which origins have not been confirmed. Performed risk assessment suggested possible risk to children in vulnerable windows of development which should not be underestimated. Also employment of fractionation with subsequent combined biological and untargeted chemical analysis has been investigated and provided an insight into the possible origin of biological activity while analysis of milk for the presence of masked EDs revealed higher concentrations of contaminants resulting in an enhanced estrogenic load. Overall, the thesis underlines the importance of constant screening of food commodities for ED contamination and highlights the advantages of employing combined biological and chemical assays to facilitate accurate risk assessment.

Antivenoms, produced by immunising animals with appropriate snake venom or venoms, are the only available specific treatment for snake envenomation. In this thesis monospecific ovine antivenoms were raised against venom fron the South American rattlesnake, Crotalus durissus terrificus, using a novel immunisation procedure (in respect of antivenon production) whereby small doses of venom were given at monthly intervals.

Bacillus cereus is one of the known causes of diarrhoeal food poisoning. In their natural environment of soil surviving as spores facilitates their colonisation of raw food ingredients enabling their access to the food chain. Recently psychrotrophic strains of B. cereus have been reclassified based on divergent cold shock gene (cspA) sequences and renamed B. weihenstephanensis. It is the modified cspA gene that is thought to confer the psychrotolerant phenotype witnessed by these strains. Aside from cspA, B. cereus and B. weihenstephanensis are closely related, leading to questions about its pathogenicity and ability to mediate diarrhoeal food poisoning outbreaks. Food producers use a variety of processes to limit microbial contamination within food products. Although effective against vegetative cells, spores are often resistant and as such can persist within this environment. Chilled temperatures (4°C) are often used to limit the growth of any contaminating microbes. Under such conditions B. cereus spores would remain dormant however B. weihenstephanensis spores have been shown to germinate and outgrow under refrigerated conditions. This could result in the consumption of both B. cereus and B. weihenstephanensis spores and vegetative cells. The effect that the human gastro-intestinal tract (GI) has on B. cereus and B. weihenstephanensis vegetative cells and spores is unclear. This study showed no difference in the viability of B. cereus or B. weihenstephanensis strains to survive and grow within a simulated human GI tract. Vegetative cells were revealed to die quickly in the stomach. Spore viability was shown to reduce in the stomach environment by approximately 10⁴-fold. With a larger initial inoculum, 10⁷ spore/ml, viable spores were still recorded after 4 hours. These spores subsequently germinated within the small intestinal simulation and the resulting vegetative cells rapidly proliferated. Mass spectrometry illustrated the ability of vegetative cells from both B. cereus and B. weihenstephanensis to produce an array of secreted proteins whose function were predominately related to virulence and pathogenesis. B.weihenstephanensis strain 10202 was shown to produce the potent cytotoxin, CytK-1, while other B. weihenstephanensis and B. cereus tested strains possessed either or both Nhe and Hbl toxins. The primary diarrhoeal virulence factor/haemolysin BL was shown to be present in the supernatant of each strain through western blotting. Significantly smaller concentrations of each protein were detected, however, under simulated human GI tract conditions when compared to optimal conditions. The effects of the simulated human GI tract on virulence gene expression were monitored through real time PCR. No pattern between B. cereus and B. weihenstephanensis strains was found confirming that virulence gene expression is strain specific. Some genes were shown to be significantly upregulated such as fur, (the ferric iron uptake regulator and groEL, encoding a molecular chaperone. The expression of others however was reduced such as haemolysin BL components, hblA and hblC. Overall there were no significant differences detected between B. cereus and B. weihenstephanensis strains in their ability to survive the human GI tract and express virulence factors associated with diarrhoeal food poisoning.

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