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1
Fonseca, Aline Simoneti. "Assinatura de mRNA entre adenoma e adenocarcinoma colorretal." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/17/17135/tde-18122014-095654/.
Full textAbstract:
O câncer colorretal está entre as principais neoplasias malignas sendo a quarta causa de morte por câncer no mundo e a terceira no Brasil. Mutações nos genes APC, DCC, K-RAS e TP53 foram originalmente associadas com a progressão do câncer colorretal (CCR) esporádico. Entretanto, estudos de genoma completo e exoma têm revelado outros genes relacionados com o CCR. Como consequência dessas mutações, um conjunto de genes alteram sua expressão modulando vias gênicas em cada estágio da progressão tumoral. Nesse sentido, há um grande esforço para definir assinaturas gênicas que auxiliem na classificação dos tumores quanto ao diagnóstico e prognóstico dos pacientes. Portanto, o objetivo deste projeto foi analisar a expressão gênica em escala genômica de amostras de adenoma e adenocarcinoma colorretal visando identificar novos marcadores genéticos ligados a transição adenoma-adenocarcinoma. Para isso, dez amostras pareadas de adenoma e adenocarcinoma do mesmo paciente foram submetidas à análise de expressão gênica pela técnica de microarranjos. Análises de bioinformática, revelaram uma assinatura de 689 genes diferencialmente expressos (fold-change>2, p<0.05), que permitiram a classificação genética entre o adenoma e o adenocarcinoma. Oitos genes (IL6, IL8, OSM, SFRP4, ETV4, ESM1, SIM2 e RETNLB) foram escolhidos para validação com base na sua função e valor de expressão no tecido tumoral. A análise in silico dos genes hiperexpressos realizada no programa MetaCore (análise de dados e vias gênicas) destacou diversas vias gênicas ligadas à tumorigênese, incluindo as de adesão celular e Transição Epitélio-Mesenquimal (TEM), importantes na fase avaçada da progressão tumoral. O gene ETV4 foi selecionado para realização dos ensaios funcionais em virtude de seus altos níveis de expressão nas dez amostras de adenocarcinoma e participação nos mecanismos de proliferação celular e no TEM. Ensaios in vitro de siRNA para o gene ETV4 resultou na diminuição da proliferação celular e no potencial clonogênico da linhagem HT29. Adicionalmente, foram investigadas mutações nos genes APC, K-RAS e TP53, nas amostras pareadas de adenoma, adenocarcinoma, tecido normal e sangue periférico dos dez pacientes. Todos os pacientes apresentaram mutação germinativas nos três genes. No entanto, apenas os genes K-RAS (40%) e TP53 (30%) apresentaram mutações somáticas e patogênicas, exclusivamente nos adenocarcinomas. Esses resultados demonstraram que, na nossa coorte, mutações nos genes TP53 e K-RAS podem estar contribuindo para a progressão em uma parcela do câncer colorretal do tipo esporádico. Em resumo, o presente estudo aponta que o gene ETV4 pode contribuir para ativar o mecanismo de proliferação celular em adenocarcinoma colorretal. Além disso, o estudo demonstra a importância da combinação da análise de mutação com o perfil de expressão para melhor compreensão da base molecular do câncer colorretal.Colorectal cancer is among the main malignant neoplasia, it is the fourth leading cause of death in the world and the third in Brazil. Mutations in APC, DCC, KRAS and TP53 genes have been originally associated with the progression of sporadic colorectal cancer (CRC). However genome wide and exome studies have revealed other genes related to CRC. As a consequence of these mutations, a set of genes alters their expression modulating gene pathways in every stage of tumor progression. In this regard, there is great effort to define gene signatures that help to classify tumors in relation to patients diagnosis and prognosis. Therefore, the objective of this project was to analyze gene expression in genomic scale of colorectal adenoma and adenocarcinoma samples aiming to identify new genetic markers linked to adenoma- adenocarcinoma transition. For this purpose, ten paired adenoma and adenocarcinoma samples of the same patient were subjected to gene expression analysis by microarrays technique. Bioinformatics analyses revealed a signature of 689 genes differentially expressed (fold-change>2, p<0.05), which allowed the genetic classification between adenoma and adenocarcinoma. Eight genes (IL6, IL8, OSM, SFRP4, ETV4, ESM1, SIM2 and RETNLB) were chosen for validation based on their function and expression value in tumor tissue. In silico analysis of hyperexpressed genes, done in the program MetaCore (data analysis and gene pathways), highlighted diverse gene pathways linked to tumorigenesis, including the ones of cell adhesion and Epithelial-Mesenchymal Transition (EMT), important in the advanced phase of tumor progression. ETV4 gene was selected for functional assays due to its high expression levels in the ten samples of adenocarcinoma and due to its participation in cell proliferation mechanisms and in EMT. In vitro siRNA assays for ETV4 gene resulted in the decrease of cell proliferation and in the clonogenic potential of HT29 line. In addition, mutations in APC, KRAS and TP53 genes were investigated in paired samples of adenoma, adenocarcinoma, normal tissue, and peripheral blood from ten patients. All patients showed germline mutations in the three genes. However, only KRAS (40%) and TP53 (30%) genes showed somatic and pathogenic mutations, exclusively in adenocarcinomas. These results demonstrated that, in our cohort, mutations in TP53 and KRAS genes might be contributing to progression in a portion of sporadic-type colorectal cancer. In summary, the present study points out that ETV4 gene might contribute to activate cell proliferation mechanism in colorectal adenocarcinoma. Moreover, the study demonstrates the importance of combining the mutation analysis with expression profile in order to better understanding the molecular basis of colorectal cancer.
2
Dumortier, Mandy. "Étude du rôle des facteurs de transcription ETV4 et ETV1 de la famille ETS dans le processus tumoral de cancers hormono-dépendants : le cancer du sein, la progression métastatique du cancer de la prostate." Thesis, Lille 1, 2017. http://www.theses.fr/2017LIL10181.
Full textAbstract:
Les facteurs ETV1, 4, 5 sont souvent associée au développement de cancers. Le 1èr projet porte sur ETV4 et MMP13 en tant que gène cible et relais potentiel de l’effet pro-tumorigène d’ETV4 dans la tumorigenèse mammaire. ETV4 est surexprimé dans le cancer du sein et est associé à un mauvais pronostic, mais les événements impliqués sont encore peu connus. ETV4 contrôle l’expression de nombreux gènes comme MMP13. Cette étude a permis de montrer que MMP13 est un gène cible d’ETV4. En effet, la surexpression de MMP13 contribue aux effets pro-tumorigène et l’inhibition de MMP13 dans un contexte de surexpression de ETV4 diminue son impact. Enfin, l’étude effectuée dans la cohorte de patiente associe la surexpression d’ETV4 et de MMP13 à un cancer de mauvais pronostic. Le 2ème projet porte sur l’implication d’ETV1 dans la progression métastatique du cancer de la prostate (CaP). Les fusions de gènes impliquant les facteurs ERG et ETV1 sont présentes dans ≈50% et 10% des cas respectivement, ETV1 est présent à 50% sous sa forme pleine longueur et à 50% sous une forme tronquée. Les études ne présageant pas de différences entre les fusions ERG et ETV1. Dans ce contexte, mon 2ème sujet d’étude porte sur la recherche de l’implication du facteur ETV1 (pleine longueur ou tronquée) dans la formation des métastases du CaP et la recherche de gènes cibles impliqués, le tout en comparaison avec les données sur ERG. Cette étude nous a permis de mettre en évidence une différence en terme d’agressivité entre les 2 formes d’ETV1 in vitro et in vivo. Cependant la fusion étant peut représentée, les résultats récolté dans la cohorte ne peuvent permettre de confirmer cette différence d’agressivitéETV1, 4, 5 transcription factor are overexpress in various cancer. The first part is focus in MMP13 like target gene, potentially implicated in the mammary tumorigenesis induced by ETV4. Thus we show, the regulation of MMP13 expression by ETV4. Next we show that ETV4 promotes the mammary tumorigenesis and that MMP13 is a relay. In fact, the overexpression of MMP13 is implicate in pro-tumorigenesis effect and the repression of MMP13 in context of ETV4 overexpression decreases this effect. This approach was completed by the injection of cells in mice and show MMP13 mediate the pro-tomorigene effect of ETV4. We analysed expression of MMP13 and ETV4 in primary breast tumors and show the overexpression concomintant of ETV4 and MMP13 are associated with a poor prognosis. The second part of our study is about the ETV1 factor, in the progression of metastasis of prostate cancer (PCa) and the research of involved target genes. Gene fusion involving ERG and ETV1 and their overexpression are frequent in PCa and occur in 50% and 10% of cases respectively. To understand the role of ETV1, in comparison with available data on ERG, differents PCa cells overexpress or repress ETV1 were used. We have shown that the ETV1 factor enhance tumorigenesis capacities PCa cell lines and her truncated form has opposite effects. ETV1 full-lenght (FL) induces more bones metastasis formation than her truncated form. The expression of ETV1, and his truncated form was study in PCa samples. We confirmed the pro-tumorigenic statute of ETV1 factor in PCa and we defined differences beetwen FL and truncated length and complet the comprehension of the ETV1 function in the formation of bones metastasis of PCa
3
Cavazzola, Luciane Rostirola. "Análise da expressão do mRNA das enzimas timidina fosforilase e uridina fosforilase 1 e 2 e dos fatores de transcrição PDEF e ETV4 em tumores de próstata e de rim." Pontifícia Universidade Católica do Rio Grande do Sul, 2014. http://hdl.handle.net/10923/6884.
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Previous issue date: 2014Prostate cancer is the most important urological tumor and the second most common in men. The incidence and mortality of this multifactorial disease are varied and take into account behavioral factors and genetic predisposition. The main predisposing factors are age, race and family history. Among the various histological subtypes, the acinar adenocarcinoma is the most common. The classification of this tumor, from transrectal ultrasonography and biopsy needle, is made for degrees of Gleason score and TNM classification that provides for tumor staging. The prostatic carcinoma has a propensity to metastasize and is regulated by various molecular signaling pathways through genes, proteins, enzymes, receptors and transcription factors. The RCC is the third most common urological tumor. The wide variation in incidence correlates with the geographical area. Risk factors can be various as smoking, genetic diseases and genetic alterations. The vast majority of cases occur in adults and in the renal parenchyma, and comprise five histological subtypes, the clear and sporadic cells being the most common. The histological differentiation is made by Fuhrman grade and staging by TNM classification. As the clinical manifestations are varied, most patients are diagnosed incidentally and by ultrasonography. The enzymes thymidine phosphorylase and uridine phosphorylase 1 and 2 as well as the transcription factors PDEF and ETV4 are reported as components of pathways leading to tumorigenesis and / or metastasis. The objective of this work is to analyze these enzymes and transcription factors and their correlation with clinical and pathological variables in prostate and kidney cancers as well as in bening tissue, by RT-PCR in real time. In prostate cancer, the geometric mean of the relative expression of transcription factors PDEF and ETV were greater in the tumor than in the benign tissues These factors also had a significant correlation between them in tumor tissue and benign tissue. The geometric mean of relative expression of the enzyme thymidine phosphorylase and uridine phosphorylase 1 strongly and significantly correlated between them in benign and in malignant prostate samples. The geometric mean of relative expression of thymidine phosphorylase also correlated moderately and significantly with the ETV4 in benign tissues. The geometric mean of relative expression of uridine phosphorylase in tumor T3 was significantly lower than in T1 and T2 prostate tumors. Kidney carcinoma TNM Classification strongly and significantly correlated with the relative expression of the enzyme thymidine phosphorylase and negatively correlated very strong and significantly with the enzyme uridine phosphorylase. The relative expression of the transcription factor ETV4 correlated negatively and significantly stronger with the TNM classification, and correlated with strength and significance with the relative expression of uridine phosphorylase in tumor tissues of the kidney.
O câncer de próstata é o mais importante tumor urológico e o segundo mais frequente no homem. As taxas de incidência e de mortalidade desta doença multifatorial são muito variadas e leva em conta fatores comportamentais e prédisposição genética. Os principais fatores predisponentes são idade avançada, raça e história familiar. Dentre os vários subtipos histológicos, o adenocarcinoma acinar é o mais frequente. A classificação deste tumor, a partir da ultra-sonografia transretal com biópsia por agulha, é feita pelos graus de escore de Gleason e pela Classificação TNM que fornece o estadiamento do tumor. O carcinoma de próstata, além de ter grande propensão em formar metástases, é regulado por várias vias moleculares de sinalização por meio de genes, proteínas, enzimas, receptores e fatores de transcrição.O carcinoma de células renais é o terceiro mais comum tumor urológico. A grande variação na taxa de incidência se correlaciona com a área geográfica. Os fatores de risco podem ser vários como tabagismo, doenças genéticas e alterações gênicas. A grande maioria dos casos ocorre em adultos no parênquima renal e, além de compreender cinco subtipos histológicos, o de células claras e esporádico é o mais frequente. A diferenciação histológica é feita pelo grau de Fuhrman e o estadiamento pela Classificação TNM. Como as manifestações clínicas são variadas, a maioria dos pacientes são diagnosticados incidentalmente e por ultra-sonografia. As enzimas timidina fosforilase e uridina fosforilase 1 e 2, bem como os fatores de transcrição PDEF e ETV4, são relatados como componentes das vias que levam à tumorigênese e/ou a metastização. O objetivo deste trabalho é analizar estas enzimas e fatores de transcrição, correlacionando-as com variáveis clínicas e patológicas nos tumores de próstata e de rim, bem como nos tecidos adjacentes a estes tumores, pela técnica de RT-PCR em tempo real. No câncer de próstata, a média geométrica da expressão relativa dos fatores de transcrição PDEF e ETV4 entre os tecidos benignos e tumorais, apresentaram correlação significativa com uma maior expressão relativa no tumor quando comparado ao seu benigno. Estes fatores também apresentaram uma correlação significativa entre eles no tecido tumoral e no tecido benigno. As médias geométricas da expressão relativa das enzimas timidina fosforilase e uridina fosforilase 1 se correlacionaram fortemente e significativamente entre os tecidos benignos e tumorais da próstata. A média geométrica da expressão relativa da timidina fosforilase também se correlacionou de forma moderada e significativa com a do ETV4, entre os tecidos benignos. A média geométrica da expressão relativa da uridina fosforilase nos tumores T3 foi significativamente menor do que nos tumores T1 e T2 da próstata. No carcinoma de rim a Classificação TNM se correlacionou fortemente e significativamente com a expressão relativa da enzima timidina fosforilase, e se correlacionou de forma negativa, muito forte e significativamente com a enzima uridina fosforilase. A expressão relativa do fator de transcrição ETV4 se correlacionou de forma negativa, forte e significativamente com a Classificação TNM, e se correlacionou de forma forte e significativamente com a expressão relativa de uridina fosforilase nos tecidos tumorais de rim.
4
Cavazzola, Luciane Rostirola. "An?lise da express?o do mRNA das enzimas timidina fosforilase e uridina fosforilase 1 e 2 e dos fatores de transcri??o PDEF e ETV4 em tumores de pr?stata e de rim." Pontif?cia Universidade Cat?lica do Rio Grande do Sul, 2014. http://tede2.pucrs.br/tede2/handle/tede/1788.
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Previous issue date: 2014-08-25Prostate cancer is the most important urological tumor and the second most common in men. The incidence and mortality of this multifactorial disease are varied and take into account behavioral factors and genetic predisposition. The main predisposing factors are age, race and family history. Among the various histological subtypes, the acinar adenocarcinoma is the most common. The classification of this tumor, from transrectal ultrasonography and biopsy needle, is made for degrees of Gleason score and TNM classification that provides for tumor staging. The prostatic carcinoma has a propensity to metastasize and is regulated by various molecular signaling pathways through genes, proteins, enzymes, receptors and transcription factors. The RCC is the third most common urological tumor. The wide variation in incidence correlates with the geographical area. Risk factors can be various as smoking, genetic diseases and genetic alterations. The vast majority of cases occur in adults and in the renal parenchyma, and comprise five histological subtypes, the clear and sporadic cells being the most common. The histological differentiation is made by Fuhrman grade and staging by TNM classification. As the clinical manifestations are varied, most patients are diagnosed incidentally and by ultrasonography. The enzymes thymidine phosphorylase and uridine phosphorylase 1 and 2 as well as the transcription factors PDEF and ETV4 are reported as components of pathways leading to tumorigenesis and / or metastasis. The objective of this work is to analyze these enzymes and transcription factors and their correlation with clinical and pathological variables in prostate and kidney cancers as well as in bening tissue, by RT-PCR in real time. In prostate cancer, the geometric mean of the relative expression of transcription factors PDEF and ETV were greater in the tumor than in the benign tissues These factors also had a significant correlation between them in tumor tissue and benign tissue. The geometric mean of relative expression of the enzyme thymidine phosphorylase and uridine phosphorylase 1 strongly and significantly correlated between them in benign and in malignant prostate samples. The geometric mean of relative expression of thymidine phosphorylase also correlated moderately and significantly with the ETV4 in benign tissues. The geometric mean of relative expression of uridine phosphorylase in tumor T3 was significantly lower than in T1 and T2 prostate tumors. Kidney carcinoma TNM Classification strongly and significantly correlated with the relative expression of the enzyme thymidine phosphorylase and negatively correlated very strong and significantly with the enzyme uridine phosphorylase. The relative expression of the transcription factor ETV4 correlated negatively and significantly stronger with the TNM classification, and correlated with strength and significance with the relative expression of uridine phosphorylase in tumor tissues of the kidney.
O c?ncer de pr?stata ? o mais importante tumor urol?gico e o segundo mais frequente no homem. As taxas de incid?ncia e de mortalidade desta doen?a multifatorial s?o muito variadas e leva em conta fatores comportamentais e pr?-disposi??o gen?tica. Os principais fatores predisponentes s?o idade avan?ada, ra?a e hist?ria familiar. Dentre os v?rios subtipos histol?gicos, o adenocarcinoma acinar ? o mais frequente. A classifica??o deste tumor, a partir da ultra-sonografia transretal com bi?psia por agulha, ? feita pelos graus de escore de Gleason e pela Classifica??o TNM que fornece o estadiamento do tumor. O carcinoma de pr?stata, al?m de ter grande propens?o em formar met?stases, ? regulado por v?rias vias moleculares de sinaliza??o por meio de genes, prote?nas, enzimas, receptores e fatores de transcri??o. O carcinoma de c?lulas renais ? o terceiro mais comum tumor urol?gico. A grande varia??o na taxa de incid?ncia se correlaciona com a ?rea geogr?fica. Os fatores de risco podem ser v?rios como tabagismo, doen?as gen?ticas e altera??es g?nicas. A grande maioria dos casos ocorre em adultos no par?nquima renal e, al?m de compreender cinco subtipos histol?gicos, o de c?lulas claras e espor?dico ? o mais frequente. A diferencia??o histol?gica ? feita pelo grau de Fuhrman e o estadiamento pela Classifica??o TNM. Como as manifesta??es cl?nicas s?o variadas, a maioria dos pacientes s?o diagnosticados incidentalmente e por ultra-sonografia. As enzimas timidina fosforilase e uridina fosforilase 1 e 2, bem como os fatores de transcri??o PDEF e ETV4, s?o relatados como componentes das vias que levam ? tumorig?nese e/ou a metastiza??o. O objetivo deste trabalho ? analizar estas enzimas e fatores de transcri??o, correlacionando-as com vari?veis cl?nicas e patol?gicas nos tumores de pr?stata e de rim, bem como nos tecidos adjacentes a estes tumores, pela t?cnica de RT-PCR em tempo real. No c?ncer de pr?stata, a m?dia geom?trica da express?o relativa dos fatores de transcri??o PDEF e ETV4 entre os tecidos benignos e tumorais, apresentaram correla??o significativa com uma maior express?o relativa no tumor quando comparado ao seu benigno. Estes fatores tamb?m apresentaram uma correla??o significativa entre eles no tecido tumoral e no tecido benigno. As m?dias geom?tricas da express?o relativa das enzimas timidina fosforilase e uridina fosforilase 1 se correlacionaram fortemente e significativamente entre os tecidos benignos e tumorais da pr?stata. A m?dia geom?trica da express?o relativa da timidina fosforilase tamb?m se correlacionou de forma moderada e significativa com a do ETV4, entre os tecidos benignos. A m?dia geom?trica da express?o relativa da uridina fosforilase nos tumores T3 foi significativamente menor do que nos tumores T1 e T2 da pr?stata. No carcinoma de rim a Classifica??o TNM se correlacionou fortemente e significativamente com a express?o relativa da enzima timidina fosforilase, e se correlacionou de forma negativa, muito forte e significativamente com a enzima uridina fosforilase. A express?o relativa do fator de transcri??o ETV4 se correlacionou de forma negativa, forte e significativamente com a Classifica??o TNM, e se correlacionou de forma forte e significativamente com a express?o relativa de uridina fosforilase nos tecidos tumorais de rim.
5
Huang-Hobbs, Helen. "Dissecting the mechanism of ETV6 polymerization." Thesis, University of British Columbia, 2013. http://hdl.handle.net/2429/45691.
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ETV6 (or TEL), a member of the ETS family of eukaryotic transcription factors, normally functions as a transcriptional repressor and putative tumor suppressor. ETV6 is modular, containing a SAM (or PNT) domain and a DNA-binding ETS domain joined by a flexible linker sequence. The ETV6 SAM domain self-associates in a head-to-tail fashion, forming helical polymers proposed to generate extended repressive complexes at target DNA sites. ETV6 is also frequently involved in chromosomal translocations yielding unregulated chimeric oncoproteins with the SAM domain fused to the catalytic domain of a tyrosine receptor kinase such as NTRK3. Cellular transformation likely results from SAM domain-mediated polymerization and constitutive activation of the kinase domain. In the case of the ETV6- NTRK3 fusion (EN), this transformation is linked to congenital fibrosarcomas. Our goal is to investigate via mutations within its SAM domain, the thermodynamic and dynamic mechanisms underlying the altered transformation properties of ETV6-NTRK3. These studies have been carried out using monomeric variants of the isolated SAM domains with "head" or "tail" point mutations that prevent self-association, yet allow for formation of a mixed dimer with a native binding interface. Specifically, we used a combination of NMR spectroscopy and isothermal titration calorimetry to study the effects of additional mutations on their dimerization. Consistent with its involvement in a crystallographically-observed interdomain salt bridge, mutation of Lys99 was found to weaken the association of ETV-SAM monomers in solution, and to disrupt cellular transformation by EN. This supports the role of the SAM domain self-association in the activation of ETV6-NTRK3, and helps define the mechanisms underlying cellular transformation by similar chimeric oncoproteins.
6
Hart, Stephen Michael. "In vivo analysis of the ETV6-CBFA2 fusion gene." Thesis, University College London (University of London), 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.392499.
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Jomrich, Gerd, Florian Maroske, Jasmin Stieger, Matthias Preusser, Aysegül Ilhan-Mutlu, Daniel Winkler, Ivan Kristo, Matthias Paireder, and Sebastian Friedrich Schoppmann. "MK2 and ETV1 Are Prognostic Factors in Esophageal Adenocarcinomas." IVyspring International Publisher, 2018. http://dx.doi.org/10.7150/jca.22310.
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Background. Esophageal cancer is ranked in the top ten of diagnosed tumors worldwide. Even though
improvements in survival could be noticed over the last years, prognosis remains poor. ETS
translocation variant 1 (ETV1) is a member of a family of transcription factors and is phosphorylated
by mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2). Aim of this study was
to evaluate the prognostic role of MK2 and ETV1 in esophageal cancer.
Methods. Consecutive patients that underwent surgical resection at the department of surgery at the
Medical University of Vienna between 1991 and 2012 were included into this study. After
microscopic analysis, tissue micro arrays (TMAs) were created and immunohistochemistry was
performed with antibodies against MK2 and ETV1.
Results. 323 patients were included in this study. Clinical data was achieved from a prospective
patient data base. Nuclear overexpression of MK2 was observed in 143 (44.3%) cases for nuclear
staining and in 142 (44.0%) cases a cytoplasmic overexpression of MK2 was observed. Nuclear and
cytoplasmic ETV1 overexpression was detected in 20 cases (6.2%) and 30 cases (9.3%), respectively.
In univariate survival analysis, cMK2 and nETV1 were found to be significantly associated with
patients' overall survival. Whereas overexpression of cMK2 was associated with shorter, nETV1
was associated with longer overall survival. In multivariate survival analysis, both cMK2 and nETV1
were found to be independent prognostic factors for the subgroup of EAC as well.
Discussion. Expression of MK2 and ETV1 are prognostic factors in patients, with esophageal
adenocarcinoma.
8
Kaulfuß, Katja [Verfasser]. "Identifizierung von Zielgenen des chimären Transkriptionsfaktors ETV6/RUNX1 / Katja Kaulfuß." Berlin : Freie Universität Berlin, 2018. http://d-nb.info/1196805180/34.
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Favier, Marie. "Les thrombopénies héréditaires rares : implications des gènes ETV6, ITGA2B, ITGB3." Thesis, Aix-Marseille, 2017. http://www.theses.fr/2017AIXM0559.
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L’identification des gènes impliqués dans les thrombopénies apporte des éléments importants pour la compréhension des voies de régulation de la production et des fonctions des plaquettes voire de l’hématopoïèse. Notre laboratoire a développé une stratégie d’identification de gènes à l’origine de thrombopénies sans hypothèse a priori par séquençage d’exomes. Cette stratégie a permis de mettre en évidence des mutations touchant les gènes ETV6, ITGA2B et ITGB3.Les thrombopénies à volume plaquettaire normal sont particulièrement importantes à détecter en raison d’un risque d’évolution vers une pathologie onco-hématologique. L’origine génétique de cette catégorie de thrombopénie s’est pendant longtemps limitée aux mutations du gène runx1. Le travail que j’ai effectué au cours de ma thèse a contribué à impliquer le gène etv6 dans ce groupe de thrombopénies.Concernant le gène etv6 6 familles ont présenté des mutations pathogènes. Toutes ces mutations sont à l’origine d’une perte de l’activité répressive du gène et un nombre élevé de cellules CD34+ circulant dans le sang révélant le rôle d’ETV6 dans la prédisposition onco-hématologique. De plus la mégacaryopoïèse présente deux principales anomalies. Elles associent une augmentation du nombre de colonies progéniteurs mégacaryocytaires à une formation de proplaquettes réduites. Concernant les gènes itga2b et itgb3 3 familles ont été étudiées.Des défauts quantitatifs ou qualitatifs du récepteur αIIbβ3 conduisant à sa perte de fonction se retrouvent dans la thrombasthénie de Glanzmann (GT) caractérisée par une thrombopathie mais un nombre de plaquettes et une morphologie normaleThe identification of the genes involved in thrombocytopenia provides important elements for understanding the pathways of regulation of the production and functions of platelets or even hematopoiesis. Our laboratory has developed a strategy for identifying genes causing thrombocytopenia without a priori hypothesis by sequencing exomes. This strategy has been applied to families with autosomal dominant thrombocytopenia and has demonstrated mutations in the genes etv6, itga2b and itgb3. Normal platelet thrombocytopenia are particularly important to detect because of the risk of developing onco-hematological pathology. The genetic origin of this category of thrombocytopenia has long been limited to mutations in the runx1 gene. More recently, mutations on the ankrd26 promoter have been reported. The work I did during my thesis helped to involve the etv6 gene in this group of thrombocytopenia. Concerning this gene six families have pathogenic mutations. All these mutations are the cause of a loss of the repressive activity of the gene and a high number of CD34+ cells circulating in the blood revealing the role of ETV6 in the onco-hematological predisposition. In addition, megakaryopoiesis has two main anomalies. They associate an increase in the number of megakaryocytic progenitor colonies with the formation of reduced proplatelets.Concerning the itga2b and itgb3 genes, 3 families were studied. These genes encode the αIIbβ3 integrin. Integrin αIIbβ3 is a platelet receptor for fibrinogen and Von Willebrand factor, and plays a crucial role in thrombosis and hemostasis
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Craig, Michael P. "Transcriptional Regulation of Developmental and Tumor-Induced Angiogenesis by Etv2 and Fli1b." University of Cincinnati / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1427982504.
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Moore, John C. "The Role and Regulation of Etv2 in Zebrafish Vascular Development: A Dissertation." eScholarship@UMMS, 2013. https://escholarship.umassmed.edu/gsbs_diss/672.
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Etv2 is an endothelial-specific ETS transcription factor that is essential for endothelial differentiation and vascular morphogenesis in vertebrates. However, etv2 expression dynamics during development and the mechanisms regulating it are poorly understood. I found that etv2 transcript and protein expression are highly transient during zebrafish vascular development, with both expressed early during development and then subsequently downregulated. Inducible knockdown of Etv2 in zebrafish embryos prior to mid-somitogenesis, but not later, causes severe vascular defects, suggesting a role for Etv2 in specifying angioblasts from the lateral mesoderm. I further demonstrate that the 3’UTR of etv2 is post-transcriptionally regulated in part by the let-7 family of microRNAs. Ectopic expression of let-7a represses endogenous Etv2 transcript and protein expression with a concomitant reduction in endothelial cell gene expression. Additionally, overexpressed Etv2 in HEK293T cells is ubiquitinated and degraded by the proteasome. Accordingly, endogenous zebrafish Etv2 protein is rapidly degraded in the presence of the translation inhibitor cycloheximide in vivo. Taken together, our results suggest that etv2 acts during early development to specify endothelial lineages and is subsequently downregulated through post-transcriptional and post-translational mechanisms, to allow normal vascular development to proceed.
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Moore, John C. "The Role and Regulation of Etv2 in Zebrafish Vascular Development: A Dissertation." eScholarship@UMMS, 2005. http://escholarship.umassmed.edu/gsbs_diss/672.
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Etv2 is an endothelial-specific ETS transcription factor that is essential for endothelial differentiation and vascular morphogenesis in vertebrates. However, etv2 expression dynamics during development and the mechanisms regulating it are poorly understood. I found that etv2 transcript and protein expression are highly transient during zebrafish vascular development, with both expressed early during development and then subsequently downregulated. Inducible knockdown of Etv2 in zebrafish embryos prior to mid-somitogenesis, but not later, causes severe vascular defects, suggesting a role for Etv2 in specifying angioblasts from the lateral mesoderm. I further demonstrate that the 3’UTR of etv2 is post-transcriptionally regulated in part by the let-7 family of microRNAs. Ectopic expression of let-7a represses endogenous Etv2 transcript and protein expression with a concomitant reduction in endothelial cell gene expression. Additionally, overexpressed Etv2 in HEK293T cells is ubiquitinated and degraded by the proteasome. Accordingly, endogenous zebrafish Etv2 protein is rapidly degraded in the presence of the translation inhibitor cycloheximide in vivo. Taken together, our results suggest that etv2 acts during early development to specify endothelial lineages and is subsequently downregulated through post-transcriptional and post-translational mechanisms, to allow normal vascular development to proceed.
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Meichsner, André [Verfasser], and Martin [Akademischer Betreuer] Maier. "Herstellung, Charakterisierung, Modellierungsansätze und Simulation von edelstahltextilverstärktem Polypropylen (ETV-PP) und Langglasfaserthermoplasten mit PP-Matrix (ETV-PP/GF) / André Meichsner ; Betreuer: Martin Maier." Kaiserslautern : Technische Universität Kaiserslautern, 2017. http://d-nb.info/1138234222/34.
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Pedrola, Montero Núria. "Molecular pathways regulated by the ETV5 transcription factor in the invasion of endometrial cancer." Doctoral thesis, Universitat Autònoma de Barcelona, 2013. http://hdl.handle.net/10803/117328.
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El càncer d’endometri és la malaltia més freqüent del tracte genital femení en països desenvolupats. L’aparició de símptomes en estadis primerencs [1], contribueix a la detecció de la malaltia en estadis no avançats i amb un alt índex de supervivència. No obstant, en el 20 % dels casos, el diagnòstic es més tardà i els pacients pateixen infiltració miometrial i/o afectació limfàtica. La invasió miometrial, que indica invasió tumoral, és el factor pronòstic més important i determina l’augment de l’índex de recurrència després del tractament quirúrgic i la disminució de l’índex de supervivència als 5 anys. Conseqüentment, el descobriment dels passos inicials associats amb la infiltració miometrial és fonamental per identificar nous agents terapèutics per la prevenció de la disseminació del càncer.
Per assolir aquest propòsit, el principal objectiu del nostre grup durant els últims anys ha estat la identificació de mecanismes moleculars involucrats en la invasió i la disseminació del càncer d’endometri. Hem identificat el factor de transcripció ETV5 com el principal agent de la invasió miometrial en les cèl·lules tumorals endometrials. ETV5 està específicament sobreexpressat en el tumor d’endometri invasiu [2] i és capaç d’activar la metaloproteassa MMP2 promovent la migració i la invasió cel·lular in vitro i in vivo [3]. A més, hem demostrat el paper d’ETV5 en la inducció de l’EMT (transició epiteli-mesènquima) que provoca l’adquisició de capacitats invasives i migratòries del tumor.
El principal objectiu d’aquesta tesi és la identificació de noves molècules involucrades en la invasió miometrial. En particular, ens hem focalitzat en la identificació de noves dianes d’ETV5 que participen en l’adquisició de propietats migratòries i invasives de les cèl·lules tumorals en el carcinoma endometrial.
Per caracteritzar els passos inicials de la invasió miometrial regulada per ETV5, vam analitzar mitjançant l’estudi diferencial d’expressió gènica aquells gens que estaven alterats en cèl·lules de càncer d’endometri Hec1a amb una sobrexpressió estable de la proteïna de fusió GFP-ETV5, comparat amb les cèl·lules Hec1a control.
En particular, vam identificar NID1 i NUPR1 com a directes dianes transcripcionals d’ETV5. NID1 és una proteïna que forma part de la matriu extracel·lular juntament amb la laminina [4]. NUPR1 és una proteïna del grup de les HMG i la seva principal funció és unir-se a la cromatina dels promotors per modificar l’accessibilitat de la cromatina durant la regulació gènica [5]. Vam voler examinar el paper de NID1 i NUPR1 coma mediador de les funcions d’ETV5 en les cèl·lules Hec1a de càncer d’endometri in vitro i in vivo utilitzat un model animal [6].
Junts, els nostres resultats suggereixen que la regulació de NID1 mitjançant ETV5 genera la invasió cel·lular i l’adhesió a la matriu extracel·lular, i la regulació de NUPR1 mitjançant ETV5 provoca la migració cel·lular en la línia cel·lular de càncer d’endometri in vitro. D’altra banda, proposem que NID1 i NUPR1 regulen la migració i la invasió, cel·lular en part, mitjançant la regulació indirecte de la N-cadherina. També vam poder observar que els ratolins injectats amb cèl·lules que presentaven NID1 silenciat generaven tumors de mida inferior que els injectats amb cèl·lules control. D’altra banda, els ratolins injectats amb cèl·lules amb NUPR1 o NID1 silenciats generaven menys metàstasis que els injectats en cèl·lules control suggerint que NID1 i NUPR1 mitjançant ETV5 estan involucrats en la progressió i la disseminació tumoral in vivo.
Finalment, per tal de confirmar la regulació de NID1 i de NUPR1 per ETV5, vam examinar el paper d’ambdós gens en mostres humanes de càncer d’endometri i la seva associació amb les característiques clíniques i patològiques. En estudis previs, vam descriure la sobreexpressió d’ETV5 en el carcinoma d’endometri, concretament el front d’invasió del tumor [7]. En aquest estudi, hem trobat que l’expressió de NID1 i d’ETV5 està clínicament correlacionada en mostres humanes de càncer d’endometri tan a nivell de mRNA com de proteïna. A més a més, em pogut observar una sobreexpressió de NID1 i de NUPR1 en el front d’invasió del tumor tant a nivell de mRNA con de proteïna.
Com conclusió, aquestes dades presentades en aquesta tesi contribueixen al descobriment de mecanismes moleculars involucrats en la disseminació del càncer d’endometri. El descobriment de les bases moleculars de la invasió en el carcinoma endometrial contribuirà en el desenvolupament d’estratègies terapèutiques més específiques i efectives.Endometrial cancer is the most frequent malignancy of the female genital tract in Western countries. The early appearance of symptoms [1], contributes to the earlier detection of this malignancy and results in better survival rates. However, in 20% of cases the diagnosis is delayed and patients present with myometrial infiltration and/or lymph node affectation. Myometrial invasion, an initial event that signals tumour invasion is one of the most valuable prognostic factors and it determines increased recurrence rates after the first surgical treatment and decreased 5-year survival. Consequently, unravelling the initial steps associated with myometrial infiltration is fundamental to identify new therapeutic agents for the prevention of cancer dissemination. To this end, the major scientific aim of our group during the past few years has been the identification of the molecular mechanisms involved in endometrial cancer invasion and dissemination. We identified ETV5 transcription factor as an important agent of myometrial invasion by endometrial tumour cells. ETV5 is specifically upregulated in invasive endometrial tumours and is able to [2] activate the metalloproteinase MMP2, thus promoting cell migration and invasion in vitro and in vivo [3]. In addition, we demonstrated the role of ETV5 on the induction of EMT, which result in the acquisition of migratory and invasive capabilities. The main objective of this thesis is to identify new molecules involved in the myometrial invasion of endometrial cancer. In particular, we have focused on the identification of new ETV5 downstream targets that mediate the role of ETV5 in the endometrial tumour cells’ acquisition of migratory and invasive properties. To further characterise the initial steps of myometrial invasion regulated by the ETV5 transcription factor, we analysed by gene expression microarray technology those genes whose expression was altered in Hec1A endometrial cancer cells with stable overexpression of a fusion GFP-ETV5 protein, compared with Hec1A control cells. In particular, we have identified NID1 and NUPR1 as direct transcriptional targets of ETV5. NID1 is a ubiquitous protein component of the BM and a partner of laminin [4]. NUPR1 is a HMG protein and its principal role in the cell is to bind to the chromatin promoters to modify the accessibility of chromatin in gene regulation [5]. We wanted to examine the role of NID1 and NUPR1 as a mediator of ETV5 functions in Hec1A endometrial cancer cells in vitro and in vivo using an orthotopic mouse model [6]. Our results suggest that NID1 regulation by ETV5 enhances cell invasion and cell adhesion to the extracellular matrix, and that NUPR1 regulation by ETV5 enhances cell migration in endometrial cancer cells in vitro. We propose that NID1 and NUPR1 regulation of cell migration and invasion may be in part mediated by the regulation of N-cadherin. We also observed that the mice injected with cells with NID1 silenced, generated a small size tumours compared with mice injected with control cells. Moreover, we found that mice injected with cells with NID1 or NUPR1 inhibition produced a decreased number of metastases compared with mice injected with control cells. These results suggest that the regulation of NID1 and NUPR1 by ETV5 contributes to the tumour progression and dissemination in vivo. Finally, in order to confirm the regulation of NID1 or NUPR1 by ETV5, we wanted to examine the role of both NID1 and NUPR1 genes in human endometrial tumour samples and their association with clinical and pathological characteristics In previous studies, ETV5 was upregulated in human endometrial tumour samples compared with control tissue, in particular in the invasion front of endometrial tumours [7]. We found that the expression of NID1 and ETV5 were clinically correlated in human endometrial tumour samples at mRNA level and at the protein level. Moreover, we observed a upregulation of NID1 and NUPR1 in invasion front of the tumour. In conclusion, the data presented in this thesis contributes to the elucidation of the molecular mechanisms involved in endometrial cancer dissemination. Understanding the molecular basis of myometrial invasion in endometrial cancer will contribute to the development of more specific and more effective therapeutic strategies.
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Lobitz, Stephan [Verfasser]. "Posttranskriptionelle Expressionshemmung von ETV6/RUNX1 durch lentivirale Transduktion mit dem Ribozym buRz28 / Stephan Lobitz." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2012. http://d-nb.info/1030381100/34.
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Saultier, Paul. "Pathologies plaquettaires constitutionnelles associées aux défauts des facteurs de transcription FLI1, ETV6 et GATA1." Thesis, Aix-Marseille, 2018. http://www.theses.fr/2018AIXM0262.
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Les thrombopénies constitutionnelles (TC) sont des maladies encore incomplètement caractérisées. Ce travail de thèse concerne les TC liées à 10 variants des gènes codant les facteurs de transcription FLI1, ETV6 et GATA1, dont 9 n’avaient jamais été décrits. Ces pathologies ont été étudiés à partir de patients recruté dans un réseau national (Centre de Référence des Pathologies Plaquettaires CRPP) et un réseau international (BRIDGE consortium).Nous montrons qu’il existe un déficit sévère en granules denses dans les plaquettes des patients porteurs de variants FLI1 du fait d’un probable défaut de biogénèse. Ce travail, et d’autres études publiées récemment, ont permis de définir la TC liée aux variants ETV6 en tant que nouveau syndrome de prédisposition aux hémopathies malignes. Les variants FLI1 s’associent à une diminution d’activité transcriptionnelle et d’accumulation nucléaire de la protéine et à des anomalies de la différenciation mégacaryocytaire. Les variants ETV6 s’associent à un défaut d’activité répressive et les mégacaryocytes dérivés des patients montrent un excès de prolifération et un défaut marqué de formation des proplaquettes. Les plaquettes de patients porteurs de variants GATA1 ont montré une expression anormale de la protéine MYH10, ce qui suggère un défaut de répression du gène MYH10 au cours de la mégacaryopoïèse. Une analyse in silico de données de ChIP-seq a ainsi montré l’existence d’une fixation de GATA1 dans le promoteur et dans un intron de MYH10 dans le mégacaryocyte.Ce projet a permis d'apporter des connaissances sur les causes génétiques, le phénotype, le diagnostic, le pronostic et les mécanismes physiopathologiques des TCConstitutional thrombocytopenia (CT) is a group of diseases incompletely characterized. This thesis focused on CTs due to 10 variants in genes encoding the transcription factors FLI1, ETV6 and GATA1, of which 9 had never been described. These diseases were studied in French and European patients recruited using national (French national reference center for inherited platelet disorders CRPP) and international (BRIDGE consortium) networks.We showed that the platelets of patients carrying FLI1 variants harbored a severe dense granule defect probably due a biogenesis defect. Our work, associated with data published by other groups, has defined ETV6-related CT as a new hematological malignancy predisposition syndrome. FLI1 variants are associated with a decreased transcriptional activity, a decreased nuclear accumulation of the protein and abnormal megakaryocyte differentiation. ETV6 variants led to a decreased repressive activity and the megakaryocytes derived from patients showed increased proliferation and a marked defect in proplatelet formation. The platelets of GATA1 variant carriers showed aberrant expression of MYH10 protein suggesting a defective silencing of MYH10 gene during megakaryopoiesis. Consistently, in silico analysis of ChIP-seq data showed that GATA1 binds the promoter and an intronic region of the MYH10 in megakaryocytes.This project has provided insights into genetic causes, phenotype, diagnosis, prognosis and pathophysiological mechanisms of CTs
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Jakobczyk, Hélène. "Rôles de RUNX1 dan la pathogenèse des leucémies aiguës lymphoblastiques à réarrangement ETV6-RUNX1." Thesis, Rennes 1, 2018. http://www.theses.fr/2018REN1B033.
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Les leucémies aiguës lymphoblastiques de la lignée B (LAL-B) sont les cancers pédiatriques les plus fréquents. Dans ce type de leucémie, l'une des anomalies génétiques les plus fréquentes est la translocation t(12 ;21) aboutissant à la protéine de fusion ETV6-RUNX1. Cette pathologie est décrite comme un modèle à deux « hits ». Le premier, se produit in utero et génère la protéine de fusion. Le second, correspond à l’acquisition d’anomalies génétiques après la naissance. Ces réarrangements génomiques aberrants ont été décrits comme provenant d’une activité anormale de la recombinasse RAG. Notre travail a consisté dans un premier temps à compléter le modèle de leucémogénèse à plusieurs « hits ». En continuant notre étude des LAL B à translocation ETV6-RUNX1, nous nous sommes concentrés sur le rôle de RUNX1, gène dérégulé dans ce type de leucémie.L’ensemble de nos résultats confirme le rôle prépondérant de RUNX1 dans l’hématopoïèse et la leucémogenèse grâce à sa capacité à s’associer à des protéines aux fonctions différentes et grâce à son implication dans la transcription de gènes clé en hématologie. Nos résultats ouvrent donc de nouvelles perspectives dans la compréhension du contrôle de l’activité transcriptionnelle de RUNX1 et dans son rôle dans les hémopathies malignesB-cell precursor acute lymphoblastic leukemia (B-ALL) is the most common pediatric cancer. In this type of leukemia, one of the most common genetic abnormalities is the ETV6-RUNX1 rearrangement. This malignancy is described as a two "hits" model. The first event occurs mainly in utero and generates the fusion gene ETV6-RUNX1. The second event consists in the acquisition of additional genetic abnormalities after birth. These aberrant genomic modifications have been described as resulting from abnormal activity of the RAG recombinase. Our work consisted initially in completing the leukemogenesis model. In continuing our study of ETV6-RUNX1 B-ALL, we focused on the role of RUNX1, an upregulated gene in this type of leukemia. All results confirm the predominant role of RUNX1 in hematopoiesis and leukemogenesis thanks to its ability to associate with proteins with different functions and its involvement in the transcription of key genes in hematology. Our results therefore open new perspectives in understanding the control of transcriptional activity of RUNX1 and its role in malignant hematology
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Rost, Megan S. "The roles of Vegf and Stabilin-2 signaling during arterial-venous differentiation." University of Cincinnati / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1427883233.
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Koenig, Andrew L. "Novel Mechanisms of Blood and Lymphatic Vessel Development." University of Cincinnati / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1522413280582749.
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Ng, Po-mo, and 吳寶武. "An evaluation of ETV teaching materials in the integrated science subject." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1996. http://hub.hku.hk/bib/B42574523.
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Ng, Po-mo. "An evaluation of ETV teaching materials in the integrated science subject." Click to view the E-thesis via HKUTO, 1996. http://sunzi.lib.hku.hk/hkuto/record/B42574523.
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Zhang, Chang-Dong. "Analysis of the interaction of TIP60β and PIN1 with the ets family transcription factor ETV6." Diss., lmu, 2005. http://nbn-resolving.de/urn:nbn:de:bvb:19-38996.
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Arnaud, Marie-Pierre. "Physiopathologie des leucémies aigues lymphoblastiques de la lignée B à remaniement ETV6/RUNX1 : rôle de la protéine CD9." Thesis, Rennes 1, 2015. http://www.theses.fr/2015REN1S064/document.
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Malgré l'amélioration des traitements, environ 20% des patients atteints de leucémie aigue lymphoblastiques (LAL) rechutent dans la moelle osseuse ou dans des sites extra-médullaires tels que les ovaires et les testicules, ce qui est particulièrement fréquent dans les rechutes tardives de LAL-B présentant un remaniement ETV6/RUNX1. Les travaux réalisés par Virginie Gandemer en 2007, ont montré que l'expression de CD9 permettait de distinguer les leucémies ETV6/RUNX1 des autres types de leucémie. Le gène CD9 code pour une protéine de la famille des tétraspanines dont l'expression a été corrélée avec le risque métastatique et la survie des patients. Par ailleurs il a été démontré que la protéine CD9 était impliquée dans le homing et la prise de greffe des cellules souches hématopoïétiques et leucémiques. Nous avons donc émis l'hypothèse qu'à travers ses propriétés fonctionnelles sur la migration et le homing, CD9 pourrait être un acteur clé des rechutes de LAL-B. Le but de ce travail de thèse était donc premièrement de déterminer le mode de régulation de CD9 dans les LAL-B ETV6/RUNX1 et deuxièmement de déterminer les effets de l'expression de CD9 sur la motilité et la prise de greffe des LAL-B. Les analyses préalablement réalisées au laboratoire avaient suggéré que CD9 pouvait être régulé par des miARNs. Nous avons identifié un cluster de 3 miARNs potentiellement impliqués dans la régulation de CD9 dans les LAL-B ETV6/RUNX1. Ces résultats doivent cependant être complétés par d'autres analyses fonctionnellles afin d'être confirmés. Nous avons étudié le rôle de la protéine CD9 dans la dissémination des cellules de LAL-B. Nous avons démontré que CD9 était un régulateur potentiel de l'adhésion et un nouveau facteur impliqué dans la migration et le homing dépendants de CXCR4 en favorisant l'activation de RAC1 et les réarrangements de l'actine en réponse au CXCL12. Enfin, nous avons décrit pour la première fois l'influence de CD9 sur la migration et le homing dans les testicules via RAC1. Nos résultats montrent donc que CD9 favorise la dissémination des cellules de LAL-B dans les testicules et suggèrent que cette protéine pourrait constituer un acteur majeur des rechutes tardives de LAL-B dont les mécanismes d'apparitions sont peu connusDespite improvements in survival rates, approximately 20% of children suffering from acute lymphoblastic leukemia (B-ALL) present relapses from bone marrow or from B-extramedullary sites, such as the testes or ovaries, particularly in cases of late relapse of ETV6/RUNX1-ALL. Virgine Gandemer showed in 2007, that the expression of CD9, a protein from the tetraspanin superfamily, can be used to distinguish ETV6/RUNX1 lymphoblastic leukemia from other types of ALL. CD9 expression has been correlated with the risk of metastasis and is associated with a poor clinical outcome in various types of cancer. Moreover CD9 has been implicated in hematopoietic and leukemic stem cell homing. We hypothesized, that CD9 protein, through its functional properties on migration and homing, could be a key actor of B-ALL relapses. The purpose of our study was then to investigate, first the transcriptional regulation of CD9 in ETV6/RUNX1 B-ALL and secondly, the effect of CD9 expression on motility and engrafment of B lymphoblasts. The analysis of CD9 transcriptional regulation previously made in the team, suggested that it could be regulated by miRNAs. We identified a cluster of 3 miRNAs potentially implicated in the regulation of CD9 expression in ETV6/RUNX1 B-ALL. This result has to be confirmd by more functional analysis. We investigated the role of CD9 in the dissemination of B-ALL. We identified CD9 as a potential regulator of B-ALL cell adhesion and a new factor involved in CXCR4-mediated migration and homing, through the promotion of actin rearrangement in response to CXCL12. We also characterized the effect of CD9 protein expression on RAC1 activation, which had an impact on blast migration and engraftment. Finally, we described, for the first time, the influence of CD9, mediated by RAC1 signaling, on B-cell chemotactic migration and homing in the testis. Our work provides evidence for an impact of CD9 on the ability of pre-B leukemic cells to disseminate to testes, through its effects on migration and homing, and suggests that CD9 may be a key player in late relapses of B-ALL, which are currently poorly understood
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Schäfer, Daniel [Verfasser], and William F. [Gutachter] Martin. "Establishment of a Novel Technique Termed GIPFEL to Determine the Frequency of ETV6-RUNX1 Fusions in Healthy Newborns and Analysis of Cooperating Oncogenic Lesions Leading to ETV6-RUNX1 Positive Childhood Leukemia / Daniel Schäfer ; Gutachter: William Martin." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2018. http://d-nb.info/1150477407/34.
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Schäfer, Daniel Verfasser], and William F. [Gutachter] [Martin. "Establishment of a Novel Technique Termed GIPFEL to Determine the Frequency of ETV6-RUNX1 Fusions in Healthy Newborns and Analysis of Cooperating Oncogenic Lesions Leading to ETV6-RUNX1 Positive Childhood Leukemia / Daniel Schäfer ; Gutachter: William Martin." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2018. http://d-nb.info/1150477407/34.
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Cordeiro, Diana Cristina de Oliveira Gomes. "Estudo da reutilização de uma água residual tratada na rega paisagística. Caso estudo: ETAR da ETVO - Valorsul." Master's thesis, Faculdade de Ciências e Tecnologia, 2012. http://hdl.handle.net/10362/7522.
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Dissertação para obtenção do Grau de Mestre em
Engenharia do Ambiente, perfil SanitáriaA presente dissertação teve como principal objectivo avaliar a possibilidade de reutilização da água residual tratada da ETAR da ETVO-Valorsul, na rega dos espaços verdes, através da realização de ensaios laboratoriais. Foi realizada a caracterização química do efluente tratado relativamente a parâmetros essenciais à rega e a espécies químicas que podem causar toxicidade nas plantas regadas. Esta caracterização permitiu definir factores de diluição do efluente, no caso dos parâmetros que apresentaram valores superiores aos VMA ou VMR para rega, de acordo com o Decreto-Lei nº 236/98. O efluente da ETAR da ETVO apresentou elevados teores médios de salinidade (16,1 dS/m), nitratos (662 mgNO3-/L) e cloretos (2420 mg Cl-/L). O primeiro ensaio foi realizado em placas de Petri, com areia e com a aplicação de uma rega diária com concentrações do efluente de 0%, 1%, 5%, 10%, 25%, 75% e 100% (v/v). Foram utilizadas as espécies vegetais Sorghum saccharatum, Sinapis alba, Lepidium sativum e uma mistura de diferentes espécies de plantas herbáceas habitualmente aplicadas na relva de jardins. Este ensaio permitiu observar a ocorrência de efeito inibidor do efluente na germinação das quatro plantas, quando foi aplicado em concentrações de 75% e 100% (v/v). Posteriormente foi efectuado um ensaio de fitotoxicidade em “Phytotoxkit” da empresa Microbiotests, com solo padrão, com a duração de três dias, tendo a rega sido realizada apenas no primeiro dia. Foram utilizadas concentrações do efluente de 0%, 6,25%, 12,5%, 25% e 50% (v/v). As espécies utilizadas neste ensaio foram também as anteriormente referidas. Os resultados obtidos permitiram verificar um ligeiro efeito inibidor do efluente no comprimento de raízes das plantas em estudo, assim como na sua germinação, quando foi aplicado numa concentração de 50% (v/v). Por fim realizaram-se ensaios de germinação e crescimento da mistura de relva no solo da ETVO, com rega diária utilizando o efluente tratado da ETAR desta unidade. Estes ensaios foram realizados em placa e em vaso, durante 15 dias e 30 dias, respectivamente. Obtiveram-se taxas de germinação superiores a 50%, nos ensaios em placa, e superiores a 80%, nos ensaios em vaso, quando foram efectuadas regas com concentrações de efluente até 25% (v/v). Quando foi efectuada a irrigação com uma concentração do efluente de 50% (v/v), foram obtidas taxas de germinação de 30%. Esta obesrvação permitiu concluir que, para concentrações iguais ou superiores a 50% (v/v), o efluente teve um efeito inibidor na germinação da relva. Após a conclusão do ensaio de germinação e crescimento com a duração de 30 dias, foram analisadas amostras do solo e da relva, com o objectivo de se avaliar a acumulação de elementos químicos. Registaram-se acréscimos dos teores de sódio, cloretos, cobre, ferro e arsénio, no solo, e de sódio, zinco e arsénio, na relva, relativamente aos vasos em que foi utilizada a concentração do efluente de 50% (v/v).Concluiu-se que a reutilização do efluente tratado na rega dos espaços verdes das instalações da ETVO poderá ser uma opção viável, quando aplicado numa concentração inferior a 25% (v/v). Contudo, esta prática terá que ser acompanhada de um plano de monitorização da qualidade das plantas e do solo, e terá que ser garantida a protecção de eventuais aquíferos existentes na zona da ETVO.
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Hasse, Kerstin [Verfasser]. "Untersuchung des Beitrags von ETV6/RUNX1 zur Entstehung akuter lymphatischer Leukämie (ALL) im Kindesalter / Kerstin Hasse." Berlin : Freie Universität Berlin, 2013. http://d-nb.info/1031099581/34.
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Roche, Emmanuel. "Etude des mécanismes de résistance des cancers de prostate aux inhibiteurs de topoisomérases I de la famille des camptothécines." Thesis, Bordeaux, 2014. http://www.theses.fr/2014BORD0423/document.
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Les ADN-Topoisomérases (Topo) de type I et II sont des enzymes essentielles à la suppression des surenroulements de engendrés par la plupart des transactions de l’ADN. Elles sont des cibles de médicaments anticancéreux très utilisés en clinique. Parmi eux, les inhibiteurs de Topo1 de la famille des camptothécines (CPT) exercent leur cytotoxicité en produisant des cassures double-brin de l’ADN provenant de la collision des fourches de réplications avec les complexes ADN-Topo1 stabilisés par ces inhibiteurs. Les dérivés de CPT sont approuvés pour le traitement des cancers coliques, de l’ovaire et du poumon, mais il existe de multiples mécanismes de résistance à ces agents qui sont à l’origine de l’échec du traitement. Les cancers de la prostate sont réfractaires aux CPT, mais très peu d’études ont été réalisées pour expliquer cette résistance « intrinsèque ». Ce travail de thèse visait à identifier les mécanismes de cette résistance en nous appuyant (1) sur des résultats antérieurs de l’équipe montrant que l’interaction entre la DNA-PKcs, une kinase impliquée dans la réparation de l’ADN par recombinaison non-homologue, et la Topo1 pouvait réguler la sensibilité aux CPT de manière indépendante de la réparation de l’ADN et (2) sur une étude ayant mis en évidence une interaction entre DNA-PKcs et le facteur de transcription ERG dont le gène est remanié dans plus de 50% des tumeurs de prostate. Nos résultats montrent pour la première fois que ERG est effectivement impliqué dans la régulation de la réponse aux CPT dans la lignée VCaP présentant un gène de fusion TMPRSS2-ERG. La répression de ERG dans la lignée VCaP induit une sensibilisation à la CPT mais pas à l’étoposide (un inhibiteur de Topo2) et est accompagnée d’une augmentation du nombre de complexes ADN/Topo1. Ce mécanisme peut-être soit lié à un effet de ERG sur l’interaction DNA-PKcs/Topo1 ou à une régulation transcriptionnelle de gènes impliqués dans la réponse aux CPT incluant la Topo1 elle-même. Nous avons confirmé cette deuxième hypothèse, en démontrant que ERG régule la transcription du micro ARN miR-24 et que l’expression de Topo1 est également sous contrôle de miR-24 dans la lignée VCaP. Des résultats similaires ont été obtenus dans la lignée LNCaP (présentant le gène de fusion TMPRSS2-ETV1) dans laquelle la répression de ETV1 confère aussi une sensibilisation à la CPT. Au cours de notre travail nous avons également recherché des inhibiteurs de l’interaction entre la DNA-PKcs et Topo1 afin de pouvoir utiliser ces composés comme agents de potentialisation des dérivés de CPT en clinique. Les résultats du criblage d’une banque de 320 composés naturels réalisé par la technologie AlphaScreen n’ont malheureusement pu identifier que des inhibiteurs catalytiques de Topo1. Nous avons néanmoins pu montrer que l’un d’entre eux, la mahanimbine, présentait une forte activité cytotoxique vis-à-vis de lignées résistantes aux dérivés de CPT et vis-à-vis de la lignée VCaP ce qui permet d’envisager le développement de nouvelles classes d’inhibiteurs catalytiques Topo1 pouvant contourner la résistance des dérivés de CPT en cliniqueType I and II DNA Topoisomerases (Top) are essential enzymes involved in the removal of DNA torsional constraints induced by most DNA transactions. They are the targets of various anticancer agents used in the clinic. Among them, Top1 inhibitors from the camptothecins (CPT) family exert their cytotoxicity by producing DNA double-strand breaks that are generated by the collision of advancing replication forks with DNA-Top1 complexes that are stabilized by these inhibitors. CPT derivatives are approved for the treatment of colon, ovary and lung cancers but resistance mechanisms are developed and lead to treatment failure. Prostate cancers are refractory to CPT, but few studies have addressed the mechanisms of such “intrinsic” resistance. This work was aimed at identifying such mechanisms based on (1) previous results from the laboratory showing that interaction of Top1 with DNA-PKcs, a kinase that is essential for non-homologous end-joining, could regulate cell sensitivity to CPT independently of DNA repair and (2) a study that showed an interaction of DNA-PKcs with ERG, a transcription factor from the ETS family which is rearranged in more than 50% of prostate tumors. Our results show for the first time that ERG is indeed involved in the regulation of prostate cancer cell response to CPT as its repression sensitized VCaP cells displaying the TMPRSS2-ERG gene fusion to CPT but not to the Top2 inhibitor etoposide. This effect is accompanied with an increase in Top1-DNA complexes. This could be due to either an effect of ERG on DNA-PKcs/Top1 interaction, or to the transcriptional regulation of genes involved in cell response to CPT, including Top1 itself. We confirmed the latter hypothesis by showing that ERG can regulate the transcription levels of the microRNA miR-24 and that Top1 expression relies, at least in part, on miR24 levels in VCaP cells. We obtained similar results in LNCaP cells (characterized by a TMPRSS2-ETV1 gene fusion), in which ETV1 repression also sensitizes cells to CPT. In parallel, we also searched for inhibitors of DNA-PKcs/Top1 interaction in order to use these compounds to potentiate CPT derivatives in the clinic. We screened a chemical library of 320 natural compounds using the AlphaScreen technology. The results were disappointing as we only identified compounds that are catalytic inhibitors of Top1. Nevertheless, we could show that among them, mahanimbine displayed a potent cytotoxic activity towards CPT-resistant colon cancer cell lines and could efficiently inhibit the growth of VCaP cells that are highly resistant to CPT. This opens new avenues for the development of new classes of Top1 catalytic inhibitors that could be used to circumvent the clinical resistance to CPT derivatives
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Al-Shehhi, Halima. "A molecular cytogenetic investigation of secondary abnormalities and clonal evolution in ETV6-RUNX1 positive acute lymphoblastic leukaemia." Thesis, University of Newcastle upon Tyne, 2015. http://hdl.handle.net/10443/2807.
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The bacterial cell wall surrounds the cytoplasmic membrane and protects the cell against osmolysis in addition to providing shape. The cell wall is comprised of peptidoglycan, repeating units of N-acetly glucosamine and N-acetyl muramic acid form glycan strands and are crosslinked by short peptides that contain both L- and D-amino acids. Owing to the unique nature of peptidoglycan, and its absence in eukaryotic organisms, the cell wall has become an important target for many antibiotics, including the β-lactams and glycopeptides. Newly synthesised peptidoglycan contains pentapeptides, which extend from the lactyl moiety of the MurNAc sugar. These chains consist of L-alanine-D-γ- glutamate/glutamine-L-lysine/meso-diaminopimelic acid-D-alanine-D-alanine. The terminal D-alanine is often lost during cell wall maturation, either as a result of the crosslinking reaction, in which the penultimate D-alanine is attached to the side-chain of a neighbouring L-lysine or meso-diaminopimelic acid by an isopeptide bond, or as a consequence of the activities of DD-carboxypeptidases, and results in a tetrapeptide. The tetrapeptide can then be trimmed further to form a tripeptide by the action of LD-carboxypeptidases. Although many DD-carboxypeptidases have been well characterised, the majority of LD-carboxypeptidases that have been studied are active only against peptidoglycan fragments and so cannot be responsible for producing the tripeptides found in the cell wall. Of the LD-carboxypeptidases active against the mature cell wall, DacB (Streptococcus pneumoniae), Csd6 (Helicobacter pylori) and Pgp2 (Campylobacter jejuni), each has been shown to be essential in maintaining cell morphology. It should be noted, however, that neither Csd6 nor Pgp2 share any sequence similarity with DacB and belong to different peptidase families. This thesis concerns the structural and biochemical characterisation of DacB, herein renamed to LdcB (LD-carboxypeptidase B). The crystal structures of the apo form of LdcB from both S. pneumoniae and Bacillus subtilis were solved, revealing a single domain, globular protein with 2 sub-domains forming a V-shaped cleft in which the active site is located. LdcB binds one zinc ion per monomer, located at the bottom of the active site, and is a member of the LAS (lysostaphin, D-Ala-D-Ala peptidases, sonic hedgehog) family of metalloproteins. Additionally, the activity of LdcB as an LDcarboxypeptidase was confirmed and the crystal structure of LdcB from S. pneumoniae ii was solved in complex with a product mimic, M-Tri-Lys(D-Asn), revealing the molecular basis for peptidoglycan recognition in this family of enzymes. Finally, the affinity of LdcB for zinc and copper has been determined and it has been shown that catalysis is not inhibited by the substitution of zinc by copper or cobalt.
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BENEFORTI, LINDA. "Role of Bone Marrow-Mesenchymal Stromal Cells and inflammation in the pre-leukemic phase of ETV6-RUNX1-positive childhood Acute Lymphoblastic Leukemia (ALL)." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2019. http://hdl.handle.net/10281/241325.
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La traslocazione t(12;21) è il riarrangiamento cromosomico più frequente nei tumori pediatrici e si associa esclusivamente alla leucemia linfoblastica acuta (LLA) a precursori B. La traslocazione avviene in utero nelle cellule staminali-progenitrici ematopoietiche ma è insufficiente per la leuchemogenesi, poiché il gene di fusione ETV6-RUNX1 (E/R) che ne deriva genera un clone pre-leucemico clinicamente silente; mutazioni secondarie sono quindi necessarie per completare la trasformazione. Queste ultime avvengono nel periodo post-natale verosimilmente in seguito ad una risposta immunitaria disregolata a infezioni /infiammazioni comuni. Le recidive E/R+ non sono molto frequenti ma è plausibile che siano determinate dall’accumulo di nuove mutazioni nel clone pre-leucemico chemioresistente. In passato abbiamo dimostrato che TGFβ, una citochina prodotta durante l’infiammazione, limita la proliferazione delle cellule normali pro-B mentre favorisce il clone pre-leucemico che è insensibile al suo effetto; in più, TGFβ seleziona cellule pre-leucemiche staminali in progenitori CD34+ derivati da sangue cordonale umano. Abbiamo anche precedentemente dimostrato che cellule pro-B murine E/R+ mostrano alterazioni in molecole di adesione e nella migrazione verso CXCL12, suggerendo un loro possibile comportamento anomalo all’interno della nicchia midollare. Le cellule mesenchimali stromali (MSC) sono regolatori chiave sia delle cellule ematopoietiche nella nicchia che dell’infiammazione. È stato inoltre dimostrato che alterazioni delle MSC attivano pathways infiammatori nelle cellule staminali/progenitrici del sangue promuovendo la loro trasformazione a leucemia mieloide acuta in sindromi genetiche predisponenti. Grazie a due modelli cellulari esprimenti E/R (la linea cellulare murina proB Ba/F3 e cellule CD34+ di sangue cordonale umano), il presente studio dimostra che le cellule pre-leucemiche sono avvantaggiate dalla copresenza di MSC e infiammazione in termini di migrazione, sopravvivenza e potenziale progressione. Ba/F3 E/R+ mostrano un profilo di espressione genica pro-infiammatorio, con una particolare signature migratoria e pro-mieloide. Inoltre, sia Ba/F3 che CD34+ esprimenti E/R migrano di più verso i surnatanti di MSC infiammate rispetto alle cellule controllo; nel primo caso, la migrazione dipende dal recettore CXCR2. Molto importante, Ba/F3 E/R+ sono favorite rispetto al controllo quando coltivate su MSC e infiammazione, poiché questa condizione diminuisce molto proliferazione e sopravvivenza delle Ba/F3 normali mentre ha effetti minori o assenti sulle pre-leucemiche. Il vantaggio è mediato da fattori solubili, ma né TGFβ né CXCR2 sono implicati. In aggiunta, MSC e infiammazione aumentano il danno genotossico sia nelle Ba/F3 controllo che E/R+, come indicato dagli aumentati livelli di fosforilazione dell’istone H2AX e di espressione dell’enzima AID, il quale è stato dimostrato favorire la transizione da pre-leucemia a leucemia E/R+. Tuttavia, mentre le Ba/F controllo vanno incontro ad apoptosi, le pre-leucemiche resistono accumulando danni genetici che possono favorirne la trasformazione. Infine, infiammazione e MSC cooperano nel far emergere il compartimento CD34+IL7R+ all’interno della popolazione di CD34+ pre-leucemiche, mentre sfavoriscono quello della popolazione normale. Questa osservazione è particolarmente importante alla luce del fatto che tale compartimento rappresenta lo stadio dell’ematopoiesi fetale che è suscettibile all’azione pre-leucemica di E/R. Concludendo, il presente lavoro dimostra che cellule mesenchimali stromali di midollo osseo e infiammazione cooperano nel favorire la persistenza e la possibile progressione del clone pre-leucemico ETV6-RUNX1+. L’elucidazione dei meccanismi che sottendono tale azione favorente potrebbe fornire strategie innovative per l’eradicazione del clone pre-leucemico chemioresistente.Translocation t(12;21) is the most frequent chromosomal rearrangement in pediatric cancers, exclusively leading to B-cell Precursors Acute Lymphoblastic Leukemia (BCP-ALL). Translocation occurs in utero in stem-progenitor cells (HSPC) but it is insufficient for leukemogenesis, since the consequent ETV6-RUNX1 (E/R) fusion gene only generates a silent B-progenitor pre-leukemic clone; additional mutations are thus required for transformation. These latter occur in the post-natal period likely due to dysregulated immune response to common infections/inflammation. Our published data demonstrated that TGFβ, a cytokine produced during inflammation, limited the proliferation of normal pro-B and cells while favoring the insensitive E/R+ clone; moreover, TGFβ selected putative pre-leukemic stem cells (preLSC) in umbilical cord blood (UCB) CD34+ progenitors transduced with the oncogene. On the other hand, we previously showed that ETV6-RUNX1+ murine B-progenitors were altered in adhesion molecules expression and CXCL12-directed migration, suggesting possible dysregulated interactions within the bone marrow (BM) niche. Mesenchymal Stromal Cells (MSC) are key regulators of both HSPC and inflammation in the niche. Importantly, it has been shown that mesenchymal inflammation promotes secondary myeloid leukemia in predisposing syndromes by increasing DNA damage in HSPC, while MSC/BCP-ALL blasts cross-talk profoundly modifies cytokine and chemokine signalling within the niche excluding normal hematopoiesis in favor of leukemia. Taking advantages from two ETV6-RUNX1-expressing cell models (Ba/F3, a murine pro-B cell line, and ETV6-RUNX1-expressing human UCB-CD34+ progenitors) the present PhD study demonstrates that ETV6-RUNX1-expressing cells take advantage from mesenchymal inflammation in terms of migration, persistence and potential progression. In particular, we have found that pre-leukemic Ba/F3 show a peculiar pro-inflammatory gene expression profile characterized by a marked migratory and myeloid signature. Concordantly, ETV6-RUNX1+ Ba/F3 and CD34+ cells preferentially migrate toward inflamed compared to unstimulated BM-MSC supernatants; in case of the first, migration is CXCR2-dependent. Moreover, ETV6-RUNX1+ Ba/F3 are favored compared to controls in presence of BM-MSC and inflammatory cytokines, as they decrease normal cells proliferation and survival while minimally affecting pre-leukemic cells. The effect is mediated by soluble factors, but neither TGFβ nor CXCR2 axis are implicated. Importantly, the inflamed mesenchymal niche increases genotoxic stress in both control and E/R+ Ba/F3, as indicated by high levels of H2AX phoshorilation, as well as transcription of the activation-induced cytidine deaminase (AID) enzyme (which is implicated in ETV6-RUNX1+ pre-leukemia to leukemia transition). However, while control cells go through apoptosis, pre-leukemic Ba/F3 are resistant to this fail-safe mechanism, increasing chance to accumulate secondary mutations and malignantly transform. Finally, an inflamed MSC favor the emergence of CD34+ILR7+ compartment within ETV6-RUNX1+ UCB-CD34+ population while decreasing its frequency in the normal counterpart; of note, such differential effect doesn’t occur in case of unstimulated MSC. This observation is particularly important as the CD34+ILR+ compartment seems to represent the critical developmental stage during early fetal hematopoiesis for ETV6-RUNX1 pre-leukemic activity. Concluding, our work demonstrated that BM-MSC and inflammation cooperate in favoring the persistence and transformation of ETV6-RUNX1+ pre-leukemic clone. Elucidating mechanisms that underlay such promoting action could provide novel strategies for the pre-leukemic clone eradication.
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Silva, André Fernando Pessoa. "Optimização do doseamento de uma fonte de carbono a um processo de desnitrificação biológica." Master's thesis, Faculdade de Ciências e Tecnologia, 2011. http://hdl.handle.net/10362/6221.
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Dissertação para obtenção do Grau de Mestre em
Engenharia do AmbienteA presente dissertação pretendeu estudar a eficiência do Hidrolisado (efluente do tanque de Hidrólise), enquanto fonte de carbono, na melhoria do rendimento de desnitrificação numa unidade piloto que simulava, à escala laboratorial, a ETAR da ETVO. O efluente final da ETAR da ETVO,apresentava dificuldade no cumprimento das concentrações limite de alguns parâmetros de controlo, constantes na licença de descarga. Os parâmetros de controlo nos quais se identificaram incumprimentos foram os seguintes: nitritos, nitratos e azoto total. Neste contexto, pretendeu-se que o presente estudo, fosse capaz de optimizar o processo de desnitrificação, que ocorre no sistema, por recurso ao Hidrolisado, enquanto fonte de carbono a aplicar na etapa de desnitrificação. Nesta dissertação, o trabalho foi desenvolvido numa unidade piloto, que foi desenvolvida propositadamente para este trabalho e que estava instalada no laboratório de ensaios biológicos do DCTB/FCT/UNL. A unidade piloto operou em fluxo contínuo com a água residual a tratar, designada por Centrifugado, e com a fonte de carbono ensaiada (Hidrolisado). Ambas as águas residuais foram colhidas na ETVO e resultam dos processos de valorização orgânica aplicados à fracção orgânica dos Resíduos Sólidos Urbanos que são recepcionados na ETVO. A unidade piloto foi sujeita a cinco ensaios. Em cada ensaio foram testadas diferentes condições de operação da unidade piloto. Dos resultados obtidos foi possível concluir que o Hidrolisado possui propriedades que o tornam capaz de ser utilizado como fonte de carbono para a desnitrificação biológica, já que, no ensaio II, onde se registou o melhor desempenho por parte da unidade piloto, obtiveram-se elevadas remoções de nitritos, nitratos, azoto amoniacal e azoto orgânico.
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Chinabaashi, Kazuhisa. "Direct binding of Grb2 has an important role in the development of myeloproliferative disease induced by ETV6/FLT3." Kyoto University, 2013. http://hdl.handle.net/2433/174807.
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Richter, Ulf. "Entwicklung von Anwendungsmöglichkeiten der Slurry-GFAAS und Slurry-ETV-ICP-MS in der Umweltanalytik." [S.l. : s.n.], 1998. http://www.sub.uni-hamburg.de/disse/63/inhalt.html.
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Bassalert, Cécilia. "Influence des voies de signalisation IGF et MAPK sur la spécification des lignages de l'embryon de souris préimplantatoire." Thesis, Université Clermont Auvergne (2017-2020), 2018. http://www.theses.fr/2018CLFAC029.
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Au cours de la préimplantation, l'embryon de souris produit deux lignages cellulaires, le trophectoderme (TE), et la masse cellulaire interne (MCI) qui elle-même se différencie en épiblaste (Epi) et en endoderme primitif (EPr), caractérisés respectivement par l'expression exclusive de Nanog et de Gata6. La voie FGF/MAPK joue un rôle critique dans l’acquisition de l’identité EPr. J’ai examiné l’expression de pERK, DUSP4 et ETV5 qui permettent de visualiser l'activité des MAPK. Ces analyses ont été effectuées en activant ou inhibant la voie FGF/MAPK, ainsi que dans des embryons mutants pour Nanog et/ou Gata6. Ceci a permis d’observer l’activation de la voie FGF/MAPK dès E3,25. Un autre volet de mon travail a été d'analyser la voie de l’IGF dans les embryons préimplantatoires afin de comprendre l’influence de cette voie dans les différents lignages. J’ai montré que le récepteur activé pIGF1R est exprimé de manière différentielle dans le TE, l’EPr et l’Epi au cours du développement. Une supplémentation d’IGF1 induit une augmentation du nombre de cellules en deux phases, d'abord de l’Epi puis de l’EPr. A l’inverse, une perte de fonction d’IGF1R induit une diminution du nombre de cellules entre E3,75 et E4,25During preimplantation, mouse embryo produces two cellular lineages, the trophectoderm (TE), and the inner cell mass (ICM), which differentiates in epiblast (Epi) and primitive endoderm (PrE), characterized respectively by the complementary expression of Nanog and Gata6. FGF/MAPK pathway plays a critical role in the acquisition of a PrE identity. I examined the expression of the markers of MAPK activity pERK, DUSP4 and ETV5. The analyze was performed with activation or inhibition of FGF/MAPK pathway and in mutant embryos for Nanog or Gata6. This showed that FGF/MAPK pathway is activated as soon as E3,25. I have also analyzed the IGF pathway in preimplantation embryos in order to understand the role of this pathway in embryonic lineages. I showed that active receptor pIGF1R is differentially expressed in TE, PrE and Epi during embryonic development. Supplementation with IGF1 induces an increase in cell number in two phases, first in Epi then in PrE. Conversely, loss of function of IGF1R induces a decrease in cell number between E3,75 and E4,25
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Monge, i. Azemar Marta. "Caracterització del factor de transcripció ERM/ETV5 durant la infiltració miometrial i aproximacions proteòmiques al procés d'invasió en càncer d'endometri." Doctoral thesis, Universitat de Barcelona, 2009. http://hdl.handle.net/10803/1037.
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El càncer d'endometri és la malaltia ginecològica més comuna i representa la quarta neoplàsia més freqüent en la dona en els països desenvolupats. Actualment és possible diagnosticar el 80% dels casos de EEC en estadiatge I FIGO, per tant, poder aplicar cirurgia i presentar un bon pronòstic. L'estadiatge I FIGO inclou tres subtipus: afecta l'endometri (IA), infiltra el miometri <50% (IB) i >50% (IC). El nostre grup es va centrar en la identificació de nous gens implicats en el EEC mitjançant l'estudi dels patrons d'expressió gènica diferencial entre teixit endometrial sa, hiperplàsic i tumoral, així com la cerca de nous gens associats al fenotip carcinomatós endometrial amb potencial valor diagnòstic i/o pronòstic. Es va trobar que els dos gens majorment sobreexpressats eren RUNX1/AML1 i ERM/ETV5 i alhora, es troben sobreexpressats en la fase en la qual el EEC esdevé invasiu. Partint d'aquí, vam plantejar la caracterització del mecanisme d'acció del factor de transcripció ERM/ETV5 en el EEC durant els esdeveniments inicials d'invasió i disseminació.
La sobreexpressió d'ERM/ETV5 a la línia cel·lular de càncer d'endometri Hec-1A indueix dispersió cel·lular que correlaciona amb un augment de l'activitat gelatinasa de la MMP2. Tant els experiments de ChiP com amb iRNA o l'ús d'un inhibidor específic de MMP2 mostren un nexe funcional entre la sobreexpressió d'ERM/ETV5 i l'activació de MMP2. En el model animal ortotòpic de EEC es demostra que l'augment de l'activitat de MMP2 associat a la sobreexpressió d'ERM/ETV5 confereix major capacitat invasiva als tumors endometrials i aquests mostren un patró més agressiu i infiltrant. Es va confirmar que ERM/ETV5 tenia un paper en els passos inicials de la disseminació endometrial ja que es van localitzar ERM/ETV5 i MMP-2 en el front d'invasió de mostres de EEC humanes amb infiltració miometrial. Per tant podem proposar que en el EEC, ERM/ETV5 actua a través de l'activitat gelatinolítica de MMP-2 per a donar major capacitat invasiva, associat al punt d'inici de la infiltració miometrial.
Tot seguit vam plantejar determinar les alteracions moleculars associades a la sobreexpressió del factor de transcripció ERM/ETV5 durant la infiltració miometrial en el EEC, identificant nous marcadors moleculars o dianes terapèutiques que podrien estar sota el control de l'activitat transcripcional d'ERM/ETV5. L'anàlisi comparatiu mitjançant 2D-DIGE i espectrometria de masses dels patrons d'expressió proteica diferencial de la línia cel·lular que sobreexpressa de forma estable GFP-ERM/ETV5 anvers la línia cel·lular sense transfectar i la que presenta el vector GFP buit, determina un llistat de proteïnes diferencialment expressades que es troben implicades en la regulació d'actina i la senyalització per TGF-beta i progesterona. Vam caracteritzar la sobreexpressió específica de la proteïna Hep27 que presenta localització mitocondrial depenent d'ERM/ETV5. Estudis funcionals van demostrar associació amb l'estrès oxidatiu. L'anàlisi dels resultats obtinguts reforcen el paper d'ERM/ETV5 com a factor regulador de la migració i invasió tumoral, i assenyala la seva implicació en la resposta a l'estrès oxidatiu associat a l'inici de la invasió en el carcinoma endometrial. Fins ara, la immunohistoquímica ha estat l'eina utilitzada per a la identificació de proteïnes implicades en la invasió del carcinoma endometrial, sense tenir en compte la percepció global del front d'invasió. En aquest últim treball hem dut a terme una aproximació proteòmica per a caracteritzar components específics del front d'invasió o l'estroma reactiu mitjançant la comparació de l'àrea invasiva d'un carcinoma endometrial anvers l'àrea tumoral superficial no invasiva i el teixit normal provinents de la mateixa pacient. Així hem pogut identificar proteïnes diferencialment expressades en el front d'invasió i que es troben implicades en la morfologia cel·lular, acoblament i moviment, així com els mecanismes moleculars relacionats amb la senyalització i interacció cèl·lula-cèl·lula i la resposta moduladora a l'estrès oxidatiu.
We have recently described the Ets family transcription factor, ERM/ETV5, specifically up-regulated in EEC, and associated with myometrial infiltration. Ets family members have been correlated to tumor progression by up-regulating the expression of matrix-degrading proteases. We investigated the possibility that in EEC, ERM/ETV5 may induce the expression of genes involved in extra-cellular matrix remodeling.
The overexpression of ERM/ETV5 induced scattering in the EEC cell line Hec-1A, correlating to increased MMP-2 gelatinase activity. Both ChIP and iRNA experiments and specific MMP-2 inhibitor demonstrated a functional link between ERM/ETV5 overexpression and MMP-2 activation. Orthotopically implanted overexpressing ERM/ETV5 tumors presented a more aggressive and infiltrative pattern of myometrial invasion. The specific localization of ERM/ETV5 and MMP-2 at the invasive front of myometrial infiltrating human EEC reinforced the hypothesis of a role for ERM/ETV5 in the early steps of endometrial dissemination.
To understand the role of ETV5 during myometrial infiltration, we analysed by 2D-DIGE technology those proteins whose expression was altered in endometrial cell lines stably over-expressing ERM/ETV5. Pathway analysis pointed to actin regulation and TGF-beta and progesterone signalling as processes regulated by ERM/ETV5. We characterized the specific up-regulation of the nuclear dehydrogenase/reductase Hep27, its ERM/ETV5-dependent mitochondrial localization, and functional studies demonstrated a link with oxidative stress.
Overall, the ETV5-related proteomic approach performed in the Hec-1A cell line reinforces a role of this transcription factor in the regulation of the migratory and invasive tumour behaviour, and points to a modulated response to oxidative stress associated with the promotion of invasion in endometrial cancer.
To date, the identification of proteins involved in endometrial carcinoma invasion has been essentially conducted by immunohistochemical methods, without a global perception on the invasive front. In this work we attempted a proteomic approach to characterise specific components of the invasive front or reactive stroma by comparing the invasive area of an endometrial carcinoma with the non¬invasive superficial area and normal tissue from the same patients. This led us to identify proteins involved in cellular morphology, assembly and movement, differentially expressed at the invasive front, as well as pathways like cell-to-cell signalling and interaction and a modulated response to oxidative stress as events related to endometrial carcinoma invasion. In conclusion, we describe a novel proteomic approach that specifically deals with endometrial carcinoma invasion front, allowing the identification of new players of myometrial infiltration.
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Casie, Chetty David S. "Single-cell transcriptomic analysis of vascular progenitors and the roles of Vegf signaling and Ets1 in vascular development." University of Cincinnati / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ucin159326667315738.
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Martin, Matthew Joseph. "The role of the insulin-like growth factor signaling axis in ETV6-NTRK3- mediated anchorage-independent growth and transformation." Thesis, University of British Columbia, 2006. http://hdl.handle.net/2429/30949.
Full textAbstract:
The insulin-like growth factor signaling axis is an important regulator of normal cell
growth and proliferation, and is frequently dysregulated in cancer. Most dominantly
acting oncoproteins tested to date require the type-1 insulin-like growth factor receptor
(IGF-IR) for cellular transformation. ETV6-NTRK3 (EN) - the product of a
t(12;15)(p13;q25) translocation that occurs in human pediatric spindle cell sarcomas
and secretory breast carcinoma - is one such protein, as it fails to transform IGF-IR-null
fibroblasts. EN binds and tyrosine phosphorylates the insulin-receptor substrate (IRS)-1,
a major substrate of IGF-IR, which links EN to activation of the PI3K/Akt survival
pathway and the Ras/Erk proliferative cascade. Here we show that EN specifically
interacts with the phosphotyrosine-binding (PTB) domain of IRS-1, and in the absence
of IRS-1 can bind its closely related homolog IRS-2. Disruption of these EN«IRS
interactions through overexpression of the IRS-1 PTB domain, or depleting the cell of
IRS-1 and IRS-2 together, inhibit EN-induced anchorage-independent growth. Further,
we find that IGF-IR, through its ability to bind IRS molecules, serves to localize EN to
the plasma membrane, which leads to acivation of the PI3K/Akt pathway. Nontransformed
IGF-IR-null fibroblasts fail to activate this pathway in response to EN
expression when plated under anchorage-indpendent conditions, and undergo
detachment-induced cell death. Chemical inhibition of PI3K, or its downstream effector
mTOR, significantly impairs EN-mediated transformation. Finally, I demonstrate that EN
expression induces the ligand-independent tyrosine phosphorylation of IGF-IR. Both
IGF-IR IRS-binding and kinase activity are required for this phenomenon, and IGF-IR
mutants lacking either function do not display EN-mediated PI3K/Akt activation or
subsequent oncogenesis. These observations point to EN as a regulator of a novel
multicomponent membrane-localized signaling complex which potently stimulates the
PI3K/Akt survival cascade, and they suggest that blocking the formation or activity of
this complex would be a promising way to target EN-expressing tumors in vivo.Medicine, Faculty of
Medicine, Department of
Experimental Medicine, Division of
Graduate
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Gaudichon, Jérémie. "Effets de l’hypoxie sur la régulation de l’expression et la fonction de la tétraspanine CD9 dans les leucémies aiguës lymphoblastiques de l’enfant." Thesis, Rennes 1, 2018. http://www.theses.fr/2018REN1B026.
Full textAbstract:
Les leucémies aiguës lymphoblastiques (LAL) sont le cancer le plus fréquent chez l’enfant et dérivent le plus souvent de précurseurs lymphoïdes B. D’importants progrès thérapeutiques ont permis d’améliorer considérablement le pronostic. Néanmoins, 15 à 20 % des enfants rechutent encore. Ces rechutes peuvent survenir de façon isolée ou combinée dans la moelle osseuse, le site primitif des lymphoblastes, et/ou dans des organes extramédullaires tels que le testicule ou le système nerveux central. Notre équipe a montré que la protéine transmembranaire CD9 jouait un rôle majeur dans la migration des blastes dans ces sites et notamment le testicule, par l’activation de la voie RAC1 en réponse à la stimulation des cellules par le CXCL12. Ici, nous avons mis en évidence qu’un faible niveau d’oxygène, caractéristique commune aux niches médullaire et extramédullaires, régulait positivement l’expression de CD9 aux niveaux transcriptionnel et protéique, via la voie majeure de réponse à l’hypoxie, dépendante du facteur de transcription Hypoxia Inducible Factor 1a (HIF1a). Nous montrons que HIF1a se fixe directement sur le promoteur de CD9 pour induire sa transcription. Nous montrons aussi que la protéine CD9 est essentielle aux propriétés d’adhérence et de migration des blastes dans des conditions de basse oxygénation, et que son action pourrait s’exercer à travers RAC1 comme en normoxie. Nos résultats dans des expériences de xénogreffe à des souris indiquent que la voie HIF1a favorise la dissémination des blastes, possiblement à travers la régulation qu’elle exerce sur CD9. Ainsi, ce travail contribue à mieux comprendre le rôle de CD9 dans la pathogenèse des LAL de l’enfantAcute lymphoblastic leukemia (ALL) are the most frequent cancer in children and derive most often from B-cell precursors. Huge therapeutic improvements have allowed to reach high survival rates near 90% at 10 years from diagnosis. However, 15-20% of children still relapse with a significant risk of death. Relapses can occur in bone marrow and/or extramedullary sites such as testis or central nervous system, usually referred as “sanctuary sites”. Our previous work showed that the transmembrane protein CD9 plays a major role in lymphoblasts migration into these sites, especially in testis, through the activation of RAC1 signaling upon blasts stimulation with C-X-C chemokine ligand 12 (CXCl12). Here, we addressed the question of putative common factors shared by bone marrow and extramedullary niches which could upregulate CD9 expression and function. Consequently, we found that low oxygen levels could actually enhance CD9 expression both at mRNA and protein levels. We further determined that Hypoxia Inducible Factor 1a (HIF1a), the master transcription factor involved in hypoxia response, binds directly CD9 promoter to induce its transcription. We also showed that CD9 protein is crucial for leukemic cell adhesion and migration at low oxygen levels, possibly through its action on RAC1 signaling. Mouse xenograft experiments indicate that HIF1a signaling pathway favors ALL cells dissemination, which may involve CD9 as well. The present work increments our understanding of CD9 implication in ALL pathogenesis
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Palencia, Desai Sharina. "Transcriptional Regulation of Early Endocardial Development." University of Cincinnati / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1378195299.
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CAZZANIGA, VALERIA. "Impact of PAX5 alterations on gene expression and signaling pathways in ALL." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2014. http://hdl.handle.net/10281/51260.
Full textAbstract:
Recently, PAX5 has been identified as a frequent target of mutation in BCP-ALL (around 30%). Among them 25% of the cases report deletions, 7% point mutations and 2-3% translocations that juxtapose PAX5 to several partner genes.
To dissect the real frequency of PAX5 alterations in the Italian Cohort of BCP-ALL pediatric cases, we screened patients enrolled into AIEOP-IBFM-ALL2000 protocol, identifying alterations in the PAX5 locus, including deletions and translocations. To this purpose, we used different techniques in order to:
a) select the patients carrying alterations on the chromosome 9p by cytogenetic analysis (e.g. karyotype analysis);
b) describe the exact locus of the alterations and, in case of translocations, identify the partner genes;
c) detect additional events, providing further elements to characterize PAX5 alterations as driver or passenger lesions.
To study the functional properties of PAX5 fusion genes, we established an in vitro murine model of PAX5/ETV6 protein, as the most frequently reported example of PAX5 translocations. In particular, we aimed to widely characterize PAX5/ETV6 as an aberrant transcription factor, identifying the alterations induced in the gene expression profile and in the signaling pathway of primary populations of wt pre-BI cells co-cultured on OP9 BM stroma.
Finally, to investigate the potential role of PAX5 as a tumor suppressor gene in ALL, we approached to study PAX5 deletions, in a xenograft model of primary BCP-ALL cells transplanted in NSG mice. We focused the attention on the Ph+ subgroup, because of the high frequency of PAX5 deletions in BCR/ABL1 positive patients. To this purpose we used different approaches:
a) establishment of xenografts of 14 childhood BCP-ALL patients (Ph+ and Ph-, as control group);
b) ChIP sequencing in order to identify PAX5 target genes in leukemic settings;
c) analysis of the phosphorylation status;
d) generation of an in vitro inducible vector model, in order to better study the effect of PAX5 and PAX5/ETV6 in human leukemic cells.
Taken together, this study aimed to contribute to a better understanding of the role of PAX5 lesions in BCP-ALL leukemia with a comprehensive approach, from patients samples to the establishment of murine in vitro and human ex vivo models.
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Budry, Lionel. "A transcriptome analysis of mouse pituitary development: implication of Etv1 and Pax7 transcription factors in POMC transcription and cell differentiation." Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=96892.
Full textAbstract:
As the key organ in the endocrine system, the pituitary has long been the subject of intense scientific questioning. From the characterization of pituitary hormones to the understanding of the mechanisms controlling their release, the study of the pituitary yielded great discoveries, some being awarded a Nobel prize. More recently, the transcriptional control of genes encoding pituitary hormones was the object of much attention. In that context, our laboratory described the function of Tpit and NeuroD1 in the control of POMC-expressing cell differentiation and POMC cell-specific transcription and regulatory mechanisms. These two important genes do not suffice however to explain every aspect of POMC cell differentiation. We thus undertook the systematic screening of the developing and adult pituitary transcriptome in a search for novel transcriptional regulators. Using state of the art bioinformatic tools, we observed the concerted variations of biologically relevant gene groups as the organ develops and matures. Using a candidate gene approach, we identified new transcription factors expressed in POMC lineages. We describe for the first time the expression of Etv1, an Ets-domain containing factor, in the pituitary. We showed that Etv1 expression is specific to POMC cells and that it is important for activation of Pomc transcription in collaboration with Tpit and Pitx1. Furthermore, we demonstrated the melanotroph-specific expression of Pax7 in the pituitary, establishing a clear distinction between the two Pomc-expressing lineages at the transcriptional level. Using Pax7 knock-out mice, we determined the impact of this transcription factor on the genetic program of Pomc cells. Pax7 plays a major role in activating melanotroph genes and repressing corticotroph genes. In summary, our bio-informatic analysis yielded extremely relevant data with regard to pituitary development, particularly through the elucidation of key critical roles for Etv1 and Pax7 in Pomc cell biology.Organe clé du système endocrinien, l'hypophyse est depuis longtemps le centre d'un inépuisable intérêt scientifique. De la caractérisation des hormones qu'elle produit à la compréhension des mécanismes qui contrôlent leur libération, les intenses investigations dont elle a été l'objet ont par le passé fait l'objet d'un prix Nobel.Plus récemment dans ce domaine, l'attention s'est concentré sur l'étroit contrôle transcriptionnel auquel sont assujettis les gènes qui codent pour les hormones hypophysaires. Notre laboratoire a précédemment défini les rôles de Tpit et NeuroD1 dans la transcription du gène de la Pomc et dans la différenciation des cellules qui l'expriment. Ces deux régulateurs transcriptionnels n'expliquent cependant pas à eux seuls tous les mécanismes de la différentiation des cellules Pomc. Nous avons donc entrepris un crible systématique du transcriptome hypophysaire durant le développement et dans la glande adulte à la recherche de nouveaux régulateurs transcriptionnels. À l'aide d'outils bio-informatiques, nous avons observé à l'échelle génomique des variations concertées de groupes de gènes biologiquement pertinents pour le développement et la maturation de l'hypophyse. Une approche de type « gène candidat » nous a permis d'identifier de nouveaux facteurs de transcription propres aux cellules Pomc-positives. Tout d'abord, nous avons démontré pour la première fois l'expression de Etv1, un facteur à domaine Ets préférentiellement exprimé dans les cellules Pomc dans l'hypophyse. Etv1 s'est révélé être un important activateur transcriptionnel de Pomc en collaboration avec Tpit et Pitx1. Par ailleurs, nous avons démontré l'expression spécifique de Pax7 dans les cellules mélanotropes de l'hypophyse, établissant ainsi les bases transcriptionnelles de la différence entre les deux lignées qui expriment la Pomc. À l'aide de la souris inactivée pour Pax7, nous avons montré l'impact de ce facteur sur le programme génétique des cellules Pomc. Pax7 joue un rôle majeur à la fois dans l'activation des gènes mélanotropes et dans la répression des gènes corticotropes. En résumé, notre analyse bio-informatique s'est révélée riche en données extrêmement pertinentes pour la compréhension de la biologie hypophysaire, comme le montre la description du rôle de Etv1 et de Pax7 dans la biologie des cellules Pomc.
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Linka, Yvonne Verfasser], Arndt [Akademischer Betreuer] [Borkhardt, Ursula [Akademischer Betreuer] Fleig, and J. H. [Akademischer Betreuer] Hegemann. "Identifizierung von Zielgenen des chimären Transkriptionsfaktors TEL-AML1 (ETV6/RUNX1) / Yvonne Linka. Gutachter: Arndt Borkhardt ; Ursula Fleig ; J. H. Hegemann." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2013. http://d-nb.info/1035274124/34.
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Bokemeyer, Almut [Verfasser]. "Häufigkeit und prognostische Relevanz von zusätzlichen genetischen Veränderungen bei ETV6/RUNX1-positiven Rezidiven der akuten lymphoblastischen Leukämie im Kindesalter / Almut Bokemeyer." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2013. http://d-nb.info/104294069X/34.
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Hoffmann, Jana [Verfasser]. "Der genomische Bruchpunkt der ETV6-RUNX1 Fusion als Marker zum Response Monitoring bei der akuten lymphoblastischen Leukämie im Kindesalter / Jana Hoffmann." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2021. http://d-nb.info/1241541906/34.
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Noack, Ivo [Verfasser]. "Induzierbare ETV6-RUNX1 Repression im humanen Zellmodell akuter lymphoblastischer Leukämie : Auswirkungen auf das Wachstumsverhalten und den JAK-STAT Signalweg / Ivo Noack." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2021. http://d-nb.info/1241541280/34.
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Senger, Toralf. "Untersuchungen zur Metallhomöostase in Arabidopsis thaliana." Phd thesis, Universität Potsdam, 2007. http://opus.kobv.de/ubp/volltexte/2007/1323/.
Full textAbstract:
Alle Organismen sind für ihr Überleben auf Metalle angewiesen. Hierbei gibt es für jedes Metall einen Konzentrationsbereich, der das Optimum zwischen Metallmangel, -bedarf und -toxizität darstellt. Es gilt mittlerweile als erwiesen, dass alle Organismen zur Aufrechterhaltung des Metallgleichgewichts ein komplexes Netzwerk von Proteinen und niedermolekularen Verbindungen entwickelt haben. Die molekularen Komponenten dieses Netzwerks sind nur zu einem Teil bekannt und charakterisiert: In den letzten Jahren wurden einige Proteinfamilien identifiziert, deren Mitglieder Metalle durch Lipidmembranen transportieren. Eine dieser Metalltransporterfamilien ist die Cation Diffusion Facilitator (CDF)-Familie: Alle charakterisierten Mitglieder exportieren Metalle aus dem Zytoplasma – entweder in zelluläre Kompartimente oder aus der Zelle heraus. Von den zwölf Mitgliedern dieser Familie in Arabidopsis thaliana (A. thaliana) – Metall Toleranz Protein (MTP)-1 bis -12 – wurden bisher AtMTP1 und AtMTP3 charakterisiert.
In dieser Arbeit wird die Charakterisierung von AtMTP2 beschrieben. Wie die homologen Proteine AtMTP1 und AtMTP3 führt AtMTP2 zu Zn-Toleranz, wenn es heterolog in Zn-sensitiven Hefemutanten exprimiert wird. Mit AtMTP2 transformierte Hefemutanten zeigten darüber hinaus erhöhte Co-Toleranz. Expression von chimären AtMTP2/GFP Fusionsproteinen in Hefe, A.thaliana protoplasten und in stabil transformierten A.thalinana Planzenlinien deutet auf Lokalisation of AtMTP2 in Membranen des Endoplasmatischen Retikulums (ER) hin, wenn GFP an den C-Terminus von MTP2 fusioniert wird. Fusion of GFP an den N-Terminus von AtMTP2 führte zu Lokalisation in der vakuolären Membran, was wahrscheinlichsten auf Fehllokalisierung durch Maskierung eines ER-Retentionsmotivs (XXRR) am N-Terminus von AtMTP2 zurückgeht. Dies legt nahe, dass AtMTP2 die erwähnten Metalle in das Endomembransystem der Zelle transportieren kann. Eine gewebespezifische Lokalisierung wurde mit Pflanzen durchgeführt, die das β-Glucuronidase (GUS)-Reporterprotein bzw. chimäre Fusionsproteine aus EGFP und AtMTP2 unter Kontrolle des nativen pMTP2-Promotors exprimierten. Diese Experimente bestätigten zum einen, dass der pMTP2-Promotor nur unter Zn-Defizienz aktiv ist. GUS-Aktivität wurde unter diesen Bedingungen in zwei Zonen der Wurzelspitze beobachtet: in den isodiametrischen Zellen der meristematischen Zone und in der beginnenden Wurzelhaarzone. Darüber hinaus konnte gezeigt werden, dass die EGFP-Fusionsproteine unter Kontrolle des nativen pMTP2-Promotors nur in epidermalen Zellen exprimiert werden. Für eine homozygote Knockout- Linie, mtp2-S3, konnte bisher kein eindeutiger Phänotyp identifiziert werden. Auf Grundlage der bisher durchgeführten Charakterisierung von AtMTP2 erscheinen zwei Modelle der Funktion von AtMTP2 in der Pflanze möglich: AtMTP2 könnte essentiell für die Versorgung des ER mit Zn unter Zn-Mangelbedingungen sein. Hierfür spricht, dass AtMTP2 in jungen, teilungsaktiven und damit Zn-benötigenden Wurzelzonen exprimiert wird. Die auf die Epidermis beschränkte Lokalisation könnte bei diesem Modell auf die Möglichkeit der zwischenzellulären Zn-Verteilung innerhalb des ER über Desmotubules hindeuten. Alternativ könnte AtMTP2 eine Funktion bei der Detoxifizierung von Zn unter Zn-Schock Bedingungen haben: Es ist bekannt, dass unter Zn- Mangelbedingungen die Expression der zellulären Zn-Aufnahmesysteme hochreguliert wird. Wenn nun die Zn-Verfügbarkeit im Boden z. B durch eine pH-Änderung innerhalb kurzer Zeit stark ansteigt, besteht die Notwendigkeit der Entgiftung von Zn innerhalb der Zelle, bis der starke Einstrom von Zn ins Zytoplasma durch die Deaktivierung der Zn-Aufnahmesysteme und einer geringeren Expression in der Pflanze gedrosselt ist. Ein ähnlicher Mechanismus wurde in der Bäckerhefe S. cerevisae beschrieben, in der darüber hinaus ein Zn-Transporter verstärkt exprimiert wird, der Zn durch Transport in die Vakuole entgiften kann. Es ist durchaus möglich, dass in Arabidopsis AtMTP2 die Zn-Detoxifizierung unter diesen speziellen Bedingungen durch Zn-Transport in das ER oder die Vakuole vermittelt.
Zur Identifikation weiterer Komponenten des Metallhomöostasenetzwerks sind verschiedene Ansätze denkbar. In dieser Arbeit wurde in Hefe ein heterologer Screen durchgeführt, um Interaktoren für vier Mitglieder der Arabidopsis-CDF-Familie zu identifizieren. Unter den 11 im Hefesystem bestätigten Kandidaten befindet sich mit AtSPL1 ein AtMTP1-Interaktionskandidat, der möglicherweise eine Rolle bei der Cu-,Zn-Homöostase spielt. Als wahrscheinliche AtMTP3-Interaktionskandidaten wurde die c”-Untereinheit der vakuolären H+-ATPase AtVHA identifiziert sowie mit AtNPSN13 ein Protein, das vermutlich eine Rolle bei Fusionen von Vesikeln mit Zielmembranen spielt.
Ein anderer Ansatz zur Identifikation neuer Metallhomöostasegene ist die vergleichende Elementanalyse von natürlichen oder mutagenisierten Pflanzenpopulationen. Voraussetzung für diesen Ansatz ist die schnelle und genaue Analyse des Elementgehalts von Pflanzen. Eine etablierte Methode zur simultanen Bestimmung von bis zu 65 Elementen in einer Probe ist die Inductively Coupled Plasma Optical Emission Spectrometry (ICP OES). Der limitierende Faktor für einen hohen Probendurchsatz ist die Notwendigkeit, Proben für die Analyse zu verflüssigen. Eine alternative Methode der Probenzuführung zum Analysegerät ist die elektrothermale Verdampfung (ETV) der Probe. Zur weitgehend automatisierten Analyse von Pflanzenmaterial mit minimiertem Arbeitsaufwand wurde eine Methode entwickelt, die auf der Kopplung der ETV mit der ICP OES basiert.All organisms require for their survival essential metals. For each required metal exists an optimal concentration between metal deficiency and -toxicity. It has become evident that all organisms developed a complex network of proteins and low molecular compounds to maintain the equilibrium between all metals. Only few molecular components of this metal-homeostasis network are characterized in detail: A number of protein families whose members transport metals over the barrier of lipid-membranes have been identified during the last couple of years. One of those metal-transport families is the Cation Diffusion Facilitator (CDF) family. All characterized members export metals from the cytoplasm – either into cellular compartments or outside the cell. From the 12 Arabidopsis thaliana (A.thaliana) members – Metal Tolerance Protein (MTP)-1 to 1-2 – only MTP1 and MTP3 have been characterized yet. In this work, characterization of MTP2 is described. As was found for the homologous proteins AtMTP1 and AtMTP3, heterologous expression of AtMTP2 in Zn-sensitive yeast mutants leads to enhanced Zn-tolerance. Less pronounced, enhanced tolerance was also found for Co when AtMTP2 was expressed in Co sensitive yeast mutants. Expression of chimeric AtMTP2/GFP fusion proteins in yeast, A.thaliana protoplasts and in stably transformed A.thalinana plant lines indicated localization of MTP2 in membranes of the endoplasmic reticulum, when GFP was fused to the C-terminal end of MTP2. Fusion of GFP to the N-terminal end of MTP2 lead to vacuolar localization that is most likely explained as mistargeting due to masking of an ER retrieval motive (XXRR) found at the N-terminus of MTP2. This suggests that AtMTP2 mediates the transport of Zn and Co into the endomembrane system of the cell. Tissue specific localization was performed with plant lines expressing the β-Glucuronidase (GUS) reporter protein and with plant lines expressing chimeric fusions of GFP with AtMTP2 under control of the native pMTP2 promoter. Those experiments confirmed Affymetrix Genechip® data suggesting activity of the pMTP2 promoter only under Zn-deficiency. GUS activity was only found under Zn-deficiency in two zone of root tips – the meristematic zone, characterized by isodiamtric cells, and in the beginning differentiation zone, characterized by appearing root hairs. Confocal microscopy with plant lines expressing chimeric MTP2 /GFP fusions demonstrated that expression of AtMTP2 is restricted to epidermal cells. A phenotype for the homozygous mtp2-S3 knockout mutant could not be identified yet. Based on the data obtained as yet
two mode of action of AtMTP2 in planta seam likely: AtMTP2 could be essential for delivery of Zn to the ER under Zn-deficiency. This is supported by the fact, that AtMTP2 is active in young, dividing (and therefore Zn-requiring) zones of the root. The epidermal-restricted expression of AtMTP2 points towards a distribution of Zn in these root zones of Zn within desmotubules. Alternatively, AtMTP2 could have a Zn-detoxifying function under Zn-shock. It is known that in yeast under Zn-deficiency not only the expression of an Zn-uptake transporter is up-regulated, but also the expression of a vacuolar Zn-transporter. It mediates Zn-detoxification of surplus Zn that enters cells upon Zn-resupply before shut down of the Zn uptake system. AtMTP2 could exert this function when soil Zn-availability raises suddenly, for example due to rain after a drought. Different means/methods are perceivable to identify further components of the metal homeostasis network. In this work, a heterologuos screen was performed in yeast to identify interacting proteins for four members of the Arabidopsis CDF-family. Among 11 candidates identified and confirmed in the Split Ubiquitin System (SUS, a Yeast-2-Hybrid variant) is with AtSPL1 an AtMTP1 interaction candidate, which plays putatively a role in Zn,Cu homoestasis. The c” subunit of the vacuolar H+-ATPase AtVHA was found as likely AtMTP3-interaction candidate, as well as AtNPSN13, an protein that plays putatively a role in fusion of vesicles with target-membranes. Another method to identify new metal homeostasis genes is the comparative elemental analysis of natural and mutagenized plant populations. Prerequisite for this approach is the fast and accurate analysis of the elemental composition of plants. An established method for elemental analysis is Inductively Coupled Plasma Optical Emission Spectrometry (ICP OES). The limiting factor for high thoughput is the requirement for laborious wet digest of plant samples before analysis. An alternative mean of sample delivery to the ICP OES is electrothermal vaporization (ETV). For faster, less laborious analysis of plant material, a method based on the established coupling of ETV with ICP OES was developed, which is optimized for plant material and automated as far as possible.
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Planagumà, i. Valls Jesús. "Anàlisi de l'expressió gènica diferencial en el càncer d'endometri." Doctoral thesis, Universitat Autònoma de Barcelona, 2006. http://hdl.handle.net/10803/3551.
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Lu, Karyn Y. "Interaction Design Principles for Interactive Television." Thesis, Georgia Institute of Technology, 2005. http://hdl.handle.net/1853/6962.
Full textAbstract:
Interactive television (iTV) is an umbrella term used to cover the convergence of television with digital media technologies such as computers, personal video recorders, game consoles, and mobile devices, enabling user interactivity. Increasingly, viewers are moving away from a "lean back" model of viewing to a more active "lean forward" one. When fully realized on a widespread scale in the United States, our current experience of watching television will be dramatically transformed. Because iTV is a new medium in its own right, however, standards for iTV programming and interaction in the United States remain undefined.
This document identifies and articulates interaction design principles for interactive television programming in the United States. Chapter one presents a brief survey of the field as it stands in 2005. In chapters two and three, I categorize iTV by platforms and by persistent television genres, and present representative examples for each category. In chapter four, I provide an overview of existing design standards in related areas. Insights from chapters two, three, and four all serve to inform chapter five, in which I propose principles for iTV interaction design by looking closely at existing designs (both deployed and prototyped), conventions, and patterns of interaction. My analyses are rooted in visual culture and human-computer interaction design principles, and the design principles I offer are abstracted from the applications I analyze within this framework. Finally, in chapter six, I offer some conclusions and thoughts for future directions.
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Silva, Alessandra Furtado da. "Desenvolvimento de métodos para a determinação de mercúrio e tálio em amostras ambientais usando GF AAS e ETV-ICO-MS." Florianópolis, SC, 2004. http://repositorio.ufsc.br/xmlui/handle/123456789/87275.
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Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Físicas e Matemáticas. Programa de Pós-Graduação em Química.Made available in DSpace on 2012-10-21T17:35:42Z (GMT). No. of bitstreams: 1 204136.pdf: 947494 bytes, checksum: 8d45a223926fb8b99323ef00e885e939 (MD5)
Métodos para a determinação de mercúrio e tálio em amostras ambientais por GF AAS e ETV-ICP-MS foram desenvolvidos, usando a análise direta de sólidos e suspensão. Foram investigados modificadores permanentes como irídio, paládio, ródio e rutênio para a determinação de mercúrio em materiais certificados: cinza, esgoto, sedimento marinho e sedimento de rio, usando a GF AAS com amostragem sólida. O desempenho do paládio foi o mais consistente, tendo a massa característica como o maior critério. Uma quantidade de mercúrio foi perdida da solução aquosa durante a etapa de secagem, e essa perda pode ser contornada após a adição de permanganato de potássio, que finalmente tornou possível o uso de calibração com padrões aquosos para a análise direta de sólidos. Os resultados obtidos para mercúrio foram satisfatórios para um procedimento de rotina, apresentando um limite de detecção de 0,2 mg kg-1. Foi proposto também um método para a determinação de mercúrio e tálio em amostras ambientais usando a ETV-ICP-MS com amostragem por suspensão. O permanganato de potássio em solução, bem como o potássio e o manganês em solução, foram estudados como modificadores e carreadores. A melhor sensibilidade e estabilização térmica foram obtidas com permanganato de potássio em um tubo não tratado, especialmente para tálio. A exatidão do método foi confirmada pela análise de oito materiais certificados, usando calibração externa com padrões aquosos preparados da mesma maneira que as suspensões. Os limites de detecção nas amostras foram 0,18 mg g-1 para Hg e 0,07 mg g-1 para tálio. A precisão encontrada para as diferentes amostras, como desvio padrão relativo, foi de 0,8-11 % para mercúrio e 1-9% para tálio (n = 3). O último desenvolvimento de método foi a determinação de tálio usando a GF AAS de alta resolução com fonte contínua. Os resultados obtidos sem modificador, com paládio adicionado em solução e rutênio como modificador permanente, para 11 amostras de carvão e uma de cinza de carvão foram concordantes, com um nível de confiança de 95%, usando calibração com padrões aquosos. A precisão, como desvio padrão relativo, foi melhor que 5% e o limite de detecção foi de 0,01 mg g-1 de tálio.
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FRASSINE, ANDREA. "ZEBRAFISH AS A MODEL TO STUDY THE ROLE OF PAX5/ETV6 FUSION GENE IN ACUTE LYMPHOBLASTIC LEUKEMIA AND THE FUNCTION OF THE SINGLE WILD TYPE GENES IN NORMAL HEMATOPOIESIS." Doctoral thesis, Università degli Studi di Milano, 2016. http://hdl.handle.net/2434/412959.
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B cell precursors Acute Lymphoblastic Leukemia (BCP-ALL) is a tumor characterized by the malignant expansion of B cell progenitors and it is the most common childhood cancer. About 30% of BCP-ALL cases are characterized by mutations, deletions or translocations of the PAX5 gene. Among PAX5 translocations, the most recurrent is t(9;12) which determines the generation of PAX5/ETV6 fusion gene. The PAX5 transcription factor is essential for B cell lineage commitment and differentiation. PAX5 fulfills its function mainly through the activation of B lineage-specific genes and the repression of inappropriate ones. ETV6 transcription factor is specifically required for adult hematopoiesis in mice. Moreover, ETV6 accelerates erythroid differentiation of cell lines and it stimulates hemoglobin synthesis in adult mice. PAX5/ETV6 is able to down-regulate the expression of some PAX5-activated-genes, as well as to up-regulate some PAX5-repressed-genes. Accordingly, PAX5/ETV6 caused the alteration of B cell differentiation and conferred survival advantages to mouse B cell precursors in vitro. The acquisition of survival advantages by PAX5/ETV6 expressing cells is due to the activation of the Lck-STAT5 pathway leading to the upregulation of STAT5 effectors Ccnd2 and cMyc. When we started this project very little was known about the conservation of pax5 and etv6 roles in zebrafish hematopoiesis. We thus analyzed their expression pattern and we found that these genes are expressed in several hematopoietic tissues. Indeed, pax5 is expressed in pancreas, which has been proposed to be a transient embryonic site of B cell lymphopoiesis in zebrafish. pax5 knockdown caused the alteration of the expression of several B cell markers. Otherwise, etv6 is expressed in tissues related to both primitive and definitive hematopoiesis. etv6 loss of function determined the reduction of primitive mature erythrocytes and we found that this alteration is due to the defective differentiation of primitive erythrocytes. Moreover, we tried to shed light into the mechanism through which etv6 regulates primitive erythrocytes maturation. Interestingly, we found that etv6 is a potential modulator of Notch signaling in primitive erythrocytes. Furthermore, etv6 is involved, directly or indirectly, in the repression of klf1, klf3, klf6a and klf17 genes, which are essential for primitive erythrocytes maturation. Overall, our findings suggest that the roles of pax5 and etv6 in hematopoiesis could be, at least in part, conserved in zebrafish. Interestingly, etv6 knockdown caused the alteration of the expression of some B cell markers which are impaired also by pax5 loss of function, thus, we investigated if the contemporary pax5 and etv6 knock-down could cooperate to alter the expression of these genes. However, the coinjection of pax5 and etv6 morpholinos did not cause significant alteration of igh and cd79a, thus excluding a possible cooperation between pax5 and etv6 in the regulation of their expression. Finally, we transiently expressed PAX5/ETV6 in zebrafish embryos and, despite it did not caused the deregulation of B cell markers expression, this aberrant gene is able to activate the same pathway which is altered in vitro, inducing the phosphorylation of Lck and Stat5 proteins as well as the upregulation of Stat5 effectors, suggesting that zebrafish could be a suitable model to study BCP-ALL related to PAX5/ETV6 fusion gene.