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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.
21

Comparison of the effects of dietary flavonoids and statins on lipopolysaccharide-induced vascular inflammation

Alshalmani, Salmin Khalid January 2011 (has links)
Numerous epidemiological studies indicate that flavonoid intake as part of a balanced diet confers beneficial health effects in man, including improved cardiovascular function, reduced incidence of cancer and amelioration of symptoms associated with inflammatory disorders (Boots et al., 2008). A recent area of interest that may be fruitful is the study of anti-inflammatory effects of flavonoids in combination with statins. Porcine coronary artery (PCA) segments were incubated overnight at 37°C in modified Krebs-Henseleit solution with or without 1µg/ml lipopolysaccharides (LPS), with either (0.1–10µM) quercetin, or 10µM quercetin 3′-suphate and 10µM quercetin-3-glucuronide, or with (0.01-10µM) epicatechins, 10µM catecchin and10µM epigallocatechin gallate. (0.03-3µM) simvastatins and 10µM pravastatin are also used in this study. In addition, since many quercetin-rich foods also contain significant amounts of myricetin, this flavonoid has also been examined. After 16 to 18 hours, segments were prepared for isometric tension recording in Krebs-Henseleit solution. The segments were then exposed to cumulatively increasing concentrations of KCl and then U46619. Responses are shown as milliNewton or calculated as the concentration causing 50% of the maximum effect (-log EC50). For nitrite measurement, segments of the PCA were incubated in DMEM at 37°C for 24 hours, with or without 1μg/mL LPS. The nitrite content (nmol) of the bathing medium was determined by spectrophotometry using the Griess reaction, while inducible nitric oxide synthase was identified immunohistochemically. Differences between mean values were assessed by ANOVA (post-hoc Dunnett test). Prolonged exposure to LPS caused hyporesponsiveness of the PCA associated with increase in nitrite production by a mechanism that appears to involve the induction of nitric oxide synthase. Nitrite content of the incubation medium increased 3 to 10-fold following exposure to LPS and inducible nitric oxide synthase was detected in the adventitia. The results indicated that all of the tested flavonoids and statins are able to suppress LPS-induced changes in vascular responses, nitrite production and expression of inducible nitric oxide synthase. While 10µM myricetin was inactive. In conclusion I have demonstrated that quercetin, and its principal human metabolites and catechins oppose pro-inflammatory events in both endothelial cells and vascular smooth muscle cells. Possibly through a mechanism involving inhibition of NFkB. Since pre-treatment of the PCA with statins reduced LPS-induced changes in vasoconstrictor responses, suppressed the induction of nitric oxide synthase caused by LPS and the associated increase in nitrite production. It is unlikely that the effect of the statin involves direct inhibition of NOS. These findings are consistent with clinical studies suggesting that prior use of statins may afford protection against bacterial sepsis.
22

Structural and genetic analyses of the RdgC protein in Escherichia coli

Yu, Jing January 2009 (has links)
Previous studies found that RdgC protein plays a role in the DNA repair system in Escherichia coli. In recBC sbcBC strains, loss of rdgC made growth of the strains dependent upon recombination, hence Recombination Dependent Growth. RdgC was also found to regulate the activity of RecA, a key protein in recombination, both in vivo and in vitro. The function of the protein, however, remains unknown. In this study, I purified and crystallised the RdgC protein. The crystal structure of the protein was then revealed as a homo-dimer, with a head to head, tail to tail organisation, resembling a ring structure. To further investigate how RdgC binds DNA and its in vivo functionality, point mutations and chunk deletions were designed and constructed; and I examined all the mutant proteins in DNA binding shift assays in vitro and in synthetic lethality assays in vivo. A DNA binding model was then proposed based on the results of the DNA binding shift assays. The mutant studies in vivo reinforce the idea that the DNA binding activity is crucial for RdgC’s function in Escherichia coli.
23

The evolution of transposable elements in humans and Drosophila

Styles, Pamela January 2010 (has links)
The different genomic environments in which transposable elements reside in the Great Apes and the Drosophila result in substantial differences between the evolution of transposable elements in these two groups of organisms. In the Great Apes, where deletion of transposable elements is relatively rare, elements tend to be retained in the genome to the extent that complete sets of elements belonging to a particular transposable element family can be obtained. In Drosophila, there is a rapid turnover of transposable elements, imposing strong selection pressure on transposable elements to be able to infect new hosts. This study investigates the evolution of transposable elements in these two genomic environments. Complete sets of elements belonging to young Alu subfamilies in humans and closely-related species are used to investigate factors involved in their evolutionary history, such as mutation and gene conversion. The application of the master gene model, and other proposed models of the proliferation of young Alu subfamilies, are considered in light of the results obtained. The evolution of the AluYg, Yh and Yi lineages are investigated using a C++ program to simulate their evolutionary history. The results of the simulations are compared to statistics such as theta and pi, as well as the number of shared mutations and the proliferation time, in order to determine possible, and likely, values for parameters such as the retrotransposition rate and the number of source elements for each subfamily. The results suggest that although the master gene model may apply to some lineages, it is not the best model to explain the evolutionary history of all young Alu subfamilies. The selection pressure on transposable elements in Drosophila results in a high level of horizontal transfer of these elements among species of the Drosophila genus. In this study, the twelve sequenced Drosophila genomes are used to investigate the frequency of horizontal transfer within these twelve species using a large dataset of transposable element sequences from the DNA transposons, as well as LTR, and non-LTR, retrotransposons. Horizontal transfer is inferred where identity between transposable elements of the same family in different species exceeds that between the coding regions of the Adh gene in the relevant species. Cases are further supported by evidence from the distribution of the transposable element family across the Drosophila genus, and phylogenetic incongruence, which in many cases elucidates likely directions of transfer. The results suggest that horizontal transfer may be even more common than previously thought, and appears to be most common for the LTR retrotransposons. The possibility that possession of the env gene may result in higher rates of horizontal transfer of LTR retrotransposons is investigated, and the env open reading frame is found to be under selective constraint.
24

The induction and effects of Substance P and its receptor in human immune cells and neurons : potential relevance in multiple sclerosis

Vilisaar, Janek January 2012 (has links)
INTRODUCTION: Substance P (SP) has well-established roles in neurogenic inflammation and pain transmission, however recently, a number of SP immunomodulatory effects have been shown. In this thesis SP and its neurokinin-1-receptor (NK1R) role in autoimmune inflammation was investigated with an applicability to multiple sclerosis (MS). In the four experimental chapters the role of SP and its receptor was studied in human immune cells and neurons with a focus on the relationship with Th17 and Th1 pathways as the main pro-inflammatory arms in autoimmune pathology. AIMS: To quantify the effects of SP on inflammatory cytokine induction in peripheral blood mononuclear cells (PBMC); to measure Th17 and Th1 pathway effects on SP and NK1R expression in T cells and NT2 neurons; to compare NK1R expression and relevant parameters in peripheral immune cells of relapsing-remitting MS patients and healthy controls. METHODS: Real-time PCR, flow cytometry, ELISA, Western blotting and promoter studies were used to measure the expression of target genes under different stimulation conditions. Cells were isolated from consented healthy controls, relapsing-remitting MS patients, or differentiated as specified. RESULTS: In PBMC, treatment with SP significantly increased the relative quantity of IL-12/IL-23 subunit p40, IL-23 p19 and IL-12 p35 mRNA showing that SP can signal induction of IL-12 and IL-23. As part of the reciprocal mechanism in T cells, NK1R and SP expression was strongly upregulated by Th17 cytokines and significantly less by Th1 cytokines. These effects for NK1R were confirmed at promoter and protein levels. The Th17 effects were prevalent at earlier stages compared to the Th1 effects. As a novel finding, IL-17 (IL-17A) had direct effects on neurons via its functionally expressed receptor. Neuronal NK1R mRNA-level expression was subject to regulation by IL-17, whereas SP precursor was considerably less upregulated by IL-17. In MS patients in a relapse NK1R mRNA in peripheral immune cells was strongly downregulated as compared to controls. This finding is likely associated with the inflammatory activity in an acute MS relapse. CONCLUSIONS: Mutual interactions exist between SP and Th17, Th1 responses with SP showing involvement in Th17 and less in Th1 pathway effects. This supports NK1R role in mediating autoimmune activity as occurs in an acute MS relapse. The results also show direct neuronal involvement in immune interactions involving SP and Th17 pathway.
25

Vascular actions of oleamide in health and disease

Hopps, Jamie January 2013 (has links)
Oleamide, an endocannabinoid-like mediator, is a fatty acid that shares structural similarities with anandamide. Oleamide induces cannabimimetic responses and is a potent vasodilator of rat small mesenteric arteries. The cardiovascular actions of oleamide have received relatively little attention in comparison to those of anandamide, the prototypical endocannabinoid. The aim of this study was to examine the vascular effects of oleamide in both health and disease, making a comparison with those of anandamide. This study demonstrated that oleamide caused vasorelaxation of the rat isolated aorta. The vasorelaxant actions of oleamide were found to be tissue dependent as oleamide did not evoke vascular responses in the porcine mesenteric and coronary arteries. Anandamide did not produce similar responses to oleamide in any of these vessels, displaying marked differences between the two compounds. Oleamide-induced vasorelaxation of the rat aorta was abolished by capsaicin pre-treatment but this was independent of sensory-nerve activity. This demonstrates a potential additional site of action for oleamide and prompted further investigations into the vascular actions of capsaicin. Oleamide also caused relaxation of the rat perfused whole mesenteric arterial bed. This response was diminished by a depolarising concentration of extracellular K+, implicating the involvement of K+ channels. Capsaicin evoked relaxation of both rat aortae and porcine coronary arteries. The vasorelaxant effect of capsaicin was insensitive to capsaicin pre-treatment and the presence of capsazepine, a TRPV1 antagonist. It was also found that the presence of capsaicin inhibited the uptake of Ca2+ in depolarised porcine coronary arteries and rat aortae on reintroduction of calcium. In porcine coronaries, capsaicin abolished the contractile response to Bay-K 8644, a L-type calcium channel activator. Therefore, it is proposed that capsaicin inhibits L-type calcium channels to drive vasorelaxation, demonstrating a TRPV1-independent mechanism of action for capsaicin. Having described the vasorelaxation of Wistar aortae, the effects of hypertension on the vascular actions of oleamide were determined. Oleamide-induced vasorelaxation was significantly enhanced in aortae from spontaneously hypertensive rats (SHR) compared to those from normotensive Wistar Kyoto (WKY) controls. Oleamide caused approximately 40% relaxation of the SHR aorta compared to 15% in the WKY isolated aorta. Similarly, responses to anandamide were also increased in aortae from hypertension causing 30% relaxation compared to 10% in arteries from normotensive controls. Augmented vasorelaxation to oleamide and anandamide was opposed by pre-treatment of vessels with capsaicin, an effect independent of TRPV1 receptors. Inhibition of cyclooxygenase with indomethacin potentiated responses to oleamide in WKY aortae to a level comparable to responses in SHR aortae. Thus, this thesis suggests that changes in the cyclooxygenase pathway are important in regulating responses to oleamide in hypertension and may represent an adaptive change in the early stages of established hypertension in SHR rats. In summary, this study provides further evidence of the vasorelaxant nature of oleamide, which can be enhanced in arteries from hypertension. Augmented responses in hypertension may relate to alterations in the cyclooxygenase pathway during the early stages of established hypertension in the SHR. This investigation also documents the capsaicin-sensitive nature of oleamide responses in aortic rings, which exists independently of sensory-nerve mediated activity. The observation of a non-TRPV1 capsaicin-sensitive mechanism may ultimately lead to the uncovering of an alternative mechanism of action for capsaicin in conduit arteries and a novel site of action for oleamide.
26

Metabolic and cellular effects of carbohydrate-based preconditioning drinks

Awad, Sherif January 2010 (has links)
This thesis investigates the metabolic and cellular effects of carbohydrate-based preconditioning drinks in humans. Previous studies have demonstrated that preoperative carbohydrate loading, as opposed to overnight fasting, attenuated the development of postoperative insulin resistance by up to 50% and led to clinical benefits. Preconditioning with carbohydrate-based drinks was incorporated into enhanced recovery after surgery programs. The latter included interventions that aimed to minimise ‘metabolic-stress’ and hasten recovery after major surgery. However, the cellular mechanisms underlying the adverse effects of preoperative fasting and the beneficial effects of preconditioning with carbohydrate-based drinks were hitherto unknown. In healthy volunteers, short-term fasting (up to 24 hours) reduced liver volume, depleted liver glycogen (-50%) and lipid reserves, and increased intramyocellular lipid concentrations (+23%), as measured by magnetic resonance spectroscopy. Changes in liver glycogen were partially reversed following ingestion of a carbohydrate-based drink that also contained glutamine and antioxidants (ONS, Fresenius Kabi, Germany). Fasting also led to significantly decreased blood mononuclear cell mitochondrial complex activity. In patients undergoing laparoscopic cholecystectomy, preoperative conditioning with ONS, compared to ingestion of a placebo-drink, significantly increased intraoperative liver glycogen by 50%, increased intraoperative plasma glutamine and antioxidant concentrations, led to lower expression of skeletal muscle pyruvate dehydrogenase kinase 4 mRNA and protein expression, and finally, reduced cellular oxidative stress, as indicated by a 1.5-fold lower expression of metallothionein-1A in the ONS group. Ingestion of ONS led to markedly differing hormonal and metabolic responses compared to those following a clear carbohydrate drink (preOp®, Nutricia Clinical Care, UK), with ‘blunted’ postprandial glucose and insulin responses following ONS. Supplementing preOp® with glutamine ‘blunted’ postprandial insulin and glucose responses but this was not due to differences in glucagon-like peptide-1 concentrations. Finally, the gastric emptying of these drinks was more dependent on carbohydrate content than macronutrient composition or osmolality.
27

Agonist stimulus trafficking by human prostanoid CRTH2 (DP2) receptors

McArthur Wilson, Richard John January 2007 (has links)
Agonists of hormone receptors possess affinity (the ability to bind) & efficacy (the ability to stimulate effect). In this thesis, alternative expressions of efficacy by recombinant prostanoid Chemoattractant Receptor Homologous molecule of TH2 cell (hCRTH2) receptors have been studied using a variety of assays and pharmacological techniques. When expressed in CHO cells, either with or without co-expression of chimeric G alpha 16z49 G-proteins, CRTH2 receptor-mediated calcium mobilisation pharmacology was found to be as published. Coupling of receptor activation to calcium elevation involved G beta gamma i/o mediated PLC beta -dependent mobilisation of both intra- & extra- calcium. In chimera-expressing cells, an additional coupling mechanism was observed which was presumably G alpha 16z49-mediated. The relative expression of receptor and G-protein molecules in both cell types was investigated but because of deficiencies in the methods employed the relative expression is essentially unknown. Because G alpha 16z49 & G beta gamma i/o represent different classes of PLC beta -activating G-proteins, simultaneous activation of them may have produced a synergistic response in chimera-expressing cells which may have affected the observed receptor pharmacology. When the G alpha 16z49 component was isolated in PTX-treated chimera-expressing CHO G alpha 16z49 cells, reversals of potency order were observed with respect to responses in untreated cells. These were most striking for 17 phenyl PGD2, 15 R 15 methyl PGF2 alpha, 15 deoxy delta 12,14 PGJ2 and 15 R 15methyl PGF2 alpha. Alterations of potency order were also observed in non-chimeric cells (G beta gamma i/o coupling) compared with PTX treated chimera-expressing cells. These were most striking for indomethacin, 16,16 dimethyl PGD2, delta 12 PGJ2 and 9,10 dihydro 15 deoxy delta 12,14 PGJ2. In [35S]-GTP gamma S accumulation assays using membranes prepared from non-chimeric cells and presumably reporting G alpha i/o coupling, agonist pharmacology was similar to G alpha 16z49 mediated calcium mobilisation data. However, the data were markedly different from G beta gamma i/o-mediated calcium mobilisation data generated in non-chimeric cells. These differences were most apparent for 13,14 dihydro 15 keto PGD2, 15 deoxy delta 12,14 PGJ2 and indomethacin. Desensitisation of agonist-stimulated calcium mobilisation was also studied. PGD2 produced rapid & long-lasting desensitisation of hCRTH2 receptors in a biphasic manner suggesting that two desensitisation mechanisms may operate. At low concentrations of PGD2 desensitisation was PTX-insensitive suggesting that a non-Gi/o-protein mediated mechanism may be responsible. Other CRTH2 receptor agonists inhibited responses to subsequent PGD2 EC80 exposure in calcium mobilisation assays. Interestingly, a group of molecules devoid of agonism in the calcium assay also inhibited PGD2 responses. This group of molecules included 19 hydroxy prostaglandins A2, E2 & F2 alpha , and PGE2 and appeared to mediate their effects through a mechanism that did not involve a competitive interaction with PGD2. The data generated here show that CRTH2 receptor agonist pharmacology is critically dependent on G-protein coupling partner and assay methodology, and are strongly indicative of agonist-directed stimulus trafficking. The data are consistent with the notion that G beta gamma subunit activation is not a passive "on-off" event but is rather an active event triggered by agonist- and GTP-dependent conformation changes in both receptor and G alpha subunit molecules.
28

Effect of diet, insulin and exercise on the regulation of carbohydrate metabolism in health and type 1 diabetes

Chokkalingam, Kamalakkannan January 2007 (has links)
The objective of this thesis was to further the understanding of the effect of diet, insulin and exercise on the regulation of carbohydrate metabolism in health and type 1 diabetes. Three studies were undertaken in both non-diabetic healthy volunteers and patients with type 1 diabetes. The first study determined the influence of high fat diet on biochemical and molecular regulators of whole body and muscle metabolism in healthy volunteers. The second study examined the influence of insulin on whole body and muscle metabolism in patients with type 1 diabetes during moderate exercise. The final study compared the influence of insulin and a high carbohydrate diet on liver glycogen concentrations and substrate oxidation during exercise between patients with type 1 diabetes and healthy volunteers. The main results were, a) 6 days of high fat/low carbohydrate dietary intake did not induce whole-body insulin resistance but caused a shift in intramuscular glucose metabolism from oxidation to glycogen storage when compared to a normal balanced diet. Insulin-stimulated carbohydrate oxidation and muscle PDCa activity were blunted after the high fat diet and this was associated with an up regulation of muscle PDK4 mRNA and protein expression, b) Exercise under hyperinsulinaemic conditions in patients with type 1 diabetes did not spare muscle glycogen utilisation at a time of high exogenous glucose utilization and oxidation, and finally c) Changes in liver glycogen concentration and substrate oxidation during exercise occurred at comparable rates in patients with type 1 diabetes and in healthy controls despite the presence of relative hyperinsulinaemia in the former compared to the latter group. The key conclusions are, 1) in healthy humans short-term high fat feeding does not induce whole body insulin resistance but impairs basal and insulin-stimulated carbohydrate oxidation, most likely as a result of fat-induced upregulation of muscle PDK4 protein expression. The precise signaling mechanisms involved in the chronic regulation of PDK4 need to be determined. 2) Contrary to previous observation in non-diabetic individuals, it appears that hyperinsulinaemic conditions in patients with type 1 diabetes do not suppress the exercise-induced changes in muscle and liver glycogen stores. The underlying physiological mechanism(s) behind this apparently divergent response remains to be elucidated.
29

Generation of diversity at the human beta-defensin copy number

Abu Bakar, Suhaili January 2010 (has links)
Submicroscopic structural genomic variation includes copy number variation (CNV) that can result changes in DNA dosage, and the impacts can be observed on common disease, metabolism, and heritable traits such as colour vision and rhesus blood group. Human beta-defensins form a cluster of at least seven genes on human chromosome 8p23.1, with a diploid copy number commonly ranging between 2 and 7 copies. They encode small secreted antimicrobial peptides with cytokine-like properties which are found expressed at high levels in psoriasis patients, and copy number at this locus has been found to be associated with inflammatory bowel disease, particularly colonic Crohn’s disease. The focus of this thesis has been divided into two studies; looking for the origin of diversity at the human beta-defensins copy number, and development of a multiplex PRT measurement system for accurately typing the beta-defensin region in large case association study. The origin of diversity at the human beta-defensin copy number has been followed by using segregation in CEPH families. Three out of 416 meiotic transmissions changed the copy number by simple allelic recombination between two distinct loci for these genes. Deducing haplotype copy number from microsatellite and multiallelic length polymorphism have allowed this study to map the beta-defensin repeats in two locations at the original location distally REPD and about 5 Mb away at proximally REPP. We have demonstrated our multiplex PRT system is a powerful technique to determine the association of the beta-defensin genes in Crohn’s disease even though we did not produce any convincing support for associations reported from previous studies.
30

Investigation into the ion channels and plasma membrane properties of white adipocytes

Bentley, Donna C. January 2013 (has links)
Ca2+ is a ubiquitous intracellular signalling molecule that is involved in the regulation of numerous cellular functions. To date Ca2+ influx pathways present in white fat adipocytes have not been characterised. Additionally impaired [Ca2+]i management is implicated in the induction of the insulin resistant state in adipocytes. As adipocytes have a prominent role in the management of energy homeostasis, the presence of Ca2+ influx pathways was examined. Initial [Ca2+]i measurements confirmed the presence of functional Ca2+ influx and efflux pathways in adipocytes. Further [Ca2+]i measurements identified the Cav1.3 Voltage-gated Ca2+ channel (VGCC). The presence of the α1 subunit of Cav1.3 channel protein in adipocytes was confirmed by Western blotting, the expression of which was reduced in adipocyte samples sourced from Zucker obese rats. Initial [Ca2+]i imaging experiments utilising conditions of elevated extracellular K+ (50mM) did not stimulate Ca2+ influx. The plasma membrane potential (Vm) regulates many physiological processes, including cellular Ca2+ influx by VGCCs, with dysregulations in Vm underlying functional pathologies. K+ is widely believed to be the predominant ion that controls Vm for many cell types, however, whether K+ regulates adipocyte Vm is also unknown, prompting, investigation into the ionic species involved in the regulation of Vm in primary and differentiated 3T3-L1 adipocytes. As insulin and β-adrenoceptors regulate adipocyte function, their effect on Vm was also explored. The Vm of primary and 3T3-L1 adipocytes were -34.14mV (n=68) and -28.5mV (n=88) respectively. Elevation of extracellular K+ from 5.6mM to 50mM had no significant effect on the Vm of either type of adipocyte. The role of Cl- on adipocyte Vm was then investigated. Reduction of extracellular Cl- from 138 to 5mM, by equimolar substitution with Gluconate significantly depolarised the Vm of both primary and 3T3-L1 adipocytes. Patch clamp investigations also revealed a role of Na+ in adipocyte Vm. Neither insulin (100nM) or the β-adrenocpetor agonist isoprenaline (10µM) significantly changed adipocyte Vm. The role of Cl- in adipocyte Vm is indicative of the presence of Cl- channels, however electrophysiological studies failed to characterise the Cl- currents underlying adipocyte Vm. Overall, further investigations are required to characterise not only the Ca2+ influx pathways in adipocytes, and the roles thereof, but also the means by which they are regulated.

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