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1
Miller, Aaron. "Black Band Disease: Elucidating Origins and Disease Mechanisms." FIU Digital Commons, 2012. http://digitalcommons.fiu.edu/etd/558.
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Coral diseases were unknown in the scientific community fifty years ago. Since the discovery of a coral disease in 1965, there has been an exponential increase in the number of known coral diseases, as the abundance, prevalence, distribution, and number of host species affected has also significantly increased. Coral diseases are recognized as contributing significantly to the dramatic losses of coral cover on a global basis, particularly in the Caribbean. The apparent sudden emergence of coral diseases suggests that they may be a symptom of an overall trend associated with changing environmental conditions. However, not much evidence has been gathered to address this question. The following studies were designed to build a comprehensive argument to support this hypothesis for one important coral disease – black band disease (BBD).
A meta-analysis of clone libraries identifying the microbial communities associated with BBD reveal important information including that a single cyanobacterial operational taxonomic unit (OTU) was by far the most prevalent OTU in diseased samples, and that the alphaproteobacteria, which include some of the most common bacteria in marine waters, were the most diversely represented. The analysis also showed that samples exhibited regional similarities. An fine and ultrastructural characterization of the disease revealed that the cyanobacteria are prolific borers through the coral skeleton, and that the cyanobacteria penetrate coral tissue, leading to their presence ahead of the main migrating disease band. It was further found that apparently healthy corals exposed to toxins found in BBD, exhibited similar tissue degradation to those infected with BBD. Comparing the disease progression to biofilm formation, it was determined that scouting cyanobacteria may contribute to the migration of the disease through progressive biofilm development over intact coral tissue.
Together, these studies provide significant evidence for the hypothesis that BBD is an opportunistic disease, caused by common environmental bacteria, facilitated by the changing environmental conditions associated with climate change.
2
Palmer, Samantha Jane. "Compensatory mechanisms in Parkinson's disease." Thesis, University of British Columbia, 2010. http://hdl.handle.net/2429/22661.
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Parkinson’s disease (PD) is a common movement disorder, affecting 1% of the population over the age of 65. Pathologically, PD results from degeneration of nigral dopaminergic neurons, however symptoms do not appear until an estimated 50% of these cells are lost, suggesting compensatory mechanisms exist which mask disease onset, and may later delay progression of the disease. Compensation may take place over various spatial and temporal scales, from changes in synaptic dopamine release and synthesis that take place over a period of minutes, to recruitment of novel, widespread networks of brain regions for a specific task, which may require formation of new connections over an extended period of time. Neuroimaging techniques have recently allowed the investigation of regional and network changes in activation related to motor performance in PD, however the question of whether such changes represent a downstream effect of basal ganglia degeneration, or a compensatory change, remains difficult to determine. Here, we applied an approach from research into Alzheimer’s Disease, where abnormal activation patterns are studied in the context of tasks of increasing difficulty, such that inferences regarding their compensatory nature can be made. We show that individuals with PD are able to increase the recruitment of normal networks for a motor task (motor reserve) as a form of compensation, in addition to compensatory recruitment of novel networks to accomplish the same task as healthy controls. In particular, we observe a switch from striato-thalamo-cortical (STC) motor loops to cerebello-thalamo-cortical (CTC) loops as a compensatory strategy. This compensatory recruitment involves changes in the amplitude, spatial extent, and connectivity of regions within the CTC pathway. However, this compensation does not come without a price, since we show that compensatory CTC recruitment involving disconnection between the STC and CTC loops occurs in subjects with tremor-dominant PD, but not akinetic-rigidity-dominant PD, supporting a growing body of evidence that suggests the cerebellum plays an important role in the generation of PD tremor. Together, this body of research has implications for treatments that target the symptom of tremor in PD, as therapies which minimize tremor might also reduce beneficial aspects of compensation.
3
Bourne, Nathan T. "Molecular mechanisms of Alzheimer's disease." Thesis, University of Sheffield, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.521906.
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Freeburn, Jennifer. "Haemostatic mechanisms in vascular disease." Thesis, University of Ulster, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.390118.
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Zhang, Li. "Immunological mechanisms in autoimmune disease." Thesis, University of Cambridge, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.308276.
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Freese, Andrew. "Excitotoxic mechanisms in Huntington's disease." Thesis, Massachusetts Institute of Technology, 1991. http://hdl.handle.net/1721.1/17295.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Whitaker College of Health Sciences and Technology, 1992.Includes bibliographical references (leaves 191-250).
by Andrew Freese.
Ph.D.
7
Vadnal, Jonathan. "Epigenetic Mechanisms in Neurodegenerative Disease." Kent State University / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=kent1353955013.
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Harcombe, Alun Andrew. "Immunological mechanisms in cardiac disease." Thesis, University of Edinburgh, 1995. http://hdl.handle.net/1842/20552.
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The aim of the work presented in this thesis was to examine cell and antibody-mediated immune mechanisms in heart muscle disease. The two conditions studied were acute rejection of cardiac allografts as an example of cell-mediated immune damage, and alcoholic heart muscle disease as a potential example of antibody-mediated disease. In the first study, experiments were performed on 94 endomyocardial biopsies (EMB) from 73 patients to determine the relationship between the ability to grow lymphocytes from EMB in culture and (i) grade of concurrent or future rejections, (ii) the presence of endocardial lymphocytic infiltrates (ELI), and (iii) donor/recipients HLA mismatches. These studies showed that outgrowth of lymphocytes was not closely related to the degree of rejection, not influenced by the use of polyclonal activators in the medium but it was affected by the presence to two HLA mismatches at the DR locus. The presence of ELI was related to rejection but not to outgrowth of the lymphocytes. It was concluded that, contrary to predictions in the literature, the ability to grow lymphocytes from EMB did not indicate presence of impending injection. In the second study, experiments were performed to test the hypothesis that alcoholic heart muscle disease may be caused by autoantibodies to acetaldehyde-modified myocardial proteins. Sera from 61 subjects were tested by Western analysis for antibodies against acetaldehyde-treated human myocardial proteins. No such antibodies were detected in 11 healthy controls subjects, or 28 patients with non-alcoholic heart diseases. In contrast, 4 out of 14 patients with alcoholic heart disease (28%) had detectable antibodies, suggesting a role for these antibodies in alcohol-induced heart muscle disease. To isolate potential antigenic myocardial proteins from small samples of myocardium a technique was developed for rapidly electroeluting proteins separated on a polyacrylamide gel. This system was validated by electroeluting endothelin receptors from human myocardium.
9
O'Regan, David. "Mechanisms of glucocorticoid programmed disease." Thesis, University of Edinburgh, 2004. http://hdl.handle.net/1842/27148.
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This thesis investigates the role of the renin-angiotensin (RAS), and sympathetic nervous systems (SNS) in determining programmed hypertension, and further seeks to determine whether programming effects are sexually dimorphic. DEX administration in the last week of gestation reduces offspring birth weight and programmes adult cardiovascular and metabolic physiology in a sex specific manner. In male offspring, prenatal glucocorticoid exposure programmes elevated basal circulating corticosterone, elevated PEPCK activity, and produces adulthood postglucose hyperglycaemia and hyperinsulinaemia. Whilst in female offspring, prenatal DEX programmes elevated hepatic angiotensinogen mRNA expression, elevated plasma angiotensinogen and renin activity, and produces hypertension, when measured by tail-cuff plethysmography. Unlike previous studies, offspring blood pressure was subsequently assessed with radiotelemetry, which is unmarred by any stress artefact. We no demonstrate that prenatal DEX-treated male and female offspring actually display lower basal blood pressure in adulthood; with the commonly expected hypertensive phenotype only being noted when these offspring are subjected to any stressor. Moreover, DEX-treated offspring sustain their stress-induced hypertension for longer. These hypertensive responses are mediated by alterations in the responsitivity of the sympathetic nervous system, being ameliorated by the inhibition of catecholamine synthesis, and further exaggerated by the promotion of systemic catecholamine release. Additionally, we demonstrate that DEX-treated offspring display greater sensitivity to various vasoconstrictors in the isolated mesenteric vasculature. These findings demonstrate that in utero over-exposure to glucocorticoids actually results in stress-induced hypertension, and support a role for both RAS and SNS in mediating this. Furthermore, it appears that the programming of cardiovascular physiology may reflect distinct processes in each gender, whilst the programming of metabolic physiology is male specific.
10
James, V. M. "Molecular insights into the disease mechanisms of startle disease." Thesis, University College London (University of London), 2012. http://discovery.ucl.ac.uk/1379645/.
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This study describes an in-depth investigation into the pathogenic mechanisms of inherited mutations that lead to disorders of inhibitory glycinergic transmission, primarily the rare human disorder known as startle disease/hyperekplexia. I also investigated mutations causing a similar startle phenotype in cows, mice and zebrafish. Using molecular genetics techniques, I identified pathogenic mutations in the genes that encode for proteins involved in glycinergic neurotransmission, specifically the postsynaptic glycine receptor (GlyR) subunits and the presynaptic glycine transporter GlyT2. Using homology modelling and other computational biology methods, I examined the structural and functional impacts of mutations on protein function, revealing key motifs and amino acids crucial for receptor and transporter activity. Using cDNA cloning and site-directed mutagenesis, I also generated expression constructs for wild-type and mutant proteins that were used in functional tests to measure the impact of pathogenic mutations on glycine receptor and transporter function. For certain animal models of startle disease, I was also able to develop diagnostic PCR tests for pathogenic mutations, which can be used to alleviate further animal suffering by preventing ‘at risk matings’ of carrier animals. Taken together, my findings reveal several new pathogenic mechanisms of GlyR and GlyT2 mutations in startle disease in humans and animals, revealing insights into receptor and transporter function that may be applicable to other neurological disorders.
11
Duncan, Lesley Alexandra. "Disease avoidance mechanisms and their implications." Thesis, University of British Columbia, 2009. http://hdl.handle.net/2429/13155.
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Pathogens and parasites posed significant fitness threats to our ancestors. As a consequence of these enduring threats, there are likely to have evolved a set of cognitive and affective systems designed to facilitate the behavioral avoidance of disease-causing pathogens and their carriers – including individuals who are already infected. The behavioural immune system is a suite of attentional, affective, cognitive, and behavioural responses which function to decrease the probability of contracting pathogens by activating aversive responses to indirect cues that heuristically connote the presence of infectious agents. These cues, however, are at best probabilistically related to the actual presence of pathogens, because the majority of pathogens are too small to be detected. Specific hypotheses were developed to test the influence of pathogen avoidance motivations on three aspects of social cognition. The first focuses on individual variation in concerns with pathogens. The second topic addressed pertains to the types of physical features that act as triggers to activate the behavioural immune system. The third topic addressed the extent to which pathogen avoidance mechanisms play a role in the way we learn cues which connote pathogen presence. In conclusion, this thesis provides evidence which is consistent with the operation of a psychological system which functions to prevent the transmission of infectious threats. The results reported here represent both a substantial contribution to our understanding of the subtle effects of these processes on early cognitive process and a starting point for the application of our existing knowledge to solving real world problems that have great potential for providing social and theoretical rewards.
12
Cvetkovic, ́. Jasmina. "Immune mechanisms in atherosclerotic vascular disease /." Stockholm, 2002. http://diss.kib.ki.se/2002/91-7349-268-X.
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Sitarz, Kamil Sebastian. "Disease mechanisms in mitochondrial maintenance disorders." Thesis, University of Newcastle Upon Tyne, 2012. http://hdl.handle.net/10443/1679.
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OPA1 and MFN2 are two critical mitochondrial membrane proteins required for mitochondrial fusion. OPA1 mutations account for approximately 60% of cases of autosomal-dominant optic atrophy (DOA) and up to 20% of mutational carriers develop a more severe multi-systemic neurological phenotype (DOA+) in addition to visual failure. MFN2 mutations result in Charcot-Marie-Tooth disease type 2A (CMT-2A) and in a subgroup of patients, the peripheral neuropathy is complicated by optic atrophy, highlighting a degree of phenotypic overlap with DOA+. POLG1 encodes the catalytic subunit of DNA polymerase gamma (POLG) and POLG1- related diseases are clinically highly heterogeneous, ranging from early-onset Alpers- Huttenlocher syndrome to late-onset isolated chronic progressive external ophthalmoplegia. OPA1, MFN2 and POLG1 mutations all result in disturbed mitochondrial DNA (mtDNA) maintenance, with both quantitative (depletion) and qualitative (point mutations and deletions) mtDNA abnormalities having been identified in patient tissue samples. In this PhD project, the disease mechanisms underpinning these nuclear mitochondrial disorders have been studied further. OPA1 and MFN2 mutations were found to result in significant mtDNA proliferation as a likely compensatory mechanism to impaired mitochondrial oxidative phosphorylation. Using a previously-validated repopulation assay, mtDNA replication in cultured POLG1-mutant fibroblasts was severely depressed following a period of ethidium bromide-induced mtDNA depletion. A similar observation was made with OPA1-mutant fibroblasts, but this effect was not as marked as for POLG1-mutant fibroblasts. Significant reorganisation of the mitochondrial network was also apparent for both groups of mutant fibroblasts. Although neuromyelitis optica (NMO) shares some clinical features with DOA, genetic variations within OPA1 are not associated with the risk of developing NMO. Finally, research into DOA and other mitochondrial optic neuropathies have been severely restricted by the lack of the access to retinal ganglion cells (RGCs), precluding direct studies to be performed on the cell type which is preferentially affected in this group of disorders. To circumvent this limitation, human induced pluripotent stem cell (hiPSC) lines have been generated from patient-derived fibroblasts harbouring confirmed OPA1 mutations. The future differentiation of these induced pluripotent stem cell lines into RGCs will hopefully provide a powerful and versatile tool for disease modelling and the development of targeted therapeutic strategies.
14
Arsenescu, Razvan I. "NOVEL MECHANISMS IN INFLAMMATORY BOWEL DISEASE." UKnowledge, 2011. http://uknowledge.uky.edu/gradschool_diss/211.
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Inflammatory Bowel Diseases, Crohn's Disease and Ulcerative colitis, are idiopathic chronic conditions with multifactorial determinants. In general, terms, intestinal inflammation results from abnormal host-microbe interactions. Alterations in homeostasis involve host genetic factors, environmental cues and unique luminal microbial niches. We have examined the coordinated expressions of several molecular targets relevant to the mucosal immune system and identified signature biomarkers of IBD. Qualitative and quantitative changes in the composition of microbiota can be related to unique immuno-phenotypes. This in turn can have more systemic effects that involve energy metabolism. Adiponectin, an adipose tissue derived adipokine, can restore cellular ATP levels and fulfills innate immune functions. We have concluded that IBD might represent a state of adiponectin resistance relating to chronic inflammation and obesity status.
Lastly we hypothesized that activation of xenobiotic pathway (AHR-aryl hydrocarbon receptor) can further modulate host immune and metabolic responses, and thus contribute to IBD phenotypes. We found that IBD is associated with robust mucosal, aryl hydrocarbon receptor pathway and related to proinflammatory cytokine secretion. We conclude that IBD heterogeneity is reflected through distinct immunophenotypes. Furthermore, environmental cues that involve the AhR receptor and adipose tissue derived adiponectin are important regulators of the inflammatory process in IBD.
15
Baddeley, Susan Maureen. "Cellular mechanisms in allergic eye disease." Thesis, University of Southampton, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.295267.
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Padayachee, Eden Rebecca. "Biochemical mechanisms towards understanding Alzheimer's disease." Thesis, Rhodes University, 2014. http://hdl.handle.net/10962/d1011092.
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The start of the amyloidogenic pathway in Alzheimer’s disease (AD) begins with the deposition of the Aβ₁₋₄₂ peptide surrounded by astrocytes. High levels of arginine and low amounts of neuronal nitric oxide synthase (nNOS) are associated with AD. These astrocytes store reserve arginine that is eventually metabolized by nNOS, within the vicinity of the Aβ₁₋₄₂ peptide. We propose the existence of an association vs. dissociation equilibrium between Aβ and nNOS such that nNOS is an amyloidogenic catalyst for fibrils. When Aβ binds to nNOS, it inhibits the activity of the enzyme (association phase). However when the amyloid peptide dissociates into a form that can no longer bind, later deduced as a fibril, the activity is restored. Thus, the interaction of Aβ with nNOS could serve to regulate the interaction between nNOS and arginine by restoring activity of the enzyme but at the same time promoting fibrillogenesis. Given this event occurring with the neuron, both nNOS and amyloid can serve as a biomarker for the early onset of AD. The enzyme nNOS catalyzed the formation of fibrils in the presence of Aβ peptides, while Ag nps were shown to reverse the fibril formation from Aβ peptides more so than Au and curcumin either through electrostatic or π-π stacking (aromatic) influences. Our studies have shown that the fragments of Aβ₁₋₄₂ i.e. the pentapeptide (Aβ₁₇₋₂₁) and the three glycine zipper peptides (Aβ₂₅₋₂₉, Aβ₂₉₋₃₃, Aβ₃₃₋₃₇) and the full length glycine zipper stretch (Aβ₂₅₋₃₇) all inhibited nNOS activity to varying degrees. The peptides Aβ₁₇₋₂₁ and Aβ₂₉₋₃₃ with their respective Ki values of 5.1 μM and 7.5 μM inhibited the enzyme the most. The Ki values for reversed sequenced peptides (Aβ₁₇₋₂₁r and Aβ₂₉₋₃₃r) were two fold greater than that of the original peptides while the Ki values for the polar forms (Aβ₁₇₋₂₁p and Aβ₂₉₋₃₃p) were between 3-4 fold greater than that of the original peptides. It was also found that Ag nps (Ki = 0.12 μM) inhibited the activity of nNOS the most compared to Au nps; (Ki = 0.15 μM) and curcumin (Ki = 0.25 μM). At 298K, all the ligands bound at a single site on the enzyme (n=1) and a single Trp residue (θ =1), (later identified as Trp678) was made available on the enzyme surface for quenching by the ligands. Increasing the temperature from 298K-313K, increased the value of Ksv and pointed to a dynamic quenching mechanism for Aβ peptides, nps and curcumin interaction with nNOS. The positive signs for entropy and enthalpy for all Aβ peptides nps and curcumin pointed to hydrophobic–hydrophobic interaction with the enzyme. The fact that Kd increased with temperature emphasized the endothermic nature of the binding reaction and the requirement of thermal energy to aid in diffusion of the ligand to the active site. It was concluded that the binding reaction between the ligands and nNOS was non-spontaneous and endothermic at low temperatures (+ΔG) but spontaneous at high temperatures (-ΔG). The two amino acids Tyr706 and Trp678 moved from their original positions, subject to ligand binding. Trp678 moved a minimum distance of 5 Å toward the heme while Tyr706 moved a maximum distance of 14 Å away from the heme. AutoDock 4.2 was a valuable tool in monitoring the distance of Trp678 within the enzyme interior and fluorescence resonance energy transfer (FRET) was efficient in monitoring the distance moved by Trp residues on the enzyme surface.
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Zaid, Ali, and n/a. "IMMUNE EVASION AND DISEASE MECHANISMS IN ROSS RIVER VIRUS INFECTION." University of Canberra. Biomedical Sciences, 2008. http://erl.canberra.edu.au./public/adt-AUC20091216.122508.
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Ross River virus (RRV) is an Alphavirus distributed throughout Australia. It is transmitted by
mosquitoes and is known to cause moderate to severe disease symptoms in humans. Along with
other alphaviruses such as Sindbis virus and Chikungunya virus, RRV is known to cause arthritic
symptoms, characterised by muscle and joint inflammation. Several investigations have established
the role of macrophage cells and pro-inflammatory host factors in the development of RRV-induced
disease.
In this study, we attempted to determine differences between RRV passaged in mammalian and
mosquito cells. There is strong evidence that arthropod-borne viruses are able to display enhanced
infectivity when passaged into arthropod cell line. We showed that mosquito cell-derived RRV
(mos-RRV) was able to replicate to higher titres than mammalian cell-derived RRV. We also
showed that mos-RRV failed to induce Type I IFN-associated antiviral responses.
The second aim of this study was to investigate the role of TNF-ᬠa pro-inflammatory cytokine
implicated in arthritic diseases, in the development of RRV disease. We treated RRV-infected
C57BL/6J mice with a commercially available TNF-ᠩnhibitor drug and monitored disease signs.
We found that the TNF-ᠩnhibitor does not ameliorate RRV disease (RRVD) symptoms, and that it
does not prevent muscle and joint inflammation. We analysed histological sections of muscle and
joint tissue of Enbrel-treated and untreated, RRV-infected cells. We also determined and compared
host cytokine expression profiles.
Finally, we sought to determine the requirement for natural killer (NK) cells in RRV disease. NK
cells have been detected in the synovium of RRV-infected patients since early studies, but their role
in disease pathogenesis remains unclear. Using a NK-dysfunctional mouse (C57BL/6J-Lystbg), we
showed that mice lacking a functional NK system are more susceptible to RRV disease than wildtype,
C57BL/6J mice. We monitored disease symptoms following RRV infection and assessed
muscle and joint inflammation in Lystbg and C57BL/6J mice.
This thesis examines mechanisms of viral infection and immune evasion employed by RRV, as well
as into the role of host cells and cytokines in RRVD pathogenesis disease mechanisms. We showed
that a functional NK cell system is required for the regulation of RRV-induced muscle and joint
inflammation. Our characterisation of the use of a commercial TNF-ᠩnhibitor in RRV-induced
disease in mice may provide information on the role of TNF-ᠩn viral arthritis, and may help
towards developing safe and effective treatment.
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Urb, Mirjam. "Mechanisms of «Aspergillus fumigatus» chronic airway disease." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=110500.
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Colonization of airways by Aspergillus fumigatus hyphae in immunocompetent patients with chronic lung disease leads to progressive decline in pulmonary function. While a minority of these patients develop a severe allergic response to the fungus, the majority of patients show a decline in lung function and airway hyperreactivity despite the absence of elevated IgE levels, eosinophilia or other evidence of hypersensitivity. Treatment of these non-allergic patients with antifungal drugs improves the symptoms, suggesting that pulmonary complications might be directly caused by A. fumigatus. The mechanisms underlying the acquisition of A. fumigatus colonization and the pathogenesis of pulmonary inflammation have not been studied. Our central hypothesis is that A. fumigatus interacts directly with elements of the immune system to facilitate its own colonization and induces ineffective inflammatory responses that damage host airways. To begin to investigate this hypothesis we used two complementary approaches to examine the interactions of A. fumigatus with elements of the pulmonary immune system. First, we studied the in vitro interactions of A. fumigatus with mast cells, key effector cells involved in orchestrating inflammatory responses and airway reactivity. We found that A. fumigatus triggers mast cell degranulation, while suppressing cytokine expression in an IgE-independent manner. While both degranulation and cytokine transcription required direct contact with mature hyphae, cytokine suppression could also be induced in part by A. fumigatus culture supernatant. Further studies revealed that A. fumigatus hyphae modulated mast cell function by downregulating protein tyrosine phosphorylation and in part by cleavage-dependent activation of protein tyrosine phosphatase 1B (PTP1B). The cleavage of PTP1B was caused by A. fumigatus serine proteases. In parallel, we developed a murine model of A. fumigatus colonization, where airways of healthy mice were colonized with live A. fumigatus by intratracheal injection of conidia embedded within agar beads. Unlike previously described models that induce airway hyperreactivity by repeated challenge with antigen or A. fumigatus spores, infection with this approach resulted in chronic colonization with fungi for up to 28 days. Fungal lesions within the airways were surrounded by a robust neutrophilic inflammation and peribronchial infiltration of lymphocytes. During the first 2 weeks of infection, low level Th2 type responses were seen in the infection, including increased pulmonary IL-4 levels, elevated serum IgE, and a mild increase in airway responsiveness. In addition, significant levels of pro-inflammatory cytokines and chemokines including TNF-α and MIG were observed, suggesting a mixed inflammatory picture. Furthermore, elevated IL-17 levels and presence of RORγ-positive mononuclear cells during late phase of the infection indicated the development of a Th17 response in association with reduction in pulmonary fungal load. Collectively, these results provide insight into the pathogenesis of A. fumigatus-induced chronic airways disease in patients without allergic bronchopulmonary aspergillosis, and suggest a possible role for mast cells in the pathogenesis of this condition.La colonisation des voies respiratoires par les hyphes d'Aspergillus fumigatus chez des patients immunocompétents, mais avec une maladie pulmonaire chronique, entraîne le déclin progressif de la fonction des poumons. Alors qu'une minorité de ces patients développe une réponse allergique aigue au champignon, la majorité montre un déclin dans la fonction des poumons et une hyperréactivité des voies respiratoires malgré l'absence d'une augmentation du niveau des IgE, des éosinophiles ou d'autres indications d'hyper-sensibilité. Le traitement antifongique chez ces patients améliore les symptômes suggérant que le champignon peut être la cause directe des complications observées. Les mécanismes qui sous-tendent la colonisation par A. fumigatus et la pathogenèse de l'inflammation des voies respiratoires restent largement indéterminés. Notre hypothèse centrale stipule que le champignon interagit directement avec des éléments du système immunitaire pour faciliter la colonisation et induire une réponse inflammatoire inefficace qui cause des dégâts aux voies respiratoires de l'hôte. Pour vérifier cette hypothèse, nous avons développé deux approches complémentaires visant à tester l'interaction d'A. fumigatus avec des éléments du système immunitaire pulmonaire. D'abord, nous avons étudié l'interaction in vitro entre A. fumigatus avec les mastocytes, une population clé de cellules impliquées dans la réponse inflammatoire. Nous avons montré que le champignon déclenche la dégranulation des ces cellules, tout en bloquant l'expression des cytokines indépendamment des IgE. Les processus de dégranulation et de transcription des cytokines nécessitent un contact direct avec les hyphes matures, alors que la suppression des cytokines quant à elle peut être induite en partie par le surnageant de culture d'A. fumigatus. Une étude plus approfondie nous a permis de montrer que les hyphes d'A. fumigatus peuvent moduler la fonction des mastocytes via une régulation négative de la phosphorylation d'une protéine tyrosine kinase et, en partie, via l'activation clivage-dépendante de la protéine tyrosine phosphatase 1B (PTP1B). Ce clivage de PTP1B est causé par la serine protéase du champignon. Dans une seconde approche, nous avons développé un modèle murin pour la colonisation par A. fumigatus, où nous avons fait coloniser les voies respiratoires de souris saines avec le champignon par injection intra-trachéale de conidies encapsulées dans des billes en agar. Ce modèle, au contraire des précédents, qui induisent l'hyperréactivité des voies respiratoires par exposition répétée à un antigène ou des spores de A. fumigatus, permet une colonisation chronique par le champignon allant jusqu'à 28 jours. Après ce traitement, les lésions des voies respiratoires, causées par le champignon, se retrouvent entourées par une inflammation neutrophilique robuste ainsi qu'une infiltration péri-bronchiale des lymphocytes. Durant les deux premières semaines qui suivent l'infection, nous avons détecté des niveaux bas d'une réponse type Th2, y compris une augmentation des niveaux des IL-4 pulmonaires, élévation des IgE dans le sérum, et une augmentation légère de la réponse des voies respiratoires. En plus, une augmentation significative des cytokines et des chémokines pro-inflammatoires, y compris les TNF-α et MIG, a été observée suggérant une réponse inflammatoire mixte. Les niveaux élevés des IL-7 et la présence des cellules mononucléaires RORγ-positives durant la phase tardive de l'infection indiquent le développement d'une réponse de type Th-17 associée à une réduction de la charge fongique pulmonaire. Collectivement, ces résultats nous permettent de mieux comprendre le processus de pathogenèse lié aux maladies chroniques des voies respiratoires induites par A. fumigatus chez les patients sans aspergillose bronchopulmonaire allergique, et suggèrent que les mastocytes peuvent jouer un rôle dans cette pathogenèse.
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Lim, Andrew Yih-Fan. "Mechanisms of immune regulation in HIV disease." University of Western Australia. School of Surgery and Pathology, 2008. http://theses.library.uwa.edu.au/adt-WU2008.0081.
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[Truncated abstract] HIV infection compromises the ability of the host to mount effective immune responses. In untreated HIV disease, immune activation drives high rates of cell turnover and apoptosis, ultimately leading to abnormal and dysregulated cellular function. Immune activation may also induce the expansion of CD4+ regulatory T (Treg) cell populations capable of suppressing anti-HIV responses. Treatment with antiretroviral therapy (ART) allows the recovery of CD4+ T cell numbers in most patients. Persistent deficiencies in the number and function of CD4+ T cells seen in a proportion of individuals may reflect elevated numbers of Treg cells or an imbalanced regulatory-to-effector cytokine milieu. Furthermore, some patients develop paradoxical illnesses associated with the recovery of cellular function, known as immune restoration disease (IRD). The first part of this thesis addresses the role of CD4+ Treg cells in untreated and treated HIV disease. The second part addresses the phenotype of immune cells that express IL-10 and its receptor in untreated and treated patients, and the role of IL-10 in mycobacterial IRD. Firstly, several cell surface markers were evaluated to find a flow cytometry assay that could be used routinely to identify CD4+ Treg cells in HIV-infected patients. I tested CD25, GITR, CTLA-4, NRP-1 and LAG-3, but their expression did not mirror the expression of FoxP3, an intracellular transcription factor specific to CD4+ Treg cells (Chapter 2). Two published studies then described the use of CD127 to identify CD4+FoxP3+ Treg cells in humans. Using CD127, I determined the proportions and numbers of CD4+ Treg cells in untreated HIV-infected patients and in patients in their first year of ART. Proportions of CD4+ Treg cells correlated with the proportions of activated (HLA-DRHI) CD4+ T cells and with plasma HIV RNA levels in untreated patients, but showed an inverse correlation with CD4+ T cell count. In both untreated and treated patients, the proportions and numbers of FoxP3+ cells that expressed CD8 were significantly higher than in uninfected donors. This was clearest in patients with CD4+ T cell counts below 300/'L (Chapter 3). This body of work suggests that the frequencies of CD4+ Treg cells are directly related to the level of HIV-associated immune activation. Phenotyping of FoxP3+CD4+ Treg cells in untreated and treated patients and in uninfected donors revealed that co-expression of CD45RO, CD28, CTLA-4 and markers of activation were similar in all HIV-infected patients and controls. ii FoxP3+CD8+ T cells exhibit lower levels of CD45RO, CD28 and CTLA-4, but higher expression of PD-1 and CD57 (Chapter 4). This suggests that FoxP3+CD8+ T cells may have a reduced functional capacity. It is unclear whether they have regulatory activity by virtue of FoxP3 expression. ... Both patients produced higher levels of IFN? compared with IL-10 in response to mycobacterial antigens. In contrast, patients who experienced uneventful immune reconstitution produced higher levels of IL-10 (Chapter 6). Part 1 of this thesis highlights the importance of using specific cellular markers to identify CD4+ Treg cells, and confirms CD127 as a valuable marker for routine monitoring of blood Treg cells. Part 2 of this thesis demonstrates the important regulatory role of IL-10 in patients receiving ART.
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Matthews, Stephen Paul. "Immune mechanisms of respiratory syncytial virus disease." Thesis, Imperial College London, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.406348.
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Cross, Rebecca Lee. "Biological mechanisms of depression in chronic disease." Diss., Restricted to subscribing institutions, 2007. http://proquest.umi.com/pqdweb?did=1563274391&sid=1&Fmt=2&clientId=1564&RQT=309&VName=PQD.
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Köchert, Karl. "From high-dimensional data to disease mechanisms." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2011. http://dx.doi.org/10.18452/16297.
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Die aberrante Aktivierung des NOTCH Signalweges trägt entscheidend zu verschiedensten malignen Erkrankungen im Menschen bei. Basierend auf der Analyse von hochdimensionalen Microarray-Datensätzen von klassischen Hodgkin Lymphoma Fällen und nicht-Hodgkin Fällen, haben wir eine Hodgkin Lymphoma-spezifische NOTCH Signatur identifiziert. Diese wird von dem essentiellen NOTCH-Koaktivator Mastermindlike 2 (MAML2) signifikant dominiert. Auf der Grundlage dieses Resultates haben wir die Rolle von MAML2 im Kontext des Hodgkin Lymphoma-spezifischen, aberrant regulierten NOTCH Signalweges weiter untersucht. Die signifikante Überexpression von MAML2 im Hodgkin Lymphom konnte in verschiedenen Hodgkin Lymphom Zelllinien und auch durch die immunhistochemische Analyse von primären Hodgkin Lymphom Fällen verifiziert werden. Mit Hilfe des Knockdowns von MAML2 bzw. der Inhibition des NOTCH Signalweges durch die Verwendung einer kompetitiv, dominant-negativ wirkenden, trunkierten Variante von MAML1 konnte daraufhin gezeigt werden, dass die Überexpression von MAML2 der limitierende Faktor für die Hodgkin Lymphomaspezifische, pathologische Deregulation des NOTCH Signalweges ist. Die MAML2- vermittelte Überaktivierung des NOTCH Signalweges ist darüber hinaus essentiell für die Proliferation von Hodgkin Lymphom Zelllinien und die aberrante Expression der NOTCH Zielgene HES7 und HEY1. Das konstitutive Vorhandensein von aktiviertem, intrazellulären NOTCH1 in Hodgkin Lymphom Zelllinien impliziert darüber hinaus,dass der Signalweg im Hodgkin Lymphom zellautonom aktiviert ist. In dieser Arbeit wird damit ein neuer, pathologisch hochwirksamer Mechanismus der NOTCH Signalweg-Deregulation aufgedeckt.Inappropriate activation of the NOTCH signaling pathway, e.g. by activating mutations, contributes to the pathogenesis of various human malignancies. Using a bottom up approach based on the acquisition of high–dimensional microarray data of classical Hodgkin lymphoma (cHL) and non-Hodgkin B cell lymphomas as control, we identify a cHL specific NOTCH gene-expression signature dominated by the NOTCH co-activator Mastermind-like 2 (MAML2). This set the basis for demonstrating that aberrant expression of the essential NOTCH co-activator MAML2 provides an alternative mechanism to activate NOTCH signaling in human lymphoma cells. Using immunohistochemistry we detected high-level MAML2 expression in several B cell-derived lymphoma types, including cHL cells, whereas in normal B cells no staining for MAML2 was detectable. Inhibition of MAML protein activity by a dominant negative form of MAML or by shRNAs targeting MAML2 in cHL cells resulted in down-regulation of the NOTCH target genes HES7 and HEY1, which we identified as overexpressed in cHL cells, and in reduced proliferation. In order to target the NOTCH transcriptional complex directly we developed short peptide constructs that competitively inhibit NOTCH dependent transcriptional activity as demonstrated by NOTCH reporter assays and EMSA analyses. We conclude that NOTCH signaling is aberrantly activated in a cell autonomous manner in cHL. This is mediated by high-level expression of the essential NOTCH coactivator MAML2, a protein that is only weakly expressed in B cells from healthy donors. Using short peptide constructs we moreover show, that this approach is promising in regard to the development of NOTCH pathway inhibitors that will also work in NOTCH associated malignancies that are resistant to -secretase inhibition.
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Saqlain, Saba. "Investigating Friedreich ataxia disease mechanisms and therapy." Thesis, Brunel University, 2018. http://bura.brunel.ac.uk/handle/2438/15853.
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Friedreich ataxia (FRDA) is an autosomal recessive, neurodegenerative disease caused by the excessive pathological expansion of an unstable GAA trinucleotide repeat within intron 1 of the FXN gene. FRDA occurs due to a pathological GAA repeat ranging from 70-1200 repeats, whereas normal individuals have up to 40 repeats. The prevalence of FRDA in Caucasians is 1:50,000 and it is the most common inherited ataxia. The presence of this large GAA repeat leads to silencing of the FXN gene, resulting in severely diminished levels of the essential mitochondrial protein, frataxin. Frataxin is required throughout development. Therefore, decreased frataxin levels affect the onset and progression of FRDA due to mitochondrial iron accumulation and an increase in oxidative damage, leading to detrimental cellular defects. The pathology of FRDA predominantly affects the cerebellum. Various FRDA cellular and mouse models have been developed to represent the characteristics of this debilitating disease. Data has revealed a positive correlation to exist between an increase in the number of GAA repeats and the severity of the disease. The analysis of these models has shown the expansion of the GAA repeat to be unstable, resulting in both somatic and intergenerational instability. In recent times, a single nucleotide polymorphism (SNP), Asn/Ser46, in the Sirt6 protein of FRDA patients has been associated with a better outcome of the disease. Therefore, Sirt6 heterozygote knockout mice obtained from the Jackson Laboratory were bred with YG8sR FRDA mice, to observe the effects of Sirt6 status on expanded GAA repeat instability, compared to Sirt6 wild type YG8sR FRDA mice. Densitometry analysis showed decreased levels of somatic GAA repeat instability and an increase in FXN protein in 6-month old heterozygous Sirt6 KO mice. However, increased levels of GAA repeat instability and decreased levels of FXN protein was seen in 12-month heterozygous Sirt6 KO mice. The SNP and decreased Sirt6 function may be beneficial to begin with, but not in the long run. A major aim of scientific research is to pave way for the generation of effective therapy. Therefore, attention turned to therapy to increase frataxin protein levels in YG8sR transgenic mice. The effects of the Src kinase inhibitor dasatinib, which had previously been shown to increase frataxin in cell culture, were investigated in YG8sR mice. Dasatinib was administered over a 3-month period, during which time motor coordination ability and frataxin protein levels were investigated. No effect on motor coordination was seen over the course of the treatment. Although, there was a non-significant decrease in frataxin protein levels in the brain in comparison to the vehicle-treated mice. In addition, further analysis by immunohistochemistry showed a decrease of frataxin protein expression in the dorsal root ganglia (DRG). Furthermore, the mean body weight of the dasatinib-treated mice was shown to decrease over time in comparison to vehicle-treated mice. Overall, these studies do not support the use of dasatinib for FRDA therapy. Further studies investigated a new line of GAA repeat expansion-containing FRDA transgenic mice, designated 'YG8LR'. These mice contain approximately 410 GAA repeats, which is significantly more than the previous 'YG8sR' line of mice which contained approximately 220 GAA repeats. As result of this larger GAA repeat, a further decrease of frataxin mRNA and protein was detected in the cerebellum and heart of YG8LR mice compared to YG8sR mice. Further investigations were undertaken to study the epigenetic status and the effects of lowered frataxin protein on the phenotype of the YG8LR model. Increased levels of histone deacetylation and methylation, together with DNA methylation, were detected at the FXN transgenic locus. The YG8LR mice also showed somatic and intergenerational GAA repeat instability as previously detected in earlier FRDA mouse models. Immunohistochemistry analysis of DRG sections showed a decreased level of frataxin protein in YG8LR mice. However, there was an absence of vacuoles in the DRG of the YG8LR mice as previously seen in YG8sR mice, although this particular phenotype is not actually seen in FRDA patients. Motor coordination ability and locomotor activity were significantly decreased as assessed by accelerating rotarod, beam-walk and beam-breaker open-field locomotor analysis. YG8LR mice showed increased glucose intolerance and insulin hypersensitivity in comparison to YG8sR and Y47R mice, investigated by glucose and insulin tolerance tests. Moreover, the most common cause of death in FRDA patients is caused by cardiomyopathy, therefore heart weights were obtained from B6, Y47R, YG8sR and YG8LR mice and a slight increase in mean heart weight was observed in YG8LR mice compared to the other models. This could suggest the lower levels of frataxin leads to the pathology of the heart as seen in FRDA patients. These investigations may help to provide an insight into and confirm molecular mechanisms of the disease, as well as providing a great mouse model for testing future FRDA therapies.
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Smith, Karen. "Cellular and humoral mechanisms of allergic disease." Thesis, University of Brighton, 2012. https://research.brighton.ac.uk/en/studentTheses/57df8daa-8006-4dff-8d44-ba39572913aa.
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CD 154 is a T cell activation marker, transiently expressed following ligation of the T cell receptor, therefore providing direct access to an antigen-specific T cell population. Using peripheral blood samples taken from allergic individuals and healthy non-atopic controls, this project identified and phenotyped CD 154 + T helper cells following ex vivo stimulation with native allergen extracts (birch pollen, cat dander, grass pollen). Peripheral blood mononuclear cells were stimulated with allergen extract for 16 hours in the presence of Brefeldin A. Responding CD 154 + T cells were identified and phenotyped using multiparametric flow cytometry. Activated CD154+ TH1, TH2 and TRl-Iike cells, that co-expressed IFNy, IL4 and ILIO respectively, were identified in allergic and non-allergic participants. A close correlation was observed between TH1, TH2 and TR1-like cell frequency in non- allergic participants, such that the three parameters increased together to maintain a low TH2:TH1 ratio. The relationship between TH1, TH2 and TR1-like responses was dysregulated in allergic individuals, with abrogation of the IL10 response and a higher TH2:THI ratio. A close correlation was observed between Th2 cell frequency and the absolute concentration of birch-specific IgE. This work confirms previous reports of a more differentiated T cell phenotype in allergic subjects with regard to seasonal allergens. The detection of CD154+ T cells after short-term antigen stimulation may be a useful method for the detection of T cell responses to allergens when cost, speed and convenience are priorities. The CD 154 assay was also used to investigate allergen-specific T cells in two situations: [1] during allergoid immunotherapy and [2] at peak pollen season compared to out of season. This preliminary data suggests an increased frequency of TH2 cells in allergic individuals during the birch pollen season compared to healthy non-allergic controls, and a decrease in the TH2:THI ratio following successful immunotherapy. A proliferation assay utilising the cell surface PKH dye was also optimised to investigate proliferative responses to native allergen extracts in allergic and non- allergic subjects. Colonisation with superantigen-producing staphylococci is common in atopic diseases and may contribute to the initiation and maintenance of allergic sensitisation. However, little is known regarding T cell responses to superantigens in atopic individuals. This project sought to investigate T helper cell responses to the superantigen Staphylococcal Enterotoxin B (SEB) from Staphylococcus aureus in non-atopic individuals and highly atopic polysensitised individuals. Peripheral blood mononuclear cells were stimulated with SEB for 16 hours in the presence of Brefeldin A. Responding TH1, TH2, TH17, TR1-like and naturally occurring regulatory T cells were identified using multiparametric flow cytometry. This preliminary study identified a downregulated T H 17 response to the superantigen SEB in highly atopic individuals that does not relate to T Reg cell frequency or TCRVP3 expression. The Pollen-Food Syndrome (PFS) is caused by sensitisation to homologous panallergens within aeroallergens and food proteins. Information regarding the sensitisation profiles of individuals with PFS in the UK is limited and investigations into causative panallergens are not routinely performed. In a small study, patients with symptoms suggestive of PFS were recruited. from the Allergy Clinic at Brighton and Sussex University Hospital NHS trust. A standardised food allergy questionnaire was completed and serum analysed by component-resolved diagnosis using ImmunoCAP ISAC technology. This study cohort conformed to the Northern European pattern of birch-pollen associated PFS with cross-reactivity between the major birch pollen allergen Bet v 1 and homologues in food proteins. Sensitisation to LTPs and profilins was also noted, but the clinical relevance of these remains to be elucidated. Profilin sensitisation was associated with reactions to a significantly larger number of foods compared to PR-10 mono sensitised individuals.
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Aman, Yahyah. "Investigating mechanisms of pain in Alzheimer's disease." Thesis, King's College London (University of London), 2018. https://kclpure.kcl.ac.uk/portal/en/theses/investigating-mechanisms-of-pain-in-alzheimers-disease(2bbc4948-3804-4e23-baa3-cddb8b4a6321).html.
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Experience of pain is a key contributor to challenge of care in Alzheimer’s disease (AD) individuals and is often associated with age-related medical comorbidities, commonly musculoskeletal conditions such as osteoarthritis (OA). Significant alteration in perception of pain is an important clinical issue in patients with AD and its effective treatment is increasingly recognised as a critical unmet clinical need. Currently, management of pain has been hindered by difficulties in the assessment of pain partly due to the impaired ability to communicate sensations as well as the inadequate understanding of the underlying mechanisms of pain in this susceptible patient group. Animal models of AD recapitulate many of the clinical and pathological features of the human AD and thereby offer to be a powerful tool in order to delineate mechanisms in AD. This thesis aims to investigate possible alterations of nociceptive sensitivity, in acute and chronic pain models, using the transgenic double-mutant APPswe×PS1.M146V (TASTPM) mouse model of AD. Furthermore, it aims to elucidate the associated plastic changes along the pain pathway (i.e. spinal cord and thalamus) of the preclinical TASTPM model and evaluate its translational implication using human post-mortem tissue obtained from AD patients with chronic pain conditions. This thesis provides preclinical evidence for alterations in nocifensive behaviour that are associated with dysfunction of the opioidergic system in TASTPM mice compared to age- and gender-matched wild-type (WT) controls. Specifically, TASTPM mice display reduced sensitivity to acute noxious thermal stimulation and impaired persistent pain-like behaviour in a model of OA induced by an intra-articular administration of monosodium iodoacetate (MIA). These changes coincide with impairment of cognition, development of amyloid plaques in the brain, and intraneuronal accumulation of amyloid precursor protein/β-amyloid (APP/Aβ) in the spinal cord of TASTPM mice. Increase in expression of endogenous inhibitory peptides: enkephalins in the dorsal horn and β-endorphins in plasma correlate with the attenuated nociceptive sensitivity to noxious thermal stimulation and persistent pain, respectively, in TASTPM mice. Administration of naloxone, an opioid antagonist, re-establishes normal sensory thermal thresholds and unmasks the reduced MIA-induced mechanical allodynia exhibited by the TASTPM mice. Subsequent administration of the analgesic morphine, an opioid agonist, induces heightened responsiveness in the TASTPM mice compared to WT controls. Together, these findings implicate the disruption of the opioidergic system as a common mechanism underlying the reduced acute nocifensive and impaired persistent pain-like behaviour in TASTPM model of AD. In parallel to these observations, alteration in the neuro-immune plasticity along the pain pathway is also identified as a possible mechanism that underlies impaired persistent pain-like behaviour exhibited by AD mice. In particular, diminished spinal microgliosis in response MIA administration in the periphery coupled to inability of gabapentin to induce analgesia were evident in TASTPM. These data indicate blunted central sensitisation in the model of AD. Intriguingly, increase in the extent of neuroinflammation is observed in the thalamus of TASTPM mice compared to WT, in the model of OA. However, no change in APP/Aβ pathology was detected in both the spinal cord and brain of TASTPM mice. Analyses of human post-mortem tissue obtained from AD individuals lend support to preclinical observations in TASTPM mice of intraneuronal accumulation of APP/Aβ and amyloid plaque deposition in the spinal cord. The other pathological hallmark of AD, neurofibrillary tangles (NFT), was not detected in the spinal cord of AD patients. Assessment of supraspinal structures involved in pain processing, including the thalamus, reveal presence of both amyloid plaques and NFT, which increased in abundance with progression of AD. Moreover, evaluation of AD-associated pathology in AD individuals with a clinical history of chronic pain and/or persistent analgesic use displays no alteration in deposition of amyloid plaques and NFT. However, increase in microglial activation was observed in both the spinal cord and supraspinal regions compared to AD subjects with no clinical pain record. These data provide additional support for the exacerbation of ongoing neuroinflammation identified in the preclinical TASTPM mice in a model of OA. Such observations from human post-mortem tissue reinforce the advantage of utilising the TASTPM model of AD in order to delineate mechanisms of pain in AD as it recapitulates some of the key features that are observed in human AD patients. Taken together, the findings of this thesis have important clinical implications for the care of patients with AD who have deteriorating cognitive function along with reduced sensitivity to pain. Altered pain sensitivity could be considered in assessing clinical risk as it may be associated with neuropsychiatric symptoms as well as an increased risk of injury during daily routine activities. Finally, these data highlight the need to re-evaluate current treatments, such as opioids, or develop novel therapeutic strategies for management of pain in individuals with AD.
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Singhal, Sumeet. "Disease modifiers, mechanisms and monitoring in CADASIL." Thesis, St George's, University of London, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.418294.
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Beadle, Nina Elizabeth. "Mitochondrial DNA defects and human disease : the molecular mechanisms underlying disease expression." Thesis, University of Newcastle Upon Tyne, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.544209.
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Lane, Fiona Mary. "Defining mechanisms of neurodegeneration associated with protein misfolding diseases." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/19542.
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Protein misfolding diseases (PMDs) are a broad group of disorders including Alzheimer’s, Parkinson’s and prion diseases. They are characterised by the presence of aggregated, misfolded host proteins which are thought to cause cell death. Prion diseases are associated with misfolded prion protein (PrPSc), which has a tendency to form fibrillar aggregates. By contrast, Alzheimer’s disease (AD) is associated with misfolded amyloid beta (Aβ), which aggregates to form characteristic Aβ plaques. A feature which is common across PMDs is that small assemblies (oligomers) of the misfolded proteins are thought to be the important neurotoxic species, and it has been proposed that there may be a shared mechanism leading to cell death across PMDs caused by oligomers. In this study, the toxicity of different misfolded forms of recombinant PrP (recPrP) and recombinant Aβ (recAβ) and the mechanisms leading to cell death were investigated using a primary cell culture model. In addition, the importance of the disulphide bond in recPrP in relation to oligomer formation was explored using size exclusion chromatography and mass spectrometry, the toxicity of the different resulting oligomer populations were also investigated. Both recPrP oligomers and fibrils were shown to cause toxicity to mouse primary cortical neurons. Interestingly, oligomers were shown to cause apoptotic cell death, while the fibrils did not, suggesting the activation of different pathways. By contrast, recAβ fibrils were shown to be non-toxic to cortical neurons, Aβ oligomers, however, were shown to cause toxicity. Similar to recPrP, my data showed that it is likely that recAβ 1-42 oligomers also cause apoptosis. However, by contrast this seemed to be caused by excitotoxicity, which was not found to be the case for recPrP. Additionally, I have shown that the presence or absence of the disulphide bond in PrP has a profound effect on the size of oligomers which form. RecPrP lacking a disulphide bond leads to the formation of larger oligomers which are highly toxic to primary neurons. Findings from this study suggest that structural properties such as the disulphide bond in PrP can affect the size and toxicity of oligomers, furthermore, whilst oligomers have been shown to be important in both AD and prion diseases, they may not trigger the same pathways leading to cell death.
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Schoultz, Ida. "Mechanisms of bacterial-epithelial interaction in Crohn’s disease." Doctoral thesis, Linköpings universitet, Kirurgi, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-21104.
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Crohn’s disease (CD) is believed to be initiated when an individual, who has agenetic predisposition either leading to a disturbance in the barrier functionand/or the innate immune system is exposed to triggering environmentalfactors, the most important being intraluminal bacteria. Genetic and functionalstudies have confirmed the Pattern-recognition receptors (PRRs), Nod2, TLR4and NALP3, as important mediators of the inflammatory process associatedwith disease progression. However, the mechanisms that link enteric bacteriaand barrier function in a background of genetic predisposition to CD are justbeginning to emerge. The general aim of this thesis was therefore to morethoroughly investigate the mechanisms of bacterial-epithelial interaction in CD. Here we present evidence suggesting that the small bowel is able to inducetranscytosis of antigens after short term exposure to Yersinia pseudotuberculosis. This suggests that small bowel enterocytes are able toattain follicle associated epithelial (FAE) abilities and contribute to the barrierdysfunction observed in CD. Furthermore we report a positive effect of anti-TNFα treatment (infliximab) on the translocation of adherent invasive E.coli (AIEC) across the colonic mucosa of patients suffering from severe CD. We also confirm the importance of the Nod-like receptors (NLRs) in thepathogenesis of CD by showing that combined polymorphisms in the genesencoding NALP3 and CARD8 confer susceptibility to CD among Swedish menand in addition to previous published results add a gender aspect on thegenotype-phenotype relationship in CD. Finally, we show that Nod2 is rapidlysubjected to ubiquitination followed by proteasomal degradation, henceproviding important clues about how NLR regulation might occur in the cell,suggesting that the ubiquitin-proteasome pathway is an important factor toconsider in the development of the disease. In conclusion we report novel insights into the bacterial-epithelial interactionsoccurring in CD and contribute important clues about the origin of this disease.ISBN: 978-
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Fang, Zhi You. "Mechanisms and therapeutic implications of diabetic heart disease /." [St. Lucia, Qld], 2004. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe18240.pdf.
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Shaw, James A. (James Alexander) 1968. "Mechanisms of plaque stability in coronary artery disease." Monash University, Dept. of Medicine, 2001. http://arrow.monash.edu.au/hdl/1959.1/9016.
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Ostrowski, Stephen M. "Pleiotropic mechanisms of statin action in Alzheimer's Disease." Cleveland, Ohio : Case Western Reserve University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=case1190669698.
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Pan, Beiqing. "Mechanisms of skeletal disease mediated by haematological malignancies /." Title page, table of contents and abstract only, 2004. http://web4.library.adelaide.edu.au/theses/09PH/09php1871.pdf.
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Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine and The Hanson Centre, Institute of Medical and Veterinary Science, 2004."August 2004" Errata inside front cover. Bibliography: leaves 126-159.
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Lax, Nichola Zoe. "Understanding the mechanisms of neurodegeneration in mitochondrial disease." Thesis, University of Newcastle Upon Tyne, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.544206.
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Chakrabarti, Shubro. "Mechanisms of fibrosis in feline chronic kidney disease." Thesis, Royal Veterinary College (University of London), 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.572451.
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Babetto, Elisabetta. "Axon degeneration mechanisms in Alzheimer's disease and injury." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609249.
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Bentham, James Robert. "Genetic & molecular mechanisms of congenital heart disease." Thesis, University of Oxford, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.496824.
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Bunn, L. M. "Sensory mechanisms of balance control in cerebellar disease." Thesis, University College London (University of London), 2011. http://discovery.ucl.ac.uk/1306178/.
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A wealth of evidence exists to suggest that the cerebellum has an important role in the integration of vestibular, proprioceptive and visual sensory signals. Human bipedal balance depends on sensory integration and balance impairment is a common feature of cerebellar disease. I test the hypothesis that disrupted sensori-motor processing is responsible for balance impairment in cerebellar disease. Balance control in subjects with pure cerebellar disease (SCA6) was compared with matched healthy subjects using a mix of traditional clinical and laboratory-based tests. Sensory processing was explored using a novel combination of tools designed to deliver single-sensory channel balance perturbations. The vestibular, proprioceptive and visual channels were stimulated with galvanic vestibular stimulation, vibration and visual scene motion respectively. Standing balance was explored using 3D whole body motion analysis. Sway speed when standing quietly with eyes open was significantly increased in those with SCA6 and strongly correlated with disease severity scores. Responses to isolated vestibular stimulation suggest largely normal vestibulo-motor processing in SCA6 subjects. Responses had normal latency and magnitude. Response direction followed head position in the normal way suggesting intact vestibulo-proprioceptive integration. Vision had a normal attenuating effect on response magnitude suggesting intact vestibulo-visual integration. Responses to isolated vestibular, proprioceptive and visual stimuli responses were compared to investigate whether there might be a predominant deficit in any one channel. Vestibular and proprioceptive stimuli evoked largely normal responses. In contrast, visual stimuli consistently evoked abnormally large responses with significant timing delays. Increases in SCA6 response magnitudes to moving visual stimuli strongly correlated with disease severity scores. This finding is the first to point to a specific change in sensori-motor processing in cerebellar disease. This finding could contribute to balance impairments but is unlikely to explain balance impairment observed with the eyes closed. Overall sensory processing for balance control in SCA6 is largely intact.
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De, Asis Kathleen Gayle. "Novel mechanisms of fibrinolysis in health and disease." Thesis, University of British Columbia, 2014. http://hdl.handle.net/2429/46746.
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This is a two-part thesis, focusing first on a clinical and then on a biochemical aspect of fibrinolysis, the process that dissolves blood clots.
Hyperfibrinolysis: An explanation for reduced cardiovascular disease in hemophilia patients
Hemophilia is a coagulation disorder where factor (F) VIII or FIX deficiency results in prolonged bleeding. Therapeutic FVIII or FIX replacement has lengthened hemophilia patient life expectancy. Interestingly, retrospective studies have demonstrated a lower standard mortality risk from cardiovascular disease (CVD) compared to the normal population and enhanced fibrinolytic capability has been proposed as an explanation for this relative protection from CVD. Through the analysis of tissue-type plasminogen activator (tPA), the initiator of fibrinolysis and two inhibitors of fibrinolysis: plasminogen activator inhibitor-1 (PAI-1) and thrombin activatable fibrinolysis inhibitor (TAFI), the current study showed that some hemophilia patients have reduced inhibition of the fibrinolytic pathway compared to age and cardiovascular risk matched controls. A trend of hyperfibrinolysis was also seen in hemophilia patients through accelerated plasma clot lysis with 50% of the patients having at least a 2-fold enhancement, and 22% with at least a 10-fold enhancement.
Regulation of Clotting Factor Xa Auxiliary Cofactor Function in Fibrinolysis through β-Peptide Excision
We have shown that clotting factor Xa (FXa) cleaved by the fibrinolysis protease, plasmin (Pn), produces consecutive fragments, called FXaβ and Xa33/13. These both have newly exposed C-terminal lysines (Lys) that accelerate tPA in purified clot lysis assays. However, in plasma Xa33/13 rapidly loses this fibrinolytic function due to degradation. It is therefore important to define the role of four possible basic amino acid residues in generating FXaβ; Lys(K)427, Arg(R)429, K433 and K435. Using site directed mutagenesis, K435 was defined as the preferred cleavage site, while R429 was unfavourable. K433 and K435 were found to be important for RVV-X activation and β-peptide cleavage facilitates the production of Xa33/13. Interestingly, when all four basic residues were mutated to Gln (Q), preventing production of FXaβ, an unexpected distal cleavage site was demonstrated to also enhance Pn generation. Replacing the R429 with lysine generated a hyperfibrinolytic species suggesting a potential novel therapeutic approach.
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Cartwright, Lindsay Sarah. "Nicotinamide : neuroprotection and neurodegeneration mechanisms in Parkinson's disease." Thesis, University of Birmingham, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.433503.
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Chaves, Alysia Anne. "Mechanisms of AIDS and cocaine related cardiovascular disease." Columbus, Ohio Ohio State University, 2003. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1056031201.
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Thesis (Ph.D.)--Ohio State University, 2003.Title from first page of PDF file. Document formatted into pages; contains xx, 401 p.; also includes graphics (some col.). Includes abstract and vita. Advisor: John A. Bauer, Dept.of Pharmacy. Includes bibliographical references (p. 387-391).
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Binda, Caroline Sophie. "Molecular mechanisms of synaptic dysfunction in Alzheimer's disease." Thesis, University of Bristol, 2018. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.752765.
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Muhammed, Kinan. "Mechanisms underlying apathy in health and Parkinson's disease." Thesis, University of Oxford, 2018. http://ora.ox.ac.uk/objects/uuid:930d41d1-c815-4494-8c32-1439947db899.
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Apathy or lack of motivation is increasingly recognized to be a major factor affecting quality of life and prognosis in many neurological conditions. It is particularly prevalent in Parkinson's disease, impacting on every disease stage, including de novo cases, and has been reported to affect up to 70% of cases. Despite the pervasiveness of apathy, challenges remain in its detection, clinical assessment and treatment. Several lines of evidence have implicated fronto-striatal reward related neural pathways in the genesis of apathy but the precise processes remain to be fully explained. This thesis examines the potential mechanisms of apathy using Parkinson's disease as a model to study the condition. Novel oculomotor tasks that used eye movement and pupillary responses were developed to help assess if insensitivity to incentives could be an underlying component of apathy. This was examined in healthy young and elderly participants as well as in patients with Parkinson's disease. Patients were tested both ON and OFF their normal dopaminergic medication so that the effect of dopamine could be assessed and the association with apathy determined. This was also performed in a pharmacological study in young participants with the use of Haloperidol, a dopaminergic D2-selective antagonist. Insensitivity to rewards modulated by dopamine was regarded to be a contributory mechanism of apathy in Parkinson's disease and also applicable to general mechanisms of motivation in healthy populations.
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Ngandu, Mpoyi Elie. "Engineering biointerfaces to reveal collagen IV disease mechanisms." Thesis, University of Glasgow, 2017. http://theses.gla.ac.uk/9032/.
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Basement Membranes (BMs) are specialised extracellular matrix (ECM) structures that underlie all endothelial and epithelial cells, and provide structural support to tissues as well as influence cell behaviour and signalling. Mutations in the BMs major component collagen IV cause eye, kidney and cerebrovascular disease including intracerebral haemorrhaging (ICH). Haemorrhagic stroke accounts for 15% adult stroke and 50% paediatric stroke, and carries the worst prognosis and there are no therapeutic strategies. Mutations in the genes COL4A1/COL4A2 (collagen IV alpha chain 1 and 2) cause BM defects due to mutant protein incorporation in the BM or its absence by ER retention, and ER-stress due to intracellular accumulation of collagen IV. Despite this, the mechanism(s) of collagen IV mutations disease remain poorly characterised. To provide novel insights into mechanisms of collagen diseases, this study investigates the effect of defined engineered biointerfaces on cell behaviour/signalling, collagen secretion in COL4A2 mutant and wild-type cells. Atomic force microscopy and spectroscopy were employed together with confocal and biochemical analyses of cells cultured on engineered synthetic polymers, poly(ethyl acrylate) and poly(methyl acrylate), coated with ECM proteins, namely laminin, collagen IV and fibronectin. This enabled us to address the hypothesis that biomaterials may alter the behaviour of COL4A2+/G702D mutant cells by overcoming some of the defects caused by the mutation and rescuing the downstream effect of the ER stress. Of the ECM proteins that were used, only fibronectin was observed to undergo a drastic structural change depending on the substrate chemistry. On poly(ethyl acrylate), fibronectin was assembled into fibrillary networks upon adsorption, and these nanonetworks induced increased secretion of Col4a2 in COL4A2+/G702D cells than on poly(methyl acrylate) or control glass. The behaviour of the mutant cells appeared to be influenced by the underlying biointerface, increased levels of molecular chaperones and reduced ER area suggested an increased collagen IV folding capacity when the cells were cultured on the FN nanonetworks compared to the other surfaces. COL4A2+/G702D cells interacted with the adsorbed proteins and were able to mechanically translocate them. Enhanced formation of focal adhesions was also seen on FN-coated polymers, where ligand density and actin-myosin contractility accounted for the observed increase in cell adhesion strength. The stiffness of the mutant fibroblasts and of their ECMs was found to be 10 times lower than that of the wild-type cells; interestingly, mutant cells cultured on FN nanonetworks on poly(ethyl acrylate) were able to deposit a protein matrix with significantly higher Young modulus than on glass or poly(methyl acrylate). These findings suggest that biomaterials are able to influence the behaviour of these mutant cells through changes in the interfacial layer of adsorbed proteins presented to them. Collectively, these data provide an understanding of the effect of mutations on cell characteristic and a basis of concept that material may be employed to modulate effects of mutations of collagen/ECM molecules. Understanding the mechanisms through which these surfaces trigger a change in cell response will prove valuable for the development of new therapeutic approaches to address pathologies due to collagen IV mutations. In this respect, further investigation is needed to dissect the signalling pathways involved.
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Ostrowski, Stephen M. "Pleiotropic Mechanisms of Statin Action in Alzheimer's Disease." Case Western Reserve University School of Graduate Studies / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=case1190669698.
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Hill, Rebecca Maree. "Biological mechanisms of disease relapse in childhood medulloblastoma." Thesis, University of Newcastle upon Tyne, 2015. http://hdl.handle.net/10443/2796.
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Over 30% of patients diagnosed with a medulloblastoma experience disease recurrence. Relapse is almost universally fatal, only infants who receive delayed radiotherapy at disease recurrence typically survive long term. Consequently relapse is the single leading cause of mortality disease-wide. Improved understanding of medulloblastoma at diagnosis has led to the identification of four distinct molecular subgroups with differing biology and outcome. These comprise of medulloblastomas associated with WNT and SHH pathway disruption (MBWNT and MBSHH respectively), and Group 3 and Group 4 tumours (MBGroup3 and MBGroup4). In contrast, very little is understood about the disease at recurrence, and at present there are only two published studies interrogating the biology of relapsed medulloblastoma. However, improved understanding of the biology at relapse is critical to improving treatment. Events at disease relapse could be explored as therapeutic targets or, if predictive of disease recurrence, provide an opportunity to escalate upfront therapy with the aim of preventing relapse. This study compiled a cohort of medulloblastoma tumours sampled at relapse (n=29), paired with their diagnostic counterparts. All clinicopathological and molecular features, with an established relationship to disease prognosis at diagnosis, were interrogated in this paired relapse cohort. With the exception of molecular subgroup, all features investigated displayed evidence of alteration and predominantly acquisition at recurrence. Most strikingly, the emergence of combined p53-MYC defects was commonly observed at relapse and these features were associated with locally aggressive, rapidly progressive disease following relapse. Through collaborative work, this discovery was explored further, with the development of a novel GTML/Trp53 KI/KI mouse model which faithfully recapitulated the clinicopathological and molecular features of the p53-MYC human tumours, and demonstrated the dependency of tumourigenesis and maintenance on this genetic interaction. Moreover, therapeutic inhibition of Aurora A kinase using MLN8237 in these mouse tumours led to degradation of MYCN, tumour reduction and prolonged survival. v A novel genome-wide DNA methylation analysis was next undertaken in the paired relapse cohort, focusing on MBGroup4 tumours, to interrogate maintained and acquired DNA methylation events between diagnosis and relapse, which may play a role in tumour development. Individual CpG sites on the Infinium DNA methylation 450K array were assessed for changes in their DNA methylation status between diagnosis and relapse. Fifteen candidate genes demonstrated tumour-specific methylation states that emerged at relapse and correlated with gene expression. The T-box and Homeobox gene families accounted for 8/15 (53%) candidates identified. Both these families are reportedly important for tumour development in other cancers. In addition, several studies suggest that epigenetic mechanisms, such as DNA methylation, play a regulatory role in their gene expression. Finally, a large cohort of medulloblastoma tumours (n=206), sampled at diagnosis, from patients who are known to go on and recur, was assembled to investigate any subgroup-specific patterns and timings of relapse. MBWNT rarely relapsed, whereas MBSHH frequently relapsed at both local and distant sites, but were the tumour subgroup most readily salvaged by radiotherapy in patients who were not treated with craniospinal irradiation (CSI) at diagnosis (8/12, 67%). Both MBGroup3 and MBGroup4 were widely metastatic at recurrence (34/41 (83%) and 52/61 (85%)) but contrastingly MBGroup3 relapsed quickly (p=0.0022), whereas MBGroup4 relapsed more slowly (p=0.0008). In patients who did not receive upfront CSI, MYC amplification at diagnosis was associated with rapid disease progression after relapse (p=0.0003). No diagnostic feature was significantly associated with time to death following relapse in the cohort of patients who received upfront CSI. This finding was supported by data from the paired relapse cohort where, in patients who received upfront CSI, it was the biological features of the tumour at relapse and not diagnosis, which were associated with disease course. In summary, this study has discovered emergent combined p53-MYC defects at medulloblastoma relapse which are associated with disease behaviour, identified potentially epigenetically regulated candidate genes in relapsed MBGroup4 tumours, and shown that the patterns of disease relapse are associated with radiotherapy and molecular subgroup. Together these findings demonstrate that medulloblastoma tumour biology is significantly different at relapse and that the timings and location of vi disease recurrence should be considered in the context of molecular subgroup and treatment. Biopsy at disease recurrence is now essential to validate and expand on these novel findings, interrogate all molecular subgroups at disease recurrence, and translate these discoveries into improved outcomes for the patients suffering from this devastating diagnosis.
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Harry, Rachael Alexandra. "Mechanisms underlying the vasodilation of human liver disease." Thesis, King's College London (University of London), 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.421761.
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Mutsaers, Chantal. "Mechanisms of disease pathogenesis in Spinal Muscular Atrophy." Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/9774.
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Low levels of survival motor neuron (SMN) protein cause the autosomal recessive neurodegenerative disease spinal muscular atrophy (SMA), through mechanisms that are poorly defined. SMN protein is ubiquitously expressed, however the major pathological hallmarks of SMA are focused on the neuromuscular system, including a loss of lower motor neurons in the ventral horn of the spinal cord and atrophy of skeletal muscle. At present there is no cure for SMA. Most research to date has focused on examining how low levels of SMN lead to pathological changes in motor neurons, therefore the contribution of other tissues, for example muscle, remains unclear. In this thesis I have used proteomic techniques to identify intrinsic molecular changes in muscle of SMA mice that contribute to neuromuscular pathology in SMA. I demonstrate significant disruption to the molecular composition of skeletal muscle in pre-symptomatic SMA mice, in the absence of any detectable degenerative changes in lower motor neurons and with a molecular profile distinct from that of denervated muscle. Functional cluster analysis of proteomics data and phospho-histone H2AX labelling of DNA damage revealed increased activity of cell death pathways in SMA muscle. In addition robust up-regulation of VDAC2 and down-regulation of parvalbumin was confirmed in two mouse models of SMA as well as in patient muscle biopsies. Thus intrinsic pathology of skeletal muscle is an important event in SMA. I then used proteomics to identify individual proteins in skeletal muscle of SMA that report directly on disease status. Two proteins, GRP75 and calreticulin, showed increased expression levels over time in different muscles as well as in skin samples, a more accessible tissue for biopsies in patients. Preliminary results suggest that GRP75 and calreticulin can be detected and measured in SMA patient muscle biopsies. These results show that proteomics provides a powerful platform for biomarker identification in SMA, revealing GRP75 and calreticulin as peripherally accessible potential protein biomarkers capable of reporting on disease progression in muscle as well as in skin samples. Finally I identified a role for ubiquitin-dependent pathways in regulating neuromuscular pathology in SMA. Levels of ubiquitin-like modifier activating enzyme 1 (UBA1) were reduced in spinal cord and skeletal muscle tissue of SMA mice. Dysregulation of UBA1 and subsequently the ubiquitination pathways led to the accumulation of β-catenin. I show here that pharmacological inhibition of β-catenin robustly ameliorates neuromuscular pathology in animal models of SMA. Interestingly, downstream disruption of β-catenin was restricted to the neuromuscular system in SMA mice. Pharmacological inhibition of β-catenin failed to prevent systemic pathology in organs. Thus disruption of ubiquitin homeostasis, with downstream consequences for β-catenin signalling, contributes to the pathogenesis of SMA, thereby highlighting novel therapeutic targets for this disease.
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Thornton, Catherine Clare. "Mechanisms of vascular protection in chronic inflammatory disease." Thesis, Imperial College London, 2014. http://hdl.handle.net/10044/1/26595.
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Premature cardiovascular disease (CVD) complicates chronic systemic inflammatory disease, and is initiated by endothelial dysfunction. Despite an ever-improving array of medications to treat these diseases, how to prevent CVD is not known. Studying the actions of endogenous mediators of endothelial cytoprotection and of disease-modifying drugs might establish how best to protect patients with systemic inflammatory diseases from atherosclerosis, if specific beneficial effects on vascular endothelium could be demonstrated. High flow laminar shear stress (LSS) and vascular endothelial growth factor (VEGF) are two well known endogenous drivers of endothelial cytoprotection. Both can induce expression of protective genes but it is not known how confluent endothelial cells (EC) respond to VEGF when conditioned with LSS. Methotrexate (MTX) is the most widely prescribed treatment for rheumatoid arthritis and clinical data suggests that it reduces CV mortality. Known mechanisms of action of MTX result in intracellular accumulation of activators of AMP-activated protein kinase (AMPK). AMPK regulates cytoprotective genes in EC and its activation is associated with diverse desirable effects. I hypothesised that EC conditioned with LSS would be primed to respond to VEGF, resulting in a synergistic induction of cytoprotective genes. Secondly I investigated whether MTX exerts beneficial protective effects on vascular endothelium via activation of AMPK, which enhance endothelial resistance to injury. Studies of human umbilical vein EC (HUVEC) exposed to LSS showed that responses to VEGF are not enhanced in these conditions. However, MTX phosphorylated AMPKαThr172 and induced expression of several cytoprotective genes, notably manganese superoxide dismutase (MnSOD). The addition of folic acid did not alter this; and it was also preserved when HUVEC were pre-treated with TNFα to mimic dysfunctional endothelium. siRNA depletion of AMPK attenuated MTX-mediated MnSOD induction. MTX treatment led to AMPK-dependent phosphorylation of the transcription factor CREBSer133. siRNA depletion of CREB also reduced MnSOD induction by MTX, and chromatin immunoprecipitation demonstrated binding of CREB to the MnSOD promoter in MTX-treated samples. Moreover, MTX protected HUVEC against apoptosis induced by glucose deprivation, demonstrating the functional importance of this pathway. Finally, treatment of the murine (BXSB x NZW)F1 SLE model of inflammatory vasculopathy with MTX improved the intramyocardial arterial vasculopathy and reduced end-organ damage, increased aortic MnSOD and phosphorylated AMPKαThr172, and reduced ICAM-1 expression. I have shown that MTX activates an AMPK-CREB pathway in vascular endothelium leading to enhanced expression of cytoprotective genes and protection against apoptosis in vitro and inflammatory vascular injury in vivo. This novel mechanism may explain its observed benefits in reducing CVD in chronic systemic inflammation.
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Nitoiu, Daniela. "Insights into molecular and functional mechanisms behind inherited heart and skin disorders." Thesis, Queen Mary, University of London, 2015. http://qmro.qmul.ac.uk/xmlui/handle/123456789/8911.
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Desmosomes are macromolecular, dynamic and adaptable complexes that connect intermediate filaments of neighboring cells in a variety of tissues, generating a large mechanically resilient structure. The importance of maintaining desmosome homeostasis for tissue integrity and optimal organ function has been revealed through the identification of desmosome-associated disorders and mechanistic studies into desmosome regulation. This thesis focuses on inherited skin and heart conditions linked to mutations in desmosomal genes or in genes believed to be implicated in desmosome regulation. Part of this thesis is focused on the molecular analysis and identification of novel desmosomal mutations in patients clinically diagnosed with Arrhythmogenic Right Ventricular Cardiomyopathy, and the genetic diagnosis of patients with hypotrichosis, hypotrichosis and PPK or acral peeling skin syndrome. Patients were analysed using a number of different genetic techniques including custom capture array, HaloPlex targeted resequencing, exome capture and Sanger sequencing. Both novel and previously reported mutations were identified in DSP, DSC2, DSG2, PKP2, DSG4 or CSTA in patients diagnosed with these disorders. The molecular mechanisms behind mutations in the protease inhibitors cystatin A and calpastatin, leading to the skin disorders exfoliative ichthyosis and PLACK syndrome, were also investigated. In vitro analysis, using siRNA-mediated knockdown in the immortalised keratinocyte cell line HaCaT, demonstrated that these mutations, affecting the structure and function of the protease inhibitors, lead to deficient intercellular adhesion, possibly through the indirect regulation of desmosomal complexes through their target proteases.