FERREIRA FILHO, Luiz Ivando Pires. Alterações citogenômicas na medula óssea de trabalhadores rurais expostos a agrotóxicos. 2013. Dissertação (Mestrado Ciências Médicas) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2013. / Submitted by denise santos (denise.santos@ufc.br) on 2014-02-17T12:21:59Z No. of bitstreams: 1 2013_dis_lipferreira filho.pdf: 2103165 bytes, checksum: 7ca3a3bd505574da57104d00a78b404d (MD5) / Approved for entry into archive by denise santos(denise.santos@ufc.br) on 2014-02-17T12:22:47Z (GMT) No. of bitstreams: 1 2013_dis_lipferreira filho.pdf: 2103165 bytes, checksum: 7ca3a3bd505574da57104d00a78b404d (MD5) / Made available in DSpace on 2014-02-17T12:22:47Z (GMT). No. of bitstreams: 1 2013_dis_lipferreira filho.pdf: 2103165 bytes, checksum: 7ca3a3bd505574da57104d00a78b404d (MD5) Previous issue date: 2013 / Os pesticidas são produtos químicos de uso disseminado no controle das pragas nas lavouras agrícolas. Estes produtos são classificados em herbicidas, inseticidas e fungicidas e podem acumular-se no meio ambiente. Um grande número de estudos epidemiológicos em trabalhadores rurais sugeriu um aumento do risco de câncer no grupo exposto a pesticidas. O objetivo deste estudo é avaliar a presença de anormalidades cromossômicas em células da medula óssea e alterações do gene TP53 por FISH em trabalhadores rurais expostos a pesticidas. Para o nosso conhecimento, este é o primeiro estudo a avaliar estas alterações utilizando células-tronco hematopoéticas coletadas por aspirado da medula óssea. Foram avaliados 43 trabalhadores rurais do sexo masculino e detectadas anormalidades cromossômicas através de citogenética por Banda G em 11 (25%). A maioria destas anomalias era relacionada com aneuploidias (6/11). Aneuploidias podem ocorrer devido a um cromossomo extra ou ausente. De extrema importância, encontramos quatro casos com anormalidades estruturais relacionadas aos cromossomos 4, 5, 7 e 11, como deleções do braço longo dos cromossomos 5, 7 e 11. Detectamos supressão do gene TP53 por Hibridização por Fluorescência in situ (FISH) em 4/31 e de amplificação em 3/31 casos. As outras alterações por FISH foram relacionadas à aneuploidia (6/31). Entre as anormalidades numéricas, encontramos tetrassomia em 3 casos, trissomia em dois casos e monossomia em um caso. Nosso trabalho é o primeiro a apresentar anomalias citogenéticas em células da medula óssea de trabalhadores rurais expostos a agrotóxicos. Nosso estudo destaca a importância da prevenção primária, pois apenas 23% dos trabalhadores rurais relatou medidas de proteção. Todas essas anormalidades citogenéticas aqui detectados são descritas como responsáveis por aumentar o risco de desenvolvimento de neoplasias. Como o uso de agrotóxicos está disseminado em nossa agricultura, os trabalhadores rurais devem evitar o uso impróprio devido ao risco de desenvolver neoplasias.

VIANA, Daniel de Araújo. Polimorfismos do gene TP53 no linfoma de Hodgkin. 2007. 85 f. Dissertação (Mestrado em Patologia) - Universidade Federal do Ceará. Faculdade de Patologia, Fortaleza, 2007. / Submitted by denise santos (denise.santos@ufc.br) on 2011-12-21T11:21:30Z No. of bitstreams: 1 2007_dis_daviana.pdf: 2983687 bytes, checksum: a560463a3e70aa3380fd982718cb146f (MD5) / Approved for entry into archive by Eliene Nascimento(elienegvn@hotmail.com) on 2012-02-01T16:10:20Z (GMT) No. of bitstreams: 1 2007_dis_daviana.pdf: 2983687 bytes, checksum: a560463a3e70aa3380fd982718cb146f (MD5) / Made available in DSpace on 2012-02-01T16:10:20Z (GMT). No. of bitstreams: 1 2007_dis_daviana.pdf: 2983687 bytes, checksum: a560463a3e70aa3380fd982718cb146f (MD5) Previous issue date: 2007 / Hodgkin lymphoma is a hematololgic B neoplasm occurring at patients within any age, however more likely to affect those from 15 to 40 years-old. The TP53 gene is 20kb length gene with 11 exons that encodes the p53 protein, which main function is related to the conservation and integrity of the genetic code. Most cancers show point mutations in the TP53 sequence. The analysis of these mutations allows a better understanding of the function of the diverse domains of the protein and its relationship to tumor suppression. There is only a few data about TP53 polymorphisms and Hodgkin lymphoma. In this manner, in our study we try to detect polymorphisms within the codons 272, 273, 278, 282, 306 of the exon 8 of the TP53 gene in Hodgkin lymphoma. In our survey we analyzed 42 paraffin-embedded tissues from 2000 to 2006. These samples were prepared for DNA extraction and PCR isolation and amplification of the 137bp fragment of the exon 8 of the TP53 gene, using exclusive primers specially designed to our experiment: PFw8 e PRv8. After PCR amplification, the products of the reaction were purified to the Sequencing reaction. The last part of the experiment encoded the bioinformatics analysis of sequences. DNA extraction and PCR amplification were successfully obtained in our study in all the samples. However, the DNA sequencing was only obtained in 32 samples, but there was no characteristic electropherogram of the analyzed region of the gene. Therefore, it was not possible to determine the presence or absence of SNPs in the TP53 exon 8. / O Linfoma de Hodgkin é uma hemopatia linfóide pode ocorrer em qualquer faixa etária; no entanto, é mais comum na idade adulta jovem, dos 15 aos 40 anos. O TP53 é um gene de 20kb de comprimento que possui 11 éxons situado no cromossomo 17 e codifica a proteína p53, uma proteína cuja principal função está relacionada à preservação da integridade do código genético, e, durante o ciclo celular faz verificação quanto à eventual ocorrência de uma mutação na seqüência do código genético. Na presença dessas mutações, impede que esta célula entre em processo de mitose e complete a divisão celular. A maioria dos cânceres apresenta mutações pontuais na seqüência do TP53. A análise dos padrões dessas mutações é de grande valia uma vez que o conhecimento dessas mutações leva a um melhor entendimento das funções dos vários domínios da proteína p53 e seu envolvimento com o mecanismo de supressão tumoral, além de permitir que essas mutações sejam utilizadas como biomarcadores para desvendar a oncogênese humana. Na literatura vigente, poucos trabalhos abordam a identificação dessas mutações em relação ao linfoma de Hodgkin – forma clássica. Dessa forma, este trabalho se propõe a investigar quantitativa e qualitativamente os padrões de polimorfismos existentes nesta forma do linfoma de Hodgkin. A primeira etapa do nosso experimento constou da seleção de 42 casos de linfonodos diagnosticados como linfoma de Hodgkin entre os anos de 2000 e 2006, arquivados em blocos de parafina. Os casos foram selecionados de pacientes com diagnóstico de linfoma de Hodgkin, sem predileção por idade, sexo ou raça. Em seguida, o DNA foi extraído das amostras selecionadas para reação de PCR, realizada para isolar e amplificar, o fragmento de 137pb correspondente ao éxon 8 do gene TP53, através de primers exclusivos desenhados para o experimento: PFw8 e PRv8. Após a reação, os produtos de PCR foram purificados para reação de Seqüenciamento de DNA, utilizando o seqüenciador automático de DNA da marca ABI Prism® 3100 de 16 capilares (Applied Biosystems). A última etapa aconteceu em laboratório de bioinformática – dry lab, onde as seqüências de DNA obtidas foram analisadas qualitativa- e quantitativamente. Os processos de extração do DNA genômico, amplificação do éxon 8, purificação do produto de PCR foram realizadas com sucesso em todas as amostras obtidas de material parafinizado. No seqüenciamento do DNA parafinizado foi possível determinar, com segurança e confiabilidades previstas em parâmetros convencionais, a seqüência de pelo menos 32 amostras; contudo, não se obteve distinção suficiente dos picos de eletroferogramas para a determinação de eventuais polimorfismos na região analisada. Não foi possível portanto, verificar com margem de segurança razoável, a presença ou ausência de SNPs nas amostras seqüenciadas. Houve, contudo, uma qualificação e competência laboratorial instalada localmente (em Fortaleza), a partir da experiência desenvolvida com o esforço deste trabalho, para continuar a investigação molecular em prol da determinação, em futuro breve, da ocorrência ou não de SNPs no exon 8 do TP53 em linfoma de Hodgkin.

Protein p53 je jednou z nejdůležitějších molekul v lidském těle. P53 reguluje celou řadu procesů v buňce, jako je například oprava DNA, buněčný cyklus nebo indukce apoptózy. Protein p53 je známý i jako „strážce genomu“. DNA vazebné schopnosti proteinu p53 jsou důležité pro normální vývoj a růst buňky. Mutace genu pro p53 mohou vést ke ztrátě jeho DNA vazebných vlastností a funkce nádorového supresoru, což muže způsobit rozvoj rakoviny. Teoretická část této diplomové práce je zaměřena na popis vlastností, funkce a mechanismus aktivace proteinu p53 a popis lokálních sekundárních struktur DNA. Hlavním cílem experimentální části byla produkce čtyř mutantních forem proteinů p53 a wild-type p53 proteinu a studium jejich vazebných vlastnosti s různými lokálními sekundárními strukturami DNA. Pomoci Gateway klonovacího systému byly připraveny čtyři expresní vektory, které byly použity pro produkci proteinů v bakteriálním expresním systému. Celkem byly úspěšně připraveny čtyři mutantní formy a wild-type p53 protein. Jejich vazebné vlastnosti byly studovány gelovou retardační analýzu. Výsledky naznačují různé DNA-vazebné vlastnosti wild-type p53 a studovaných mutantních forem tohoto proteinu. Všechny mutantní proteiny ztratily schopnost sekvenčně specificky vázat DNA, zatímco nespecifická interakce s DNA byla pozorována u tří ze čtyř mutantních forem. Jeden ze studovaných mutantních proteinů se vázal jenom na superhelikální formu DNA.

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Die Therapiemöglichkeiten für Patienten im Melanom Stadium IV sind nach wie vor begrenzt und die Erkrankung nur selten heilbar. Eine mögliche Ziel¬struktur in der Melanom¬therapie der Zukunft könnte das im Melanom häufig genetisch wild¬typisch vorliegende p53 sein. Für vorliegende Arbeit wurden humane Melanomzelllinien, welche stabil mit einem p53-Reportergenkonstrukt transduziert waren, hinsichtlich ihrer p53-Expression, -Akti-vität und -Akti¬vierbarkeit untersucht. Alle verwendeten Melanom¬zell¬¬¬linien exprimierten p53 un¬ab¬hängig vom p53-Mutations¬status. Drei der sieben untersuchten p53-wild¬typischen Melanomzelllinien zeigten keine oder nur sehr niedrige p53-Reporter¬gen¬aktivität. Die anderen vier p53-wildtypischen Zelllinien dagegen waren durch hohe, mittels p53-Knockdown unterdrückbare Reportergen¬expression gekennzeichnet. Die Proliferation dieser Zellen in Gegenwart von aktivem p53 belegt, dass Melanomzellen eine hohe Toleranz gegenüber diesem Tumor¬suppressor besitzen können. Eine weitere Steige¬rung der p53-Expression und -Aktivität durch die Hemmung von MDM2 (mouse double minute 2) mit der Substanz Nutlin-3a führte in den Zellen mit messbarer p53-Aktivität jedoch zu einem G1-Zell¬zyklusarrest. Dies belegt die prinzipielle Eignung von p53 als mögliche thera¬peutische Zielstruktur. Aufgrund ihrer schlechten Biover¬füg¬barkeit und hohen Toxizität gelten MDM2-Inhibitoren bisher aber als ungeeignet für den klinischen Einsatz. Die Reduktion hoher therapeutischer Nebenwirkungen könnte durch eine Melanom-spezifische Reaktivierung von p53 gelingen. Eine mögliche negativ-modulierende Wirkung des Mela¬¬nom¬¬markers TRP2 (tyrosinase-related-protein 2) auf p53 wurde im Jahr 2010 von Sendoel et al. nach Unter¬suchungen am Fadenwurm C. elegans vorgeschlagen. TRP2 wird beim metastasierten Melanom in mehr als 80 % der Fälle exprimiert und wäre, handelte es sich beim Melanom um einen weit verbreiteten Regulationsmechanismus, ein interessantes Zielprotein, um die Aktivität von p53 zu steigern. Die dargestellten Ergeb¬nisse zeigen, dass TRP2 zwar in vier von fünf Melanomzelllinien exprimiert wurde, die Unterdrückung der TRP2-Expression jedoch weder spezifischen Einfluss auf die p53-Expression noch auf die p53-Reportergenaktivität zeigte. Auch das veränderte Wachs¬tums¬¬¬verhalten der Zellen nach Unterdrückung von TRP2 mittels drei unterschiedlicher shRNAs konnte im Rescue-Experiment, bei dem TRP nach seinem Knockdown ektop exprimiert wurde, keinem spezifischen Effekt von TRP2 auf die p53-Expression oder p53-Reporteraktivität zugeordnet werden. Auch in der TRP2-negativen Zelllinie führte die ektope TRP2-Expression nicht zu einer verminderten p53-Expression oder -Aktivität. Für das im Gegensatz zu MDM2 deutlich melanomspezifischere TRP2 konnte demensprechend kein sicherer regulatorischer Zusammenhang mit p53 dargestellt werden. Weitere Untersuchungen müssen zeigen, welche Bedeutung wildtypischem p53 im Melanom zukommt und ob sich weitere mögliche p53-Regulatoren als therapeutische Angriffspunkte eignen. / The aim of this study was to investigate the role of wildtype and transcriptional inactive p53 and tyrosinase related protein 2 (TRP2) as potential regulator of p53 in melanoma cell lines. Nine human melanoma cell lines were transduced with a lentiviral pGreenFire™ reporter construct encoding luciferase and GFP under the control of a p53 responsive element. Western blots, luciferase and green fluorescent protein (GFP) assays were used to analyze p53-expression, -activity and -upregulation before and after knockdown of p53 and TRP2 by specific shRNAs. Moreover, melanoma cells were treated with Nutlin-3a, a specific inhibitor of the p53 ubiquitinase mouse double minute 2 homolog (mdm-2), to study its effect on cell proliferation using propidium iodide cell cycle FACS-analysis. Growth of melanoma cells in the presence of active p53 suggests that they tolerate high levels of p53. Furthermore this study shows that preexisting p53 activity can be upregulated by treating the cells with Nutlin-3a. This upregulation was associated with a clear cut cell cycle arrest while the low level p53 activity melanoma cell lines did not show any comparable effect. In contrast regulation of p53 by TRP2 seems not to be common in melanoma cell lines. This study could not depict specific effects on p53 activity alteration after TRP2 knockdown. The role of TRP2 as a potential regulator of p53 in melanoma stays questionable. In principle enhancement of preexisting p53 activity by treating melanoma cells with Nutlin-3a would be a suitable therapeutic target. However, due to its toxicity in humans it might be inappropriate for clinical applications.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

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La réussite de la thérapie cellulaire à l'aide des cellules souches embryonnaires, nécessite une bonne compréhension des mécanismes moléculaires qui contrôlent leur différenciation. Une des voies de signalisation, impliquée dans le contrôle de la différenciation des cellules souches, est la voie p38MAPK. Dans le but de comprendre les mécanismes moléculaires impliqués dans le contrôle de la différenciation précoce des cellules ES, nous avons réalisé un criblage sur puces à ADN. Les résultats du criblage ont montré que certains gènes régulés différentiellement, comme le gène Bcl2, sont des cibles communes de p38MAPK et de p53. En plus de son rôle de répresseur de tumeur, p53 a été impliqué dans le développement embryonnaire et dans la biologie des cellules ES. Ainsi, il est connu que p53 agit comme un répresseur du gène de pluripotence, Nanog. Il est également connu que p53 interfère avec le processus de reprogrammation des cellules adultes ; et que son activité transcriptionnelle augmente avec l'entrée des cellules ES en différenciation. En revanche, son rôle dans la différenciation et dans la formation des différents lignages issus des cellules ES, reste inconnu. Nous avons trouvé que le traitement des cellules ES sauvages avec l'inhibiteur spécifique de p53, la pifithrine-α, durant le processus de différenciation, inhibe les lignages mésodermiques, et à l'inverse, stimule la neurogenèse. De plus, la transfection transitoire des cellules ES avec des siRNAs spécifiques, dirigés contre p53 ainsi que l'utilisation des cellules ES déficientes pour le gène p53, montrent que l'absence de p53, affecte les lignages cardiaques, du muscle lisse et du muscle squelettique. / Embryonic stem cells (ESCs) differentiate in vitro into all cell lineages. We previously found that p38MAPK controls two independent successive steps during the early mesodermal commitment of ESCs. The first one is Brachyury dependent, a master gene of mesoderm formation whereas the second one is not. In order to understand the molecular mechanism implicated in the second step, we treated ESCs with the p38 specific Inhibitor PD169316 and performed microarray experiments on mRNAs extracted from treated versus untreated cells. Our results show that many regulated genes are common targets of p38MAPK and p53 transcription factor. In addition to its role as a tumor suppressor and cell cycle checkpoint control, p53 has been involved in embryonic development, but its role in ESC differentiation is still unknown. We found that treatment of wild type ESCs with the p53 specific inhibitor pifithrin α during the differentiation process inhibits mesodermal lineages and, by contrast, stimulates neurogenesis. Likewise, ESCs Transfected with p53 siRNAs and p53 KO ESCs show an inhibition of cardiac, endothelial, smooth muscle and skeletal muscle lineage formation. Furthermore, p38MAPK inhibition by PD169316 for 24h induces a strong decrease of p53 protein level. Our results suggest that p53 mediates the p38MAPK control of the commitment of ESCs towards mesodermal lineages. The involvement of the various p53 isoforms in this process will be discussed.

O objetivo deste estudo foi comparar leiomiomas e leiomiossarcomas de boca por meio de análise histológica e imunoistoquímica, utilizando os marcadores Ki67, p53 e PTEN. A actina de músculo liso foi utilizada para confirmar o diagnóstico. Foram examinados 13 tumores de músculo liso disponíveis nos arquivos do Departamento de Estomatologia da Faculdade de Odontologia da USP, sendo 7 leiomiossarcomas, 4 angioleiomiomas e 2 leiomiomas sólidos. As lâminas foram avaliadas de acordo com o grau de atipia, índice mitótico e presença de necrose. Foi realizada a análise imunoistoquímica para o ki67, p53 e PTEN, por meio da contagem de células positivas em 500 células nas áreas mais representativas. A maioria dos angioleiomiomas não apresentou atipia, mitose ou necrose; atipia discreta foi encontrada nos dois leiomiomas sólidos, um deles com 1 mitose por 10 CGA. Atipia foi observada em todos os leiomiossarcomas, o índice mitótico variou de 0 a 6 mitoses por 10 CGA, e apenas um caso apresentou necrose. Houve positividade para o ki67 em apenas um angioleiomioma e em 5 leiomiossarcomas, o restante dos casos foram negativos. Em relação ao p53, ocorreu leve positividade para um caso de leiomioma sólido e moderada para o outro; a maioria dos angioleiomiomas foram levemente positivos e apenas um foi negativo; todos os leiomiossarcomas foram positivos, 2 com marcação leve, 4 moderada e um intensa. A expressão do PTEN foi negativa em um leiomioma sólido e intensamente positiva em outro; todos os casos de angioleiomioma foram positivos, sendo que 2 demonstraram positividade leve e 2 moderada; 3 leiomiossarcomas apresentaram positividade moderada e 3 intensa, apenas um foi negativo. Nossos resultados sugerem que a marcação para ki67 e p53 são úteis na diferenciação entre leiomiomas e leiomiossarcomas. O mesmo não foi observado para o anticorpo PTEN. / This study aimed to compare oral leiomyomas and leiomyosarcomas by means of histological and immunohistochemical analysis. Cases from the Stomatology Department, School of Dentistry at University of São Paulo were retrieved. For immunohistochemistry Ki67, p53 and PTEN markers were used. Smooth muscle actin was used to confirm the diagnosis. There were 13 smooth muscle tumors: 7 leiomyosarcomas, 4 angioleiomyomas and 2 solid leiomyomas. For morphological analysis, sections were evaluated for atypia, mitotic index and presence of necrosis. Immunohistochemical analysis of Ki67, p53 and PTEN expression was performed by counting 500 positive cells in the most representative areas. Most angioleiomyomas did not present atypia, mitosis or necrosis; mild atypia was found in two solid leiomyomas, one with 1 mitosis per 10 HPF. Atypia was found in all leiomyosarcomas, the mitotic index varied from 0 to 6 mitosis per 10 HPF, and necrosis was found in only one case. Ki67 expression was positive in one angioleiomyoma and 5 leiomyosarcomas, the remaining cases were negative. For p53, one solid leiomyoma was mildly positive and the other showed moderate positivity; most of angioleiomyomas were mildly positive and only one was negative; all leiomyosarcomas were positive, 2 with mild expression, 4 moderate and one intense. PTEN expression was negative in one solid leiomyoma and intensely positive in the other; all angioleiomyomas were positive, with 2 mildly positive and 2 moderately positive; 3 leiomyosarcomas presented moderate positivity and 3 intense, only one was negative. Our results suggest that ki67 and p53 are useful in the differentiation between leiomyoma and leiomyosarcoma. The same was not found for PTEN.

by Poon Hong Kit. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 129-161). / Abstract also in Chinese. / ACKNOWLEDGEMENTS --- p.i / ABSTRACT (ENGLISH/CHINESE) --- p.ii / ABBREVIATIONS --- p.vi / LIST OF TABLES --- p.vii / LIST OF FIGURES --- p.viii / TABLE OF CONTENTS --- p.x / INTRODUCTION --- p.1 / LITERATURE REVIEW --- p.4 / Chapter I. --- Models for studying photoreceptor cell death --- p.4 / Chapter II. --- Photic retinopathy --- p.5 / Chapter II.1. --- Proposed Mechanisms of Photic Retinopathy --- p.5 / Chapter II.2. --- Factors Affecting Photic Retinopathy --- p.6 / Chapter II.3. --- Pathologic Processes of Photic Retinopathy --- p.7 / Chapter III. --- P53 --- p.9 / Chapter III.1. --- Historical Perspective --- p.9 / Chapter III.2. --- Structure of p53 --- p.10 / Chapter III.2.1. --- The p53 Gene & mRNA --- p.10 / Chapter III.2.2. --- The p53 Protein --- p.10 / Chapter III.3. --- Modifications of p53 Protein --- p.13 / Chapter III.4. --- Functions of p53 --- p.14 / Chapter III.4.1. --- p53 & Tumors --- p.14 / Chapter III.4.2. --- p53 & DNA Damage --- p.15 / Chapter III.4.3. --- p53 & Apoptosis --- p.16 / Chapter III.4.3.1. --- p53-dependent Apoptosis --- p.16 / Chapter III.4.3.2. --- p53-independent Apoptosis --- p.20 / Chapter IV. --- clusterin --- p.22 / Chapter IV. 1. --- Historical Perspective --- p.22 / Chapter IV.2. --- Structure of Clusterin --- p.22 / Chapter IV.2.1. --- Clusterin Gene & mRNA --- p.22 / Chapter IV.2.2. --- Clusterin Protein --- p.22 / Chapter IV.3. --- Functions of Clusterin --- p.22 / Chapter IV.3.1. --- Clusterin & Apoptosis --- p.23 / OBJECTIVES --- p.24 / MATERIALS AND METHODS --- p.26 / Chapter V. --- Sample collections --- p.26 / Chapter V.l. --- Induction of Photic Injury in Rat --- p.26 / Chapter V.2. --- Tissue Processing --- p.26 / Chapter V.2.1. --- Paraffin-embedded Tissues --- p.26 / Chapter V.2.2. --- Epoxy-embedded Tissues --- p.27 / Chapter V.3. --- Cell Culture --- p.28 / Chapter VI. --- Light-microscopic study --- p.29 / Chapter VI. 1. --- Histopathology --- p.29 / Chapter VI.1.1. --- Toluidine Blue --- p.29 / Chapter VI. 1.2. --- Morphometry of the ONL Thickness --- p.29 / Chapter VI.2. --- In situ TUNEL --- p.29 / Chapter VI.2.1. --- Morphometry of TUNEL --- p.29 / Chapter VI.3. --- Immunohistochemistry --- p.30 / Chapter VI.3.1. --- Single-labeling --- p.30 / Chapter VI.3.1.1. --- Immunolabeling of p53 Protein --- p.31 / Chapter VI.3.1.2. --- Immunolabeling of p21 Protein --- p.31 / Chapter VI.3.1.3. --- Immunolabeling of bax Protein --- p.31 / Chapter VI.3.1.4. --- Immunolabeling of c-fos Protein --- p.32 / Chapter VI.3.1.5 --- Immunolabeling of Clusterin Protein --- p.32 / Chapter VI.3.2. --- Double-labeling --- p.32 / Chapter VI.3.2.1. --- TUNEL & Fluorescent-labeling of p53 --- p.32 / Chapter VI.3.2.2. --- TUNEL & Fluorescent-labeling of p21 --- p.33 / Chapter VI.3.3. --- Grading of Immunoreactivities --- p.33 / Chapter VI.3.4. --- Morphometry of c-fos Immuno-positive Nuclei --- p.33 / Chapter VI.4. --- In situ RT-PCR --- p.34 / Chapter VI.4.1. --- Isolation of Retinal DNA --- p.34 / Chapter VI.4.2. --- Polymerase Chain Reaction (PCR) --- p.34 / Chapter VI.4.3. --- In situ Reverse Transcriptase 226}0ؤ PCR (RT-PCR) --- p.35 / RESULTS --- p.37 / Chapter VII. --- LIGHT-MICROSCOPY --- p.37 / Chapter VII.1. --- Histopathology & Morphometry --- p.37 / Chapter VII. 1.1. --- Histopathology --- p.37 / Chapter VII.1.2. --- Morphometry of the ONL Thickness --- p.49 / Chapter VII.2. --- DNA Nicks Detection --- p.42 / Chapter VII.2.1. --- In situ TUNEL --- p.42 / Chapter VII.2.2. --- Morphometry of TUNEL --- p.42 / Chapter VII.3. --- p53 --- p.51 / Chapter VII.3.1. --- Immunohistochemistry of p53 --- p.51 / Chapter VII.3.2. --- Grading of p53 Immunoreactivities --- p.51 / Chapter VII.3.3. --- p53 in situ RT-PCR --- p.51 / Chapter VII.3.4. --- Double-labeling ofp53 with in situ TUNEL --- p.59 / Chapter VII.4. --- p21 --- p.74 / Chapter VII.4.1. --- Immunohistochemistry of p21 --- p.74 / Chapter VII.4.2. --- Grading of p21 Immunoreactivities --- p.74 / Chapter VII.4.3. --- Double-labeling of p21 with in situ TUNEL --- p.74 / Chapter VII.5. --- bax --- p.84 / Chapter VII.5.1. --- Immunohistochemistry of bax --- p.84 / Chapter VII.5.2. --- Grading of bax Immunoreactivities --- p.84 / Chapter VII.5.3. --- bax in situ RT-PCR --- p.84 / Chapter VII.6. --- c-fos --- p.96 / Chapter VII.6.1. --- Immunohistochemistry of c-fos --- p.96 / Chapter VII.6.2. --- Morphometry of c-fos immuno-positive nuclei & TUNEL --- p.96 / Chapter VII.7. --- Clusterin / Chapter VII.7.1. --- Immunohistochemistry of Clusterin --- p.108 / Chapter VII.7.2. --- Grading of Clusterin Immunoreactivities --- p.108 / discussion --- p.116 / Chapter VIII. 1. --- Summary --- p.116 / Chapter VIII.1.1. --- Histopathology & Morphometry --- p.118 / Chapter VIII.1.2. --- In situ TUNEL --- p.119 / Chapter VIII.1.3. --- p53 & c-fos --- p.119 / Chapter VIII.1.4. --- p27 --- p.124 / Chapter VIII.1.5. --- bax --- p.124 / Chapter VIII. 1.6. --- Clusterin --- p.124 / Chapter VIII.2. --- Hypothesis --- p.125 / Chapter VIII.3. --- Conclusion --- p.127 / bibliography --- p.129