RSS 1.0Theses
This collection contains the Doctoral and Masters by Research theses produced within the department.
This collection is licenced under a Creative Commons licence. The collection may be reproduced for non-commercial use and without modification, providing that copyright is acknowledged.
Recent Submissions
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Heat shock proteins as danger signals to the immune systemAbstract available in hard copy
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The effect of long-term post-operative cardiac rehabilitation: a comparative intervention study in Kingdom of Saudi ArabiaAbstract available in hard copy
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The ethical dimensions of the HIV/AIDS pandemicAbstract available in hard copy
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An investigation of the effects of repurposed drugs on human neuroblastoma and glioblastoma cell linesNeuroblastoma is an aggressive and highly metastatic extracranial tumour of the sympathetic nervous system commonly seen in children, whereas, glioblastoma multiforme is an aggressive and highly proliferative intracranial tumour of the central nervous system seen more in adults. The cellular heterogeneity and molecular pathogenesis of both of these tumours have limited the development of successful treatments. The combined effects of the lipid-lowering drug, bezafibrate (BEZ) and the contraceptive drug, medroxyprogesterone acetate (MPA) (combined as BaP) has shown promising anti-cancer effects on blood cancers like myeloid leukaemia, Burkitt’s lymphoma and chronic lymphoid leukaemia, and osteosarcoma, with elevated levels of reactive oxygen species and down-regulation of lipogenic enzymes implicated in the mechanisms of action for these drug treatments. The aim of this study was to determine the effects of these combined drugs on neuronal cancers, using the SH-SY5Y neuroblastoma and U-87MG glioblastoma cell lines as model systems. BEZ treatment alone was shown to have a significant and BEZ concentration-dependent inhibitory effect on SH-SY5Y and U-87MG viable cell proliferation, whereas MPA treatment alone was shown to have very little effect on the cells. The combination of the drugs was shown to have a significant and drug concentration-dependent inhibitory effect on SH-SY5Y and U-87MG viable cell proliferation to a greater extent than BEZ treatment alone. These anti-cancerous effects were associated with increased cell death, and elevated levels of reactive oxygen species (ROS) in both cell lines after 24 hours of treatment. Levels of the lipogenic enzymes, stearoyl CoA desaturase-1 and fatty acid synthase were seen to be significantly lower in SH-SY5Y and U-87MG cells than in blood cancer cell lines. Further, oleic acid supplementation rescued SH-SY5Y neuroblastoma cells, but in serum not serum free conditions only. Oleic acid supplementation of U-87MG cells did not rescue their susceptibility to BaP treatment. Inclusion of valproic acid combined with BaP (termed VBaP) enhanced the effects of BaP treatment on SH-SY5Y and U-87MG cells, as well as U266 multiple myeloma cells. This is important, as higher concentrations of BEZ are nephrotoxic. The effects of VBaP on U-87MG cells also were explored in a 3D alginate culture system to mimic the microenvironment of the brain. In this situation, VBaP treatment still inhibited U-87MG cell growth and also was effective at inhibited established tumour spheroids. Hence, this thesis has examined whether drug repurposed drug treatments that have been established in blood cancers may also have application in neuronal cancers, and demonstrated that this is possible.
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Predicted Structure and Potential Binding of the Nematode Tetraspanin 7 Protein (tsp-7) With Human Tissue Inhibitor Metalloprotease 1 (TIMP-1): a Possible Role In Parasite- Host InteractionsHuman parasitic nematodes evade the host immune system through cross communication by unknown mechanisms. The proteins of the parasitic nematodes are still poorly annotated. CD63 is a human tetraspanin and binds to TIMP-1 a metalloproteinase inhibitor, which has recently been found to play an active role in inflammation by acting as a cytokine when bound to CD63. Caenorhabditis elegans is a free-living nematode, a model organism for both human and parasitic nematodes. C. elegans tetraspanin protein -7 (tsp-7) is an orthologue of human CD63. The model organism used can provide information about potential tetraspanin proteins from parasitic nematodes, which are believed to play a role in the cross communication between parasite and host. The tsp-7 protein sequence from C. elegans shares a high percentage similarity, identity, and homology with a variety of human parasitic nematode proteins. Using a number of computer-based programmes including ‘Alpha-Fold,’ which is currently the most powerful Artificial Intelligence protein structure prediction programme, the structure of the C. elegans tsp-7 was determined. From the predicted structure, the tsp-7 protein has one highly likely, membrane exposed tyrosine phosphorylation site, with implications of its potential involvement in signalling pathways and immune responses. Using multiple protein docking databases and programmes, tsp-7 has been shown to theoretically bind to human TIMP-1. Two mutant strains of C. elegans were obtained from Caenorhabditis Genetics Centre, University of Minnesota, USA, with amino acid deletions within the tsp-7 protein (mutant tm5046 and mutant tm5761). These tsp-7 mutant strains were shown to have an overall longer life span than the wild type C. elegans (N2). The mutant tm5761 showed a greater stress response compared to the N2 wild-type, when exposed to various chemical stimuli, however, mutant tm5046 was more similar to wild type C. elegans. Under heat stress, mutant tm5761 and wild type N2, were less tolerant to heat compared to the tm5046 mutant. In general, mutant tm5046 was more stable and displayed a reduced stress behavioural response compared to mutant tm5761. A primary polyclonal antibody was produced against the tsp-7 large extracellular loop domain, believed to be the key active site in protein-protein interactions. The expression of tsp-7 in mutant tm5761 was detected at lower concentrations than the wild type and mutant tm5046 in an ELISA assay and it also could not be detected by western blotting. Mutant tm5046 was detected weakly by western blotting. Cos-7 cells transfected with the tsp-7 protein-GFP tagged plasmid were incubated with active human TIMP-1. The tsp-7 tagged cells showed co-localisation with TIMP-1, using immuno-fluorescence microscopy, thus adding to the evidence that tsp-7 could interact with human TIMP-1. Such an interaction may well play a role in parasite-host interactions and its effects on the immune response. As C. elegans tsp-7 shares high similarity with many parasitic nematodes that are known to infect humans, it is a good protein candidate to further explore the potential interactions of parasitic nematodes with humans, in terms of cross communication and interaction with the host immune system.
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Lysophosphatidic Acid Signalling in Diabetes MellitusDriven by obesity, the incidence of type 2 diabetes mellitus (T2DM) has escalated, reaching unprecedented levels worldwide. This has led to a change in approach from treatment to prevention or delaying the onset of the disease. While current treatment of the disease has been successful in increasing patients’ quality of life, it has substantially increased the financial burden on healthcare systems worldwide. In view of this, the NHS, in partnership with Diabetes UK and Public Health England, has launched a diabetes prevention programme to target people at risk of the disease and provide targeted support to prevent its development. A prelude to this strategy is to identify robust risk factors that will enable the capture of people at increased risk of developing the disease. The phospholipid lysophosphatidic acid (LPA), upregulated during obesity, has been shown to be a potent regulator of glucose homeostasis and insulin secretion in animal models. This thesis is the first to assess circulating levels of LPA in human T2DM patients versus non-DM control. The thesis aims to assess the usefulness of LPA as a biomarker for T2DM screening and has three major objectives: (1) to determine plasma LPA concentration in T2DM patients compared with non-diabetic controls and assess LPA correlation with current diagnostic markers for T2DM. (2) to determine LPA correlation with pathological markers known to lead to DM complications; (3) to develop an immunofluorescence assay to assess LPA signalling of glucose homoeostasis using a mammalian cell line. (4) to assess LPA regulations of GLUT4, PKC and α2a adrenergic receptor (α2aAR) using the developed assay.
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An Investigation of Interactions between Heat Shock Proteins and the Immune SystemThe human body initiates an inflammatory immune response following exposure to certain danger signals. The Danger Model proposes that Antigen-presenting cells (APCs) can be activated by danger or alarm signals. These danger signals come from exogenous molecules such as pathogens, toxins, or even mechanical cell damage. Damage due to cellular stress can initiate the extracellular release of endogenous molecules, such as heat shock proteins (HSPs) into the extracellular environment. HSPs can act as danger signals triggering the immune response. This immune response is based on antigen recognition by specific cell surface receptor proteins, and T-cells, which then determine the type of inflammatory (pro or anti-inflammatory) cytokines produced or expressed. This thesis investigates the effects of Hsp72 and Hsp27 in the activation of immune cells to secrete cytokines, which are upregulated during inflammatory responses. In determining the role of HSPs in affecting inflammatory immune responses, U937 cells, U937 macrophages, and peripheral blood mononuclear cells (PBMCs) were used. To determine whether the effects observed with recombinant HSPs used in these studies, were due to bacterial contamination: U937 macrophages were exposed either to denatured HSPs, polymyxin B (which can neutralise bacterial lipopolysaccharides), anti-HSPs antibodies, or receptor proteins blocking peptides, and their ability to induce cytokine (ie IL-1β, TNF-α, and IL-10) secretion were analysed using enzyme-linked immunosorbent assays (ELISA). Western blotting was used to confirm the presence of Hsp72, Hsp27, and Hsp60 in U937 cells and U937 macrophage. Flow cytometry was used to identify the expression of immune responsive receptor proteins such as CD14, CD36, CD11b, TLR2, TLR4, TLR5, and TLR7, on U937 cells and U937 macrophages. The result presented in this thesis also showed that Hsp72 and Hsp27 can stimulate immune responses independent of bacterial contamination. These HSPs are able to induce an inflammatory immune response, possibly through interactions with a number of immune responsive receptor proteins, which were identified in U937 cells and U937 macrophages. Further evidence using CD14, CD36, CD11b, TLR2, TLR4, TLR5, and TLR7 blocking peptides, also confirmed that an interaction between cytokine secretion caused by, Hsp72 and Hsp27 were likely due to their interactions with specific immune responsive receptor proteins. Interestingly, some of the receptor proteins identified are not activated by lipopolysaccharide (LPS), again highlighting the fact that the results presented in this thesis, are unlikely to be artifacts caused by bacterial contamination. Furthermore, it is clear that HSPs are interacting with numerous receptor proteins, and possibly more than even demonstrated in this thesis. This therefore highlights the promiscuous nature of HSPs interaction with different signalling pathways through the different receptor proteins. The promiscuous property of HSPs could thus be used for the treatment of diseases, since HSPs have been linked with many diseases, including cancer. Therefore, understanding the full relationship between HSPs and these signalling pathways, may prove promising in using HSPs for therapeutic purposes in future.
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The influence of CLA on obesity, lung function, adipokines and inflammationObesity is currently widespread in the world; the epidemic and pathogenesis of the disease negatively affect several body systems including cardiovascular, endocrine and respiratory systems. Obesity influences the respiratory functions and this effect could be challenging for women, because the air way and lungs are smaller in women compared to men, as well as obesity itself exerts a negative mechanical effect on the women’s airway. Since inflammation was proposed asthe main link between obesity and lung functions, a natural supplement like conjugated linoleic acid (CLA), which has been proposed as an antiinflammatory and anti-obesity food component, could be a potential supplement that can improve the lung functions in obese women. Therefore, the aim of this thesis is to explore the effect of CLA on obesity, lung function, adipokines and inflammation. Additionally, the effect of CLA on inflammation in the current thesis was explored using novel inflammatory markers, such as adhesion molecules (CD11b and CD62L) and heat shock proteins (HSPA1A and HSPB1). Investigating the evidence about the effect of CLA supplementation on obesity in women was conducted via a systematic review with meta-analysis. The meta- analysis searched randomised control trials (RCTs) supplemented CLA mixture in form of oral capsules for less than 6 months. Two search strategies were applied, and eight eligible trials were included with 330 women. CLA significantly reduced body weight (BW; 1.2±0.26 kg, p<0.001), body mass index (BMI; 0.6 ±0.13 kg/ m², p <0.001) and total body fat (TBF; 0.76± 0.26 kg, p=0.003) when it was supplemented for short durations (6- 16 weeks). Moreover, subgroups meta-analyses were conducted which were based on obesity level, menopausal age and life style of the participants. This meta-analysis suggested a mild anti-obesity effect of CLA. However, it was not clear whether the anti-obesity effect is enough to modulate obesity-induced inflammation and lung functions. Therefore, initially a crosssectional trial was conducted to assess the direct associations between the circulating level of CLA and obesity markers, lung functions and inflammations. To the best of Knowledge, this was the first cross-sectional trial that explored these direct associations. The cross-sectional trial recruited 77 women with average age 39 years old with forced expiratory volume in one-second (FEV1) ≥70%. The level of CLA in plasma was assessed by gas chromatography; the expression of the CD markers and HSPs were assessed using flow cytometry; body composition was assessed using bioelectric impedance; and lung functions were assessed using spirometer. Interestingly, the trial revealed significant positive associations between CLA and BW (R=0.4, p<0.001), BMI (R=0.4, P<0.001) and TBF (R=0.34, P<0.001) in the overall population, and in perimenopause women. A significant inverse correlation between t10, c12-CLA and TBF was detected in overweight women (R=- 0.42, p<0.05). A significant positive association (R=0.45, P<0.04) was detected between the c9, t11-CLA and percentage peak of flow predicted (PEF %) in postmenopausal women, meanwhile t10, c12-CLA was negatively associated with peak of flow (R=-0.44, P<0.04). CLA was inversely associated with adiponectin in both obese (R=-0.55, p<0.1) and morbidly obese (R=0.48, P<0.004) women. C9, t11-CLA was positively associated with the expression of HSPA1A inside the lymphocytes in postmenopausal women (R=0.58, p=0.04). HSPB1 expression in the monocytes were associated with both c9, t11-CLA (R=0.58, p<0.05) and total CLA (R=0.71, p<0.001). The level of expression of CD11b on the pro-inflammatory monocytes (CD14++ CD16+ ) was negatively associated with CLA (R=-0.36, p<0.05). Ultimately, the study did not provide strong evidence regarding the direct relationship between CLA and obesity markers or lung functions. However, it showed a potential immunomodulatory effect of CLA on obesity-induced chronic inflammation, which subsequently could influence multiple obesity compilations. The lack of strong evidencewas primarily due to the nature of the study design (observational study). Therefore, in chapter 5 a randomised double-blind placebo control trial was conducted, for more powerful evidence based. The aim of the RCT was to look at the effect of 12-week CLA supplementation on obesity, lung function, adipokines and inflammation in obese and overweight women. The RCT recruited 56 overweight and obese women with a mean age of 42 years old, participants were randomly assigned either to receive 4.5gm/day of CLA or placebo (High Oleic Safflower oil). Participants had to attend three clinics at base line, after 6 weeks and after 12 weeks. In each clinic body composition, lung functions and inflammatory markers were assessed. The study revealed a significant 1.8% reduction in %BF in the CLA group compared to the baseline. No significant effect of CLA on the lung functions was detected, however, this study found a significant reduction in the expression of CD11b on the stimulated pro-inflammatory monocytes after 12 weeks compared to baseline in the CLA group. CLA caused a significant reduction in the expression of intracellular HSPA1A in PBMCs at week 12 compared to baseline. The results might suggest a limited anti-obesity effect of CLA, and a potential positive effect on obesity induced chronic inflammation. Ultimately, no evidence was demonstrated on the direct effect of CLA on lung functions or adipokines. The effect of CLA on adhesion molecules and HSPA1A could suggest an indirect impact on the lung function, but more research in clinically diagnosed patients with pulmonary dysfunctions could help to confirm the effect of CLA on the lung function and adipokines.
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The effects of targeted therapy on cell viability and apoptosis on CML and AML cell linesTyrosine kinase inhibitors (TKIs) are currently the first therapy option for chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML) patients. However, many patients affected by CML and AML may develop resistance to TKIs or may not recover under this treatment regime. New potential and more effective treatments are recently emerging. Heat shock protein inhibitors (HSPIs) and the proteasome inhibitor Bortezomib are drugs which have been yet to be successfully tested on leukemic patients, despite being successful on other malignancies such as multiple myeloma (MM). The combination between HSPIs and Bortezomib could potentially be successful in killing leukemic cells, by enhancing their respective molecular mechanisms. Indeed, HSPIs would bind to HSP72 avoiding the protein to exert its ligase function to the proteasome, whilst Bortezomib could stop the ubiquitinated proteins to enter the proteasome and ultimately inducing apoptosis. To test the effects of such combination, cell viability was measured via MTS assay, apoptosis levels were tested through Annexin V\PI assays. Involvement of HSP72 and pro-survival protein Bcl-2 were measured via flow-cytometry. The cells were administered with HSPIs and Bortezomib first as single agents for 24 hours, to establish working minimal concentration. Also, the drugs were tested for a shorter time, to understand when the drugs start to be effective. It emerged that one hour is sufficient for the drugs to give an initial effect in terms of cell viability and apoptosis. Following, combination experiments of HSPIs and Bortezomib were performed; the first drug was administered for one hour, the second following one hour and the cells were incubated for 24 hours. This was repeated alternatively for both type of drugs on the different cell lines. MTS and Annexin V\PI showed that there is not a synergistic effect between drugs, but instead there is antagonism. No necrosis was found at any level of the study. The cells were then probed for HSP72 and Bcl-2, to investigate their involvement in apoptosis mechanisms. Following 6 hours of combined and single agent treatment, both type of drugs inhibit HSP72 but failed to reduce the expression of Bcl-2, particularly on AML cells. It is thus proposed that CML and AML cells may die by apoptosis following a short time of treatment with HSPIs and Bortezomib by an extrinsic pathway of apoptosis, independent from Bcl-2 involvement and from mitochondrial pathway of apoptosis. This study may be the first to indicate a potential use of HSPIs and Bortezomib on CML and AML patients for a short time of treatment, although not in combination. Future studies are needed to further investigate the mechanisms of action of these drugs, aiming to potentially give CML and AML patients another successful therapy option to overcome resistance to canonic chemotherapy.
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The role of MAPK signalling pathways in leukemic cell deathMitogen-activated protein kinase (MAPK) signalling pathways are important signalling pathways involved in mediating various cellular processes including both cell survival and cell death. The c-Jun N-terminal kinase (JNK) pathway, the p38 pathway and the extracellular signal-regulated kinase (ERK1/2) pathway are three well-studied conventional MAPK signalling pathways. Previous research has shown these MAPK signalling pathways play an important role in the development and progression of leukaemia and in the response of leukemic cells to therapy. Whilst it appears to be well established that the constitutive activation of ERK mediates leukemic cell survival, the roles of the JNK and p38 signalling pathways in leukemogenesis, in particular the role in acute myeloid leukaemia (AML), are less well understood. This thesis investigates the role of the JNK, p38 and ERK signalling pathways in leukemic death. MAPK signalling pathways were targeted in the U937 monocytic cell line using small molecule MAPK inhibitors in combination with various cell stressors: UV light, chemotherapeutic agents (doxorubicin and vincristine) and heat treatment. The effects on cell death were examined using plate-based assays, flow cytometry and fluorescence microscopy. Preliminary investigations were also performed in peripheral blood mononuclear cells (PBMCs) from healthy individuals to allow a comparison to non-leukemic cells. Results show inhibition of ERK signalling in U937 cells induced cell death and ERK signalling had little effect on UV-induced and heat treatment-induced cell death. JNK signalling and p38 signalling provided protection against UVinduced cell death in both U937 cells and in PBMCs from healthy individuals. JNK and p38 signalling mediated cell survival in response to heat treatment to a certain extent. JNK signalling was required for the induction of cell death induced by doxorubicin whereas p38 signalling provided a level of protection against doxorubicin-induced cell death. U937 cells were found to be more sensitive to vincristine treatment than PBMCs from healthy individuals and the activation of JNK and p38 signalling was essential for vincristine-induced cell death in U937 cells. Taken together, the results presented in this thesis demonstrate that the roles of the JNK, p38 and ERK signalling pathways in leukemic cell death are stimuli-specific. This highlights the importance of understanding the involvement of particular pathways in the response to specific chemotherapeutic agents, in order to provide effective leukaemia therapy. Therapeutic inhibition of MAPK signalling pathways to increase the sensitivity of leukemic cells to chemotherapy could be beneficial when MAPKs are involved in providing protection against chemotherapy-induced cell death. For chemotherapies which require MAPK activation for cell death, failure to activate MAPKs may provide a mechanism for chemoresistance. Therapeutic methods to enhance activation of the pathways provide a possible approach to increase the susceptibility of leukemic cells to death.
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Manipulation of apoptosis in cancer cellsConventional cancer therapies can have severe side effects, so new strategies to limit these needs to be investigated. Several anticancer agents induce the expression of tumour suppressor gene p21 in colorectal cancer cell line HT-29. Interestingly, the stress protein HSPA1A is also often elevated in tumour cells and has an anti - apoptotic activity. The main aim of this study was to examine whether a two - pronged approach, overexpressing p21 (using genetic approach and inhibition of HSPA1A using pifithrin - µ would be effective in inducing apoptosis in tumour cells. Chitosan or BSA based delivery systems were evaluated for cytotoxicity, with the intension of using it for plasmid DNA based cell transfections in this study. The interaction of HSPA1A protein in combination treatments involving UV radiation and hyperthermia at 42℃ were also evaluated to perceive the various roles of HSPA1A in arresting colorectal cancer cells. Colorectal cancer cell lines HT-29 and leukaemia cancer cell lines U937 were used in the study. All experiments were performed with cancer cell lines maintained in culture medium devoid of antibiotics. Cell cytotoxicity were evaluated using MTS and PI assays. The rate of apoptosis was determined using annexin V and PI staining by flow cytometry. Chitosan or BSA based microparticles or microgels were observed for size determination or morphology using scanning electron microscopy. Full length human p21 inserted plasmid DNA was a gift from Mien - Chie Hung, Addgene, USA. HT- 29 cells were subjected to p21 plasmid DNA transfection effects. Cells were treated with pifithrin - µ (15µM) prior to gene transfection to address its combined effect with p21 plasmid DNA transfection. HSPA1A and p21 protein expression studies were analysed using FITC labelled antibodies by flow cytometer. Combination studies with HSPA1A inhibitor pifithrin - µ and UV reflected enhanced cytotoxicity compared with either of the treatments independently. Hyperthermia at 42℃ induced apoptosis by MTS assay, which was confirmed by flow cytometric analysis in both the cell lines tested. Considering the cytotoxicity reflected by the chitosan or BSA delivery systems in drug free states, the p21 plasmid DNA transfection was carried out using lipofectamine 2000. Both overexpression of p21 and inhibition of HSPA1A protein with pifithrin - µ enhanced the rate of apoptosis with statistical significance of (p-<0.0001****) compared to the respected controls. The data in this thesis suggests the inhibition of HSPA1A in combination with increased p21 would be a promising therapeutic strategy for the treatment of colorectal cancers.
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Investigating the role of heatshock on diabetic wound healingThe increasing occurrence of diabetes in the general population as a result of over nutrition and increasingly inactive lifestyle has led to an obesity epidemic which is set to grow over time. With an ever increasing obese population type 2 diabetes and cardiovascular complications are set to become the major causes of human mortality. Chronic non healing wounds are a major cause of mortality and morbidity in patients with type 2 diabetes. They are predominantly caused by macrophage dysfunction and a lack of migration of fibroblasts into the wound. This study aimed to investigate diabetic wound healing through development of an artificial scratch assay. An in vitro scratch assay developed in WS1 cells. The effect of heat shock treatments from 39°C to 45° was tested to determine if cell migration increased; however, no significant difference was seen. Mitomycin C was used to determine if wound closure occurred as a result of cell proliferation and migration or migration alone. 10μg/ml of mitomycin C inhibited cell division by 79.9% without exhibiting cytotoxicity over a 12h period. The effect of hyperglycaemia and heat shock was also tested and showed no significant difference when compared to control conditions, suggesting that fibroblast migration in vivo is hindered through other factors such as debridement or macrophage dysfunction in the wound. GLUT4 is present in insulin sensitive organs (liver, adipose and muscle) and is the major glucose transporter responsible for the clearance of glucose from the blood after a meal, thus playing a central role in glucose homeostasis. Monocytes are precursors to macrophages and can easily be isolated from whole blood. They have also been shown to express GLUT4 in response to insulin and could be used as model to assess inflammation in diabetes. A glucose uptake assay was developed in U937 cells using a fluorescent glucose analogue, 2NBDG. 2NBDG fluorescence was shown to be competitively inhibited by increasing concentrations of glucose suggesting that 2NBDG enters the cell through glucose transporters. 2NBDG uptake was also assessed at different pH and in presence of membrane fluidizers (DMSO, benzyl alcohol and phenethyl alcohol). Extremes of pH significantly reduced cell viability and only at pH 4 was 2NBDG fluorescence significantly reduced. Treatment with DMSO showed that at high concentrations (≤ 1.56%) cell viability was reduced with a concurrent reduction in 2NBDG fluorescence. The effect of benzyl alcohol and phenethyl alcohol was foundto be insignificant at the concentrations and time points tested. The presence of GLUT4 was also determined by flow cytometry and Western blotting and found to be situated in the cytoplasmic region of the cell. This study indicates that monocytes and macrophages could be a potential therapeutic target to improve diabetic wound healing as they are a source of growth factors and cytokines that can bring about resolution of inflammation and it is their dysfunction in diabetic wounds that causes poor clinical outcomes.
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Psychiatric responses to traumatic eventsThe main aims and objectives of this Ph.D. by publication are: • To analyse, explore and contextualise the psychiatric response to trauma and aetiological issues • To analyse and explore the management of Post-Traumatic Stress Disorder (PTSD) • To critically analyse the wider historical, legal and political management of mental disorder. Five peer-reviewed publications from recent years are presented on the theme of psychiatric responses to traumatic events. Two papers focus on the aetiology, (where the Oxford definition of aetiology is the ‘cause, set of causes, or manner of causation of a condition’), of PTSD and therefore consider the injuries that cause PTSD and also potential vulnerability factors (Green & Griffiths, 2013). These papers contain a mixture of quantitative and qualitative methods – examining characteristics such as psychological conceptions of risk in relation to illness duration within a case series for instance and a comparative statistical analysis of birth order in differing samples. Two papers consider modern aspects of the treatment of PTSD – including pharmacological and psychotherapeutic and difficulties and use a methodology of a structured review of the literature including analysis of the evidence base for trauma-focused Cognitive Behavioural Therapy (CBT) including Numbers Needed to Treat (NNT) (Green 2013, Green 2014). A final paper looks at admissions trends for PTSD and a range of other mental disorders and uses a statistical analysis of national data looking for emerging trends against a historical and political background of changes in the management of mental disorder (Green & Griffiths, 2014). These recent papers are set in context against older papers from a career which has spanned epidemiological research into risk factors for depression over six years, writings about psychopharmacology, and planned future research into birth order and domestic violence, and an editorial for the British Journal of General Practice (Green & Gowans, 2014) seeking to promote future epidemiological research into unmet mental health needs in the community. The papers can be viewed as being within the context of a continuum of research interests and publications (represented diagrammatically below in Figure One). In the narrative text I refer to this earlier work and also explain my plans for progress in terms of future research and publications, thus setting the work in this Ph.D. by publication in context within a continuing pattern of interests.
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HSPC1 inhibitors and their use in Chronic Lymphocytic LeukaemiaHSPC1 (Hsp90), a member of the anti-apoptotic Heat Shock Protein (HSP) family appears to play a pivotal role in the development and maintenance of several tumour cell characteristics and as a result has become a target for novel anti-cancer therapies. HSPC1 inhibitors have been tested in clinical trials on a wide variety of cancer types with moderate success. However, despite recent advantages in HSPC1 inhibitor development, the effects of these drugs are not consistent. A number of factors may play a role in determining cell sensitivity to these inhibitors. As Chronic Lymphocytic Leukaemia (CLL) is such a heterogeneous disease with great variation in baseline HSP levels and other proteins amongst the patient cohort, it would not be unreasonable to assume that HSPC1 inhibitors may have varying success as a treatment strategy for this disease. The present study examined the effects of four HSPC1 inhibitors on primary CLL cells, as well as cells from healthy control subjects, and analysed a number of HSPC1 client proteins to assess the efficacy of these inhibitors. Great variation in cellular response to these drugs was observed in both CLL and healthy control subjects. Analysis of HSPC1 client proteins in these cells including ZAP-70, Akt, NF-kB and HSPA1A, revealed that HSPC1 inhibitors do not effect client protein levels in all samples. The results suggest that these inhibitors should not be considered as a universal treatment strategy for CLL and provide a basis for further study into elucidating the mechanisms behind HSPC1 inhibitor resistance. The final aim of this work was to investigate the role of the microenvironment in CLL progression, where a co-culture system was used as an in-vitro tool. Whilst consistent data was obtained using cell lines, and showed that microenvironmental factors promoted resistance to HSPC1 inhibitors, use of primary CLL cells in this model produced inconsistent data, again highlighting the heterogeneity of the disease.
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Assessing efficacy of cardiac rehabilitation exercise therapy in heart failure patientsBackground: Exercise-based cardiac rehabilitation (CR) is considered routine practice for patients following an acute cardiac event or surgical intervention. Although there is a seemingly strong evidence base supporting it for patients with chronic heart failure (CHF), provision in the UK remains poor for this patient group. In addition, data for CHF patients reported in key CR reviews and meta-analyses are not a true representation of the UKs CHF population. The transferability of current evidence into actual practice settings in the UK therefore remains incongruous. Rationale and aims: Study outcomes have typically included an increase in VO2 peak/ VO2 max, a decrease in natriuretic peptides, improved left ventricular function and improved health related quality of life (QoL). Access to facilities and equipment, such as cardiopulmonary exercise testing equipment is limited in the UK for the majority of CR services thus an alternative means of assessment and exercise prescription is required. The recommended alternative for testing CHF patients is the six-minute walk test (6MWT); this requires a given space and a full practice test, the latter which adds to valuable clinical and staff time available. Methods: The first set of studies of this thesis therefore investigated two adapted assessment procedures for use with CHF patients: i. the use of a shorter practice walk test of two minutes vs six minutes prior to a 6MWT and ii. the use of the space saving Chester step test with an adapted lower step height protocol to accommodate the anticipated lower fitness in CHF (4-inch vs 6-inch). Having determined a more practical and efficient means of assessing exercise capacity in CHF patients, this thesis then used the 6MWT to evaluate the efficacy of a typically recommended 12-week programme (for the UK) of exercise-based rehabilitation. It was the aim of this PhD to also combine the use of the Chester step test with cardiopulmonary measures as a corresponding physiological outcome in a sub-sample of participants; however due to resource problems, only validation of the low-step protocol was possible. In the main intervention study, the efficacy of a 12-week course of supervised moderate intensity exercise in CHF patients (ejection fraction <44%, NYHA class II to III) was then evaluated. For purposes of evaluating safety and recovery of any acute myocardial stress induced by exercise in CHF, a sub-group study was performed to evaluate the influence of an acute exercise session on two-day post-exercise levels of circulating NT-proBNP. Results: In this current suite of studies, participants were more representative of the UK CHF population than typically reported in the current evidence. Their profile involved a median age of 76 ± 16 years (mean: 67 years and range: 30 to 84 years). 98% of whom were prescribed beta-blockers, 66% were diagnosed with atrial fibrillation and 98% had two or more co-morbidities. Study 1 (Chapter 3a) verified the efficacy of a two-minute practice walk in comparison to the recommended six-minute practice walk prior to performing a baseline 6MWT in patients with CHF. Study 2 (Chapter 3b) demonstrated that a 4-inch Chester step test is a reliable assessment when space is an issue, but the criterion validity of the actual oxygen costs at each stage compared with those estimated in healthy populations were significantly lower than recommended estimations from healthy populations. Study 3 (Chapter 4) revealed individual variability in the acute response of NT-proBNP release to exercise that is worthy of further study. However the NT-proBNP data overall did not suggest a need for ‘rest days’ between exercise training sessions. The main intervention study (Study 4, Chapter 5) demonstrated a significant improvement in 6MWT performance responses, compared with control, where an increased walking distance of 25 m (p < .0001) was coupled with a reduction in heart-rate-walking speed index (T1 16.3 ± 7.3 vs T2 15.3 ± 8.7 beats per 10 walked; p < .0001). Perceptually, patients were walking faster for the same rating of perceived exertion (RPE 12 to 13). This improved aerobic functioning coincided with an improved NYHA class (T1 2.3 ± .5 vs T2 1.8 ± .6; p < .0001); however there was no change in resting NT-proBNP levels after 12 weeks. Patients in the “control group” who then went on to be offered the same 12-week intervention achieved similar outcomes, but delaying their commencement of an exercise programme by 12 weeks negatively impacted on participation uptake. Key findings and conclusions: These results have demonstrated that exercise training in CHF can lead to an improvement in both physical and perceived functioning (NYHA class). In light of some previous studies showing decreases in BNP following an exercise programme and others like this one showing no change, further questions are raised about the effect of different types and doses of activity being offered to CHF patients and the responsiveness to training of different types of patients (disease severity and demographics). The nature of the cross-over design of this study revealed that delayed commencement of exercise negatively affects participation uptake by patients, which supports current UK standards in aiming for early referral to CR.
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Alternative methods of treating atelectasis in post-operative patientsCardiac surgery incisional pain can decrease inspiratory effort, alter normal respiratory mechanics, and increase the potential for post-operative pulmonary complications. Post-surgical atelectasis is the most frequent complication after coronary artery bypass grafting (CABG), ranging from 54% to 92%. All types of therapy such as an incentive spirometry (IS), deep breathing exercises (DBE) or continuous positive airway pressure (CPAP) have a valuable role to play in the prevention or the treatment of post-surgical atelectasis. However, the type of therapy that should be used is not completely clear yet. The present research aims to evaluate the benefit of early use of CPAP via mask therapy to treat or prevent post-surgical atelectasis after CABG, particularly in smokers and elderly patients, as compared to regular (IS) therapy. Also, it aims to evaluate the patients' and medical staff's experience about the use of the new method of CPAP via mask therapy. The present research was conducted at King Fahd Armed Forces Hospital in Saudi Arabia between March 2010 and December 2011. It used a mixed methods approach. The first two studies were intervention quantitative studies, which investigated the benefit of CPAP via mask therapy. The others were qualitative studies that evaluated the experience of patients and medical staff regarding CPAP therapy use.A total of 180 patients (male and female) (36 in each group) participated in the two quantitative studies. Ninety two participants (male and female) participated in the qualitative studies. The first quantitative study results showed an improvement in CPAP via mask therapy for half hours every two hours group measurements as compared to IS therapy groups. IC was increased significantly in the "CPAP every two hours group" as compared to control group (IS) (baseline mean for IS group 1.34L and "CPAP every two hours group" 1.42L, post- therapy mean 1.59L and 1.88L respectively, p= 0.037). In addition, when chest physiotherapy was added to the two regimens, the improvement of CPAP therapy measurements became more than IS therapy. Moreover, the patient’s acceptance rate for CPAP therapy every two hours was 93% and the medical staff acceptance rate was 86%. CPAP via mask therapy for half hour every two hours had better outcomes in treating or preventing post-surgical atelectasis after CABG, particularly in smokers and elderly patients. Adding chest physiotherapy led to even better outcomes. The use of the new method of CPAP therapy had high acceptance rate by the participants and medical staff.
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Interactions between extracellular Hsp72 and blood cellsIn recent years, compelling evidence has accumulated suggesting heat shock proteins (HSPs) which are generally believed to be localised and functioning mainly within eukaryotic cells as cyto-protective molecular chaperones, are also localised in the extracellular milieu. Depending on their localisation, on the cell surface (membrance-bound or embedded), or in the peripheral circulation, extracellular HSPs may induce apoptotic cell death, or in contrast protect cells from cell damage and/or cell death when exposed to cellular stress, or may even elicit a stimulatory effect on the innate immune response including cell activiation and cytokine secretion. Hence, the localisation of intracellular and extracellular HSPs appears to be critical in determining their roles in terms of stimulating cell death, cyto-protection, or immune activiation under normal physiological conditions and following exposure to stress stimuli. This thesis describes the intracellular expression, up-regulation, and cell surface localisation of endogenous HSPs: HSP27, Hsp60, Hsp72 and Hsp90 by flow cytometry, florescence microscopy and Western blotting, under control conditions and in response to environmental stress using in vitro and ex vivo models with the intention of determining their physiological roles. The ability of extracellularly administered HSPs (Hsp70 and Hsp72) to protect cultured U937 cells in vitro or peripheral primary human leukogytes or erythrocytes ex vivo from various stress stimuli was demonstrated and was found to be dependent on surface binding and/or internalisation via scavenger receptors (SRs) or phosphatidylserine (PS), which could be blocked by receptor specific ligands. Extracellular HSPs were also shown to be able to stimulate an immune response through the induction of U937 monocyte differentiation into macrophages as evidenced through the up-regulation of the surface receptors: CD36, SR-A1 and CD91 analysed by flow cytometry. These proteins were able to stimulate TNF-x and IL-10 production and secretion by U937 macrophages, shown by ELISA, and chemotatic properties were demonstrated using Boyden chambers. The cyto-protective and immune regulatory effects of extracellular HSPs have potential therapeutic value as treatments in a wide variety of clinical situations.
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The regulation of osteoprotegerin and dickkopf-1 production in osteoblastic cellsBone is a highly specialised living tissue and has both mechanical and metabolical functions. Remodelling of the bone ensures a healthy bone mass and is regulated by a trio of secreted proteins, namely receptor-activator of NFKB (RANK), receptor-activator of NFKB ligand (RANKL) and osteoprotegerin (OPG). OPG, a major regulator of osteoclastogenesis, bone resorption and vascular calcification, is produced by various cell types including mesenchymally derived cells, particularly osteoblastic cells. Wnt signalling also plays a role in maintaining healthy bone mass. Dickkopf- 1 (DKK-1) is a soluble inhibitor of Wnt signalling and its excessive expression contributes to bone loss in rheumatoid arthritis and multiple myeloma. Recently, NDKK-1 has been demonstrated to be over-produced in osteoblasts of patients with Paget's disease of bone (PDB). The osteoblastic cell lines MG63 and Saos-2 were subjected to a series of different growth factors, hormones and cytokines to investigate the production of OPG, DKK-1 and the expression of various Wnt proteins. These results demonstrate that during standard culture conditions, both OPG and DKK-1 production in osteoblastic cells depend on a factor present in serum. Serum deprivation resulted in the up-regulation of Wnt4 and Wnt11, while down-regulating the expression of Wnt7b. Serum-induced OPG and DKK-1 production and Wnt expression was found to be regulated via a number of different signalling pathways. OPG production and expression was stimulated by platelet-derived growth factor-AB (PDGF-AB) not only in MG63 and Saos-2 osteosarcoma cells, but also a mouse pre-osteoblastic cell line (MC3T3-E1) and human bone marrow stromal cells (BMSC). PDGF-AB was shown to act through the PDGF receptor, PKC, PI3K, ERK and P38 and not via NFKB or JNK. PDGF isoforms AA, BB and AB demonstrated a similar stimulation of OPG production. The importance of PDGF in fracture healing suggests a role for OPG production in countering bone resorption during the early phase of this process. BIO, an inhibitor of canonical Wnt signalling resulted in the down-regulation of DKK-1 and the up-regulation of WntSa. Phorbol ester (PE), a known stimulator of PKC resulted in the up-regualtion of DKK-1, Wnt4, WntTa and Wnt16. The effects of PE were inhibited by bisindolymaleamide but not staurosporine. DKK-1 production, but not expression, was observed to be stimulated by calcium along with an up-regulation of WntTb and a down-regulation of WntWa and Wnt11. Incubation of pre-stimulated cells with Triton-X demonstrated the ability of calcium to increase DKK-1 secretion. DKK-1 was shown to be significantly elevated in the serum of PDB patients compared to healthy controls and did not correlate with ALP levels. Immunohistochemistry demonstrated that DKK-1 production is increased in both osteoblasts and fibrotic cells within the marrow cavity in PDB patients compared to fracture callus. B-catenin was found to be localised to intercellular membranes of plump osteoblasts, demonstrating its alternate role as a cell adhesion protein. DKK-1 therefore may be a useful biomarker of PDB and that Dkk-1 may play a central role in the aetiology of PDB. In summary, the results presented in this thesis have investigated the ways in which OPG and DKK-1 production in osteoblastic cells can be modulated with various effectors and the effect of Wnt signalling. These results may therefore be beneficial to increase the understanding of bone biology, improve fracture repair and generate further research into the role DKK-1 and the osteoblast in the aetiology of PDB to enable improved treatments to be developed.
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Novel anti-oxidant properties of cobalaminOxidative stress has been associated with a wide range of diseases, including cardiovascular diseases, Alzheimer's disease, atherosclerosis, Parkinson's disease and cancer. It also plays a role in the ageing process. Hyperhomocysteimia is commonly found to be associated with these diseases. The hyperhomocysteimia is a result of a deficiency in both folate and cobalamin Folate is known to reduce Hey and protect cells from apoptosis, but there are no studies investigating the impact of cobalamin on apoptosis induced by oxidative stress or the mechanism(s) of the protection. The aims of the research are to investigate the protective role of cobalamin and the possible mechanism(s) for this protection. It also examines the protective role of novel cobalamin and investigates their superior protection. The methods used in this research for apoptosis detection we used caspase-3 and the annexin-V, while for necrosis we used PI staining, where cell viability were detected using MTS assay. We also measured the generation of superoxide by Lucigenin-enhanced chemiluminescence and reactive oxygene species by using the redox active prob DCFH-DA. Moreover, the intracellular proteins were measured via staining with specific fluorescent-conjugated antibodies were detected using flowcytometry. Our result demonstrated that 25|iM of cobalamin protects cells from apoptosis. The protection by cobalamin was associated with induction of iHsp72 and iHO-1, and these are shown to be essential for the protection. Furthermore, our research demonstrated a novel mechanism of cobalamin-apoptosis protection involving induction of NfkB, ERK1/2 and AKT signal transduction pathways. In order to protect cells from apoptosis induced by oxidative stress, cobalamin induces the pNfkB which in turn regulate the iNOS and HO-1 induction. Cobalamin also induces thepERK1/2 which regulates the induction of Hps72 and Nrf2. And finally, pAKT induced by cobalamin which regulate the Nrf2 and HO-1 induction. The inhibition of any of theses pathways leads to loss the protection. The GSCbl and NACCbl provide a superior protection against oxidative stress, this protection involved induction of the signal transduction pathways and Hsps. To conclude; cobalamin provides protection against cells death induced by oxidative stress. Cobalamin achieves this by multiple pathways which include direct antioxidant stimulation and induction of signal transduction pathways. Different cobalamin derivatives have superior protections. These finding are a useful pharmaceutical tool in the treatment of the oxidative stress related diseases.
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Heat shock proteins: Interactions with bone and immune cellsHeat shock proteins (Hsps) are increasingly being seen as having roles other than those of intracellular molecular chaperones, particularly with regard to their potential to act as cytokines, and to stimulate the innate immune system. Hsps have also been found to promote bone resorption and osteoclast formation in vitro, although the mechanism has not been previously identified. The overall aims of this thesis were to determine whether Hsps could stimulate bone resorption by affecting the RANKL/OPG pathway, and to address the hypothesis that Hsps can act as a danger signal to the innate immune system. In order for Hsps to affect either the RANKL/OPG system of bone resorption or act as danger signals they would need to be actively released from cells, ideally in a controlled manner following exposure to the source of stress. Hsp60 and Hsp70 were found to be released from a range of immune cells including the cell lines Jurkat and U937, and also PBMCs, T-cells and B-cells. This release was not due to cell damage. The release of Hsp60 and Hsp70 were downregulated by inhibitors of protein secretion, in particular Hsp70 release was reduced by compounds that inhibited lysosomal pathways and Hsp60 release by classical secretion inhibitors. Hsp60, Hsp70, GroEL and LPS all affected the RANKL/OPG system of bone regulation; OPG production and release was down-regulated in the MG63 and GCT osteoblast-like cell lines following treatment with Hsp60, Hsp70 and LPS, and RANKL expression was upregulated following treatment with Hsp60, Hsp70, GroEL and LPS. This effect on the RANKL/OPG system was found to translate into an effect on osteoclast formation when conditioned media from treated osteoblasts was added to osteoclast precursors in the presence of M-CSF. A range of different factors that affected Hsp release were identified; PHA activation of PBMCs was found to upregulate Hsp60 release from PBMCs. GroEL and LPS caused an upregulation in Hsp70 release from PBMCs and GCT osteoblast like cells, and Hsp70 was found to stimulate Hsp60 release from PBMCs and GCT cells. These responses of Hsp release were used to form a theory of a cascade-like danger signal that may occur when cells are exposed to bacterial infection and which would result in activation of antigen presenting cells via previously identified receptors for Hsps such as CD14/TLR4 or by unidentified pathways. The elevated release of Hsps in response to GroEL and LPS was also identified as a mechanism that could stimulate bone loss during infection or autoimmuniry by affecting the RANKL/OPG system. hi conclusion, Hsp60 and Hsp70 can be released from immune cells under normal conditions, and from both immune and osteoblast-like cells following stimulation with LPS and other Hsps. The observed release responses provide a mechanism through which Hsps can act as danger signals to the innate immune system, and also as promoters of bone resorption via the RANKL/OPG system.
