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1
Gerlinger, Emmanuel. "Proto-oncogenes et developpement embryonnaire : etude bibliographique." Université Louis Pasteur (Strasbourg) (1971-2008), 1989. http://www.theses.fr/1989STR1M202.
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Bennett, Julie Denise. "Cell cycle regulation of B-myb transcription." Thesis, Imperial College London, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.362337.
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Ewels, Philip Andrew. "Spatial organisation of proto-oncogenes in human haematopoietic progenitor cells." Thesis, University of Cambridge, 2013. https://www.repository.cam.ac.uk/handle/1810/245861.
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The eukaryotic cell nucleus is a highly organised organelle, with distinct specialised sub- compartments responsible for specific nuclear functions. Within the context of this functional framework, the genome is organised, allowing contact between specific genomic regions and sub-compartments. Previous work has shown that genes in both cis and trans can make specific contacts with each other. I hypothesise that such a preferred juxtaposition may impact the propensity for specific cancerinitiating chromosomal translocations to occur. In this thesis, I describe how I have extended and developed a ligation based proximity assay known as enriched 4C. I have coupled this technique with high throughput sequencing to determine genomic regions that spatially co-associate with the proto-oncogenes MLL, ABL1 and BCR. In addition to further developing the laboratory protocol, I have created bioinformatics tools used in the analysis of the sequencing data. I find that the association profiles of the three genes show strong correlation to the binding profile of RNA polymerase II and other active marks, suggesting that transcribed genes have a propensity to associate with other transcribed regions of the genome. Each gene also exhibits a unique repertoire of preferred associations with specific regions of the genome. Significantly, I find that the most frequent trans association of BCR is telomeric chromosome 9, encompassing its recurrent translocation partner gene ABL1. Interestingly, ABL1 is not at the maximum point of interaction. I use DNA-fluorescence in-situ hybridisation to validate the e4C association. My data supports a hypothesis that gene transcription has a direct role on genome organisation. I suggest that preferred co-associations of genes at transcription factories may promote the occurrence of specific chromosomal translocations.
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Maxwell, Marius. "Expression of proto-oncogenes and growth factors in glioblastoma multiforme." Thesis, University of Oxford, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.259967.
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Kemble, David J. "A biochemical study on the regulation of the SRC and FGFR family of protein tyrosine kinases /." View online ; access limited to URI, 2009. http://0-digitalcommons.uri.edu.helin.uri.edu/dissertations/AAI3367994.
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Baker, David Alan. "Mutational analysis of the proto-oncogenes c-fms and c-kit." Thesis, Imperial College London, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.362390.
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Veal, Elizabeth Ann. "The role of proto-oncogenes in normal and dystrophic skeletal muscle." Thesis, University of Liverpool, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.307666.
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Cerutti, Janete Maria. "Analise do papel de c-MYC no processo de transformação das celulas foliculares da tireoide humana." [s.n.], 1995. http://repositorio.unicamp.br/jspui/handle/REPOSIP/317287.
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Orientador: Solange Bento FarahTese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Doutorado
Genetica
Doutor em Ciências Biológicas
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Lyon, Jonathan James. "The role of c-Myb in the regulation of haemopoiesis." Thesis, Imperial College London, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.307259.
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Amouyel, Philippe. "Expression des proto-oncogenes ets dans les astrocytes et dans les tumeurs astrocytaires." Lille 2, 1988. http://www.theses.fr/1988LIL2M054.
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Corcos, Daniel. "Etude de l'expression des proto-oncogenes dans le foie normal et cancereux chez le rat." Paris 7, 1988. http://www.theses.fr/1988PA077042.
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Augmentation correlee de l'expression des trois genes ras dans les tumeurs et egalement dans les parties non tumorales des foies cancereux. L'etude des variations physiologiques de l'expression des protooncogenes a revele un controle alimentaire de l'expression du gene c-myc
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Chui, Chung-hin. "Molecular characterization of C-KIT proto-oncogene in Hong Kong leukemia patients : 'culprit or bystander' /." Hong Kong : University of Hong Kong, 1998. http://sunzi.lib.hku.hk/hkuto/record.jsp?B1947037X.
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Zhu, Jiang. "HOXB5 cooperates with TTF1 in the transcription regulation of human RET promoter." Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B43278607.
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Bidshahri, Arezoo (Roza). "Novel ultra-sensitive digital PCR assays for screening and detection of rare missense mutations in (proto)-oncogenes." Thesis, University of British Columbia, 2017. http://hdl.handle.net/2429/62151.
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Somatic mutations can lead to cancer, often by altering the activity of kinases within signaling pathways that control cell growth and proliferation. Targeted cancer therapeutics are designed and used to regulate these aberrant signaling pathways in cases where somatic mutations within kinase genes predict a positive patient response to those treatments. For example, the V600E mutation in BRAF, the gene coding for the BRAF serine threonine kinase, predicts the effectiveness of vemurafenib in treating metastatic melanoma, while the mutational status of codons G12/G13 in the KRAS gene predicts likely colorectal cancer patient response to the monoclonal antibody (mAb) cetuximab.¹-³ However, FDA approved assays currently used to detect missense mutations in BRAF V600 and KRAS G12/G13 are not capable of detecting clinically actionable mutations at mutational frequencies low enough to permit their robust application to early disease detection or minimal residual disease monitoring. Moreover, detection of all clinically actionable missense mutations is not certain or generally achieved, in part due to limitations to assay specificities and the inability to unequivocally discriminate missense mutations from synonymous germline sequence variations.
This thesis addresses that limitation through the development and validation of a novel platform for creating highly sensitive assays against all possible missense mutations in an oncogenic hotspot codon or adjacent set of hotspot codons that ameliorates the known limitations to current FDA-approved assays. The platform is designed to enable development of assays against all possible missense mutations in oncogenic hotspots and, if required, unequivocally differentiate them from synonymous germline alleles. It utilizes droplet digital PCR (ddPCR) technology and chimeric wild-type specific LNA/DNA probes to create a novel “WT-negative” screening paradigm. The platform is applied to the creation of two new assays of potential clinical use in cancer diagnostics and theranostics. The first provides a reliable and sensitive screening and detection of all known clinically actionable mutations in BRAF V600, and the second achieves the same for KRAS G12/G13. Both assays show complete diagnostic accuracy when applied to formalin-fixed paraffin-embedded (FFPE) tumor specimens from metastatic colorectal cancer patients deficient for Mut L homologue-1.Applied Science, Faculty of
Graduate
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Ballantyne, Eric Sinclair. "The expression and prognostic role of proto-oncogenes and tumour suppressor genes in medulloblastoma and embryonic brain." Thesis, University of Liverpool, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.366487.
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Siqueira, Débora Rodrigues. "Influência das variantes genéticas do proto-oncogene RET na apresentação clínica da neoplasia endócrina múltipla tipo 2." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2012. http://hdl.handle.net/10183/61883.
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O carcinoma medular de tireóide (CMT) é uma neoplasia das células C ou parafoliculares da tireóide, correspondendo a 5–8% dos tumores malignos da glândula. O CMT apresenta-se como um tumor esporádico (75-80%) ou na forma hereditária (20-25%). Na forma familiar é um dos componentes de uma síndrome genética de herança autossômica dominante, apresentando-se isoladamente, como carcinoma medular de tireóide familiar (CMTF) ou como um dos componentes da neoplasia endócrina múltipla (NEM) 2A ou 2B. A síndrome genética NEM 2A caracteriza-se pela presença de CMT (95%), feocromocitoma (30 – 50%) e hiperparatireoidismo (10-20%), enquanto que pacientes com NEM 2B podem aprensentar CMT (90%), feocromocitoma (45%), ganglioneuromatose (100%) e hábito marfanóide (65%). O proto-oncogene RET, gene responsável pela NEM 2, codifica um receptor tirosinoquinase expresso nas células derivadas da crista neural. Mutações germinativas no RET são descritas principalmente nos exons 10, 11 e 16. Diversos estudos demonstraram a associação entre mutações específicas (genótipo) e idade no diagnóstico ou agressividade do CMT hereditário (fenótipo). No entanto, pacientes portadores da mesma mutação no proto-ongene RET podem apresentam quadros clínicos diferentes. Por esta razão, os polimorfismos do RET têm sido investigados como possíveis modificadores do fenótipo clínico em pacientes com NEM 2. Porém, os dados existentes na literatura sobre o papel dos polimorfismos do RET ainda são controversos. O objetivo do nosso estudo foi avaliar o papel das variantes genéticas do RET na apresentação clínica do CMT (Siqueira DR et al, Endocr Relat Cancer 2010; 17:953-963). Dentro deste contexto, realizamos uma revisão do conhecimento atual sobre a associação dos polimorfismos do RET com o risco de desenvolver ou modificar a evolução do CMT (Ceolin L et al, Int J Mol Sci 2012; 13:221-239). A melhor compreensão dos diferentes mecanismos moleculares envolvidos na patogênese tumoral permitiu o desenvolvimento de novos tratamentos, direcionados principalmente aos pacientes com doença metastática. Dentre as diversas classes de novas drogas, destacam-se os inibidores tirosina quinase; essas drogas inibem a ação de vários receptores, entre eles o RET. O crescente número de estudos publicados avaliando o efeito de inibidores tirosina-quinase no manejo de pacientes com CMT metastático determinou um estudo da literatura sobre este tema (artigo submetido à publicação).
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SENAN, FREDERIQUE. "Etude du role des proto-oncogenes ets1, ets2 et fli au cours du developpement embryonnaire de xenopus laevis." Université Louis Pasteur (Strasbourg) (1971-2008), 1995. http://www.theses.fr/1995STR13103.
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Cette these decrit le clonage et l'etude de l'expression spatio-temporelle de 3 proto-oncogenes de la famille ets chez xenopus laevis. Une strategie basee sur la conservation de sequence existant entre les differents membres de la famille, nous a permis d'isoler les adnc ets1, ets2 et fli de xenope. L'etude de l'expression temporelle du gene ets1 a ete realisee en northern blot. Parmi les 4 transcrits ets1 detectes, deux transcrits (7,5 kb et 4,4 kb) sont majoritaires. Ils apparaissent des les stades ovocytaires et restent presents lors de l'embryogenese precoce, pour disparaitre au moment de la gastrulation. Une nouvelle expression a lieu au stade neurula, puis decroit progressivement jusqu'au stade 40. Chez l'adulte, ils sont presents uniquement dans la rate et l'ovaire. La double expression maternelle et zygotique est egalement observee pour le gene ets2, tandis que l'expression du gene fli n'est mise en evidence que chez le zygote. Des anticorps polyclonaux diriges contre les proteines ets1, ets2 et fli sont capables de detecter les proteines produites in vitro ou surexprimees dans les embryons, mais pas les proteines endogenes probablement en raison de leur taux tres faible au cours de l'embryogenese. Le role des proteines ets1, ets2 et fli a ete etudie in vivo. L'injection de transcrits fli revele des malformations des le stade fin gastrula. Les phenotypes observes vont de la dysmorphie des yeux et/ou du systeme circulatoire jusqu'a l'absence totale des axes embryonnaires. Ils semblent resulter d'une perturbation des proprietes d'adhesivite cellulaire. Des experiences d'hybridation in situ montrent que les transcrits ets1 et ets2 sont presents dans le cytoplasme des ovocytes, dans le pronephros en formation, ainsi qu'au sein de plusieurs territoires issus de la migration des cellules de la crete neurale. Le gene ets1 possede par ailleurs un site d'expression important au niveau des regions angioformatrices
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Zhu, Jiang, and 朱江. "HOXB5 cooperates with TTF1 in the transcription regulation of human RET promoter." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hub.hku.hk/bib/B43278607.
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Puñales, Márcia Khaled. "Rastreamento genético do carcinoma medular de tireóide: identificação de mutações no protooncogene "ret"." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2000. http://hdl.handle.net/10183/115378.
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O carcinoma medular de tireóide (CMT) é responsável por 5 a 8% dos tumores malignos da tireóide, ocorrendo na forma esporádica (80%) ou hereditária (20%). O CMT hereditário é uma doença autossômica dominante, maligna, de difícil diagnóstico clinico-laboratorial precoce e de alta mortalidade, podendo apresentar-se como componente das síndromes de Neoplasia Endócrina Múltipla (NEM 2A e 2B) ou Carcinoma Medular de Tireóide Familiar (CMTF) ou outras formas (famílias não incluídas nas formas anteriores). A síndrome genética NEM 2A se caracteriza por CMT (95%), feocromocitoma (30-50%) e hiperparatireoidismo (10-20%). De acordo a alguns autores e baseando-se na apresentação clínica, a síndrome NEM 2A foi subdividida em 3 subtipos fenotípicos; 1) NEM 2A(1), que consiste no acometimento pelos 3 componentes da síndrome (CMT, feocromocitoma e hiperparatireoidismo), 2) NEM 2A(2), que consiste no acometimento por 2 componentes da síndrome (CMT e feocromocitoma) e 3) NEM 2A(3), que consiste no acometimento por 2 componentes da síndrome (CMT e hiperparatireoidismo). A síndrome NEM 2B se caracteriza pela presença de CMT (90%), feocromocitoma (45%), ganglioneuromatose intestinal (100%) e hábitos marfanóides (65%). O CMTF, consiste na presença de CMT isolado em pelo menos 4 membros da mesma família e as outras formas hereditárias consistem no acometimento de 2 ou 3 membros da família com CMT, sem a presença de feocromocitoma ou hiperparatireoidismo até o momento do rastreamento. Diferentes mutações no proto-oncogene rei foram identificadas como responsáveis pelo CMT e estudos recentes sugerem uma correlação entre o genótipo-fenótipo, podendo existir uma variabilidade grande de síndromes clínicas associadas as diferentes mutações, ou seja, indivíduos com um determinado tipo de mutação tem uma probabilidade maior ou menor de apresentar um determinado componente da síndrome. Visto que o CMT é uma doença autossômica dominante, maligna e de alta mortalidade, a aplicação do rastreamento genético para o manejo adequado da hereditariedade do CMT é de extrema importância, já que o diagnóstico precoce determina a conduta e o prognóstico no indivíduo afetado e em seus familiares. O presente estudo teve como objetivo a análise molecular do protooncogene ret no CMT e avaliar uma possível correlação entre as mutações identificadas e os diferentes fenótipos nos indivíduos afetados e seus familiares. Foram selecionadas para estudo molecular 21 famílias com diagnóstico íiistopatológico de CMT, provenientes de diferentes localidades e encaminhados aos ambulatórios de endocrinologia do HCPA e HC-Curitiba. O DNA genômico foi extraído de leucócitos periféricos dos indivíduos afetados e/ou familiares. Os exons 10, 11, 13 e/ou 16 do proto-oncogene ret foram amplificados pela técnica de reação polimerase em cadeia (PCR) com primers específicos. A presença de mutações foi determinada pela análise de restrição enzimática e/ou sequenciamento, seguindo a estratégia diagnostica. Das 21 família analisadas, 14 famílias apresentavam a forma hereditária do CMT e 7 a forma esporádica. Das famílias com CMT hereditário (6 NEM 2A, 2 NEM 2A associada à Líquen Amilóide Cutânea (CI_A), 1 NEM 2B, 2 CMTF e 3 outras formas hereditárias), no total de 96 indivíduos. Em todas as famílias com NEM 2A, inclusive na variante Cl_A, a mutação localizava-se no exon 11 (códon 634; TGC -» CGC ou TGC TAC) e nas famílias com CMTF, no exon 10 (códon 618, TGC AGC). No paciente portador da NEM 2B foi detectada uma mutação de novo no exon 16 (códon 918, ATG ->ACG). Nas outras formas de carcinoma hereditário, as mutações localizavam-se no exon 11 (códon 634, TGC CGC ou TGC TAC) numa família, em outra no exon 10 (códon 618, TGC -> AGC) e na última no exon 10 (códon 618 ou 620, TGC CGC). Foram identificadas mutações em todos os indivíduos com história clínica e laboratorial sugestiva de doença hereditária e/ou diagnóstico histopatológico de CMT muiticêntrico e em 8 familiares assintomáticos, destacando a importância do rastreamento genético como método diagnóstico.Medullary Carcinoma of the Thyroid (MTC), a rare thyroid malignancy originating from parafollicular C cells, represents less than 8% of ali thyroid cancers and may occur either as sporadic (80%) or hereditary (20%) disease. Hereditary MTC oan occur either alone - familial MTC (FMTC) - or as the thyroid manifestation of multipie endocrine neoplasia type 2 (MEN 2) syndromes (MEN 2A and MEN 2B) or others. MEN 2A is characterized by MTC (95%), phaeochromocytoma (30-50%) and hyperparathyroidism (10-20%). Three phenotypic subtypes have been reported. MEN 2A(1) corresponda to kindred with ai! three components. MEN 2A(2) includes kindred with MTC and phaeochromocytoma, without hyperparathyroidism. MEN 2A(3) relates to kindred with MTC and hyperparathyroidism, without phaeochromocytoma. A variant of MEN 2A, associated with pruritic skin lesions known as cutaneous lichen amyloidosis (CLA), has aiso been described in a few kindred. MEN 2B syndrome is weil characterized by having a specific phenotype, by MTC (90%), neuromas and ganglioneuromatosis (100%) and marfanoid habitus (65%). FMTC includes kindred with at least 4 members with MTC, without other components of MEN 2A or MEN 2B. Other hereditary MTCs correspond to kindred with MTC in 2 or 3 members, without phaechromocytoma or hyperparathyroidism. Germiine mutations in the ret proto-oncogene, which codes for a receptor tyrosine kinase, cause MEN 2 and recent studies suggest a relationship between specific mutations and different phenotypes in MEN 2 syndromes. Early diagnosis and treatment considerably improve the prognosis in patienís with MTC and genetic screening is a fundamental tool for the management of MTC hereditary. The purpose of this study was to identify ret proto-oncogene mutations and analyze a possible relationship between genothype-phenothype in Brazilian kindred with MTC. A total of 21 families with histopathoiogical diagnosis of MTC were included in the study, 14 with the hereditary pattern and 7 with sporadic tumors. DNA was extracted from leukocytes of the affected individuais and relatives. Exons 10, 11, 13 and 16 were amplificated by PCR, using specific primers. The presence of mutation was determined by enzymatic restriction analysis and/or automatic sequencing. The phenotypes of hereditary MTC •Já were as foilows: 6 MEN 2A, 2 MEN 2A associated with CLA, 1 MEN 2B, 2 FMTC and 3 other forms. We identified mutations at codon 634, exon 11 (TGC -> CGC or TGC TAC) in ali families with MEN 2A and MEN 2A+ CLA. In both cases of FMTC, the mutation was found in the codon 618, exon 10 (TGC --> AGC). A mutation at codon 918, exon 16 (ATG -> AGC) was identified in the only individual with MEN 2B, while in the other hereditary forms of the MTC, mutations were identified at 3 different codons, 634 (exon 11, TGC -> CGC or TGC -> TAC). 618 (exon 10, TGC AGC) and 618 or 620 (exon 10, TGC CGC). The genetic screening was abie to identified 8 assymtomatic carriers and determine the hereditary MCT pattern in 2 individuais with apparentiy sporadic tumors. In conclusion, genetic testing can identify affected and assymtomatic individuais with hereditary disease, allowing eariy diagnosis and treatment. In addition, the resuits suggest a correlation between specific mutations and phenotypes, meaning that molecular analysis could also improve the follow up of gene carriers.
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Chui, Chung-hin, and 徐宗憲. "Molecular characterization of C-KIT proto-oncogene in Hong Kong leukemia patients: 'culprit or bystander'." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1998. http://hub.hku.hk/bib/B31236790.
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Haeri, Hosseini S. Mohammad. "The effects of ectopic expression of TAL1 and LMO1 on lipoprotein lipase in NIH 3T3 cells." Virtual Press, 2003. http://liblink.bsu.edu/uhtbin/catkey/1273265.
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Childhood acute lymphoblastic leukemia (ALL) is the most common malignancy in children. Several proto-oncogenes that encode nuclear proteins are activated by various chromosomal translocations in ALL including TALI, TAL2, and LMO1 and LMO2. Ectopic TALI expression is observed in about 50 % of T-ALL and is the most common genetic anomaly associated with this pathology. Of interest to the present work is the characterization of various multiprotein complexes and protein protein interactions that drive T-ALL progression (as it relates to TALI and LMO1) and over expression of TALI and LMO1 has been shown to have inhibitory effects on apoptosis. Recent data suggests possible interactions between these two oncoproteins and the protein product of the lipoprotein lipase (LPL) gene. Lipoprotein lipase has a complex pattern of regulation and can be regulated in different ways including down-regulation upon induction of TNF-a in 3T3-L1 cells. Thus, this study was undertaken to determine if LPL is expressed in cells over expressing TAL1 and LMO1. Results from this study demonstrated an increase in LPL expression at both transcriptional and translational level in cells engineered to express TAL1 alone and TAL1 and LMO1 together. This finding is a step forward to understanding mechanisms that result in apoptosis prevention in T-ALL. Therefore, the apoptosis preventive role seen in cells that over express TAL and LMO1 and the presence of LPL in the same cell line, theorizes an apoptosis preventive role for lipoprotein lipase as well.Department of Physiology and Health Science
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CHALKIOPOULOU-MARX, MARIA. "Modification des genomes viraux et transduction de proto-oncogenes au cours de la replication des retrovirus dans les cellules aviaires." Paris 11, 1991. http://www.theses.fr/1991PA112001.
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Dans la premiere partie de cette these, nous avons etudie la generation des mutants du virus du sarcome de rous, defectifs pour la transformation (td) dans les fibroblastes embryonnaires normaux de caille, et dans une lignee de fibroblastes de caille immortalisee (lignee q3b). Nous avons montre que le phenotype de la cellule hote influence le taux de production des virions td. Les cellules q3b produisent en grand exces des mutants td avec des deletions localisees dans la partie 5 du gene v-src. Elles sont generees par des mecanismes multiples. Nous avons isole le mutant td dlpa105 qui porte une deletion de 93 acides amines situee dans la partie nd2-terminale de la proteine v-src. Nous avons montre que la partie deletee definit un domaine du v-src non essentiel pour la fonction mitogene mais indispensable pour la transformation des fibroblastes. Dans la deuxieme partie, nous avons etudie les mecanismes de transduction retrovirale in vitro dans les cellules de neuroretine de poule (nrp) infectees par le rav-1, un retrovirus depourvu d'oncogenes. Les nrp sont induites a proliferer apres infection par le rav-1. Cette proliferation resulte de l'activation et de la transduction des oncogenes c-mil et c-rmil. L'epissage entre le site donneur d'epissage des sequences leader du rav-1 et le site accepteur d'un exon des oncogenes, est un mecanisme precoce intervenant dans la transduction des proto-oncogenes
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Vieira, Alexandre Eduardo Franzin. "Rastreamento bioquimico e molecular de portadores assintomaticos de neoplasia endocrina multipla tipo 2A." [s.n.], 2001. http://repositorio.unicamp.br/jspui/handle/REPOSIP/310740.
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Orientadores: Margaret de Castro, Maricilda Palandi de MelloDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: A Neoplasia Endócrina Múltipla (NEM) tipo 2A é caracterizada pela presença de Carcinoma Medular de Tireóide (CMT), feocromocitoma e hiperparatireoidismo. Esta síndrome pode ser diagnosticada em portadores assintomáticos pertencentes a famílias de indivíduos acometidos pela síndrome utilizando-se testes periódicos de estimulação da secreção de calcitonina pela gastrina. Entretanto, a analise do DNA genomico permite a identificação destes indivíduos, possibilitando diagnostico mais precoce e tratamento preventivo. O objetivo deste trabalho foi analisar dois diferentes testes de rastreamento de CMT, com a finalidade de diagnosticar portadores assintomáticos de famílias com indivíduos com NEM 2 A: um teste bioquímico utilizando o omeprazol e a analise molecular do gene do protooncogene com NEM 2A foram submetidos ao teste de secreção de calcitonina induzida pelo omeprazol. Calcitonita e gastrina foram determinadas por imunoensaios. DNA genomico foi extraído de sangue total dos 15 indivíduos que concordaram com o estudo. Os exons 10 e 11 foram amplificados por PCR e os produtos analisados por seqüenciamento direto. O teste de estimulo da secreção de calcotonina pelo omeprazol não identificou nenhum portador assintomático. Entre os membros da família 1 encontramos três indivíduos afetados pela mutação germinativa TGC ->TAC (C634Y). Dois irmãos apresentavam CMT, sendo que na irmã associavam-se feocromocitoma e hiperparatireoidismo; o filho desta ultima, de nove anos de idade, apresentava status previamente desconhecido. O estudo da família 2 demonstrou a mutação TGC ->CGC (C634R) somente no caso-indice. Esta paciente apresentava alem do CMT, feocromocitoma, hiperparatireoidismo e líquen amiloidotico cutâneo. Seus pais e seus quatro irmãos, todos assintomáticos, não apresentaram esta mutação, sugerindo que a mutação C634R ocorreu de novo nesta paciente. Em conclusão, a especificidade do teste do omeprazol foi limitado e a eficácia deste teste permanece a ser estabelecida. As duas mutações mais freqüentemente encontradas no protooncogene RET na NEM 2A, estão presentes em famílias brasileiras. A analise molecular deverá ser o procedimento de escolha para o rastreamento de famílias com NEM 2A, desde que é preciso e dispensa testes bioquímicos repetitivos
Abstract: Patients with MEN type 2A are at risk for early medullary thyroid carcinoma (MTC), which might be diagnosed by periodical gastrin-induced calcitonin tests. However, direct DNA analysis permits the identification of asymptomatic mutant carriers in a family, providing an early and definitive diagnosis. We performed different screening tests for MTC, a recently reported biochemical screening test using omeprazole and DNA analysis. Fifteen members of two non-consanguineous Brazilian famílies with MEN 2A were submitted to the omeprazole-induced calcitonin stimulation test. Serum calcitonin and gastrin were determined by immunoassays. RET proto-oncogene analysis was carried out by direct DNA sequencing of PCR-amplified products for exons 10 and 11. Family 1 showed a TGC~ TAC (C634Y) germline mutation in three individuais. Two brothers with symptomatic MTC, one of them also associated with pheocromocytoma and hyperparathyroidism whose son was a nine-year-old boy of previously unknown status. Famíly 2 showed a TGC~CGC (C634R) mutation only in the index case, who presented cutaneous lichen amyloidosis in addition to MTC, pheocromocytoma and hyperparathyroidism. Neither her parents nor her four brothers showed this genetic mutation. The two most frequent RET protooncogene mutations in MEN 2A are present in Brazilian families. In addition, the specificity of basal and omeprazole-stimulated calcitonin is rather limited and the efficacy of the omeprazole test still remains to be systematically examined. Therefore, RET proto-oncogene analysis must be the first choice for a screening procedure to identify mutant gene carriers in MEN 2A family members and to permit early prophylactic treatment
Mestrado
Patologia Clinica
Mestre em Ciências Médicas
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Gebler, Christina [Verfasser], Frank [Gutachter] Buchholz, and Axel [Gutachter] Roers. "Developing the CRISPR/Cas-system for Inactivation of Proto-oncogenes in Human Cancer Cells / Christina Gebler ; Gutachter: Frank Buchholz, Axel Roers." Dresden : Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2018. http://d-nb.info/1227196482/34.
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LIDEREAU, WEINSTEIN ROSETTE. "La variabilite genetique des proto-oncogenes ras, myc et mos comme marqueur de predisposition et d'evolution dans le cancer du sein." Paris 7, 1987. http://www.theses.fr/1987PA077129.
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Queiroz, Julia de Souza 1982. "Atividade moduladora da alga Chlorella vulgaris sobre alterações neuroendócrinas e hematopoéticas causadas pelo estresse." [s.n.], 2012. http://repositorio.unicamp.br/jspui/handle/REPOSIP/309859.
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Orientadores: João Palermo Neto, Antonio Armario GarciaTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: A exposição do organismo a estressores psicossociais e ambientais altera de forma significativa o funcionamento do sistema imune. Os efeitos do estresse sobre a resposta imune têm sido atribuídos, principalmente, à ativação do eixo hipotálamo-pituitária-adrenal (HPA) com consequente aumento nos níveis de ACTH e glicocorticóides e à ativação do sistema nervoso autônomo simpático (SNAS), com liberação de catecolaminas. Nos últimos anos, a alga Chlorella vulgaris (CV) tem despertado o interesse da comunidade científica pelos seus efeitos moduladores sobre as defesas do hospedeiro imunossuprimido. Em estudos anteriores mostramos que o restabelecimento da geração de granulócitos-macrófagos nos órgãos hematopoéticos e a ativação das funções efetoras de fagócitos e linfócitos são cruciais na expressão da atividade imunomoduladora da alga. No entanto, nada se sabe sobre os efeitos da CV no sistema nervoso central em situações de estresse. Sendo assim, neste trabalho realizamos estudos pioneiros com relação ao efeito do tratamento com a alga sobre: 1) a ativação neuronal (c-fos) no córtex pré-frontal, septum lateral, núcleo da Rafe e lócus coeruleus; 2) a ativação do eixo HPA através da expressão do gen de hnCRF na região parvocelular do núcleo paraventricular do hipotálamo, mpdPVN, liberação de ACTH e de corticosterona e, 3) avaliação indireta da atividade do SNAS através dos níveis de glicose no plasma de animais estressados. Considerando-se a medula óssea ser o sítio de origem das células pluripotenciais das quais se originam as células do sistema hematopoético, e que este sistema é totalmente vulnerável ao controle neuroendócrino, avaliamos os efeitos do tratamento com CV sobre a hematopoese de animais estressados através dos 4) crescimento e diferenciação de precursores para granulócitos e macrófagos (CFU-GM); 5) presença de fatores estimuladores da formação de colônias do soro (colony stimulating activity - CSA); 6) quantificação de populações de células maduras e imaturas e 7) morte celular na população de células tronco. A regulação da produção de células hematopoéticas pelas células do estroma da medula óssea em camundongos estressados foi avaliada pela técnica de cultura líquida de longa duração de células da medula óssea (LTBMC), que consiste em um modelo ex vivo para o estudo das interações entre as células progenitoras hematopoéticas e as células do estroma. Nela avaliamos 8) o CFU-GM, níveis de IL-1? / IL-6 e quantificamos uma população madura e uma imatura. Nossos resultados mostraram que a aplicação do estressor produziu um aumento na expressão de c-fos em todas as áreas cerebrais avaliadas, assim como na expressão do gen de hnCRF na região mpdPVN. Os níveis de ACTH e corticosterona também estavam aumentados após o estresse, assim como os níveis de glicose. Na medula óssea observamos que a aplicação do estressor reduziu o número de CFU-GM, e aumentou os níveis de CSA no plasma. Houve um aumento na morte celular e redução no número de precursores hematopoéticos e de células maduras. Na LTBMC, um prejuízo na atividade funcional do estroma medular foi observado através: da redução do CFU-GM, dos níveis de IL-1? / IL-6 e do número de células imaturas e maduras. O aumento na expressão de c-fos após o estresse foi prevenida pelo tratamento com CV em todas as áreas avaliadas, com exceção da região magnocelular do PVN. O resultado mais acentuado do tratamento com CV foi observado na redução da expressão de c-fos no núcleo da Rafe e do gen para hnCRF no mpdPVN, que se encontrou em níveis semelhantes aos observados no grupo controle após o estresse. Todas as alterações hematopoéticas causadas pelo estresse foram prevenidas pelo tratamento com CV. Tomados em seu conjunto, nossos resultados mostraram que o efeito protetor da hematopoese pode ser devido a uma prevenção na ativação neuronal de áreas cerebrais relacionadas à decodificação do estressor do tipo emocional, reduzindo a amplitude de ativação do eixo HPA e do SNAS
Abstract: The exposition of the organism to psychosocial and environmental stressful stimuli alters the functioning of the immune system in a significant way. The effects of stress on the immune response are mainly attributed to the activation of the hypothalamic-pituitary axis (HPA) with consequent increment on ACTH and glucocorticoids levels and, to the activation of the autonomic nervous system, with the incremented levels of cathecholamines. In the last years, increasing interests about the algae Chlorella vulgaris (CV) has been demonstrated by the scientific community, due to its modulatory effects on the defenses of the immunosuppressed host. In previous studies we demonstrated that the reestablishment of the generation of granulocytes and macrophages in bone marrow and, the activation of effectors functions of phagocytes and lymphocytes, are crucial features about the immunomodulatory activity from the algae. However, nothing is known about the activity of CV in the central nervous system. Thus, pioneer investigation was made in this work about the effect of treatment with the CV on: 1) neuronal activation (c-fos) in pre-frontal cortex, lateral septum, Rafe nucleus and locus coeruleus; 2) activation of the HPA axis by analysis of expression of the gen to hnCRF in the parvocelular region from the paraventricular nucleus of the hypothalamus -mpdPVN and the release of ACTH and corticosterone) and, 3) glucose levels, as an indirect indicator of autonomic nervous system activity. Considering that the bone marrow is the site of origin from pluripotent cells from which all cells from the hematopoietic system are originated, and also that this system is vulnerable to the neuroendocrine control, we evaluated the effects of the treatment with CV on the hematopoiesis of stressed animals through 4) growing and differentiation of precursors to granulocytes and macrophages (CFU-GM); 5) colony stimulating activity from the serum (CSA); 6) quantification of population of mature and immature populations and 7) cell death. The interaction between stromal cells and hematopoietic progenitors in stressed mice was evaluated by the technique of long term bone marrow culture (LTBMC). In the culture we evaluated 8) the CFU-GM, levels of IL-1? / IL-6 and quantification of mature and immature population. The application of the stressor produced an increase in the expression of c-fos in all brain areas evaluated and in the expression of the gen to hnCRF in mpdPVN. Increased levels of ACTH, corticosterone and glucose found in stressed animals corroborate these findings. Reduced numbers of CFU-GM in the bone marrow and increase in plasma CSA, increased cell death in stem cell population (LSK) and decreased numbers of hematopoietic precursors and of mature cells was also observed in stressed group. In LTBMC we observed impairment on the functional activity from medullar stroma, which was observed by reduction of: CFU-GM, IL-1? / IL-6 levels and number of immature and mature cells. Treatment with CV partially prevented increase in c-fos activation caused by stress in the brain except in the magnocelular region from PVN. The more accentuated result from treatment with CV of stressed animals was observed in the expression of c-fos in the Raphe nucleus and in the expression of the gen to hnCRF in mpdPVN, where levels were similar to that observed in control group. All hematopoietic alterations observed after stress were prevented by the treatment with CV. Taken together, our results demonstrate that the protective effect of the treatment with CV on hematopoiesis of stressed animals may be due to a prevention of the neuronal activation in areas related to the decodification of the emotional stressful stimuli, reducing the amplitude of HPA axis and autonomic nervous system activity
Doutorado
Farmacologia
Doutora em Farmacologia
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Olum, Jimmy. "EXPRESSION OF THE THREE PROTO-ONCOGENES, EGFR, MEK AND B-RAF AFTER TRANSFECTION INTO HUMAN CELLS AND THEIR DETECTION BY MASS SPECTROMETRY." Thesis, Malmö högskola, Fakulteten för hälsa och samhälle (HS), 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:mau:diva-25604.
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When activated by growth factors and mitogens, cellular receptors like EGFR become activated and transmit signals from the cell surface through the MAPK pathway into the nucleus of the cell, in order to elicit a cellular response like growth, apoptosis, proliferation, and survival. Over activation of EGFR even without receptor binding and members of the MAPK pathways like BRAF and MEK1 have been reported in tumorigenesis. We used Gateway Technology to study the recombinant protein expressions from these 3 proto-oncogenes in HEK293T and HeLa cell lines, using pLenti 6.3/V5 as the expression system. BRAF was neither detected in the HeLa nor in the HEK 293T cell lines, by mass spectrometry after transfection. Only in the HeLa cell line was EGFR identified meanwhile MEK1 was identified both in HEK293T and HeLa cell lines. However, the results showed that the proteins identified were endogenous to these cell lines and therefore, no recombinant proteins were expressed using the pLenti 6.3/V5, as the expression system.
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Janet, Thierry. "Etudes sur le facteur de croissance fibroblastique basique (bfgf) : localisation, effets sur la proliferation cellulaire, mecanismes d'action et expression de proto-oncogenes." Université Louis Pasteur (Strasbourg) (1971-2008), 1991. http://www.theses.fr/1991STR13015.
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Nous avons montre que: 1) le bfgf non denature par la chaleur migre a une position inhabituelle (27 kd) par electrophorese en presence de sds, et conserve son activite biologique; 2) le bfgf est localise dans les neuroblastes de rat et de poulet en culture; 3) l'effet mitogenique du bfgf sur les astroblastes et les fibroblastes de meninges peut etre potentialise par le tgf1 (mitogene des fibroblastes) et par l'acide retinoique mitogene pour les deux types cellulaires. L'amp cyclique stimule sensiblement l'effet du bfgf dans certaines conditions. Dans les astroblastes, les oligodendroblastes (et les fibroblastes 3t3 et de meninges) l'effet mitogenique du bfgf ne passe pas principalement par une activation de la kinase c, ni par les proteines g sensibles a la toxine pertussique; 4) les proto-oncogenes c-fos et c-jun dont on croyait les expressions regulees de facon semblable, peuvent etre soumis a des regulations differentes. Le bfgf et les autres facteurs induisent c-fos, c-myc, c-jun et jun-b, mais a des taux differents. En particulier, le tgf1 et l'acide retinoique les induisent a un niveau a peine detectable; 5) le tgf1 est exprime dans les astroblastes et les oligodendrocytes sous deux formes d'arnm, mais sous une seule forme dans les neuroblastes. L'expression des arnm du tgf1 est stimulee par le tgf lui-meme et partiellement inhibee par l'ampc
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Dutenhefner, Simone Elisa. "Pesquisa da mutação T1799A do gene BRAF e a presença de metástases linfáticas no carcinoma papilífero da tireoide." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/5/5132/tde-24012012-163817/.
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Muitos pacientes submetidos à tireoidectomia por Carcinoma Papilífero da Tireoide (CPT) têm doença linfonodal subclínica no momento da cirurgia. A mutação BRAF T17799A (V600E) é um evento comum no CPT e alguns estudos demonstram correlação entre a mutação e características de maior agressividade tumoral, incluindo a presença de metástases linfonodais. O esvaziamento eletivo do compartimento central ganha aceitação, uma vez que alguns estudos evidenciam que a presença de metástases linfonodais aumenta o risco de recidiva e mortalidade. Devido ao grande potencial de complicações do esvaziamento do compartimento central, o objetivo deste trabalho foi avaliar a associação entre a presença da mutação BRAF T17799A (V600E), a presença de metástases linfonodais e fatores clínicos e histopatológicos de pior prognóstico. Métodos: 51 casos consecutivos de pacientes com CPT foram submetidos à tireoidectomia total e ao esvaziamento eletivo ou terapêutico do compartimento central. Em todos os pacientes foi pesquisada a mutação BRAF T17799A (V600E) no tecido tireoidiano com Carcinoma Papilífero de Tireoide. Resultados: Cinquenta e quatro por cento (54,9%) dos pacientes apresentaram metástases linfonodais. Seis pacientes apresentaram metástases laterais confirmadas por punção aspirativa por agulha fina no pré-operatório e 22 pacientes (43%) apresentaram metástases não detectadas no pré ou no intra operatório A mutação BRAF T17799A (V600E) foi encontrada em 15 pacientes portadores de CPT (29,4%). A presença da mutação não teve associação estatisticamente significante para sexo, idade, tamanho do tumor, extensão extratireoidiana, multicentricidade, embolização angiolinfática e metástases linfonodais. As metástases linfonodais se associaram à multifocalidade (p = 0,005) e invasão angiolinfática (p = 0,003) na análise univariada. Conclusão: A presença da mutação BRAF T17799A (V600E) não se associou à metástases linfonodais em nosso estudo. A multifocalidade e a detecção de invasão angiolinfática no CPT foram os fatores mais importantes na predição de metástases linfonodaisBackground: Many patients undergoing thyroidectomy for Papillary Thyroid Carcinoma (PTC) have subclinical node disease at the time of surgery. The BRAF T17799A (V600E) mutation is a common event in PTC and some studies have demonstrated a correlation between the mutation and aggressive characteristics including lymph node metastasis. Prophylactic Central Node Dissection (CND) is gaining acceptance in the treatment of PTC as studies have shown nodal disease increases local recurrence and may alter mortality. Given the potential complications of CND, the aim of this study was to determine the correlation among BRAF mutation, lymph node metastasis and clinical and histopathological factors of worse prognosis. Methods: A total of 51 consecutive cases of patients with PTC underwent total thyroidectomy and routine prophylactic (CND) or therapeutic neck dissection when metastases were found. All patients were tested for the BRAF mutation. Results: Overall, positive lymph nodes were found in Fifty four per cent9% of patients. Six patients had lateral metastases confirmed by fine needle aspirative cytology and 22 patients (43%) had occult metastases. The BRAF mutation was found in 15 patients (29.4%). BRAF was not correlated with sex, age, size of tumor, multifocality, extrathyroid extension or lymph node metastases. Lymph node metastases were correlated with multifocality (p = 0.005) and angiolymphatic invasion (p = 0.003) in univariate. Conclusions: The BRAF mutation was not correlated with lymph node metastases in our study. Multifocality and angiolymphatic invasion were important factors for predicting lymph node metastases
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MATSUYAMA, MUTSUSHI, R. KAZUHIKO UTSUMI, AKIRA MASUDA, MASAHIDE TAKAHASHI, WORAWIDH WAJJWALKUI, and YOSHIHISA SAKAI. "Expression of Proto-Oncogenes and Tumor Suppressor Genes in in vitro Cell Lines Derived from a Thymus, Thymoma, and Malignant Thymoma of Rats." Nagoya University School of Medicine, 1993. http://hdl.handle.net/2237/17542.
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Espindola, Marilia Bittencourt. "Expressão imunoistoquímica da proteína bcl-2 em metástases de melanoma cutâneo e relação com a sobrevida." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2007. http://hdl.handle.net/10183/11425.
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A morte celular programada (apoptose) tem sido implicada no desenvolvimento tumoral e no potencial metastático. O Bcl-2, um proto-oncogene inibidor da apoptose, vem sendo estudado em várias neoplasias incluindo o Melanoma Cutâneo (MC). Esse estudo avaliou a expressão imunoistoquímica da proteína bcl-2 em 35 metástases linfonodais regionais, 28 metástases subcutâneas e 17 metástases viscerais de MC e correlacionou com a sobrevida. O tempo médio de acompanhamento foi de 29,7 meses nas metástases linfonodais, 23,1 meses nas metástases subcutâneas e 22,9 meses nas metástases viscerais. A expressão de bcl-2 foi de 74,3% nas metástases linfonodais, 85,7% nas subcutâneas e 82,4% nas viscerais. Após análise uni e multivariada não houve correlação entre a positividade para bcl-2 e a sobrevida em nenhum dos tipos de metástases. Conclui-se que a avaliação imunoistoquímica da proteína bcl-2 isoladamente, em metástases, não é um marcador prognóstico no MC. Estudos posteriores das relações entre os membros da família BCL-2 poderão elucidar seu papel no desenvolvimento do Melanoma Cutâneo.
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DERAMAUDT, BERTRAND. "Role des proto-oncogenes fli-1 et erg dans l'expression du gene de l'heme oxygenase-1 humaine, caracterisation et etude de leurs sites de fixation a l'adn." Université Louis Pasteur (Strasbourg) (1971-2008), 2001. http://www.theses.fr/2001STR13089.
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L'heme oxygenase-1 (ho1) est une enzyme degradant l'heme en biliverdine, fer et monoxyde de carbone. On observe une surexpression de ho-1 dans de nombreuses pathologies. La regulation du gene ho-1 est encore mal connue, du fait des nombreux transactivateurs susceptibles de moduler son expression. Le promoteur du gene ho-1 presente de nombreux sites de liaison de differents facteurs de transcription dont certains pourraient etre reconnus par des membres de la famille des genes ets. Cette famille de genes code pour des facteurs de transcription se liant a l'adn sur des sequences caracterisees par un motif cur gga(a/t) et sont notamment impliques dans la regulation de genes intervenant dans l'angiogenese. De nombreuses etudes ont montre l'expression du gene ho-1 durant la cicatrisation, l'inflammation ou la croissance des muscles lisses, mais le role direct du gene ho-1 dans l'angiogenese est peu documente. Pour ce faire, nous avons utilise un modele d'angiogenese in vitro et montre que des cellules endotheliales transformees de maniere stable et surexprimant le gene humain ho-1, croissent plus vite et forment un reseau plus fourni que des cellules non transformees. Des essais ovocytaires nous ont permis de mettre en evidence une regulation negative du gene ho-1 entre les nucleotides-1163 et -1004 et la levee de cette inhibition par les proteines fli-1, erg et ets-1 au travers d'une region comprise entre les nucleotides -1412 et -1322. En utilisant une methode de selection et d'amplification d'oligonucleotides de sequences aleatoires un motif consensus de liaison a l'adn pour erg et fli-1 a ete determine et s'avere etre pratiquement identique pour ces deux proteines : aaccggaary. Les proteines erg et fli-1 n'ayant pas de reelle difference d'affinite et de sequence vis-a-vis de leur motif de liaison a l'adn, leur difference d'action pourrait s'expliquer par l'intervention de partenaires proteiques modulant leur affinite et leur activite.
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LEVY, RAFAEL. "Oncogenes, facteurs de croissance et cancers du sein : revue de la litterature, etude de la regulation du recepteur a l'igf 1 dans la lignee mcf-7 et des polymorphismes des proto-oncogenes c-ha-ras 1 et c-mos dans des cas familiaux." Lille 2, 1989. http://www.theses.fr/1989LIL2M316.
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Pimenta, Vanessa de Sousa Cruz. "Avaliação histoquímica e da expressão das proteínas p53 e c-KIT no mastocitoma canino." Universidade Federal de Goiás, 2012. http://repositorio.bc.ufg.br/tede/handle/tede/3576.
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
The objective of this study was to verify the pattern of staining by Bismarck Brown and evaluate the expression of p53 and c-KIT in neoplastic mast cells, quantifying the marking obtained by these antibodies using the Image J software and correlating the values found with histologic subtypes. Mast cell tumors are the most common skin neoplasms of dogs, is an excessive proliferation of neoplastic mast cells, of variable and unpredictable biological behavior, with great capacity of recurrences and metastasis. The lesions may be dermis or subcutaneous location and three grades are described. At the first, the tumor is well differentiated, moderately differentiated is the second and the third is little differentiated. Their cause is still unknown. One of the mechanisms proposed for the proliferation of mast cells is to change the nucleotide sequence of the c-KIT gene. The tumor suppressor gene TP53 is directly related to blockage of cell cycle and the protection is changed by mutations in the p53. Were reviewed 1242 protocols of histopathology exams obtained the archives of Laboratory of Animal Pathology/UFG, from January 2007 to April 2011, found 37 diagnosis of canine cutaneous mast cell tumor. Regarding the epidemiologic aspects the prevalence of 55.26% was noted in dogs in the age group 6-10 years, 63.16% female and 39.48% of the Boxer breed. The most common anatomic site was the hind-limb with 31.58% of records. Histologic evaluation and histochemistry using Hematoxylin - Eosin and Toluidine Blue allowed to classify the majority of mast cell tumors studied, although only 24.32% of the samples were identified using the color Bismarck Brown. The prevalence of Grade II was 43.24% of all cases The lowest value expressed for p53 and c-KIT, was in Grade II, the highest value in the Grade III and the highest average in grade I. Was concluded that the coloration of Bismarck Brown proved efficient to aid in the diagnostic accuracy of laboratory routine and utilization of antibodies anti p53 and anti c-kit promoted good immunostaining with important role to determine the prognosis of canine mast cell tumors.
O objetivo deste estudo foi verificar o padrão de coloração pelo Pardo de Bismarck e avaliar a expressão de p53 e c-KIT nos mastócitos neoplásicos, quantificando a marcação obtida por estes anticorpos através do software Image J e correlacionando os valores encontrados com os subtipos histológicos. O mastocitoma é a neoplasia cutânea mais frequente do cão, sendo uma proliferação excessiva de mastócitos neoplásicos, de comportamento biológico variável e imprevisível, com grande capacidade de recidivas e metástases. As lesões podem ser de localização dérmica a subcutânea e três graus são descritos. No primeiro, o tumor é bem diferenciado, no segundo é moderadamente diferenciado e no terceiro é pouco diferenciado. Sua causa ainda é desconhecida. Um dos mecanismos propostos para a proliferação de mastócitos é a alteração da sequência de nucleotídeos do gene c-KIT. O gene supressor de tumor TP53 está diretamente relacionado ao bloqueio do ciclo celular e a proteção é alterada por mutações da p53. Foram revisados 1242 protocolos de exames histopatológicos obtidos dos arquivos do Laboratório de Patologia Animal/UFG, do período de janeiro de 2007 a abril de 2011, encontrando 37 diagnósticos de mastocitoma cutâneo canino. Quanto aos aspectos epidemiológicos a prevalência notada foi de 55,26% de cães na faixa etária de 6-10 anos, 63,16% do gênero feminino e 39,48% da raça Boxer. A localização anatômica mais frequente foi o membro pélvico com 31,58% de registros. A avaliação histológica e histoquímica utilizando a Hematoxilina - Eosina e o Azul de Toluidina permitiu classificar a maioria dos mastocitomas estudados, porém 24,32% das amostras só foram identificadas com a utilização da coloração Pardo de Bismarck. A prevalência do Grau II foi de 43,24% do total dos casos. O menor valor expresso, por p53 e c-KIT, foi no Grau II, o maior valor no Grau III e a maior média no grau I. Foi possível concluir que a coloração Pardo de Bismarck mostrou-se eficiente para auxiliar na precisão dos diagnósticos da rotina laboratorial e a utilização dos anticorpos anti p53 e anti c-KIT promoveu boa imunomarcação, com papel importante na determinação do prognóstico do mastocitoma canino.
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Maino, Marcelo Marafon. "Expressão imunoistoquímica de CD117 no carcinoma epidermóide de esôfago." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2008. http://hdl.handle.net/10183/53132.
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Objetivo: Investigar a expressão imunoistoquímica de CD117 em um grupo de pacientes com carcinoma epidermóide de esôfago Pacientes e Métodos: Vinte e sete pacientes com carcinoma epidermóide de esôfago submetidos à ressecção cirúrgica no Hospital de Clínicas de Porto Alegre da Universidade Federal do Rio Grande do Sul foram avaliados para imunoreatividade do CD117. Como grupo controle, foram utilizadas biópsias de mucosa esofágica de dez indivíduos saudáveis. A avaliação imunoistoquímica dos tecidos foi realizada com anticorpo monoclonal anti-CD117 (DAKO). Resultados: Foram avaliados 21 (78%) homens e 6 (12%) mulheres com idade média de 58 anos (36 a 77). A maioria dos pacientes apresentava estadiamento TNM IIb ou III, e a sobrevida média foi de 21 meses (2 a 72). A reação imunoistoquímica em membrana produzida pelo anticorpo anti-CD117 foi considerada positiva em 4 dos 27 dos casos analisados (15%) . Conclusões: Esses achados sugerem que CD117 deve ser investigado como marcador para o carcinoma epidermóide de esôfago. Estudos adicionais são necessários em outras amostras populacionais, para melhor definir o papel do CD117 nesses tumores.Aim: To investigate the CD117 expression in specimens of patients with squamous cell carcinoma of the esophagus (SCCE). Methods: A pilot study was performed for CD177 immunoreactivity, using a monoclonal antibody against CD117 (DAKO), on 27 esophageal squamous cell carcinoma specimens from patients who underwent surgical resection at the Hospital de Clínicas de Porto Alegre University Hospital, Faculty of Medicine, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil. As a control group, specimens of esophageal mucosa obtained from 10 healthy subjects were also studied. Results: Twenty-one (78%) males and six (12%) females with median (sd) age of 58 (8) years, ranging from 36 to 77 years. Most of the patients were of TNM stage IIb or III and mean overall survival was 21 (2 to 72) months. Cytoplasmic membrane CD117 immunoreactivity was demonstrated in only 4 (15%) out of 27 tumors and in none of the controls (0%). Conclusions: These results suggest that the decreased expression of CD117 may be due to lack of control of the cell cycle in SCCE. Additional studies are needed to better define the role of the CD117 in such tumors.
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Borivoj, Sekulić. "Klinički i prognostički značaj ekspresije gena EVI1 u akutnoj mijeloidnoj leukemiji." Phd thesis, Univerzitet u Novom Sadu, Medicinski fakultet u Novom Sadu, 2015. https://www.cris.uns.ac.rs/record.jsf?recordId=95501&source=NDLTD&language=en.
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UVOD: Akutna mijeloidna leukemija (AML) predstavlja heterogenu grupu oboljenja u odnosu na morfologiju, citogenetiku, molekularnu genetiku, zbog čega se deli na različite kliničke i biološke entitete, sa različitim odgovorom na terapiju i ishodom lečenja. Humani EVI1 (ecotropic virus integration-1) gen ima ulogu multifunkcionalnog nuklearnog transkripcionog faktora, kako u normalnoj tako i u malignoj hematopoezi. Sve je više istraživanja koja ističu negativni prognostički značaj visoke ekspresije (overexpression) EVI1 gena u AML. CILJEVI: Ciljevi ovog istraživanja su da se ispita klinički i prognostički značaj ekspresije gena EVI1 u AML, kao i da se utvrdi povezanost visoke ekspresije gena EVI1 sa nalazima citogenetskog ispitivanja i molekularnim markerima: FLT3 mutacijom i nukleofozmin 1 (NPM1) mutacijom. MATERIJAL I METODE: Ovim prospektivnim istraživanjem je obuhvaćena grupa od 38 odraslih novodijagnostikovanih bolesnika sa de novo, non M3 AML, kod kojih je započeto standardno lečenje, a koji su dijagnostikovani i lečeni u Klinici za hematologiju Kliničkog centra Vojvodine u periodu od jula 2012. do marta 2014. Određivanje ekspresije gena EVI1 je vršeno pomoću real time kvantitativne PCR (qPCR) metode, tehnikom TaqMan, a relativna ekspresija EVI1 gena je određena primenom ΔΔCt metode. REZULTATI: Medijana starosti bolesnika pri postavljanju dijagnoze AML je bila 52 godine (23-80). Ustanovljena je statistički značajna razlika između ekspresije gena EVI1 kod zdravih osoba (kontrolna grupa) i obolelih od akutne mijeloidne leukemije (p=0.008). Računajući relativnu ekspresiju, 13,2 % bolesnika je imalo visoku ekspresiju (overexpression) gena EVI1. U odnosu na kliničke i laboratorijske karakteristike bolesnika (kao što su pol, starost, parametri krvne slike, nivo laktat dehidrogenaze, procenat blasta u perifernoj krvi i koštanoj srži, potom tip akutne mijeloidne leukemije, performans status, komorbiditetni indeks) nije ustanovljena statistički značajna razlika između bolesnika sa visokom ekspresijom EVI1 gena i ostalih bolesnika. Postoji statistički značajna povezanost visoke ekspresije EVI1 gena i nepostojanja NPM1 mutacije (p=0,031), kao i između visoke ekspresije EVI1 gena i prisustva monozomije 7 (p=0,047). Visoka ekspresija EVI1 gena je povezana sa kraćim preživaljvanjem bez dogaĎaja (p=0,004), kao i sa kraćim ukupnim preživljavanjem (p=0,025). ZAKLJUČCI: Postoji značajno povećana ekspresija gena EVI1 kod obolelih od AML u odnosu na zdrave kontrole. Visoka ekspresija EVI1 gena je faktor loše prognoze kod obolelih od akutne mijeloidne leukemije i u kombinaciji sa drugim prognostičkim markerima, doprinosi boljoj risk stratifikaciji ovih bolesnika.INTRODUCTION: Acute myeloid leukaemia (AML) represents a heterogenous group of diseases in terms of morphology, cytogenetics, molecular genetics, so it can be divided into distinct clinical and biological entities, with variable responsiveness to therapy and different treatment outcome. Human EVI1 (ecotropic virus integration-1) gene plays a role of multifunctional nuclear transcriptional factor, not only in normal, but also in malignant haematopoiesis. There are more and more investigations indicating high EVI1 expression (EVI1 overexpression) as a negative prognostic marker in AML. PURPOSES: The main goal of this investigation was to examine the clinical and prognostic significance of EVI1 expression in AML, as well as to investigate whether there was any association of EVI1 overexpression with cytogenetic abnormalities and other standard molecular prognostic factors, such as FLT3 mutation and nucleophosmin 1 (NPM1) mutation. PATIENTS AND METHODS: This prospective study included 38 adult newly diagnosed patients with de novo nonM3 AML, in whom a standard treatment was started at Clinic of Haematology, Clinical center of Vojvodina in the period from July 2012 to March 2014. EVI1 expression was analyzed by real-time quantitative polymerase chain reaction using TaqMan, and relative EVI1 expression was determined by ΔΔCt method. RESULTS: Median age of patients at diagnosis was 52 (aged 23-80). There has been determined statistically higher EVI1 expression in our AML patients than in healthy volunteers (control group) (p=0.008). The relative EVI1 overexpression was observed in 13.2% of the patients. No significant differences in clinical and laboratory patient data (including sex, age, whole blood counts, lactate dehydrogenase level, peripheral and bone marrow blast percentages, type of AML, performance status, comorbidity index) were observed between patients with high EVI1 expression and patients without high EVI1 expression. Our investigation revealed inverse correlation of high EVI1 expression and nucleophosmin 1 mutation (p=0,031). Also high EVI1 expression was significantly associated with monosomy 7 (p=0,047). Survival analysis revealed significantly inferior event free survival (p=0,004) and overall survival (p=0,025) for patients with high EVI1 expression compared to the other patients. CONCLUSION: EVI1 expression is significantly higher in AML patients compared to healthy controls. High EVI1 expression is a poor prognostic marker for patients with AML, and in combination with other well established prognostic markers, contributes to better risk stratification of these patients.
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Quattrochi, Brian J. "Subtle Controllers: MicroRNAs Drive Pancreatic Tumorigenesis and Progression: A Dissertation." eScholarship@UMMS, 2015. https://escholarship.umassmed.edu/gsbs_diss/776.
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Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal malignancies in the United States, with an average five-year survival rate of just 6.7%. One unifying aspect of PDAC is mutational activation of the KRAS oncogene, which occurs in over 90% of PDAC. Therefore, inhibiting KRAS function is likely an effective therapeutic strategy for this disease, and current research in our lab and others is focused on identifying downstream effectors of KRAS signaling that may be therapeutic targets. miRNAs are powerful regulators of gene expression that can behave as oncogenes or tumor suppressors. Dysregulation of miRNA expression is commonly observed in human tumors, including PDAC. The mir-17~92 cluster of miRNAs is an established oncogene in a variety of tumor contexts, and members of the mir-17~92 cluster are upregulated in PDAC, but their role has not been explored in vivo. This dissertation encompasses two studies exploring the role of miRNAs in pancreatic tumorigenesis. In Chapter II, I demonstrate that deletion of the mir-17~92 cluster impairs PDAC precursor lesion formation and maintenance, and correlates with reduced ERK signaling in these lesions. mir-17~92 deficient tumors and cell lines are also less invasive, which I attribute to the loss of the miR-19 family of miRNAs. In Chapter III, I find that Dicer heterozygosity inhibits PDAC metastasis, and that this phenotype is attributable to an increased sensitivity to anoikis. Ongoing experiments will determine whether shifts in particular miRNA signatures between cell lines can be attributed to this phenotype. Together these findings illustrate the importance of miRNA biogenesis, and the mir-17~92 cluster in particular, in supporting PDAC development and progression.
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Quattrochi, Brian J. "Subtle Controllers: MicroRNAs Drive Pancreatic Tumorigenesis and Progression: A Dissertation." eScholarship@UMMS, 2004. http://escholarship.umassmed.edu/gsbs_diss/776.
Full textAbstract:
Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal malignancies in the United States, with an average five-year survival rate of just 6.7%. One unifying aspect of PDAC is mutational activation of the KRAS oncogene, which occurs in over 90% of PDAC. Therefore, inhibiting KRAS function is likely an effective therapeutic strategy for this disease, and current research in our lab and others is focused on identifying downstream effectors of KRAS signaling that may be therapeutic targets. miRNAs are powerful regulators of gene expression that can behave as oncogenes or tumor suppressors. Dysregulation of miRNA expression is commonly observed in human tumors, including PDAC. The mir-17~92 cluster of miRNAs is an established oncogene in a variety of tumor contexts, and members of the mir-17~92 cluster are upregulated in PDAC, but their role has not been explored in vivo. This dissertation encompasses two studies exploring the role of miRNAs in pancreatic tumorigenesis. In Chapter II, I demonstrate that deletion of the mir-17~92 cluster impairs PDAC precursor lesion formation and maintenance, and correlates with reduced ERK signaling in these lesions. mir-17~92 deficient tumors and cell lines are also less invasive, which I attribute to the loss of the miR-19 family of miRNAs. In Chapter III, I find that Dicer heterozygosity inhibits PDAC metastasis, and that this phenotype is attributable to an increased sensitivity to anoikis. Ongoing experiments will determine whether shifts in particular miRNA signatures between cell lines can be attributed to this phenotype. Together these findings illustrate the importance of miRNA biogenesis, and the mir-17~92 cluster in particular, in supporting PDAC development and progression.
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Li, Ling. "Mechanisms Underlying Apoptosis Inhibition and Transcription Repression by Ski." Connect to text online, 2004. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=case1118235807.
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Bessa, Tiphany Coralie de. "Mecanismo associados à perda da regulação da nox1 NADPH oxidase pela dissulfeto isomerase proteica em células com ativação sustentada da via ras." Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/5/5131/tde-03072018-090616/.
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Dissulfeto isomerase proteica como a PDIA1 tem sido implicada na progressão do câncer, porém os mecanismos envolvidos ainda não foram claramente identificados. Previamente, nós demonstramos um importante efeito da PDIA1 induzindo a superexpressão da Nox1 NADPH oxidase, associada à geração de espécie reativas de oxigênio (ROS). Uma vez que a perda na regulação de ROS envolve o crescimento tumoral, nós propusemos que a PDIA1 atua como um mecanismo regulador proximal na produção de ROS em tumores. No presente estudo, nós focamos no câncer colorretal (CRC) com distintos efeitos na ativação de KRas. Resultados provenientes de bancos de dados de RNAsec e validação direta, indicam um significante aumento na expressão de PDIA1 em CRC com alta ativação constitutiva da Kras (HCT116) vs. ativação intermediária (HKE3) ou basal (Caco2). A PDIA1 sustenta a produção de superóxido dependente da Nox1 em CRC; entretanto, observamos pela primeira vez uma ação dupla da PDIA1 correlacionada ao nível de ativação da Ras: em células Caco2 e HKE3, experimentos de perda de função indicam que o PDIA1 sustenta a produção de superóxido dependente de Nox1; no entanto, em células HCT116, PDIA1 limita a produção de superóxido pela Nox1. Este comportamento da PDIA1 é associado ao aumento da expressão / atividade da Rac1. A transfecção do mutante constitutivamente ativo Rac1G12V em células HKE3 faz com que a PDIA1 se torne restritiva a produção de superóxido dependente de Nox1, paralelamente, em células HCT116 tratadas com inibidor da Rac1, PDIA1 se torna favorável à produção de superóxido. Um screening em importantes vias de sinalização celular em HKE3 mostrou que a perda de função da PDIA1 promove inativação da GSK3? em paralelo à diminuicão da ativacção de Stat3; em HCT116 em estado basal, GSK3beta é inativada enquanto Stat3 está ativa, já o silenciamento da PDIA1 não resulta em nenhum efeito adicional. As implicações funcionais do silenciamento da PDIA1 incluíram uma diminuição da proliferação e migração celular em HKE3, não detectável em HCT116. Além disso, a PDIA1 parece sustentar a transição epitélio-mesenquimal (EMT), uma vez que após o silenciamento da PDIA1, observamos um aumento da expressão da E-caderina em HKE3 e uma diminuição em HCT116. Assim, a superativação da Ras se associa a uma alteração no padrão de regulação da Nox1 pela PDIA1. A supressão do efeito regulador da PDIA1 pela Kras é provavelmente devido a uma ativação sustentada da Rac1. Portanto, PDIA1 pode exercer um papel redox-dependente adaptativo crucial relacionado à progressão tumoralProtein disulfide isomerases such as PDIA1 have been implicated in cancer progression, but the underlying mechanisms are unclear. We showed previously important PDIA1 effects enabling vascular Nox1 NADPH oxidase expression and associated generation of reactive oxygen species (ROS). Since deregulated ROS production underlies tumor growth, we proposed that PDIA1 acts as an upstream regulatory mechanism of tumor-associated ROS production. We focused on colorectal cancer (CRC) with distinct levels of KRas activation. Our results from RNAseq databanks and direct validation indicate significant increase in PDIA1 expression in CRC with constitutive high (HCT116) vs. moderate (HKE3) or basal (e.g. Caco2) Ras activity. PDIA1 supported Nox1-dependent superoxide production in CRC; however, we observed for the first time a dual effect correlated with Ras level activity: in Caco2 and HKE3 cells, loss-of-function experiments indicate that PDIA1 sustains Nox1-dependent superoxide production; however, in HCT116 cells, PDIA1 restricted Nox1-dependent superoxide production. This PDIA1 behavior in HCT116 is associated with increased Rac1 expression/activity. Transfection of Rac1G12V active mutant into HKE3 cells induced PDIA1 to become restrictive of Nox1-dependent superoxide; accordingly, in HCT116 cells treated with Rac1 inhibitor, PDIA1 became supportive of superoxide production. Screening of cell signaling routes affected by PDIA1 silencing showed induced GSK3beta inactivation and parallel decrease of active Stat3 in HKE3 cells; in baseline HCT116 cells, GSK3beta was inactivated and Stat3 active, whereas PDIA1 silencing had no further effect. Functional implications of PDIA1 silencing included a decrease of cell proliferation and migration in HKE3, not detectable in HCT116 cells. Also, PDIA1 may support epithelial-mesenchymal transition (EMT), since after PDIA1 silencing, E-cadherin expression increased in HKE3 and decreased in HCT116. Thus, Ras overaction associates with a switched in PDIA1 pattern regulation of Nox1. Ras-induced PDIA1 bypass may involve direct Rac1 activation. Therefore, PDIA1 may be a crucial regulator of redox-dependent adaptive processes related to cancer progression
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LE, ROUZIC ERWANN. "Activation insertionnelle du proto-oncogene c-myb dans une lignee de lymphome transformee par le virus de marek (mdv) : un exemple de cooperation des oncogenes c-myb et meq dans l'induction et le maintien du phenotype transforme ?" Paris 6, 1997. http://www.theses.fr/1997PA066437.
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Le proto-oncogene c-myb est l'homologue cellulaire de l'oncogene transformant v-myb porte par deux retrovirus leucemogenes, amv et e26. Le gene c-myb code un facteur de transcription et est preferentiellement exprime dans les cellules hematopoietiques immatures. L'alteration de son expression ou/et de son activite est etroitement associee a des leucemies chez les vertebres. Le virus de la maladie de marek (mdv) appartient a la famille des virus herpes et induit des lymphomes de type t chez les aviaires. De nombreux travaux ont permis de proposer un role pour les genes associes au fragment bamhi-h, le gene homologue icp4 et le gene meq apparente aux oncogenes fos/jun dans les mecanismes de la transformation tumorale. Cependant aucun de ces facteurs ne presente de proprietes oncogeniques intrinseques. Nous avons caracterise au laboratoire une activation insertionnelle d'un retrovirus rav-1 dans la region 5' du locus c-myb dans un lignee t (t9), isolee d'un lymphome induit par le mdv. Cette insertion resulte en une forte expression d'une proteine c-myb depourvue des acides amines correspondant aux exons 1 a 3. Cette alteration est similaire a la deletion amino-terminale du produit de v-myb de l'amv. Les analyses genomiques du mdv n'ont pas permis de mettre en evidence la presence de la region bamhi-h dans cette lignee. La perte de ce fragment rappelle les profils genomiques des souches oncogeniques attenuees. La region contenant le gene homologue icp4 est egalement absente. Toutefois, ces regions sont presentes dans une lignee anterieure (t9-85) a celle couramment utilisee au laboratoire (t9-94). Le gene meq est toujours exprime dans ces cellules. L'inoculation de la lignee t9-94 a des poulets a confirme les alterations genomiques du mdv. Cependant, l'inoculation de la lignee t9-85 induit le developpement de tumeurs plus efficacement qu'une lignee temoin. Le mdv present dans la lignee t9-85 est donc toujours tumorigene. La difference de pathogenicite observee pourrait refleter une proliferation accrue des cellules t9-85 in vivo, conferee par l'activation de c-myb. Ces resultats nous ont suggere un modele de la transformation cellulaire en deux etapes. Le mdv aurait induit la transformation cellulaire tandis que l'activation de c-myb et l'expression de meq coopereraient pour maintenir le phenotype transforme. Ces travaux ont mis pour la premiere fois en evidence l'activation de c-myb dans une lignee t aviaire.
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HUNTS, JOHN HOWARD. "ANALYSIS OF THE HUMAN EPIDERMAL GROWTH FACTOR RECEPTOR GENE AS A PROTO-ONCOGENE (SQUAMOUS CELL CARCINOMA)." Diss., The University of Arizona, 1986. http://hdl.handle.net/10150/183865.
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The epidermal growth factor receptor (EGFR) gene was examined as a proto-oncogene. Initially, the cellular homolog of the retroviral oncogene erb-B was shown to be localized to the same region of human chromosome 7 as the EGFR gene, giving support to the idea that these genes are closely related. To determine how some cells can over-express the EGFR gene, somatic cell hybrids constructed between a human EGFR-overproducing cell line and a mouse EGFR-deficient cell line were examined. EGFR gene amplification was observed in one of these hybrids along with EGFR gene rearrangement, which is thought to generate an abnormal mRNA. When examining tumor tissue, the EGF receptor (EGFR) was found to be elevated relative to normal adjacent tissue in 9 out of 15 primary human squamous cell carcinomas. Only 2 of these 9 tumors had EGFR gene amplification, suggesting alternative mechanisms potentially involved in increasing EGFR levels. Because placental tissue expresses high levels of EGFR, it was thought that some tissues may normally possess high EGFR levels and that some cancerous tissues inappropriately mimic the mechanisms active in the placenta. From the examination of several tissue samples, EGFR mRNA levels and EGFR protein half-life were also postulated as contributing factors regulating the EGFR levels. The EGFR gene was implicated as a proto-oncogene by evidence which suggests that either a qualitative or a quantitative change in the receptor may be involved in tumorigenesis. Finally, to begin to better understand what role EGFR hyperproduction plays in tumorigenesis, a DNA vector was constructed which produces antisense-RNA for inhibiting EGFR expression.
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Santos, Marcelo Augusto Cortina Gonçalves dos. "Detecção e rastreamento de mutações no proto-oncogene RET em pacientes com neoplasia endócrina múltipla tipo 2 por meio de eletroforese em gel sensível à conformação." Universidade de São Paulo, 2007. http://www.teses.usp.br/teses/disponiveis/5/5135/tde-06062007-170334/.
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A neoplasia endócrina múltipla tipo 2 (NEM-2) é uma síndrome tumoral herdada por mutações germinativas no proto-oncogene RET (RET) e transmitida por herança autossômica dominante. Atualmente, a indicação de tireoidectomia total preventiva é recomendada a indivíduos portadores de mutações no RET. Analisamos a aplicação do método Eletroforese em Gel Sensível à Conformação (CSGE) no rastreamento de mutações hot-spots do RET. Sete famílias com NEM-2 foram rastreadas pelo CSGE, seqüenciamento gênico e análise do Polimorfismo Conformacional de Cadeia Simples (SSCP). Usando o CSGE e SSCP, identificamos cinco das seis (83,3%) mutações verificadas pelo seqüenciamento: Cys620Arg, Cys634Arg, Cys634Tyr, Val648Ile e Met918Thr. Foram analisados 128 amplicons englobando mutações hot-spots do RET e 116 dentre 128 (90.6%) concordaram com o seqüenciamento genético. Os polimorfismos 691 e 769 também foram documentados pelo CSGE e SSCP. Os dados obtidos por CSGE e SSCP foram totalmente (100%) concordantes. O CSGE revelou ser metodologia sensível, rápida, fácil de ser executada e de baixo custo na detecção de mutações nos códons 620, 634, 648, e 918, as quais constituem grande maioria (~95%) dos pacientes com NEM-2. Quanto à mutação Val804Met (prevalência na população inferior a 3%), o método necessita ser otimizado. Concluímos que o CSGE é uma metodologia efetiva para o rastreamento de mutações que mais freqüentemente ocorrem no RET como causadora de NEM-2.Multiple endocrine neoplasia type 2 (MEN2) is an autosomal dominant inherited tumor syndrome caused by activating germline mutations in RET proto-oncogene (RET). Presently, the prophylactic total thyroidectomy is recommended to all RET mutations carriers. Here we tested the Conformation Sensitive Gel Electrophoresis (CSGE) as a screening method for the RET hot-spot mutations. Seven MEN2 families were studied by CSGE, as well as by Single Strand Conformational Polymorphism (SSCP) and direct sequencing analysis. Using CSGE and SSCP, we were able to detect five out of the six (83.3%) RET mutations verified by direct sequencing analysis: Cys620Arg, Cys634Arg, Cys634Tyr, Val648Ile and Met918Thr. RET polymorphisms 691 and 769 were verified by CSGE and SSCP. In our sample, data obtained using CSGE were fully concordant (100%) with SSCP findings. Thus, CSGE showed to be a sensitive, fast, low-cost, and ease procedure to detect RET mutations in codons 620, 634, 648, and 918 which are reported as the most prevalent RET variants (~95%) in large MEN2 series. As to the Val804Met mutation (prevalence in the population lower than 3%), this method still needs to be optimized. We concluded that CSGE is an effective screening method for the most frequent RET hot-spot disease-causing mutations.
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Schiavi, Susan C. "MYC and E1A Oncogenes Alter the Response of PC12 Cells to Nerve Growth Factor and Block Differentiation: A Thesis." eScholarship@UMMS, 1988. https://escholarship.umassmed.edu/gsbs_diss/259.
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PC12 rat pheochromocytoma cells respond to nerve growth factor (NGF) by neuronal differentiation and partial growth arrest. Mouse c-myc and adenovirus E1A genes were introduced into PC12 cells to study the influence of these nuclear oncogenes on neuronal differentiation. Expression of myc and E1A blocked morphological differentiation and caused NGF to stimulate rather than inhibit cell proliferation. NGF binding to cell surface receptors, activation of ribosomal S6 kinase, and ornithine decarboxylase induction were similar in myc and E1A expressing clones compared with wild-type PC12 cells, suggesting that changes in the cellular response to NGF were at a post-receptor level. The ability of myc and E1A expression to block the transcription-dependent induction of microtubule associated proteins by NGF further suggested that these genes may inhibit differentiation by interfering with NGP's ability to regulate transcription. These results illustrate that NGF can promote either growth or differentiation of PC12 cells, and that myc or E1A alter the phenotypic responses to growth factors.
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WENG, FEN-HUA, and 翁芬華. "Expression of oncogenes (Proto-oncogenes) in human esophageal carcinoma cells." Thesis, 1992. http://ndltd.ncl.edu.tw/handle/14401428271251077373.
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CAI, TING-FEN, and 蔡亭芬. "Structure and expression of oncogenes (proto-oncogenes) in human hepatoma cell lines." Thesis, 1987. http://ndltd.ncl.edu.tw/handle/24442076299052261461.
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QIU, XIAN-TAI, and 邱顯泰. "The effects of IGF-I on the transcription of nuclear proto-oncogenes." Thesis, 1992. http://ndltd.ncl.edu.tw/handle/70721849386099690157.
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ZHANG, JUN-ZHE, and 張俊哲. "Expression of several proto-oncogenes and homeogenes in bull frog liver during metamorphosis." Thesis, 1992. http://ndltd.ncl.edu.tw/handle/06803186295645365746.
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Chang, Hung-Chi, and 張宏基. "Expression of nitric oxide synthase and nuclear proto-oncogenes in Angiostrongylus cantonensis-infected mice." Thesis, 1999. http://ndltd.ncl.edu.tw/handle/25048612309578120665.
Full textAbstract:
碩士中山醫學院
生物化學研究所
87
The purpose of this study was to investigate the pathogenic mechanism of Angiostrongylus cantonensis (A.c) male ICR mice were used as experimental animals and divided into experimental group and control group. Each mouse in the experimented group was fed with 30 third stage larvae of Angiostrongylus Cantonensis certain number of animals were sacritied each week after infection till the sixth week to collect blood and brain tissues which were further analyzed by SDS-PAGE and Western blot to identify the change in protein expression the pathogenic mechanism was also dissected from the analysis of py-54、MEK、ERK and iNOS levels in frozen sections histonic changes were. Observed by HE. The result showed no significant express of stains py-54、MEK、ERK and iNOS levels in both experimental and control groups. First week however on the 2nd 3rd and 4th weeks the protein levels of these signal transductors in experimental mice in creased significantly, followed by a gradual decline on the 5th and 6 weeks. In addition, HE stain showed inflammatory reaction that was cousistant with the results of Western blot and immuno cyto chemistry. According to the result described above. It indicate that A.c infection induces the expression of some signal transductors that night leads to the inflamation of tissues.
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Gebler, Christina. "Developing the CRISPR/Cas-system for Inactivation of Proto-oncogenes in Human Cancer Cells." Doctoral thesis, 2017. https://tud.qucosa.de/id/qucosa%3A31120.
Full textAbstract:
Numerous mutations contribute to tumorigenesis of cancer cells. For most of them it remains unclear whether they are driver or passenger mutations. A classic knock-out to study their function in cancer cells used to take a lot of effort. The CRISPR/Cas-system can be used as a programmable “genome editing” tool. In this work, oncogenes have been inactivated with the CRISPR/Cas-system.
Considering off-targets, Streptococcus pyogenes sgRNAs can be designed for 88% of the known cancer mutations. The activity of 15 sgRNAs, targeting 13 mutations in proto-oncogenes (deletions, insertions and point mutations), has been tested with a RFP-GFP-reporter plasmid. For 13 sgRNAs, activity prediction scores correlated with measured activity. Furthermore, sgRNAs have shown preferential binding to mutated versions of targeted proto-oncogene sequence and did not induce double strand breaks in the wild type sequence. For 10 sgRNAs, the activity against their target sequence has been more than 4 times higher than against the wild type sequence. Most of those sgRNAs target insertions or deletions and fewer target point mutations.
Permanent knock-out of three mutated proto-oncogenes NPM1, BRAF and PIK3CA has been achieved with a lentiviral expression of CRISPR/Cas. Accordingly, effects on proliferation and phenotype have been studied.
Knock-out of NPM1 c.863_864insTCTG mutation has been studied in heterozygous mutated OCI AML3 cell line. Proliferation was strongly inhibited by the corresponding sgRNA. Cells arrested in G0/1-phase of cell cycle (77%) compared to control cells (56%), although no difference was observed for sub-G1 phase, indicating no induction of apoptosis. Cells treated with NPM1 sgRNA had 88% reduced expression of NPM1 c.863_864insTCTG mRNA as well as less cytoplasmic localization of nucleophosmin as assessed by immunostaining. The activity of sgRNA has been confirmed by deep sequencing, showing a shift of wild type to mutated allele ratio from 51:49 to 68:32. This effect was enhanced by the additional treatment with the NHEJ inhibitor SCR7.
A BRAFV600E sgRNA was tested in homozygously mutated melanoma cell lines A-375 and SK MEL-28. No differences were detected in comparison to controls. However, in the CRC cell line RKO, heterozygous for BRAFV600E and PIK3CAH1047R, proliferation was inhibited through sgRNAs against either BRAF or PIK3CA. A combination of both had no synergistic effect on proliferation. Activity and specificity of the sgRNA targeting BRAF were confirmed by deep sequencing, while the PIK3CA sgRNA showed a moderate induction of double strand breaks also in the wild type allele. The relation of wild type to mutated allele of BRAF was changed from 32:68 before treatment to 51:49 afterwards. This effect can be explained by a “re mutation” to the wild type after DSB via HDR with wild type sister chromatid as template. This effect was observed for PIK3CA sgRNA to a lesser extent.
In conclusion, these results show the applicability of the CRISPR/Cas-system for the inactivation of mutated proto-oncogenes.:List of tables III
List of figures IV
List of abbreviations V
1 Introduction 1
1.1 Cancer 1
1.2 Oncogenes 2
1.2.1 Role in cancer 2
1.2.2 Targeted therapies 3
1.2.3 NPM1 5
1.2.4 BRAF 6
1.2.5 PIK3CA 7
1.3 CRISPR/Cas-system 7
1.4 Aim and motivation 10
2 Material and Methods 11
2.1 Design of sgRNAs 11
2.2 Plasmids 11
2.3 Cell culture 12
2.4 FACS analysis 14
2.5 T7 assay 14
2.6 Cell cycle analysis 15
2.7 Immunostaining 15
2.8 Apoptosis assay 16
2.9 Quantification of mutant NPM1 transcripts 16
2.10 Deep sequencing 16
2.11 Statistical Analysis 19
3 Results 20
3.1 Design of sgRNAs targeting oncogenes 20
3.2 Evaluation of sgRNA efficacy and selectivity 23
3.3 Effects of oncogene knock-out in cancer cell lines 27
3.3.1 Targeting NPM1 in AML cells 27
3.3.2 Targeting BRAF in melanoma cells 30
3.3.3 Targeting BRAF and PIK3CA in colorectal carcinoma cells 31
4 Discussion 37
4.1 The design of sgRNAs is possible for most cancer mutations 37
4.2 sgRNAs targeting oncogenes have to be tested 37
4.3 Oncogenes can be knocked out with the CRISPR/Cas-system 37
4.3.1 NPM1 in AML cells 37
4.3.2 BRAF in melanoma cells 38
4.3.3 BRAF and PIK3CA in CRC cells 38
4.4 Advantages and disadvantages to target oncogenes with the CRISPR/Cas-system 40
4.5 Concluding remarks 41
5 Original Article 43
6 Summary 47
7 Zusammenfassung 49
List of references 51
Appendix VIIIIn Krebszellen tritt eine Vielzahl von Mutationen auf. Für den Großteil der Mutationen ist ungeklärt, ob es sich um krebsverursachende oder passagere Mutationen handelt. Ein gezieltes Ausschalten (Knock-out) dieser Gene zur Untersuchung ihrer Funktion in Krebszellen war bisher mit großem Aufwand verbunden. Das CRISPR/Cas-System lässt sich als programmierbares „Genome-editing“ Werkzeug einsetzen und wurde in der vorliegenden Arbeit verwendet, um gezielt mutierte Protoonkogene zu inaktivieren. Für 88% der bekannten, in Krebszellen auftretenden Mutationen lassen sich, unter Berücksichtigung von off-targets, Streptococcus pyogenes sgRNAs entwerfen. Mit Hilfe eines RFP-GFP-Reporter-Plasmides wurde die Aktivität von 15 sgRNAs gegen 13 Mutationen (Deletionen, Insertionen und Punktmutationen) in Protoonkogenen überprüft. Für 13 der sgRNAs zeigte sich eine Aktivität, die mit der Vorhersage durch den Algorithmus korrelierte. Außerdem wurde gezeigt, dass die sgRNAs spezifisch genug binden, um zwar bei der mutierten Sequenz eines Protoonkogens, jedoch nicht bei der Wildtyp-Sequenz Doppelstrangbrüche zu erzeugen. Unter den sgRNAs waren 10 mit mehr als 4-fach höherer Aktivität bei komplett übereinstimmender Zielsequenz gegenüber der Wildtyp-Sequenz. Diese spezifischen sgRNAs waren vor allem gegen Insertions- oder Deletionsmutationen gerichtet, einige auch gegen Punktmutationen. Durch permanente, lentivirale Expression von CRISPR/Cas wurden die Effekte eines Knock-out von drei mutierten Protoonkogenen, NPM1, BRAF und PIK3CA, auf das Wachstum und phänotypische Aspekte humaner Krebszelllinien untersucht. Ein Knock-out der NPM1 c.863_864insTCTG Mutation wurde in heterozygot mutierten OCI AML3 Zellen untersucht, es zeigte sich eine starke Proliferationshemmung. In der Zellzyklusanalyse trat ein G0/1-Arrest dieser Zellen (77%) im Vergleich mit Kontroll-Zellen (56%) auf, jedoch keine Unterschiede in der sub-G1-Analyse, sodass nicht von einer vermehrten Apoptose auszugehen ist. Die mit sgRNA behandelten OCI-AML3 Zellen zeigten sowohl eine um 88% verminderte NPM1 c.863_864insTCTG mRNA-Expression als auch verminderte zytoplasmatische Sublokalisation des Nucleophosmins in der Immunfärbung. Die hohe Aktivität der gRNA gegen mutiertes NPM1 wurde durch Deep Sequencing bestätigt, außerdem hat sich das Verhältnis vom Wildtyp- zu mutiertem Allel von 51:49 zu 68:32 verschoben. Dieser Effekt wurde durch Zugabe des NHEJ-Hemmstoffes SCR7 noch verstärkt. Die sgRNA gegen BRAFV600E wurde in den homozygot mutierten Melanom-Zelllinien A-375 und SK-MEL-28 getestet. Bei Proliferationsversuchen zeigten sich keine Unterschiede im Vergleich zu Kontrollzellen. In der kolorektalen Krebszelllinie RKO, die heterozygot BRAFV600E und PIK3CAH1047R ist, zeigte sich bei der Testung von sgRNAs gegen BRAF, PIK3CA und Kombination beider sgRNAs eine Wachstumshemmung. Jedoch lag kein synergistischer Effekt bei sgRNA-Kombination vor. Zudem bestätigten sich Aktivität und Spezifität der sgRNA gegen BRAF im Deep Sequencing, während die sgRNA gegen PIK3CA in mäßigem Umfang Doppelstrangbrüche im Wildtyp-Allel verursachte. Das Verhältnis vom Wildtyp- zu mutiertem BRAF Allel verschob sich von 32:68 ohne sgRNA zu 51:49 nach sgRNA-Behandlung. Eine mögliche Erklärung dieser Beobachtung ist die Rückmutation zum Wildtyp-Allel nach Doppelstrangbruch mit Hilfe homologer Rekombination durch das Wildtyp-Schwesterchromatid. Für PIK3CA konnte dieser Effekt in schwächerem Ausmaß ebenfalls beobachtet werden. Zusammengefasst zeigen diese Ergebnisse, dass das CRISPR/Cas-System zur Inaktivierung mutierter Protoonkogene genutzt werden kann.:List of tables III List of figures IV List of abbreviations V 1 Introduction 1 1.1 Cancer 1 1.2 Oncogenes 2 1.2.1 Role in cancer 2 1.2.2 Targeted therapies 3 1.2.3 NPM1 5 1.2.4 BRAF 6 1.2.5 PIK3CA 7 1.3 CRISPR/Cas-system 7 1.4 Aim and motivation 10 2 Material and Methods 11 2.1 Design of sgRNAs 11 2.2 Plasmids 11 2.3 Cell culture 12 2.4 FACS analysis 14 2.5 T7 assay 14 2.6 Cell cycle analysis 15 2.7 Immunostaining 15 2.8 Apoptosis assay 16 2.9 Quantification of mutant NPM1 transcripts 16 2.10 Deep sequencing 16 2.11 Statistical Analysis 19 3 Results 20 3.1 Design of sgRNAs targeting oncogenes 20 3.2 Evaluation of sgRNA efficacy and selectivity 23 3.3 Effects of oncogene knock-out in cancer cell lines 27 3.3.1 Targeting NPM1 in AML cells 27 3.3.2 Targeting BRAF in melanoma cells 30 3.3.3 Targeting BRAF and PIK3CA in colorectal carcinoma cells 31 4 Discussion 37 4.1 The design of sgRNAs is possible for most cancer mutations 37 4.2 sgRNAs targeting oncogenes have to be tested 37 4.3 Oncogenes can be knocked out with the CRISPR/Cas-system 37 4.3.1 NPM1 in AML cells 37 4.3.2 BRAF in melanoma cells 38 4.3.3 BRAF and PIK3CA in CRC cells 38 4.4 Advantages and disadvantages to target oncogenes with the CRISPR/Cas-system 40 4.5 Concluding remarks 41 5 Original Article 43 6 Summary 47 7 Zusammenfassung 49 List of references 51 Appendix VIII