No description available.

De nombreux sites industriels émettent, non-intentionnellement, depuis des années des particules ultra-fines dans l’air. De nombreuses questions se posent sur leurs effets sur les écosystèmes et la santé humaine. Dans ce contexte, nous avons axé nos recherches autour de nanoparticules de fer manufacturées (Fe-NP), représentatives de celles que l’on retrouve dans les fumées des industries métallurgiques dunkerquoises et nous avons mené en parallèle des études d’écotoxicité et de toxicité. L’objectif principal de ces recherches était d’étudier l’impact de Fe-NP manufacturées exposées par voie aérienne sur deux modèles biologiques : Physcomitrella patens (Hedw.) Bruch & Schimp. et des cultures primaires de cellules épithéliales bronchiques humaines (HBEC). Pour répondre à ces objectifs, nous avons dû passer par l’étape indispensable qu’est la caractérisation fine de notre modèle de NP. En effet, la caractérisation physico-chimique doit être la plus complète possible (8 paramètres) de façon à déterminer, au préalable, leurs propriétés de surface. Puis, nous avons vérifié leur pénétration au sein de nos modèles biologiques. Puis, des biomarqueurs liés au stress oxydant ont été dosés chez la bryophyte, exposée à des concentrations faibles en Fe-NP. Premièrement, aucune perte de vitalité chez notre plante n’a pu être observée au cours du temps aux doses testées. Nos études n’ont pas permis de mettre en évidence une augmentation significative des espèces réactives de l’oxygène et du malondialdéhyde ; ainsi qu’une modulation significative du ratio GSSG/GSH, même si un phénomène de « sur-compensation » peut être évoqué sur le long terme, conduisant à la production de GSH au sein de notre plante témoignant d’une adaptation de la plante au stress. Enfin une analyse toxicogénomique a montré des modulations de l’expression (non significatives) de tous les isoformes des gènes d’intérêt étudiés aux doses testées. Dans le cadre d’études de toxicité, nous avons caractérisé notre modèle cellulaire par coloration immunocytologique. Puis, un test de viabilité nous a permis de choisir notre dose d’exposition : 2 μg.cm-2. Les travaux sur le stress oxydant et la modulation de l’expression génique ont été réalisés sur des cultures de cellules issues de trois patients différents pour prendre en compte la variabilité interindividuelle. Contrairement à certaines publications, nous n’avons pas montré une augmentation dose-dépendante des ROS. Puis, notre étude pangénomique, nous a permis de sélectionner 10 gènes d’intérêt dont l’étude approfondie a mis en évidence des effets précoces (dès 6 h d’exposition) sur des gènes impliquées dans des phénomènes inflammatoires. Néanmoins, les Fe-NP n'ont pas causé d’augmentation significative du MDA et du ratio GSSG/GSH après plusieurs jours d'exposition. Suite à ces résultats, il est maintenant envisageable de mener des recherches sur les impacts des nanoparticules d’origine environnementale à l’aide de nos deux modèles biologiques et d’améliorer les connaissances concernant leur danger potentiel pour l’environnement et la santé humaine. / Many industries emit, unintentionally, ultra-fine particles in the air, for many years. Many questions arise about their effects on ecosystems and human health. In this context, we focused our research on the iron-engineered nanoparticles (Fe-NP), representative of industrial smoke emitted by metallurgical industries and we conducted, in parallel, toxicity and ecotoxicity studies. The main objective of this work was to study the impact of Fe-NP exposed by air in two biological models: Physcomitrella patens (Hedw.) Bruch & Schimp. and primary cultures of human bronchial epithelial cells (HBEC). To meet these objectives, we had to characterize our NP model. Indeed, the physic-chemical characterization must be as complete as possible (8 parameters) to determine, firstly, their surface properties. Then, we checked their penetration within our biological models. And, oxidative stress biomarkers were measured in the bryophyte, exposed to low concentrations of Fe-NP. Firstly, any loss of vitality could be observed over time at the doses tested. Our studies have failed to demonstrate a significant increase of reactive oxygen species and malondialdehyde, and a significant modulation of the ratio GSSG/GSH, although the phenomenon of "over-compensation" can be discussed, over the long term, leading to the production of GSH in our plant showing a plant adaptation to stress. Finally a toxicogenomic analysis showed modulation of expression (not significant) of all isoforms of the genes of interest studied at the doses tested. For toxicity studies, we characterized our cellular model by immunocytological staining. Then, a viability test allowed us to choose the exposure dose: 2 μg.cm-2. The research on oxidative stress and the modulation of gene expression were performed on cells derived from three different patients to take into account individual variability. Unlike some publications, we did not show a dose-dependent increase of ROS. Then, the pangenomic study has allowed us to select 10 genes. A detailed study of this genes showed early effects (from 6 h of exposure) in genes involved in inflammation. However, Fe-NP did not cause any significant increase of MDA and GSSG/GSH ratio after several days of exposure. Following these results, it is now possible to conduct research on the impacts of ultra-fine particles using our two biological models and improve knowledge about their potential danger on the environment and human health.

Made available in DSpace on 2014-10-09T12:50:15Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:01:54Z (GMT). No. of bitstreams: 1 10826.pdf: 3720317 bytes, checksum: 26d9f64b9d44684f557ae44f37e86002 (MD5) / Dissertação (Mestrado Profissionalizante em Lasers em Odontologia) / IPEN/D-MPLO / Intituto de Pesquisas Energeticas e Nucleares, IPEN/CNEN-SP; Faculdade de Odontologia, Universidade de Sao Paulo

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Made available in DSpace on 2014-10-09T12:50:15Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:01:54Z (GMT). No. of bitstreams: 1 10826.pdf: 3720317 bytes, checksum: 26d9f64b9d44684f557ae44f37e86002 (MD5) / Dissertação (Mestrado Profissionalizante em Lasers em Odontologia) / IPEN/D-MPLO / Intituto de Pesquisas Energeticas e Nucleares, IPEN/CNEN-SP; Faculdade de Odontologia, Universidade de Sao Paulo

Made available in DSpace on 2014-10-09T12:50:14Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:03:45Z (GMT). No. of bitstreams: 1 10825.pdf: 4609998 bytes, checksum: abaefc3f2b2d697034a947707dec7944 (MD5) / Dissertacao (Mestrado Profissionalizante em Lasers em Odontologia) / IPEN/D-MPLO / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP; Faculdade de Odontologia - USP

No description available.

Coenzyme Q10 is a lipid-soluble coenzyme, synthesized in mammalian tissue to support energy production, and also act as an antioxidant. Certain medication, stress and age may deplete the body’s endogenous Coenzyme Q10 store. Numerous disease conditions have been shown to benefit from Coenzyme Q10 supplementation. It is a lipid-soluble component of virtually all cell membranes, and is located in the hydrophobic domain of the phospholipid bilayer of cellular membranes. It is also the only known lipid-soluble antioxidant that animal cells can synthesize de novo, and for which there exist enzymatic mechanisms which can regenerate it from its oxidized product formed in the course of its antioxidant function. The aim of this study was to investigate the cellular effects of Coenzyme Q10 and Triton X-100 on primary chicken embryo heart and muscle cell cultures. Triton X-100, a well known membrane disrupter, extensively used by cell biologists for that purpose, was used to investigate whether Coenzyme Q10 might offer protection to cell membranes exposed to disruption. Due to the correlation found between the chemical structures of nonylphenol and Triton X-100, it was decided to determine whether Triton X-100 possess estrogenic properties. Using the Recombinant Yeast Screen Assay for estrogenic activity, it was found that Triton X-100 induced weak estrogenic activity. The primary heart and skeletal muscle cell cultures were established by harvesting skeletal muscle tissue and hearts from 13 day old chicken embryos. After establishment of the cell cultures, the concentrations of Coenzyme Q10 and Triton X-100 were tested for cytotoxicity using the MTT, NR, and CV assays, in the form of a combined colorimetric cytotoxicity assay. The MTT assay revealed an increase in cell viability in both cell cultures upon exposure to Triton X-100 and Coenzyme Q10, alone, and in combination. Triton X-100 and Coenzyme Q10, alone, and in combination, caused a decrease in lysosomal membrane integrity, as measured by the NR assay, and both substances, alone, and in combination, had no effect on cellular proteins, as measured by the CV assay. Scanning electron microscopy (SEM) was done to determine the cellular effect of heart and skeletal muscle cell cultures on the external surface, more specifically the membranes, of cells in culture. Triton X-100 in the concentrations used in the study, caused membrane disruption, ranging from complete membrane lyses at the highest concentrations to membrane ruptures and apoptotic blebbing in lower concentrations. SEM revealed that no adverse effects were caused by Coenzyme Q10 on the membrane structure, in dissimilarity, cell differentiation and proliferation, including myoblast formation were seen in the presence of all the concentrations of Coenzyme Q10. Numerous ion channels were observed on cellular surfaces exposed to Coenzyme Q10. Upon exposure to 0.005% Triton X-100, after pre-treatment with Coenzyme Q10, SEM revealed a “membrane patch” formation on membranes disrupted by Triton X-100. Damage to cell membranes in the presence of Triton X-100, were less severe when cells were pre-treated with Coenzyme Q10. Confocal microscopy was utilized to investigate intracellular occurrences in the presence of Triton X-100 and Coenzyme Q10. Using Mito Tracker Red to stain active respiring mitochondria and DAPI to stain nuclei, confocal microscopy confirmed the observations made by SEM, that Coenzyme Q10 enhance cell proliferation and differentiation, and that the adverse effects to cells exposed to Triton X-100 are less severe after pre-treatment with Coenzyme Q10. ROS generation was detected, using dichlorodihydrofluorescein diacetate, in cultures exposed to Triton X-100, and none in the presence of Coenzyme Q10. In the presence of Triton X-100, after pre-treatment with Coenzyme Q10, ROS generation was remarkably lower. The study provided apparent evidence that Coenzyme Q10 offer protection to cardiac and skeletal muscle cells in culture after exposure to relatively low concentrations of the membrane disrupter Triton X-100. Coenzyme Q10 also promotes the process of proliferation and differentiation in primary chicken embryonic cultures of heart and skeletal muscle cells. / Dissertation (MSc)--University of Pretoria, 2008. / Anatomy / unrestricted

No description available.

A PMAH é uma causa rara de síndrome de Cushing, cuja real prevalência parece subestimada. O processo fisiopatológico que culminaria com a PMAH ainda não foi totalmente elucidado. A produção de cortisol mediada por receptores acoplados a proteína G (ectópicos/tópicos da adrenal) geralmente hiperexpressos, bem como a regulação autócrina/parácrina do ACTH ectópico em clusters celulares dos macronódulos adrenais têm sido considerados como parte importante do processo fisiopatológico da PMAH. Além destes mecanismos, recentemente, foi demonstrado a associação de variantes patogênicas germinativas/somáticas no gene ARMC5 como uma causa frequente da PMAH. Os estudos funcionais que caracterizaram o ARMC5 como gene supressor de tumor e potencialmente envolvido na hiperplasia adrenal nodular e sua produção de cortisol foram realizados em células H295R, derivadas de carcinoma adrenocortical. Células estas que não representam em tese um modelo ideal para o estudo de uma doença absolutamente benigna. Objetivos: Obtenção e caracterização morfofuncional de culturas de células obtidas de nódulos adrenais de pacientes submetidos à adrenalectomia com diagnóstico histopatológico compatível com PMAH, como um modelo biológico para a análise funcional do gene ARMC5. Métodos: Foram utilizadas 13 culturas de células de PMAH caracterizadas do ponto de vista morfofuncional e molecular para as análises funcionais do gene ARMC5. Resultados: As culturas de PMAH apresentaram mutações germinativas no ARMC5 identificadas em 8 das 13 culturas analisadas, associadas ou não a segundos eventos moleculares. As culturas de células de PMAH apresentaram receptores eutópicos e ectópicos de uma maneira heterogênea e a presença de ACTH ectópico em clusters de células. O silenciamento do ARMC5 nas células de PMAH levou à diminuição da esteroidogênese, ao aumento de CCNE1 e ao número de células viáveis após 96h. Quando hiperexpresso, o gene ARMC5 induziu apoptose e necrose celular após 12h, diminuindo a viabilidade das células de células de PMAH. Conclusões: Legitimamos a importância do papel do ARMC5 na esteroidogênese relacionada à PMAH, bem como sua função favorecendo a apoptose celular; além disso, pela primeira vez, detectamos o envolvimento do ARMC5 na regulação do ciclo celular e proliferação, cuja importância será explorada em estudos futuros. / Background: PMAH is a rare cause of Cushings syndrome, with an apparent underestimated prevalence. The pathophysiology of PMAH is not yet fully understood, however the participation of aberrant receptors and intra-adrenal ACTH in the hyperplastic tissue are considered mechanisms that regulate hypercortisolism in PMAH. Additionally, germline ARMC5 mutations have been described as the most frequent genetic abnormality found in patients diagnosed with PMAH. Previous functional studies analyzed ARMC5 role using H295R cells, a cell line of adrenocortical carcinoma. Objectives: In this study we investigated the role of ARMC5 in cell cultures obtained from PMAH nodules. Results: We observed the presence of mutations in ARMC5 gene in 8 out of 13 PMAH cell cultures analyzed. We observed the presence of aberrant receptors and intra-adrenal ectopic ACTH, regardless the presence of mutations. ARMC5 silencing in non-mutated PMAH cell cultures decreased steroidogenesis-related genes and increased CCNE1 mRNA expression and the number of viable cells without affecting cell viability. Additionally, ARMC5 overexpression induced cell death in PMAH mutated cell cultures, thereby decreasing cell viability. Conclusions: We confirmed the role of ARMC5 as an important pro-apoptotic protein involved in PMAH-related steroidogenesis. We also report for the first time the possible involvement of ARMC5 in controlling proliferation and regulating cell cycle in PMAH cell cultures, which need to be further explored.