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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
81

Laboratory studies to investigate the efficacy and mechanism of action of copper alloys to kill a range of bacterial pathogens and inactivate norovirus

Warnes, Sarah Louise January 2014 (has links)
Contamination of dry surfaces with infectious pathogens can have a significant role in infection spread, particularly if the pathogen is resistant to environmental stressors and the infectious dose is low. The use of antimicrobial surfaces in high risk clinical and community environments could help to reduce cross contamination. Although copper alloys have been known to have medicinal properties for centuries it is only relatively recently that laboratory studies have demonstrated that alloys containing over 60% copper have antimicrobial properties. This study encompasses work done 2008-2013 which has continued to investigate this premise, looking at efficacy of copper and copper alloys to kill newly emerging pathogens which are proving to be a significant risk to global healthcare and also determining the mechanism of pathogen destruction on copper surfaces. Stainless steel which is ubiquitous, partly because of resistance to corrosion, was used as a control surface throughout. Initial work demonstrated that clinical isolates of vancomycin- resistant enterococci were rapidly killed on copper alloy surfaces within a few minutes to 2 hours dependant on the copper content of alloy, size of inoculum or aqueous content of the contamination (mimicking either wet droplet or dry fingertip touch contamination of fomites). In contrast, enterococci persisted on stainless steel for several months. Following increasing concerns about the emergence of infections caused by Gram-negative pathogenic bacteria these studies identified a rapid kill on copper alloys but not stainless steel of food-borne pathogens Escherichia coli O157 and Salmonella, and also multidrug-resistant E. coli and Klebsiella pneumoniae containing the β-lactamase genes bla CTX-M-15 and bla NDM-1, respectively (which are responsible for a wide range of community and hospital acquired infections worldwide with diminishing effective therapies). Further studies identified that release of Cu(I) and Cu(II) ionic species was requisite for copper surface antibacterial toxicity but significant differences in killing mechanism was observed between Gram-positive and Gram-negative bacteria related to their structural dissimilarities. Exposure to copper alloys inhibited respiration in all bacteria tested. Bacterial genomic and plasmid DNA was rapidly destroyed in Gram-positive cells but the cell membrane was not compromised immediately; however in Gram-negative cells the inner cell membrane was immediately depolarised on contact with copper alloys but the DNA breakdown occured more slowly. The outer membrane of Gram-negative bacteria remained intact upon initial contact with copper surfaces. Reactive oxygen species (ROS) are also generated so that in effect the bacteria ‘commit metabolic suicide’ on copper surfaces: hydroxyl radicals generated by Gramnegative bacteria suggested a role for a Fenton reaction although the importance varied between species. In enterococci short term production of superoxide was the principle ROS. The nucleic acid destruction observed in all bacteria tested could prevent the horizontal transfer of antibiotic resistance or virulence genes and allay concerns about the possibility of developing resistance to copper. This was supported when it was determined that transfer of β-lactamase genes from E. coli ST131 and K. pneumoniae to recipient E. coli did occur on stainless steel but not on copper dry surfaces and that transfer was immediate in the former. The incidence of carbapenemase gene bla NDM-1 transfer increased with time on stainless steel, highlighting concerns that persistence of viable cells not only poses an infection risk but also that contamination of the environment with intact DNA also increases the risk of gene transfer. These results support the use of copper alloys as biocidal surfaces to kill pathogenic bacteria and prevent horizontal gene transfer (HGT). The final investigation determined that copper alloys were efficacious in inactivating murine norovirus, a close surrogate for human virus, and exposure to copper surfaces destroyed the RNA genome. Copper ions were still responsible directly or indirectly for the inactivation but ROS were not part of the toxicity mechanism. All the results suggest that copper alloys are effective at destroying a diverse range of pathogenic microorganisms although the mechanisms may be different and multi-faceted. Exposure to copper alloy dry surfaces also prevented the horizontal transfer of genes conferring drug resistance and virulence which has been responsible for the continuing evolution of some of the world’s most dangerous pathogens. The results support the use of copper alloys as constantly killing surfaces in healthcare and community environments in conjunction with regular and efficient cleaning and decontamination regimes using non-chelating reagents that could inhibit the copper ion activity. Recent hospital trials now support this thesis.
82

Synaptic degeneration : a morphological study in a mouse model of prion disease

Al-Malki, Hussain D. January 2012 (has links)
Early synaptic degeneration in prion disease has developed into a subject of interest, because it is thought that it may allow therapeutic intervention to prevent the neuronal death which is often observed at the late stage of the disease. However, the events behind the synaptic degeneration in prion disease, that may ultimately lead to neuronal death, are still unclear. Studying the morphology of neuronal components, namely synaptic boutons, axons, spines, dendrites and cell bodies, of the population of origin may help in understanding the neuropathology of prion disease. Intrahippocampal injection of murine modified scrapie (ME7 homogenate) provides a model of prion disease in vivo. Animals injected with ME7 were compared to control animals (injected with normal brain homogenate, NBH) and both groups were killed at 13, 16 and 19 weeks. At each time point, Biotinylated Dextran Amine (BDA) tracer was injected into the CA3 area of the hippocampus to reveal the morphology of neurons and their components during the disease progression, using both light and electron microscopy. The results showed that at 13 weeks, the number of synaptic boutons, which are distributed along the axons in the CA1 stratum radiatum, was significantly reduced, while most of the remainder were hypertrophied. This correlated with an increase in both the synaptic spacing along the axonal segments and the presence of abnormal swellings on the axons throughout the stratum radiatum. At 13 weeks, electron microscopic studies revealed vacuole-like structures in the synaptic boutons, which differed from actual autophagic or spongiform vacuoles. The results also showed a significant reduction in the number of dendritic spines on CA3 neurons, associated with a reduction in dendritic arborizations and length. Abnormal swellings were also seen in dendrites and occasionally in the cell bodies. Changes in CA3 cell body size were not observed until 16 weeks, when the soma area had reduced. The results indicate that changes in the morphology of synaptic boutons and dendritic spines were observed at an early time point, 13 weeks the fist observation time, and progressed with time. These results showed for the first time that there is a correlation between both synaptic bouton and spine loss in the neuron of origin, which may suggest that there is continual, simultaneous degeneration between the efferent and afferent components of neuron, leading to an early impairment of the neuronal communication within the brain. This suggests the notion that the loss of communication of the neuron at the early stage may lead to neuronal dysfunction and degeneration at the late stage of the disease. The results also suggest that the synaptic vacuoles may play a crucial role in the hypertrophy or degeneration of the remaining synapses. Additionally, it has been shown for the first time that astrocytes in ME7-animals express features of pathology and degeneration, suggesting that the astrocytes may be another target of PrPSc in prion disease. The combination of these findings has opened new avenues in the field of prion studies. The possible mechanisms behind these results are discussed.
83

Effects of fatty acids on inflammatory markers studied in vivo and in vitro

Mohd Yusof, Hayati January 2008 (has links)
Inflammation involves interactions amongst many different cell types as a defense mechanism of the body. Inflammation is also involved in cardiovascular disease (CVD). The role of long chain n-3 polyunsaturated fatty acids (LC n-3 PUFAs) in modulating the inflammatory response has been proposed. The aim of these studies is to investigate the effects of modest intakes of n-3 PUFAs on CVD risk factors especially inflammatory markers, including soluble adhesion molecules, in adult humans with and without CVD and to identify the effects of selected fatty acids, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on inflammatory responses, especially adhesion molecule expression in cultured human endothelial cells of different origin (fetal vs. adults; vein vs. artery). In the first in vivo study, healthy middle-aged men aged 35-60 years were randomized to 1.8 g/d EPA plus 0.23 g/d DHA (n = 9) or placebo oil (2.6 g/day medium-chain saturated fatty acids; n = 11) for 8 weeks. In a second in vivo study, patients awaiting carotid endarterectomy were randomised to 0.8 g/d EPA plus 0.67 g/d DHA (Omacor; n = 47) or olive oil (n = 53) as placebo for between 7 and 102 days until surgery. Supplementation with fish oil in healthy men resulted in a 363% increase in EPA and only a 13% increase in DHA in plasma phosphatidylcholine (PC). On the other hand, Omacor supplementation resulted in significantly increased EPA and DHA in plasma PC by 161% and 70%, respectively. In healthy subjects, there was very little effect of n-3 fatty acids on the risk factors measured (lipid profiles and inflammatory markers), apart from a reduction in plasma soluble intercellular molecule-1 (sICAM-1) concentration compared with placebo (P = 0.05). The change in plasma sICAM-1 concentration was significantly inversely associated with the change in DHA in plasma PC (r = -0.675; P = 0.001). Supplementation with Omacor, however, significantly decreased total plasma cholesterol, triacylglycerol (TAG) and LDL-cholesterol concentrations (P < 0.001) by 13%, 14%, and 5% respectively. In terms of inflammatory markers, supplementation with Omacor significantly decreased sE-selectin by 23% (P = 0.006) and sVCAM-1 by 25% (P < 0.0001), and had no significant effects on other plasma inflammatory markers including sICAM-1 even though trends toward decreases in these markers were observed. This study suggests some anti-inflammatory actions of moderate dose of Omacor in carotid endarterectomy patients. Based on correlation analysis between mRNA expression of inflammatory markers in plaque and plasma concentrations, it seems that soluble inflammatory markers cannot be used to reflect the expression of these molecules at the cell surface, i.e. in the vasculature or in the plaque. In the in vitro experiments the inflammatory stimulus lipopolysaccharide (LPS) up-regulated all three adhesion molecules studied at the protein (as assessed by ELISA) and the mRNA (as assessed by reverse transcription and real-time PCR) levels. VCAM-1 was affected by fatty acids to a greater extent than ICAM-1 or E-selectin. Amongst the fatty acids, DHA has the greatest and the most consistent effects on adhesion molecule protein expression. EPA was also a potent fatty acid inhibitor of adhesion molecule expression at the mRNA level. Some effects of stearic, oleic and arachidonic acids on adhesion molecules were also seen. The effects of fatty acids on the adhesion molecule expression were fatty acid, adhesion molecule and endothelial cell specific. The inhibitory effects of fatty acids were more pronounced in vein endothelial cells than arterial endothelial cells. The precise underlying mechanism on how fatty acids affect adhesion molecule expression remains to be clarified.
84

Development of a sensitive cell culture system to assess prion infectivity and the efficacy of prion decontamination technologies

Secker, Thomas January 2012 (has links)
Creutzfeldt-Jakob disease (CJD) can be iatrogenically transmitted during transplants, grafts and transfusions from CJD infected donors and also contaminated surgical instruments. A variety of methods to amplify and detect the presence of infectious prions as disease markers are available. However, these techniques do not measure the infectivity potentially associated with these markers. Currently, animal-bioassays are used to detect infectivity; however, they have limitations in detectable prion strains, cost, ethical considerations and assay length. Novel cell-based infectivity assays offer the potential to overcome these limitations, lower the requirements for animal use and the application of different cell lines could detect a wider range of prion strains. This project utilised murine neuroblastoma, N2a #58 cells infected with 22L-murine scrapie to develop a highly sensitive assay for the in situ detection of amyloid-rich prion (PrPSc) accumulation as an indication of prion infectivity. The autofluorescence quenching properties of Sudan black (SB) were incorporated into a novel Thioflavin T (ThT) based protocol for amyloid staining with improved specificity and sensitivity. Cell passages were incorporated into the assay to increase incubation time, improve cell viability and subsequently improve assay sensitivity; thus, demonstrating the detection of infectivity from a final 10-10 dilution of 22L-infected brain homogenate. Introduction of 22L-inoculated, surgical grade stainless steel wires to the N2a #58 cells demonstrated the SB/ThT detection of prion infectivity pre and post decontamination, which was comparable to animal bioassay data. Furthermore, preliminary work on the incorporation of the SB/ThT detection of prion infectivity within neural stem cells (NSC’s), for prion propagation within a cell line that did not require genetic manipulation for increased prion susceptibility, highlighted problems with unspecific fluorescence of dead cells during NSC differentiation. Improvements in culture conditions of the NSC’s regarding atmospheric conditions and trophic support were addressed in preparation for their use in future prion infectivity assays.
85

The effect of smoking on the severity, and mechanisms of acute exacerbations of chronic obstructive pulmonary disease (COPD)

Bourne, Simon Charles January 2008 (has links)
COPD (Chronic Obstructive Pulmonary Disease) worldwide has a prevalence of 10% in men and 8.5% in women. Exacerbations of COPD account for approximately 10% of all acute medical admissions. Projected prevalence figures suggest that by 2020 COPD will be the third leading cause of mortality worldwide thus imposing a significant burden on healthcare resources in the future. Acute exacerbations are not only responsible for a decline in the patient’s quality of life, but have a major socioeconomic impact. Following a pilot study that showed current smokers recover lung function much more slowly from their exacerbation than ex smokers, I initiated a properly powered prospective study to investigate the difference between the two groups. A total of 58 patients admitted with acute infectious exacerbations of COPD were recruited to the study to determine the effect of smoking status on their exacerbation. Throughout the admission lung function was measured. Sputum was cultured for bacteria, and PCR used to detect viral infection. Blood and sputum cells were analyzed by flow cytometry. Serum was collected for CRP levels. Ex-smokers recovered significantly more quickly than current smokers in all spirometric parameters (P<0.01), and were discharged sooner (mean 3.08 vs 5.59 days, P<0.001). Sputum culture was positive for more pathogenic bacteria in current smokers, especially H. influenzae, which was associated with a significantly higher CRP rise (p<0.05) than any other organism. CD8+ T cells predominated in the sputum of ex-smokers while CD4+ T cells were the dominant cell type in current smokers (p<0.01). Current smoking is a risk factor for more severe exacerbations, delayed recovery and prolonged hospitalization. This may result from a variety of factors including bacterial, immune mediated responses and systemic inflammation.
86

Determinants of smoke induced lung damage and relationship with metabolic syndrome

Bagmane, Dinesh January 2008 (has links)
Smoking is the major risk factor for COPD. Smoking also has systemic effects and is considered as one of the risk factors for metabolic syndrome (MeS). It is unclear whether it is smoking per se or the systemic effects of COPD that cause metabolic syndrome in smokers. Smokers with and without COPD and non-smoking controls were studied by pulmonary function testing, skin prick tests, body composition, fasting glucose, CRP, and lipids analysis to diagnose MeS. This showed a gradual increase in prevalence of MeS, but with only difference between non-smokers on one hand and smokers with or without COPD on the other being significant. This suggested that smoking, rather than the systemic effect of COPD, was the cause of MeS. All smokers were then grouped and smokers and non-smokers compared in respect of lung function, inflammatory markers (CRP, a series of inflammatory cytokines), insulin resistance, and body composition. Smokers had increased central obesity and total body fat, which is in contrast to the common belief that smoking reduces weight. Male smokers demonstrated increased abdominal fat, while females showed an increase in total body fat. FEV1 was reduced when comparing all smokers with MeS and those without MeS, and there was a greater reduction in males who had a greater prevalence of MeS, but had better quality of life even though they smoked more. However, whilst smokers with MeS had higher levels of insulin resistance, as measured by Homeostasis Model Assessment (HOMA-R), none of the plasma inflammatory markers, except for IL-12, was raised, suggesting that these indices of inflammation were not the reason for MeS. Smoking is associated with a gradual decline in lung function in smokers with and without COPD. A previously recruited cohort of smokers with and without COPD and healthy non-smoking subjects were followed up over a period of 5 years. Amongst a whole series of measurements, including HRCT, measures of lung density/emphysema, only sputum neutrophilia (both absolute and percentage counts) predicted the annual decline in FEV1. In summary, this study suggests that there is an increased prevalence of MeS in smokers associated with insulin resistance caused by smoking but it fails to show an association between MeS and COPD. Smoking is also associated with central obesity and increased body fat, contributing to a reduction in FEV1. Sputum neutrophilia, but not smoking pack years or lung HRCT measurements, predicts the annual FEV1 decline in smokers.
87

Immunological mechanisms controlling chronic inflammatory diseases

Cexus, Olivier January 2009 (has links)
Autoimmune diseases (AID) are chronic inflammatory diseases (CID) mediated by selfreactive T and B cells and are generally the results of the breakdown of T cell tolerance to self-antigen and failure of peripheral regulatory mechanisms. In this thesis I studied different mechanisms controlling the development of CIDs. I investigated the initial events involved in the activation of self-reactive CD4+ T cells which mediate the destruction of the thyroid in a mouse model of spontaneous thyroiditis. TAZ10 transgenic mice express a human T cell receptor (TCR) specific for a cryptic epitope of thyroid peroxidise (TPO) generated upon endogenous processing by thyroid epithelial cells (TEC), and a naturally occurring antagonistic epitope presented by dendritic cells (DC) upon exogenous processing of TPO. I have characterized the function of myeloid derived suppressor cells (MDSCs) in TAZ10 mice. MDSCs accumulate in lymphoid and non-lymphoid organs of TAZ10 mice during acute phases of inflammation and their number decrease as inflammation is fading. Despite their strong inhibitory function on T cell function and proliferation, MDSCs fail to prevent the activation of self-reactive T cells. I showed that the manipulation of MDSCs generated DCs that efficiently promoted the activation of T cells from TAZ10 mice. By contrast, peripheral T cells from patients with rheumatoid arthritis (RA) and lupus had a high proliferative activity compared to controls. Further analysis revealed that RA patients had reduced amounts of inhibitory MDSCs in peripheral blood. I showed that in TAZ10 mice TEC upregulate MHC class II molecules and present the cryptic epitope to TAZ10 T cells inducing their activation. I have demonstrated that DCs are responsible for the spreading of the TPO cryptic epitope from the thyroid to draining lymphnodes (DLN) resulting in the strong activation of transgenic T cells from TAZ10 mice. By adoptive transfer experiments, I showed that the activation of naive TAZ10 T cells occurs within days both in the thyroid and draining lymph-nodes (DLN) and resulted in the destruction of the thyroid. Altogether, this work shows for the first time that in a model devoid of any environmental insults, the normal turnover of TEC is sufficient to induce the activation of self-reactive T cells and the development of AID. In this thesis, I have highlighted the potential role of tissue transglutaminse 2 (TG2) in the treatment of CIDs. TG2 contributes to the pathogenesis of celiac disease and I have showed that TG2 activity promotes inflammation in patients with cystic fibrosis (CF). Mutation of the cystic fibrosis transmembrane regulator gene (CFTR) in CF patients is associated with increased TG2 expression and activity. In CF, TG2 promoted the crosslinking of the antiinflammatory peroxisome proliferator-activated receptor (PPAR) into perinuclear agresomes. The functional sequestration of PPAR was leading to increased inflammation. The finding of this function of TG2 in CF was relevant in TAZ10 mice as in-vivo inhibition of TG2 downregulated common markers of inflammation.
88

The interaction between fibrillar beta-2 microglobulin and serum amyloid P component

Taylor, Garrick F. January 2011 (has links)
Dialysis Related Amyloidosis (DRA) is a serious complication of long term haemodialysis. Amyloid deposits accumulate in the joints causing great pain & restricting mobility of sufferers. The main constituent of these amyloid deposits is fibrillar β2-microglobulin (β2m), although additional components are found which are thought to affect the formation and stability of the β2m fibrils. β2m fibrils formed in vitro under acidic conditions appear to have the same morphology as fibrils formed in vivo under pathological conditions when studied using electron microscopy and atomic force microscopy. However, the in vitro formed fibrils are not stable at neutral pH and quickly dissociate into monomeric and low oligomeric species. This raises the question as to why fibrils do not dissociate in vivo at physiological pH. In vivo serum amyloid P component (SAP) is always found associated with β2m fibrils and thought to stabilise the fibrils by preventing dissociation. Here we present evidence from pull-down assays that SAP binds tightly to acid produced β2m fibrils. The behaviour of the acid produced fibrils with and in the absence of SAP at neutral pH has being characterised using Thioflavin T fluorescence studies and has revealed that SAP does have a small stabilising effect on acid produced fibrils at the concentrations tested. The studies also imply that ionic strength as well as free protein concentration are important determining factors into the longevity of the fibrils at neutral pH. Studies of β2m in inclusion bodies prior to refolding demonstrate that they are not identical to β2m fibrils but that NMR studies do show areas of structural homogeneity with fibrils suggesting that the inclusion bodies may have structure and are not amorphous aggregate as previously thought. Soluble β2m has been assigned using solution-state NMR to identify regions of structural transition between soluble and fibrillar forms of β2m. Solid-state NMR spectra of acid produced fibrils have been acquired at both acidic and neutral pH and reveal that at a molecular level the fibrils are structurally homogenous, giving rise to spectra with site specific resolution. Sequential assignment of fibrillar β2m has therefore been possible using specific labelling techniques to overcome spectral crowding. To identify the interaction interface between β2m fibrils and SAP we have undertaken solid-state NMR studies of β2m with and without SAP bound. Comparing the chemical shifts from these studies has allowed us to identify that SAP is interacting with the side chain carboxylates of fibril aspartates and glutamates. Subsequent chemical modification of these carboxylates to remove their charge resulted in complete inhibition of SAP binding; confirming that they are essential for SAP binding to occur. However there is no strong interaction between monomeric β2m and SAP occurring demonstrating that a collective action of these acidic side chains is needed for binding to occur. Fibrils provide this in the form of acidic strips along the fibril axis brought about by the parallel and anti-parallel beta-strand structure of fibrils.
89

Functional study of ubiquitin C-terminal hydrolase-L1 gene promoter haplotypes

Sanassy, Shane January 2007 (has links)
The Ubiquitin Conjugating System (UCS) describes a system in which the 96-amino acid residue Ubiquitin can be selectively covalently linked to intracellular proteins. This endows cells with an indispensable level of regulation to determine protein fate in a wide range of basic cellular events. The abundant, neuron specific Ubiquitin Carboxyl-Terminal Hydrolase-L1 (UCH-L1) is intimately involved with the UCS – both in a hydrolase and ligase capacity. Mutations in UCH-L1 have clearly been associated with various neurodegenerative disorders, including Alzheimer’s, Huntington’s and particularly Parkinson’s disease. The main and unique objective of this study was to identify any common Caucasian sequence variants in UCH-L1’s promoter, and to investigate whether they are associated with neurodegenerative symptoms, and any change in UCH-L1 transcriptional activity. Seven novel UCH-L1 Single Nucleotide Polymorphisms (SNPs), as well as the C54A documented coding region polymorphism (Ser18Tyr), were identified using both denaturing High Performance Liquid Chromatography (dHPLC) and DNA sequencing analysis. In relation to the translational start site, the novel SNPs elucidated were: A-307G, A-306G, G-234A, A-24G, C-16T, G12A and G21A. Restriction Fragment Length Polymorphism (RFLP) genotyping analysis was then employed within Caucasian DNA sample sets of 31 and 480 individuals, to firstly elucidate the common UCH-L1 promoter haplotypes that exist within the population, and secondly, in an attempt to uncover any association between the polymorphic alleles and general neurodegenerative symptoms - no association was uncovered. Using pGEM-T Easy as an initial ‘holding vector’, the three common UCH-L1 promoter haplotypes elucidated – AAGAC, GAGGT and AGAAC - were incorporated into a modified pGL3 vector to ascertain transcriptional activity rates. This was done by Luciferase expression analysis, and the results identified the GAGGT promoter haplotype as having a significantly increased transcriptional activity in all human cell lines tested. It is my contention, that the pronounced increase in transcriptional activity elucidated for the GAGGT UCH-L1 promoter haplotypes, potentially indicates a primary genetic risk factor for sporadic Parkinson’s disease in the Caucasian population – a novel pathogenic model of which is proposed in this thesis. The fact that RFLP genotyping analysis uncovered no association of the promoter polymorphic alleles with more general neurodegenerative symptoms, indicates the need for further studies to be focused more specifically towards Parkinson’s disease.
90

The effect of gender, pregnancy and diet upon rat tissue fatty acid composition and immune function

Childs, Caroline Elizabeth January 2008 (has links)
No description available.

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