Skewed somatic X inactivation (XCI), X-linked gene overexpression and abnormal X content have been associated with breast and ovarian cancer. Partial or complete reactivation of the inactive X in females may be a step in breast and ovarian cancer progression, leading to overexpression of some tumour enhancing gene. Markers of an X reactivation event were examined: X gene dosage, expression, and methylation in 8 ovarian cancer cell lines. Another marker of an X reactivation event, skewed XCI, was assayed in peripheral blood DNA from 106 breast and/or ovarian cancer patients (52 BRCA1 mutation carriers, 24 BRCA2 mutation carriers, 30 non-mutation carriers), 147 age-matched population controls. Combined RNA/DNA FISH was used to quantify the number of inactive Xs compared to total number of Xs. Five cell lines had increased X content. Three cell lines localized XIST to the presumptive inactive X; however the numbers of inactive Xs were variable. Expression levels of 8 X-linked genes were assessed by real-time PCR. Expression was inconsistent between different genes and among cell lines, ranging from a 2 to 300-fold increase compared to a control. Overall, expression was greatly increased for genes subject to inactivation but not increased in genes that escape inactivation for most ovarian cancer cell lines. Methylation at AR and FMR1 was quantified by a real-time PCR based assay and SNuPE respectively. Methylation was lower than expected for 7 of 8 ovarian cancer cell lines at AR or FMR1, while three cell lines had low or no methylation for both genes. Skewed XCI was evaluated using a methylation-based PCR assay at AR. There was no significant increase in skewing above 90% for any cancer group assayed. In addition, two markers of X reactivation were assayed in two low passage cultures of normal ovarian surface epithelium from BRCA1 mutation positive breast cancer patients. One sample did not localize XIST to the inactive X and three of five genes subject to inactivation were overexpressed. In summary, there is evidence for loss of X silencing or gain of active X content in ovarian cancer cell lines and normal ovarian surface epithelium from BRCA1 mutation carriers.

Skewed somatic X inactivation (XCI), X-linked gene overexpression and abnormal X content have been associated with breast and ovarian cancer. Partial or complete reactivation of the inactive X in females may be a step in breast and ovarian cancer progression, leading to overexpression of some tumour enhancing gene. Markers of an X reactivation event were examined: X gene dosage, expression, and methylation in 8 ovarian cancer cell lines. Another marker of an X reactivation event, skewed XCI, was assayed in peripheral blood DNA from 106 breast and/or ovarian cancer patients (52 BRCA1 mutation carriers, 24 BRCA2 mutation carriers, 30 non-mutation carriers), 147 age-matched population controls. Combined RNA/DNA FISH was used to quantify the number of inactive Xs compared to total number of Xs. Five cell lines had increased X content. Three cell lines localized XIST to the presumptive inactive X; however the numbers of inactive Xs were variable. Expression levels of 8 X-linked genes were assessed by real-time PCR. Expression was inconsistent between different genes and among cell lines, ranging from a 2 to 300-fold increase compared to a control. Overall, expression was greatly increased for genes subject to inactivation but not increased in genes that escape inactivation for most ovarian cancer cell lines. Methylation at AR and FMR1 was quantified by a real-time PCR based assay and SNuPE respectively. Methylation was lower than expected for 7 of 8 ovarian cancer cell lines at AR or FMR1, while three cell lines had low or no methylation for both genes. Skewed XCI was evaluated using a methylation-based PCR assay at AR. There was no significant increase in skewing above 90% for any cancer group assayed. In addition, two markers of X reactivation were assayed in two low passage cultures of normal ovarian surface epithelium from BRCA1 mutation positive breast cancer patients. One sample did not localize XIST to the inactive X and three of five genes subject to inactivation were overexpressed. In summary, there is evidence for loss of X silencing or gain of active X content in ovarian cancer cell lines and normal ovarian surface epithelium from BRCA1 mutation carriers.

Resumo: O câncer de ovário apresenta diagnóstico tardio e alta letalidade, devido à falta de biomarcadores sensíveis e específicos e à rápida progressão desse câncer, assintomático em estadios iniciais. As células imunes têm a capacidade de eliminar as células malignas e regular a progressão tumoral. Contudo, as células imunes do microambiente tumoral são disfuncionais e falham no controle da expansão tumoral podendo, inclusive, promover o crescimento da neoplasia. Apesar das inúmeras tentativas de se correlacionar o grau e o tipo de infiltrado celular com o prognóstico ou sobrevida do paciente com câncer de ovário, não há consenso sobre o real significado do infiltrado leucocitário nesses casos. Desse modo, este estudo tem como objetivo a ampliação dos conhecimentos relativos à imunidade inata em mulheres com câncer de ovário, através caracterização dos aspectos fenotípicos celulares da imunidade inata sérica. Trata-se de estudo transversal onde foram avaliadas 36 mulheres submetidas a exame clínico, ginecológico, ultrassonografia transvaginal e tratamento com laparotomia nos casos indicados de massa pélvica. De acordo com o resultado desses exames, as mulheres selecionadas foram agrupadas nos grupos: controle- ausência de neoplasia, com neoplasia ovariana benigna e neoplasia ovariana maligna. Realizou-se dosagem sérica de moléculas de expressão de superfície de células da resposta imune inata com análise através da citometria de fluxo. As diferenças entre os grupos foram avaliadas pelo teste de Mann-Whitney (dois grupos) ou Kruskal-Walis (três grupos) conforme indicados. As diferenças com valor de p<0,05 foram consideradas significativas. Foram selecionadas 36 pacientes: 10 mulheres no grupo controle, 9 no grupo de neoplasia ovariana benigna e 17 no grupo de neoplasia maligna. Mais de 70% das pacientes com câncer de ovário apresentavam-se com doença... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Introduction: Ovarian cancer presents late diagnosis and high mortality due to lack of sensitive and specific biomarkers and the rapid progression of this cancer, asymptomatic in early stages. Immune cells have the ability to eliminate malignant cells and regulate tumor progression. However, the immune cells of the tumor microenvironment are dysfunctional and fail to control the tumor growth and may even promote the growth of cancer. Despite numerous attempts to correlate the degree and type of cellular infiltrate and the prognosis or survival of patients with ovarian cancer, there is no consensus about the real meaning of the leukocyte infiltrate in these cases. Thus, this study aims to increase knowledge about the innate immunity in women with ovarian cancer through characterization of phenotypic cellular aspects of innate immunity levels. Methods: This is a Cross-sectional study evaluated 36 women who underwent clinical examination, gynecological examination, transvaginal ultrasound and treatment with laparotomy as indicated pelvic mass. According to the results of these tests, the women selected were grouped into two groups: control, absence of malignancy, with benign ovarian neoplasm and malignant ovarian neoplasm. The serum levels of serum molecules surface expression of cells of the innate immune response with analysis by flow cytometry. Differences between groups were evaluated by the Mann-Whitney (two groups) or Kruskal-Wallis (three groups) as indicated. Differences with p <0.05 were considered significant. Results: We selected 36 patients: 10 women in the control group, 9 in the group of benign ovarian neoplasm and 17 in the group of malignancy. More than 70% of patients with ovarian cancer presented with advanced disease and values of CA125 much changed. For the analysis found, there was a change between the groups for molecules expression in neutrophils... (Complete abstract click electronic access below) / Orientador: Agnaldo Lopes da Silva Filho / Coorientador: Andréa Teixeira de Carvalho / Banca: Paulo Traiman / Banca: Luciana Maria da Silva / Mestre

Made available in DSpace on 2014-06-11T19:29:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-26Bitstream added on 2014-06-13T18:59:45Z : No. of bitstreams: 1 paula_soc_me_botfm.pdf: 316334 bytes, checksum: 6b85b467e2fa0ecca5eec101c94687cd (MD5) / O câncer de ovário apresenta diagnóstico tardio e alta letalidade, devido à falta de biomarcadores sensíveis e específicos e à rápida progressão desse câncer, assintomático em estadios iniciais. As células imunes têm a capacidade de eliminar as células malignas e regular a progressão tumoral. Contudo, as células imunes do microambiente tumoral são disfuncionais e falham no controle da expansão tumoral podendo, inclusive, promover o crescimento da neoplasia. Apesar das inúmeras tentativas de se correlacionar o grau e o tipo de infiltrado celular com o prognóstico ou sobrevida do paciente com câncer de ovário, não há consenso sobre o real significado do infiltrado leucocitário nesses casos. Desse modo, este estudo tem como objetivo a ampliação dos conhecimentos relativos à imunidade inata em mulheres com câncer de ovário, através caracterização dos aspectos fenotípicos celulares da imunidade inata sérica. Trata-se de estudo transversal onde foram avaliadas 36 mulheres submetidas a exame clínico, ginecológico, ultrassonografia transvaginal e tratamento com laparotomia nos casos indicados de massa pélvica. De acordo com o resultado desses exames, as mulheres selecionadas foram agrupadas nos grupos: controle- ausência de neoplasia, com neoplasia ovariana benigna e neoplasia ovariana maligna. Realizou-se dosagem sérica de moléculas de expressão de superfície de células da resposta imune inata com análise através da citometria de fluxo. As diferenças entre os grupos foram avaliadas pelo teste de Mann-Whitney (dois grupos) ou Kruskal-Walis (três grupos) conforme indicados. As diferenças com valor de p<0,05 foram consideradas significativas. Foram selecionadas 36 pacientes: 10 mulheres no grupo controle, 9 no grupo de neoplasia ovariana benigna e 17 no grupo de neoplasia maligna. Mais de 70% das pacientes com câncer de ovário apresentavam-se com doença... / Introduction: Ovarian cancer presents late diagnosis and high mortality due to lack of sensitive and specific biomarkers and the rapid progression of this cancer, asymptomatic in early stages. Immune cells have the ability to eliminate malignant cells and regulate tumor progression. However, the immune cells of the tumor microenvironment are dysfunctional and fail to control the tumor growth and may even promote the growth of cancer. Despite numerous attempts to correlate the degree and type of cellular infiltrate and the prognosis or survival of patients with ovarian cancer, there is no consensus about the real meaning of the leukocyte infiltrate in these cases. Thus, this study aims to increase knowledge about the innate immunity in women with ovarian cancer through characterization of phenotypic cellular aspects of innate immunity levels. Methods: This is a Cross-sectional study evaluated 36 women who underwent clinical examination, gynecological examination, transvaginal ultrasound and treatment with laparotomy as indicated pelvic mass. According to the results of these tests, the women selected were grouped into two groups: control, absence of malignancy, with benign ovarian neoplasm and malignant ovarian neoplasm. The serum levels of serum molecules surface expression of cells of the innate immune response with analysis by flow cytometry. Differences between groups were evaluated by the Mann-Whitney (two groups) or Kruskal-Wallis (three groups) as indicated. Differences with p <0.05 were considered significant. Results: We selected 36 patients: 10 women in the control group, 9 in the group of benign ovarian neoplasm and 17 in the group of malignancy. More than 70% of patients with ovarian cancer presented with advanced disease and values of CA125 much changed. For the analysis found, there was a change between the groups for molecules expression in neutrophils... (Complete abstract click electronic access below)

Orientador: André Almeida Schenka / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-17T05:00:04Z (GMT). No. of bitstreams: 1 Nicolosi_JacquelineSpacagna_M.pdf: 1558921 bytes, checksum: 35ee58bf52c7629b47f27930a374f9fa (MD5) Previous issue date: 2010 / Resumo: Em neoplasias malignas, a angiogênese parece favorecer não só o crescimento celular como a disseminação sistêmica, tendo, potencialmente, valor diagnóstico, prognóstico e para o desenvolvimento de novas estratégias terapêuticas. Contudo, que este papel é variável de um tumor para outro, o que provavelmente reflete diferenças biológicas entre as neoplasias e limitações ou variações metodológicas dos estudos realizados. Nas neoplasias ovarianas, devido à escassez de trabalhos e grande variabilidade metodológica destes, o papel da angiogênese ainda não está estabelecido. Objetivos: (1) avaliar a influência de diferentes variáveis metodológicas na quantificação da angiogênese em tumores ovarianos; (2) estabelecer funções matemáticas para a conversão de resultados obtidos com variantes metodológicas. Metodologia: em tumores ovarianos diagnosticados no CAISM-Unicamp entre 1997 a 2003, a vascularização identificada por meio imunocoloração foi quantificada através da determinação de densidade microvascular e área endotelial. Foram avaliadas comparativamente as variantes metodológicas: formato de imagem (TIFF vs. JPEG) e número de campos de análise (1, 3, 5, 10, 15 e 20 campos de médio aumento) Resultados e conclusões: O impacto do formato de imagem sobre a quantificação vascular em tumores epiteliais ovarianos pode ser considerada pequena, já que a concordância entre imagens TIFF e JPEG é elevada (ICC>0,75). Constitui exceção o contraste TIFF vs. JPEGs de baixa qualidade, na variável densidade microvascular automática. Nesse caso, a interconversão de valores pode ser realizada através de funções de correção. Finalmente, no universo amostral avaliado, não foi possível caracterizar uma curva com platô de estabilização que permitisse a estimativa de N mínimo de campos capaz de garantir uma análise fidedigna de angiogênese em tumores ovarianos epiteliais / Abstract: In malignant neoplasms, angiogenesis seems to facilitate tumour growth and metastasis, thus, bearing a potential role in diagnostic/prognostic assessment, as well as in the development of new therapeutic strategies. However, the relative importance of such a role is variable from one entity to another, probably reflecting biological differences between tumours, as well as limitations or methodological variations among studies. In ovarian neoplasms, due to paucity of studies and great methodological variability among them, the role of angiogenesis is yet to be established. The present study aims: (1) to evaluate the influence of different methodological variables on angiogenesis quantitation in ovarian tumours and (2) to establish, by linear regression, functions that may be used to interconvert values obtained from different methodological variants. Methods: using ovarian tumours diagnosed in our Academic Women's Hospital (CAISM-Unicamp) from 1997-2003, tumour-related vascularity was detected by CD34 immunostaining and quantified by assessment of microvessel density and endothelial. Furthermore, the following methodological variants were tested: image file format (TIFF vs. JPEG), and total number of analyzed fields (1, 3, 5, 10 and 20 medium-power fields). Results and conclusions: the impact of image format over vascular quantitation was considered small, since the agreement between TIFF and JPEG was found to be high (ICC>0,75). The only exception was represented by the contrast TIFF vs. low quality JPEGs in automatic microvessel density assessments. Even in these cases, interconversion could be achieved using prediction models developed in this study. As for the second objective of the study, given the sampling universe used, we could not properly characterize a stabilization plateau curve which could allow for extrapolation of the minimum number of analysis fields (as required for an accurate morphometric analysis of angiogenesis in ovarian epithelial tumors) / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

Orientador: Carmen Silvia Passos Lima / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-12T08:37:32Z (GMT). No. of bitstreams: 1 Sagarra_ReginaAparecidaMartinho_D.pdf: 4367587 bytes, checksum: 8cb3760d80526b8901dd10650326920b (MD5) Previous issue date: 2008 / Resumo: Já é conhecida a dependência do crescimento, invasão e a disseminação de tumores sólidos na angiogênese, incluindo o carcinoma de ovário (CO). O CO está associado à produção de proteínas estimuladoras da angiogênese, como o fator de crescimento endotelial vascular (VEGF), produto do gene VEGF, e inibidoras da angiogênese, como a endostatina, produto do gene COL18A1. O CO está também associado à exposição da mulher ao estrogênio endógeno, que é metabolizado junto a outros carcinógenos pelas enzimas da família glutationa S-transferase (GSTM1, GSTT1 e GSTP1). Os genes VEGF, COL18A1, GSTM1, GSTT1 e GSTP1 são polimórficos em humanos e foram associados à origem de diferentes cânceres. O objetivo do estudo foi analisar os papéis dos polimorfismos C936T do gene VEGF, D104N do gene COL18A1, GSTM1, GSTT1 e IleVal do gene GSTP1 na ocorrência do CO e de suas associações a aspectos clínicos das pacientes e do tumor. Foram avaliadas 137 pacientes com CO e 137 controles. A genotipagem foi realizada por meio da reação em cadeia da polimerase e digestão enzimática em amostras de sangue periférico. O significado estatístico das diferenças entre grupos foi calculado por meio do teste da probabilidade exata de Fisher ou qui-quadrado e as determinações dos riscos de ocorrência do CO foram obtidas por meio das razões de Odds (RO). Observamos que as freqüências dos genótipos variantes do gene VEGF e do gene GSTP1 foram menores em pacientes do que em controles. Indivíduos com os genótipos variantes estiveram sob menor risco de ocorrência do CO do que aqueles com os genótipos selvagens dos genes. Freqüências similares dos genótipos distintos dos genes COL18A1, GSTM1 e GSTT1 foram observados em pacientes e controles. Entretanto, a freqüência da deleção homozigótica do gene GSTT1 foi maior em pacientes com tumor de estágios avançados do que aquelas com tumores localizados. Esses resultados sugerem que os polimorfismos C936T do gene VEGF e Ile/Val do gene GSTP1 influenciam o risco de ocorrência do CO e a deleção homozigótica do gene GSTT1 influencia a agressividade do tumor em mulheres de nossa região. / Abstract: Angiogenesis has been established as an important factor in human carcinogenesis influencing tumor growth and invasion, including ovarian carcinoma (OC). OC has been associated with angiogenic proteins like vascular endothelial growth factor (VEGF), produced by VEGF gene, and antiangiogenic agents, like endostatin, produced by COL18A1 gene. There is a clear association between the excessive exposure of endogen estrogen metabolized by glutathione S-transferase (GST) family as like others carcinogens. The VEGF, COL18A1, GSTM1, GSTT1 and GSTP1 genes are polymorphic in humans and they have been involved in the development of tumors. It was conducted a case-control study to investigate the importance of the C936T polymorphism VEGF gene, D104N polymorphism COL18A1 gene, GSTM1, GSTT1 and Ile105Val GSTP1 polymorphisms GST genes and the risk of OC. Therefore, the result was related to clinical aspects of the patients and pathological aspects of the tumor. The study included 137 OC patients and 137 healthy controls. The genomic DNA from peripheral blood was analyzed using polymerase chain reaction followed by restriction endonuclease digestion. Cases and controlswere compared using chi-squared test. Odds ratio and 95% confidence interval were calculated by logistic regression analysis. We observed a lower frequency of variants genotypes in OC patients than controls related to VEGF gene (12,5% versus 24,1% respectively) and GSTP1 gene (31,8% versus 44,5%, respectively). Women with the variants genotypes were under lower risk of OC when compared to those with the wild genotype related to VEGF gene (P= 0,01) and GSTP1 gene (P= 0,02). Similar frequencies of COL18A1 gene, GSTM1 and GSTT1 genes were seen in patients and controls. However, the homozygous deletion frequency of the GSTT1 was higher in advanced tumors patients rather than early stages. This result suggests that C936T polymorphism of VEGF gene and Ile/Val polymorphism of GSTP1 gene are associated with OC risk and homozygous deletion of the GSTT1 is associated with the aggressiveness of the tumor in women at our region. / Doutorado / Clinica Medica / Doutor em Clínica Médica

Skewed somatic X inactivation (XCI), X-linked gene overexpression and abnormal X content have been associated with breast and ovarian cancer. Partial or complete reactivation of the inactive X in females may be a step in breast and ovarian cancer progression, leading to overexpression of some tumour enhancing gene. Markers of an X reactivation event were examined: X gene dosage, expression, and methylation in 8 ovarian cancer cell lines. Another marker of an X reactivation event, skewed XCI, was assayed in peripheral blood DNA from 106 breast and/or ovarian cancer patients (52 BRCA1 mutation carriers, 24 BRCA2 mutation carriers, 30 non-mutation carriers), 147 age-matched population controls. Combined RNA/DNA FISH was used to quantify the number of inactive Xs compared to total number of Xs. Five cell lines had increased X content. Three cell lines localized XIST to the presumptive inactive X; however the numbers of inactive Xs were variable. Expression levels of 8 X-linked genes were assessed by real-time PCR. Expression was inconsistent between different genes and among cell lines, ranging from a 2 to 300-fold increase compared to a control. Overall, expression was greatly increased for genes subject to inactivation but not increased in genes that escape inactivation for most ovarian cancer cell lines. Methylation at AR and FMR1 was quantified by a real-time PCR based assay and SNuPE respectively. Methylation was lower than expected for 7 of 8 ovarian cancer cell lines at AR or FMR1, while three cell lines had low or no methylation for both genes. Skewed XCI was evaluated using a methylation-based PCR assay at AR. There was no significant increase in skewing above 90% for any cancer group assayed. In addition, two markers of X reactivation were assayed in two low passage cultures of normal ovarian surface epithelium from BRCA1 mutation positive breast cancer patients. One sample did not localize XIST to the inactive X and three of five genes subject to inactivation were overexpressed. In summary, there is evidence for loss of X silencing or gain of active X content in ovarian cancer cell lines and normal ovarian surface epithelium from BRCA1 mutation carriers. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

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