A limited range of bacterial specie were identified on cottons from across the world; the most common was Enterobacter. Fungal genera were also similar on all cotton samples analysed, the most prevalent being Aspergillus. Cotton trash material often contained 10-100 times higher contamination levels than associated lint samples. Significant positive correlations existed between the contamination parameters, particularly endotoxin and glucan. Several of these findings suggest that the cotton production environment is more hazardous than previously thought.

There is strong epidemiological evidence of association between PMi0 (particulate matter with an aerodynamic diameter less than or equal to 10 microns) and adverse health outcomes including death and increased hospital admissions for cardio-pulmonary conditions. Ambient PMi0 surrogates such as diesel exhaust particles (DEP), a common component of UK PMi0, have been shown to induce lung inflammation in both humans and rodents. To date, few studies have reported on the toxicological response of UK PM 0 in experimental animals. This thesis summarises the characterisation of the pulmonary toxicity of Cardiff urban PM10. Firstly, the pulmonary toxicological responses in male Sprague Dawley rats following the intratracheal instillation of Cardiff urban PM 0 were examined. A mild, but significant, change in lung permeability was observed in the lung post instillation of a high (10 mg) dose of the whole PMi0 as adjudged by increases in lung to body weight ratio and total acellular lavage protein. Such effects were less marked following instillation of a water-soluble fraction (80% of the total mass) but histological examination showed that lung capillaries were swollen in size with this treatment. Secondly, expression profiling of PM exposed lung tissue identified distinct genes differentially expressed as a consequence of exposure to a high dose (10 mg) of whole PM and equivalent dose of water soluble component of PM. Such changes were linked to different histopathological events within the lung. In conclusion, conventional toxicological, histological and toxicogenomic studies have indicated that Cardiff PM10 exhibits low bioreactivity in the form of mild permeability changes. In vitro toxicological assessment of the bio reactivity of the PM sample using primary cultures of epithelial type II and alveolar macrophage cells were used to identify the relative contribution of the different fractions (whole, washed "durable" fraction and water soluble component) to the overall PM toxicity. Correlations with total metal content and particle size were identified in the different cell types. Cellular analysis using advanced fluorescence labelling and microscopy identified the ability of the PM to induce subtle changes in cell and nuclear morphology that may result in increased cell death and apoptosis even at low particulate doses. Expression profiling of the PM exposed cells proved difficult and realised limited information regarding the cellular responses to the toxicant. This approach failed to identify the same gene changes reported in the lung tissue profiling, illustrating the importance of the different cell types within the lung to orchestrate a pulmonary response. In conclusion, the use of toxicogenomics and gene expression arrays in toxicological is still relatively novel approach to the assessment of lung injury. The study addresses initial problems and offers some possible solutions regarding the use of macro arrays and expression profiling in toxicology research. Expression arrays offer considerable scope as a research tool and in future will provide a powerful insight into gene changes on a global scale.

Bleomycin is a potent anti-tumour compound used in the treatment of squamous cell carcinomas. An unfortunate side effect of this drug is pulmonary toxicity. The onset of this damage manifests as mild oedema and inflammation which eventually develops into pulmonary fibrosis. The ability to correctly identify patients showing early signs of lung injury could significantly reduce the morbidity associated with bleomycin treatment. As such, this study was undertaken to identify genetic markers of early oedema and inflammation. A model of mild pulmonary injury was induced by bleomycin. Conventional quantitative analysis of broncho-alveolar lavage was used to indicate the severity of the oedematous response, whilst morphological changes were identified by histology and electron microscopy. Macroarrays were used to measure the expression of multiple genes during mild, progressive and severe oedema. Following normalisation and statistical analysis, gene expression patterns were compared from saline- and bleomycin-treated rats. A variety of genes were differentially expressed during each model, with the number increasing with the severity of the oedema. A cluster and two individual genes were consistently expressed across two of the models of oedema. The magnitude of the changes in gene expression were quantified and confirmed by quantitative PCR. In summary, complete toxicological and histological characterisation of the bleomycin-induced model of pulmonary injury successfully identified specific endpoints of injury. This model proved to be ideal for studying differential gene expression in response to drug-induced pulmonary oedema. A cluster of ion channels and trafficking genes has the potential to act as a biomarker. Two specific genetic markers (Na+/CI- betaine/GABA transporter, glucocorticoid receptor), and a protein marker (cocoacrisp) have been identified for the oedema. In addition to these genes and protein being potential biomarkers of injury, they are also prospective targets for clinical treatment.

No description available.

The risk of ototoxicity accompanying therapeutic use of aminoglycosides is clinically well recognised. However, detailed description of the incidence, degree, severity and specific patient group risk is usually poorly documented. The aim of this project was to establish the incidence and risk of ototoxicity in patients with haematological malignancies recruited during follow up within the Haematology Department at Leicester Royal Infirmary. A total of fifty patients treated for haematological malignancies were recruited; 33 who had received aminoglycoside therapy as part of their treatment and 17 who had not. Following a comprehensive review of medical history for other potential causes of hearing loss or balance disturbance, patients were then tested with: standard pure tone audiometry (PTA); high frequency pure tone audiometry (HFPTA); distortion product otoacoustic emissions (DPOAEs); computerized dynamic posturography (CDP). There was a considerable incidence of noise exposure in both treatment groups. After accounting for this, the aminoglycoside treated group PTAs returned an incidence of cochleotoxicity of 4/33 or 12%. With HFPTA, there was evidence of hearing loss in the high frequency range for both the non aminoglycoside and the aminoglycoside treated groups. The loss was more marked for the aminoglycoside treated patients. The DPOAE results were interpreted alongside the PTA results and revealed evidence of mixed sites of damage at the cochlear outer hair cells and at a secondary site at the cochlear inner hair cells and/or cochlear nerve. The CDP results returned unexpected and highly significant evidence of mixed contribution to postural control performance deficit in the visuo-vestibular system with equivalent incidence in both treatment groups. None of the above findings showed any correlation with the comparatively low exposures to aminoglycosides or to any of the other drugs these patients may have received. The PTA and DPOAE findings are novel providing physiological evidence of involvement at the inner hair cells/cochlear nerve. The evidence from the HFPTA and CDP studies is considered to reflect potential non specific broad toxic effects due to disease/other therapeutics effects manifest in the cochlea and vestibular apparatus.

Although the pro-apoptotic BCL-2 family proteins BAK and BAX play a key role in mitochondrial perturbation their transition from an inactive closed conformation to a membrane permeabilising pore remains unclear. I found that BAK in viable cells existed in a primed state which was characterized by an occluded N-terminus and an exposed BH3 domain. This conformation facilitated binding to the hydrophobic groove of BCL-XL and served as a checkpoint maintaining cell survival by preventing its further activation. Isolation of BAK by immunoprecipitation suggests that only a discrete portion is present in this primed conformation. Reconstitution of the BCL-XL BAK complex into a BAK/BAX null background rendered cells more sensitive to the BAD BH3 mimetic ABT-737 indicating that primed BAK is primarily involved in ABT-737 induced apoptosis. Primed BAK was displaced from BCL-XL by ABT-737 followed by an N-terminal conformational change and subsequent formation of dimers and higher molecular weight complexes. These sequential BAK activation steps occurred independently of cell fate and did not represent the rate limiting steps in BAK activation as a BAK BH3 mutant L78A lost proapoptotic function but still oligomerised as efficiently as wt BAK. Thus the transition from inactive BAK to a membrane permeabilising pore requires an additional activation step. I demonstrate that after 30 min of ABT-737 exposure primed BAK, after its displacement from BCL-XL, interacts with BIMEL reflecting the transient nature of this interaction. This interaction represented an additional step in BAK activation as BAK pro-apoptotic function was enhanced when BIMEL and BAK were co-expressed. However BIMEL did not induce the N-terminal conformational change nor oligomerisation of BAK and its interaction occurred downstream of both these events. In addition the pool of BIMEL involved in the further activation of BAK did not represent that sequestered by the antiapoptotic proteins BCL-2 and BCL-XL. These data suggest that BAK activation occurs in multiple steps in which a further activation event is required after the exposure of the BH3 domain, the N-terminal conformational change and the formation of high molecular weight complexes but prior to cytochrome c release. I propose that this event may be represented by interaction of N-terminal conformational changed/oligomerised BAK with BIMEL.

In neuronal systems, the degradation of intracellular proteins, controlled by the ubiquitin-proteasome system and autophagy, is of paramount importance for normal cellular homeostasis. The dysfunction of either of these pathways leads to the accumulation of protein aggregates, as seen in neurodegenerative conditions, culminating in neuronal cell death. In the current study I investigated the cleavage of the proteasome subunits, S1, S6´ and S5a in cerebellar granule neurons induced to undergo apoptosis through the withdrawal of potassium. The cleavage of S1 and S6´ and the loss of proteasomal activity corresponded with the activation of caspase-3; however the role of the proteasome was shown to be limited in this model as cells had passed the death commitment point. In addition, I developed a multiubiquitinated fluorescent sensor for the analysis of the proteasomal function on a single cell level, and characterised its use in SH-SY5Y cells. I have also constructed epitope-tagged plasmids encoding the autophagy-related proteins and examined their potential regulation by cell death proteases in an in vitro cleavage assay. Most of the autophagy-related proteins were cleaved in the in vitro model and the potential cleavage sites were identified for mutagenesis. The cleavage of Beclin 1 was also observed in apoptotic cerebellar granule neuron lysates. Finally, I investigated the mechanisms by which the HDACi, TSA, exerts a neuroprotective effect in cerebellar granule neurons. I have demonstrated that it increases the expression of a number of BCL2 family proteins, in particular MCL1, which was hypothesised to contribute to the neuroprotection observed. Taken together, I have demonstrated in this thesis that there are multiple levels of control during cell death; defining their importance is essential for the development of future drug targets.

Doxorubicin, one of the most widely used and effective anticancer drugs, is limited in its therapeutic use by cardiotoxicity. Multiple hypotheses have been advanced to explain the cardiotoxicity. In this project I utilised novel global genomic analysis of mRNA and miRNA transcription and mRNA translation to investigate the mechanism of doxorubicin cardiotoxicity in vivo. A comparator naphthoquinone was employed in parallel to specifically investigate the potential role of redox activity in the mechanism of cardiotoxicity. For both compounds mouse models were used where cardiac damage was characterised at several dose levels (acute and chronic repeat dosing for 7 weeks). A major transcriptionally and translationally affected pathway was the electron transport chain, this was further confirmed biochemically. These changes were reflected by a rapid loss of ATP and an associated increase in the AMP:ATP ratio and associated activation of AMPK indicating a change in cellular energy dynamics. In tandem mtDNA copy number and caspase 3 were rapidly increased. Comparison of all the data led to the hypothesis that the mechanism of doxorubicin toxicity was via interference with the electron transport chain, possibly through electron shuttling, leading to mitochondrial damage and activation of the intrinsic pathway of apoptosis. In further analysis miRNA alterations associated with the cardiotoxicity of both compounds were investigated. Several miRNAs appeared to be intrinsically involved and one of these, miR-181a was followed up in vitro in HL-1 cells and showed an association with susceptibility to doxorubicin cardiotoxicity. The findings provide a novel insight into doxorubicin cardiotoxicity, through the utilization of genomics and suggest the major mechanism of doxorubicin toxicity is via interference with the electron transport chain, and unrelated to the pharmacological action. These data offer the possibility of molecule alteration to retain the pharmacological profile without the associated cardiotoxicity.

The principal aim of this thesis was to begin to determine the possible functions of a novel and B-cell specific protein, FAM129C, identified from proteomic screening of purified CLL plasma membrane fractions. Bioinformatic analysis showed that FAM129C contained a pleckstrin homology domain that probably causes the protein to be associated with the plasma membrane but lacked any other obvious domains. Using quantitative RT-PCR, I showed that FAM129C was expressed from early stages of B-cell differentiation. It was expressed at high levels in chronic lymphocytic leukaemia (CLL) and in the activated subtype of diffuse large B-cell lymphoma, where it may be a useful diagnostic marker. FAM129C was also expressed at high levels in normal B-cell populations including both naïve pre-germinal centre and memory cell populations but interestingly was rapidly down-regulated following stimulation to proliferation. Similar down-regulation was also observed in CLL cells stimulated to proliferation in vitro. Similar down-regulation was also observed in CLL cells stimulated to proliferate in vitro. Subcellular fraction studies of FAM129C showed wide expression in many different cell fractions, but mainly in the cytoplasm. The pattern of FAM129C expression was similar to that of CXCR4 and, therefore I have speculated that there is a potential association between these two proteins in B cell development and in B cell maturation during germinal centre reaction.

Anaerobic bacterial metabolism in the colon produces hydrogen, methane, volatile fatty acids and carbon dioxide. Hydrogen and methane are excreted in flatus and the expired breath. Investigations in this Thesis attempted to identify factors which may influence methane excretion in man and explain why all subjects do not excrete methane. The proportions of methane producing subjects in two healthy populations studied in Edinburgh were 33% and 70% respectively. Age and sex did not significantly alter methane excretion. Dietary intakes, faecal components and bowel function were found to be similar in methane producing and non-methane producing subjects. An association was found between the ingestion of the pentose fraction of non-cellulosic polysaccharides and the concentration of breath methane in methane producing subjects. This association may be the result of a steady metabolic state in the caecum. Ingestion of pentose monomers D(+) xylose and L(+) arabinose increased methane excretion, in methane producing subjects, over five hours of study. The lack of gas production following acute complex polysaccharide administration could be due to a relatively slow metabolic response of colonic bacteria. Production of methane following the acute administration of free pentoses and the absence of methane production after the ingestion of polysaccharide sources may indicate that the release and availability of free pentose monomers from plant polysaccharides may be rate limiting steps in this process. Patient groups with clinically defined diseases appeared to have altered prevalences of methane production compared to control populations. In healthy populations there is great variation in the proportion of methane producers and the concentrations excreted. It is therefore unlikely that the methane status of an individual could be used as a diagnostic aid. Results of methane status may only be significant in population studies. The inaccessability of the human caecum necessitates the use of animal models and in vitro bacterial cultures. Caecectomy and the feeding of an elemental diet to intact rats abolished methanogenesis. A gum arabic supplemented control diet increased methanogenesis whereas methane excretion remained absent when gum arabic was added to the elemental diet. Methanogenic bacteria appear to colonise the caecum and require a fibrous residue or matrix for colonisation and as a substrate. Small but significant amounts of methane were produced by Clostridium histolryticum, C. perfrincens and C. septicum in pure culture. Simple in vitro experiments with the addition of various substrates to the growth medium of two control methanogens, Methanobrevibacter ruminantium and Methanosarcina barkeri, and C. histolyticum gave a confusing pattern of results, however L(+) arabinose increased gas production from each of the bacteria. Human methane production may result from the metabolism of other gastrointestinal organisms such as Clostridia.