Doctorat en sciences médicales / info:eu-repo/semantics/nonPublished

Die ALS ist eine fortschreitende neurodegenerative Erkrankung, deren Symptome durch den Untergang der Motoneuronen bedingt sind. Neueste zytogenetische Untersuchungen zeigen ein vermehrtes Auftreten konstitutioneller Chromosomenveränderunge bei ALS-Patienten. Dies lässt eine Verbindung zwischen dem Ausbruch der Erkrankung und der auffälligen Zytogenetik vermuten. Das Auftreten spontaner Chromosomenveränderungen als Zeichen einer chromosomalen Instabilität wurde in der vorliegenden Arbeit an ALS-Patienten untersucht. METHODE: Neben der Karyotypisierung, der Bestimmung der SCE-Rate und der Bruchrate nach Behandlung mit Bleomycin kam die Fluoreszenz in situ Hybridisierung zum Einsatz. Die Untersuchungen wurden an Patienten mit sporadischer ALS (45), an Kontrollpersonen (38) und Verwandten (9) durchgeführt. ERGEBNISSE: Die Karyotypisierung ergab bei den Patienten eine spontane Translokationsrate von 0,02 Translokationen/Zelle (t/Z), bei den Kontrollen 0,04 t/Z. Weitere numerische oder strukturelle Auffälligkeiten waren nicht signifikant verschieden. Es wurden keine konstitutionellen Chromosomenaberrationen gefunden. Die Häufigkeit der Schwesterchromatidaustausche (SCE-Rate) bewegte sich mit 7-8 SCE/Z in der Patientengruppe innerhalb der Normwerte. Durch die Zugabe von Bleomycin in die Zellkultur stieg die Zahl der Chromatidbrüche von 0,0 auf 0,8 Brüche/Z an. Dabei zeigten die untersuchten Gruppen ähnliche Progredienzen in den Bruchraten. Mit der Fluoreszenz in situ Hybridisierung werden quantitative Aussagen über spontane Translokationsraten gemacht. Sie betrug in der Kontroll-und Patientengruppe 0,03 bis 0,04 t/Z. DISKUSSION: Die vorliegenden Ergebnisse liefern keinen Anhalt für eine chromosomale Instabilität als Risikofaktor für die Entstehung der sporadischen ALS. Indizien für eine chromosomale Instabilität wie erhöhte Bruchraten und SCEs als auch vermehrtes Auftreten somatischer Aberrationen konnten bei den ALS-Patienten nicht nachgewiesen werden. Über mögliche Auffälligkeiten in den Motoneuronen lässt sich allerdings mit den Untersuchungen an Blutlymphozyten keine hinreichend sichere Aussage machen. / Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease which is characterized by the degeneration of motor neurons. Recently, a high rate of constitutional structural chromosomal rearrangements has been reported in apparently sporadic ALS patients. It remains questionable whether or not these genomic rearrangements are caused by a chromosomal instability involved in the pathogenesis of the disease. Therefore, we performed different cytogenetic studies on chromosomal instability. METHOD: We performed chromosome analyses from patients (N=45), control subjects (N=38), and relatives (N=9) after culturing blood lymphocytes. Conventional chromosome analysis after GTG-banding, chromosomal breakage test after Bleomycin treatment, the rate of sister chromatid exchange (SCE), and whole chromosome painting were used for these analyses. RESULTS: Neither karyotyping nor whole chromosome painting revealed higher levels of structural or numerical aberrations in lymphocytes of patients with sALS. After karyotyping we found 0.02 t/cell in patients and 0.04 t/cell in controls. Whole chromosome painting revealed 0.04 t/cell in patients and 0.03 t/cell in controls. The chromosomal breaks increased likewise after Bleomycin treatment in the control group and the patient group as well. Cell cultures without Bleomycin did not show any breaks while the highest Bleomycin concentration induced up to 0.08 breaks/cell. The SCE rate in patients which corresponds to the chromatid repair activity did not rise to a higher level than in the control individuals. Both groups were in the normal range of 7 to 8 SCE/cell. DISCUSSION: The pathomechanism of neurodegeneration in ALS patients is still unknown. We tried to find a cytogenetic correlative being a risk factor for the development of ALS. However, so far there is no clue for chromosomal instability being involved in the neurodegenerative process.

Doctor of Philosophy / Department of Human Nutrition / Richard C. Baybutt / Weiqun George Wang / Although omega-3 fatty acids have been a hot issue in nutrition for years, there remains a paucity of research on the topic of omega-3 fatty acid and pulmonary fibrosis and the mechanism is still unclear. The purpose of this research is to investigate the preventive effects of flaxseed oil for bleomycin-induced pulmonary fibrosis in rats and to find the possible underlying mechanisms. There are two experiments demonstrated in this dissertation, one is with various doses of flaxseed oil in the diet (0, 2.5, 5, 7.5, 10, 12.5, and 15 % (w/w)), and the other is with different times of sacrificing animals after oropharyngeal bleomycin treatment (days 7 and 21). In the first study, three proteins including transforming growth factor-[beta] (TGF-[beta]), interleukin-1 (IL-1), and [alpha]-smooth muscle actin ([alpha]-SMA), commonly associated with fibrotic inflammation in the lung, were examined by Western blot and fatty acids composition of the diets and tissues were analyzed by gas chromatography (GC). Fifteen percent of flaxseed oil group significantly reduced septal and vascular thickness and fibrosis in the lung, and significant cardiac fibrosis in the heart. The amount of IL-1 and [alpha]-SMA decreased significantly as the amount of omega-3 fatty acids increased, whereas TGF-[beta] did not change significantly. The next study further reported the time-course effect and potential underlying mechanisms. Both interleukin-6 (IL-6), a protein associated with fibrotic inflammation in the lung, and renin, an enzyme related to renin-angiotensin system, were examined by Western blot. The time-dependent increase of IL-6 in response to bleomycin treatment was reversed by flaxseed oil diet. Although renin was not significantly different in the kidney, it suggested that the renin-angiotensin system may be involved locally. In addition, the profiles of fatty acids in both liver and kidney tissues as measured by lipidomics demonstrated a significant increase of omega-3: omega-6 ratio in the flaxseed oil-fed groups. Overall, these results indicated for the first time that the omega-3 fatty acids rich in flaxseed oil inhibited the formation of pulmonary fibrosis in a dose-dependent manner - however the moderate dose of flaxseed oil was most effective - via anti-inflammatory mechanisms, which appears associated with the modulated fatty acid composition in the tissues.

Doenças pulmonares intersticiais fibrosantes são doenças que afetam homens, mulheres e crianças, tem prognóstico ruim e os pacientes possuem sobrevida estimada entre 3 e 5 anos após a confirmação diagnóstica, sobretudo os portadores de Fibrose pulmonar idiopática. Estudos recentes demonstram a capacidade das células-tronco em se diferenciar em diferentes linhagens celulares e tecidos, como já comprovado em órgãos como coração, fígado, trato gastrointestinal, sistema nervoso e pulmão. Os objetivos deste trabalho foram os de estabelecer a espécie suína como modelo experimental e utilizar a terapia celular experimentalmente como possibilidade de estudo para tratamento das doenças pulmonares intersticiais fibrosantes. Para estabelecer o modelo experimental e induzir a doença nos dois grupos de animais estudados (grupos tratado e controle) foi utilizado sulfato de bleomicina pela via intratraqueal em procedimento único. Após a instalação da doença, os animais dos grupos tratado e controle foram submetidos a tomografia computadorizada de alta resolução (TCAR); um grupo tratado com terapia celular e após noventa dias os dois grupos reavaliados com TCAR antes da eutanásia, totalizando para os dois grupos, cento e oitenta dias de doença instalada. As análises tomográficas mostraram que o tempo para que a doença intersticial seja estabelecida ocorre até três meses após a infusão de bleomicina. As provas histológicas corroboram a viabilidade do modelo testado e as análises imunohistoquímicas sugerem a migração das células mononucleares de medula óssea para os pulmões, bem como a presença de populações celulares que indicam provável reestruturação do parênquima pulmonar. / Fibrotic Interstitial pulmonary illnesses affect men, women and children, and presents bad prognostic, 3-5 years depending on the diagnostic confirmation, mainly in idiopathic pulmonary fibrosis. Recent studies demonstrate the capacity of the stem cells in differentiating into different cellular lineages and different tissues, as in heart, liver, gastrointestinal, nervous system and lung. The objectives of this study were to investigate the possibility to consider the swine as experimental model of fibrotic pulmonary disease and experimental stem cell therapy. Bleomycin sulphate was injected into the trachea to induce the pulmonary disease in control and treatment groups. High resolution computed scan (TCAR) was carried out in both groups after the confirmation of the disease. The tomographic analyses showed that the interstitial illness was established after three months of the bleomicine infusion. Histologic investigation revealed the viability of the tested model and the imunohistochemical analyses suggest the migration of the mononuclear cells to the lungs, as well as the presence of new cellular populations that would indicate probable reorganization of the pulmonary parenchyma.

La bléomycine est un antibiotique cytotoxique, son potentiel génotoxique est plus important quand elle est utilisée en combinaison avec des agents antinéoplasiques sur le cancer testiculaire, que sur les autres types qui développent souvent une résistance envers la drogue. Notre but consiste alors de mettre en évidence ce mécanisme de résistance en utilisant l’organisme modèle Saccharomyces cerevisiae. Nous avons démontré au sein de notre laboratoire, que les levures délétées au niveau de leur coactivateur transcriptionnel Imp2, présentent une hypersensibilité à la bléomycine, en raison de son accumulation toxique dans la cellule. Ceci suggère que Imp2 pourrait réguler l’expression d’une ou de plusieurs pompes à efflux, capables d’expulser la bléomycine à l’extérieur de la cellule. Pour tester notre hypothèse, nous avons recherché des suppresseurs multicopies capables de restaurer la résistance à la bléomycine chez le mutant imp2, et c’est ainsi que nous avons identifié l'activateur transcriptionnel Yap1. Ce dernier se lie à une région spécifique localisée au niveau du promoteur et permet d’activer l'expression d'un sous-ensemble de gènes, codant pour des pompes à efflux, impliquées dans la résistance aux drogues. Selon la littérature, au moins 27 pompes à efflux ont été identifiées chez la levure Saccharomyces cerevisiae, certaines d’entre elles disposent du site de liaison pour Yap1, tels que Qdr3, Tpo2 et Tpo1. Afin de déterminer si une de ces pompes expulse la bléomycine, nous avons créé des mutations simples et doubles en combinaison avec IMP2, aussi nous avons verifié si les mutants étaient sensibles à la drogue et enfin, nous avons testé si la surexpression de Yap1 pouvait restaurer le phénotype sauvage chez ces mutants, via l’activation de pompes à efflux. / Bleomycin is a cytotoxic antibiotic that, when used in combination with antineoplastic agents, has more genotoxic potential on testicular cancer than other types of cancer, which often develop resistance to the drugs. Our goal is to identify the resistance mechanism, using the organism Saccharomyces cerevisiae as a model. In our laboratory, we have demonstrated that deleted yeast strains on their transcriptional coactivator Imp2 have presented hypersensitivity to bleomycin due to the toxic accumulation inside the cell. This led us to believe that Imp2 might regulate the expression of one or more efflux pumps capable of expelling bleomycin outside the cell. To test our hypothesis, we sought multi-copy of suppressors capable of restoring bleomycin resistance in the mutant imp2. As a result we identified the transcriptional activator Yap1, which binds to a specific region within the promoter and activates the expression of subset of genes, encoding efflux pumps that are involved in drug resistance. Based on the literature, at least 27 efflux pumps have been identified in Saccharomyces cerevisiae. Some of these efflux pumps have binging sites for Yap1; such as Qdr3, Tpo2 and Tpo1. To determine whether or not one of these pumps expelled bleomycin, we proceded by single and double mutations in combination with IMP2. We also verified if these single and double mutants were sensitive to the drug, and then we have examined whether the overexpression of Yap1 could restore the wild phenotype in these mutants through the activation of efflux pumps.

Thesis (PhD.(Physiology)--Faculty of Health Sciences)-University of Pretoria, 2008. / Summary in English and Afrikaans. Includes bibliographical references.

No description available.

Doenças pulmonares intersticiais fibrosantes são doenças que afetam homens, mulheres e crianças, tem prognóstico ruim e os pacientes possuem sobrevida estimada entre 3 e 5 anos após a confirmação diagnóstica, sobretudo os portadores de Fibrose pulmonar idiopática. Estudos recentes demonstram a capacidade das células-tronco em se diferenciar em diferentes linhagens celulares e tecidos, como já comprovado em órgãos como coração, fígado, trato gastrointestinal, sistema nervoso e pulmão. Os objetivos deste trabalho foram os de estabelecer a espécie suína como modelo experimental e utilizar a terapia celular experimentalmente como possibilidade de estudo para tratamento das doenças pulmonares intersticiais fibrosantes. Para estabelecer o modelo experimental e induzir a doença nos dois grupos de animais estudados (grupos tratado e controle) foi utilizado sulfato de bleomicina pela via intratraqueal em procedimento único. Após a instalação da doença, os animais dos grupos tratado e controle foram submetidos a tomografia computadorizada de alta resolução (TCAR); um grupo tratado com terapia celular e após noventa dias os dois grupos reavaliados com TCAR antes da eutanásia, totalizando para os dois grupos, cento e oitenta dias de doença instalada. As análises tomográficas mostraram que o tempo para que a doença intersticial seja estabelecida ocorre até três meses após a infusão de bleomicina. As provas histológicas corroboram a viabilidade do modelo testado e as análises imunohistoquímicas sugerem a migração das células mononucleares de medula óssea para os pulmões, bem como a presença de populações celulares que indicam provável reestruturação do parênquima pulmonar. / Fibrotic Interstitial pulmonary illnesses affect men, women and children, and presents bad prognostic, 3-5 years depending on the diagnostic confirmation, mainly in idiopathic pulmonary fibrosis. Recent studies demonstrate the capacity of the stem cells in differentiating into different cellular lineages and different tissues, as in heart, liver, gastrointestinal, nervous system and lung. The objectives of this study were to investigate the possibility to consider the swine as experimental model of fibrotic pulmonary disease and experimental stem cell therapy. Bleomycin sulphate was injected into the trachea to induce the pulmonary disease in control and treatment groups. High resolution computed scan (TCAR) was carried out in both groups after the confirmation of the disease. The tomographic analyses showed that the interstitial illness was established after three months of the bleomicine infusion. Histologic investigation revealed the viability of the tested model and the imunohistochemical analyses suggest the migration of the mononuclear cells to the lungs, as well as the presence of new cellular populations that would indicate probable reorganization of the pulmonary parenchyma.

Idiopathic Pulmonary Fibrosis (IPF) is an interstitial lung disease with no known cause or cure. IPF has an incidence of 75/1,000,000 of the population, predominately in men over the age of 60. This relatively rare disease develops in a chronic and progressive way, ultimately leading to death within two to five years of diagnosis. Our use of translatable methodologies in the bleomycin mouse model of IPF led to the novel identification of the similarities between the average percent loss of lung function in previous human clinical trials to that of our mouse model data. There is no treatment for IPF outside of lung transplantation, therefore our goal is to develop a protein therapy to halt the progression of IPF. B6_BP_dslf is a small, 93.36 kDa minibinder protein with a nanomolar affinity to αvβ6, an integrin of therapeutic potential for IPF when inhibited by halting αvβ6/TGF-β signaling. Our hypothesis is that B6_BP_dslf will halt the progression of pulmonary fibrosis induced in a mouse model of IPF. To test this hypothesis, a de novo design method was used resulting in the B6_BP_dslf minibinder having high β unit selectivity and nanomolar affinity for αvβ6, and maintenance of its secondary structure after aerosolization. These attributes led to testing in the bleomycin mouse model for IPF as an inhaled therapy. We found that B6_BP_dslf inhalation by mice with induced pulmonary fibrosis had reduced pathogenesis through the quantification of biomarkers for αvβ6/TGF-β mediated fibrosis, lowered histopathological scores, and improved lung function. These positive results from standard biochemical analysis and clinically translatable methods show that BP_B6_dslf has clinical potential as an inhaled therapy for IPF. Additionally, we tested the use of lung function tests in an animal model of chronic obstructive pulmonary disease (COPD), using secondhand smoke exposure to induce the disease and to identify inflammatory pathways. We found that smoke exposure increased inflammatory signaling through receptors for advanced glycation end-products, and inhibition of these receptors using a novel therapy of semi-synthetic glycosaminoglycan ethers (SAGEs) reduced inflammation and improved lung function. Together, the data from two different lung disease models supports the use of lung function as a preclinical efficacy variable for experimental drugs. The combination of biochemical and functional assessments of B6_BP_dslf and SAGEs gives weight to their therapeutic potential.

No description available.