Orientadores: Jorge Humberto Nicola, Ester Maria Danielli Nicola / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-13T06:03:03Z (GMT). No. of bitstreams: 1 Hernandez_JoaoWagnerRodrigues_D.pdf: 4481265 bytes, checksum: 5351d4ae13e0c0baa8fd480610047a9b (MD5) Previous issue date: 2009 / Resumo: A pesquisa de fluorescência nativa em tecidos biológicos tem despertado grande interesse na Biologia e Medicina. A fluorescência óptica tornou-se instrumento indispensável aos diagnósticos, sendo um método eficiente e não invasivo. Neste trabalho investigamos a presença de fluorescência nativa em áreas corpóreas de ratos Wistar, subdivididos em dois grupos: um com alimentação normal e outro com dieta de glicose 5%. Em áreas internas (experimento 1), não ficou evidenciada a fluorescência nativa em animais com alimentação normal de água e ração. Nos animais submetidos à dieta de glicose, observamos a presença de fluorescência nativa vermelha, captada por espectroscopia, alcançando um pico máximo de emissão luminosa em 120 horas de dieta e retomando valores iniciais, quando sua dieta voltou ao normal. O conteúdo do lúmen intestinal foi analisado espectroscopicamente e por prova bioquímica. O gráfico obtido foi semelhante ao da PpIX e o teste bioquímico (Eales modificado ) resultou positivo à presença de substância porfirínica. A fluorescência em áreas externas, bolsa escrotal, focinho, pata e cauda, também foi pesquisada nas mesmas condições de dieta (normal e glicosada), evidenciando que animais com dieta normal, não apresentaram fluorescência e nos de dieta de glicose, deparamos com pico máximo de fluorescência em 120 horas de dieta e retomando valores iniciais, quando sua dieta voltou ao normal. A fluorescência nativa encontrada nos animais submetidos à dieta glicosada, evidencia uma nova condição fisiopatológica e que se assemelha a um quadro de porfiria reversível, bem identificada e documentada pelo experimento 2. Os resultados mostram a importância da espectroscopia óptica como um método de diagnóstico precoce, simples e confiável, para uso em diversos tipos de doenças ou disfunções com acúmulo de pigmentos fotossensíveis. Chama, ainda, a atenção para situações em que fluorescências nativas possam ser tomadas como diagnóstico falso-positivo de alterações patológicas ou não. / Abstract: The research of native fluorescence in biological tissues has aroused great interest in Biology and Medicine. The optic fluorescence became an important instrument for the diagnosis, being an efficient and non-invasive method. In this work we investigated the presence of native fluorescence in corporal areas of Wistar rats, subdivided in two groups: one with normal feeding and another one with glucose diet 5%. In the intestinal tract (Experiment 1), there was no evidence of fluorescence in animals with normal feeding of water and ration. In the animals submitted to the glucose diet, we observed the presence of red native fluorescence, caught by spectroscopy, reaching a maximum peak of luminous emission in 120 hours of diet with the return to normal values after the re-introduction of normal diet. The content of the intestinal lumen was analyzed spectroscopically and by biochemical test. The obtained graph was similar to the one of the PpIX and the test modified by Eales resulted positive to the presence of porfirinic substance. For Experiment 2, a group of 30 animals were kept at the same diet conditions (normal and glucose 5%) up to 120 hours. Four external areas were selected for the study (paw, tail, nose and scrotum). The group os animals under glucose diet presented a marked fluorescence on the selected areas with a maximum pick at 120hours. After 24 and 48 hours of reestablishment of the regular solid diet the fluorescence decreased disappearing totally at 48 hour. The native fluorescence found in the animals submitted to the glucose diet, evidences a physiopathological condition and it is similar to a condition of reversible porfiria, well identified and registered by Experiment 2. The results show the importance of the optic spectroscopy as a method of precocious, simple and trustworthy diagnosis, for use in diverse types of illnesses or dysfunctions with accumulation of photosensitive pigments. It also calls attention, for situations where native fluorescence can be taken as false-positive diagnosis of malignant alterations. / Doutorado / Ciencias Biomedicas / Doutor em Ciências Médicas

Les porphyries érythropoïétiques (PE) : Porphyrie Erythropoïétique Congénitale -PEC- et Protoporphyrie Erythropoïétique -PPE- sont caractérisées par le déficit d’une des enzymes de la voie de biosynthèse de l’hème. Le traitement curatif des formes sévères de PE est la transplantation de moelle osseuse allogénique (TMOA). La PPE est parfois compliquée d’une insuffisance hépatique majeure nécessitant une greffe hépatique. Dans un modèle murin de PPE (Fechm1Pas/Fechm1Pas), nous avons démontré l’apparition progressive de lésions hépatiques dès la 2ème semaine de vie. Une TMO précoce (nouveau-né) a permis de prévenir l’apparition de ces lésions hépatiques et de corriger la photosensibilité cutanée démontrant l’efficacité de cette approche thérapeutique pour les formes sévères de PPE. La thérapie génique par greffe de cellules souches hématopoïétiques autologues corrigées représente une alternative à la TMOA en l’absence de donneur HLA-compatible. Nous avons développé des cellules souches pluripotentes induites (iPS) à partir de cellules épidermiques issues de modèles murins de PE et d’un patient PEC. La correction génique a été obtenue par transfert du gène lentiviral (ferrochélatase ou uroporphyrinogène III synthase (UROS). La pluripotence des cellules iPS a été caractérisée in vitro par la formation de corps embryoïdes et in vivo par la formation de tératomes. In vitro, la correction métabolique a été obtenue après différenciation des cellules iPS humaines en progéniteurs hématopoïétiques. Enfin dans une dernière partie, nous nous sommes intéressés à une approche pharmacologique de la PEC. Nous avons montré que les mutations C73R et P248Q entraînaient une instabilité et une dégradation accélérée de l’UROS par la voie du protéasome. Le traitement de souris UrosP248Q par un inhibiteur du protéasome (Velcade®) a permis la correction de la photosensibilité cutanée. Ces travaux ouvrent de nouvelles perspectives pour le traitement des porphyries érythropoïétiques. / Erythropoietic porphyrias (EP) : Congenital Erythropoietic Porphyria -CEP- and Erythropoietic Protoporphyria -EPP-) are characterized by a deficit of one enzyme implicated in heme biosynthetic pathway. The curative therapy for severe cases of EP is an HLA-compatible Bone Marrow Transplantation (BMT). EPP is sometimes complicated by a major hepatic failure requiring hepatic graft. In a murine model of EPP (Fechm1Pas/Fechm1Pas), we have demonstrated that hepatic lesions progressively appear 2 weeks after birth. Early BMT (in neonates) has made it possible to prevent hepatic lesions and correct skin photosensitivity, demonstrating the efficiency of this therapeutic approach in severe cases of EPP. The gene therapy by graft of corrected autologous hematopoietic stem cells represents an alternative to BMT when HLA-compatible donors are lacking. We have developed induced pluripotent stem cells (iPSC) from epidermic cells of murine models of EP and of one PEC patient. The gene correction was obtained by lentiviral gene transfer (ferrochelatase and uroporphyrinogen III synthase -UROS). The pluripotency of iPSC was characterized in vitro by the formation of embryoid bodies and in vivo by the formation of teratomas. In vitro, the metabolic correction was obtained after differentiation of human IPSC into hematopoietic progenitors. In the last part of this thesis, we have focused on a pharmacological approach of CEP. We have shown that C73R and P248Q mutations lead to instability and accelerated degradation of the UROS protein via the proteasome. Treating UrosP248Q mice with a proteasome inhibitor (Velcade®) has allowed the correction of skin photosensitivity. These works offer new prospects for the treatment of erythropoietic porphyrias.