El sarcoma de Kaposi (SK) es una enfermedad angioproliferativa con cuatro formas clínico-epidemiológicas: clásica (SK-C), africana endémica, relacionada con fármacos inmunosupresores y relacionada con el síndrome de inmunodeficiencia adquirida (SK-SIDA). La afectación del SK es generalmente cutánea, pero en los casos agresivos puede diseminarse a localizaciones extracutáneas. En la piel, las lesiones se clasifican clínicopatológicamente en máculas, placas y tumores de acuerdo con su progresión y severidad. El SK parece iniciarse como una proliferación vascular reactiva debida a una red de citocinas alterada, mientras que en estadios avanzados, se comporta como una neoplasia multifocal.El diferente comportamiento biológico del SK de acuerdo con sus presentaciones epidemiológicas y estadios puede estar relacionado con la presencia de alteraciones específicas en los mecanismos que controlan el crecimiento y desarrollo tumoral, como la sobreestimulación de citocinas y de factores de crecimiento angiogénicos o alteraciones en las oncoproteínas que controlan la proliferación celular y la apoptosis. Estas alteraciones podrían ofrecer los estímulos neoplásicos necesarios para el desarrollo, progresión y, en definitiva, un comportamiento más agresivo del SK.El factor de crecimiento hepatocitario (HGF) es una citocina angiogénica pleiotrópica que está sobreexpresada en cánceres humanos invasivos y puede actuar como factor de progresión tumoral, estimulando la angiogénesis y la invasividad de las células tumorales. Estas respuestas son transducidas a través del receptor c-Met. El HGF estimula la proliferación de las células fusiformes cultivadas de lesiones humanas de SK, y además las células del SK sintetizan y secretan HGF y expresan c-Met, creando un rizo de retroalimentación para la proliferación tumoral y la neovascularización. Hemos estudiado la expresión del c-Met en diferentes estadios histológicos de lesiones de SK-C y SK-SIDA, observando que la intensidad de tinción de c-Met fue mayor en los tumores que en las placas, de forma que el HGF se comporta como un factor de progresión en el SK.EL HHV-8, el factor etiológico del SK, codifica la v-ciclina, que formando complejo con la cinasa dependiente de ciclina (CDK) 6, contribuye a la fosforilación y degradación mediada por proteasoma del inhibidor de las cinasas dependientes de ciclina p27KIP1. La p27KIP1 regula negativamente la proliferación celular uniéndose e inhibiendo a los complejos ciclina-CDK de fase G1. Por otro lado, la infrarregulación de la expresión de la p27KIP1 parece facilitar el desarrollo tumoral y la diseminación metastásica; por ello la p27KIP1 ha sido considerada como un factor pronóstico independiente en una gran variedad de neoplasias humanas. Aunque la naturaleza neoplásica del SK todavía es controvertida, se ha demostrado repetidas veces que en algunos pacientes el SK puede comportarse como una neoplasia maligna y seguir un curso ominoso. Para determinar si la disminución de los niveles de la p27KIP1 está también relacionada con un comportamiento más agresivo del SK, decidimos investigar la inmunoreactividad de la p27KIP1 en biopsias de SK. De este modo, intentamos determinar si la disminución de los niveles de expresión de la p27KIP1 está relacionada con la afectación extracutánea en el SK, como ocurre en otras neoplasias cuando metastatizan. La media de porcentajes de células positivas para p27KIP1 era significativamente mayor en las biopsias de lesiones cutáneas y en las máculas-placas que en las lesiones extracutáneas y en los tumores respectivamente. Estos resultados apoyan la hipótesis de que la disminución de los niveles de la p27KIP1, que pueden ser producidos por la infección por el HHV-8, facilitan la progresión del SK a través de sus estadios histopatológicos y su eventual extensión extracutáneaEstudios recientes han demostrado que la degradación de la p27KIP1 a través de la vía ubicuitin-proteasoma está mediada por el complejo SCF/ p45SKP2 y por su receptor específico de sustrato F-box p45SKP2. La p45SKP2 está frecuentemente sobreexpresada en células transformadas, induce la fase S en células quiescentes y se sospecha que es un protooncogen en tumores humanos. De hecho, se ha determinado una asociación entre el incremento de los niveles de la p45SKP2 y la disminución de los niveles de la p27KIP1 en neoplasias epiteliales. Hemos estudiado si la expresión de la p45SKP2 está alterada en las lesiones agresivas del SK y su relación con la infrarregulación de la p27KIP1, sexo e infección por el VIH. La sobreexpresión nuclear de la p45SKP2 estaba presente en todos los estadios del SK, estando significativamente incrementada en los tumores cutáneos y en las lesiones extracutáneas en comparación con la máculas y placas. No se identificaron diferencias estadísticamente significativas en relación con el sexo y estatus VIH de los pacientes, y el análisis de regresión no mostró correlación entre la p45SKP2 y la p27KIP1. Estos hallazgos sugieren que la p45SKP2 está involucrada en el SK, no sólo promoviendo la degradación de la p27KIP1, sino también a través de otros mecanismos todavía desconocidos. / Kaposi's sarcoma (KS) is an angioproliferative disease with four clinical-epidemiological forms: classic (C-KS), African-endemic, immunosuppressive drug-related and acquired immune deficiency syndrome-related (AIDS-KS). KS involvement is usually limited to the skin, but in aggressive cases it may disseminate to extracutaneous locations. In the skin, lesions are clinicopathologically classified into macules, plaques and tumours in agreement with their progression in severity. KS seems to begin as a reactive vascular proliferation due to an unbalanced cytokine network, whereas in advanced stages, it behaves as a multifocal neoplasm.The different biological behavior of KS according to its epidemiological presentations and stages might be related to the presence of specific alterations in the mechanisms controlling tumor growth and development, such as cytokine or angiogenic growth factor overstimulation or alterations of the oncoprotein networks that control cell proliferation and apoptosis. These alterations would provide neoplastic stimuli for the development, progression, and aggressive behaviour of KS. Hepatocyte growth factor (HGF) is a pleiotropic angiogenic cytokine that is overexpressed in invasive human cancers and may function as a tumor progression factor, stimulating tumour cell invasiveness and angiogenesis. These responses are transduced through the c-Met receptor. HGF stimulates proliferation of spindle cells cultured from human KS lesions, and KS cells synthesize and secrete HGF and express c-Met, thus providing an autocrine loop for tumour proliferation and neovascularization. We have studied the expression of c-Met in different histological stages of AIDS-associated and classic KS lesions. The staining intensity of c-Met was stronger in tumours than in plaques showing that HGF would be a progression factor in KS. HHV-8, the KS'etiologic factor, encoded v-cyclin, through its complexing with cyclin-dependent kinase (CDK) 6, contributes to the phosphorylation and proteasome-mediated degradation of p27KIP1, a cyclin-dependent kinase inhibitor. P27KIP1 regulates negatively cell proliferation by binding and inhibiting G1 cyclin-CDK complexes. On the other hand, down-regulation of p27KIP1 expression seems to facilitate tumour development and metastatic dissemination; then p27KIP1 has been considerated as an independent prognostic factor in a variety of human neoplasms. Although the neoplastic nature of KS remains controversial, it has been repeatedly demonstrated that in some patients KS may behave as a malignant neoplasm and follow an ominous course. To determine whether decreased p27KIP1 levels are also related to more aggressive behaviour in KS, it was decided to investigate p27KIP1 immunoreactivity in KS biopsy specimens. Thereby, we sought to determine whether the decrease in p27KIP1 expression levels is related to extracutaneous involvement in KS, as is the case in several other types of neoplasms when they metastasize. The mean percentages of p27KIP1-positive cells were significantly higher in biopsy specimens from skin lesions and in macules-plaques than in those from extracutaneous locations and tumours respectively. These results lend support to the hypothesis that decreased levels of p27KIP1, which may have been brought about by HHV-8 infection, play a role in KS progression through its various histopathological stages, to its eventual extracutaneous spread.Recent studies have demonstrated that p27KIP1 degradation through the ubiquitin-proteasome pathway is mediated by the SCF/p45SKP2 complex and by the substrate-specific receptor of this complex, the F-Box protein p45SKP2. P45SKP2 is frequently over-expressed in transformed cells, induces S phase in quiescent cells and is a suspected proto-oncogene in human tumours. In fact, there are recent reports of increased levels of p45SKP2 in association with reduced p27KIP1 levels in epithelial neoplasms. We have studied of whether p45SKP2 expression is altered in aggressive lesions of KS and its relation to p27KIP1 down-regulation, gender and HIV infection. P45SKP2 nuclear over-expression was present in all KS stages, being significantly increased in skin tumours and extracutaneous lesions as compared with macules and plaques. No statistically significant differences were found in regard to patients´ sex and HIV status and regression analysis failed to show a correlation among p45SKP2 and p27KIP1. These findings suggest that p45SKP2 is involved in KS, not only by promoting the degradation of p27KIP1 but also through other mechanisms still unknown.

Els objectius d'aquesta tesi van ser plantejats per tal d'aprofundir en l'estudi de la implicació de la proteïna p27(kip1) en processos de tumorogènesi i metàstasi, donat que els nivells baixos de p27(kip1) son cosiderats un factor de mal pronòstic en els càncers humans. Així doncs, els objectius proposats pel desenvolupament d'aquest treball són els següents: · Identificació de noves proteïnes d'unió a p27(kip1)· Caracterització funcional de les noves interaccions amb p27(kip1)· Implicació d'aquestes noves funcions de p27(kip1) amb el cicle cel·lular i càncer.

Chronic oxidative injury by allylamine induces proliferative vascular smooth muscle cell (vSMC) phenotypes in the rat aorta similar to those seen in rodent and human atherosclerotic lesions. In this study, we evaluate the potential role of cyclin dependent kinase inhibitors, p21 and p27, and extracellular regulated kinases (ERK1/2) to mediate the proliferative advantage of oxidatively stressed (i.e. allylamine injured) vSMC. Isolated rat aortic SMC from allylamine treated and control rats were cultured on different extracellular matrix (ECM) proteins. Following mitogen restriction, cultures were stimulated with serum with or without inhibitors of NF-kB or MEK. Western blot analysis was performed to identify protein differences between treatment groups. Basal levels of p21 were 1.6 fold higher in randomly cycling allylamine cells than control counterparts seeded on a plastic substrate, a difference lost when cells were seeded on collagen. p27 levels were comparable in both cell types irrespective of substrate. Basal levels of p21 and p27 were 1.4 fold higher in G0 synchronized allylamine cells compared with G0 synchronized control cells seeded on a plastic substrate. Following cell cycle progression, differences in protein levels were not detected. Treatment with 100 nM pyrollidine dithiocarbamate (PDTC) resulted in significant decreases in p21 and p27 in allylamine cells versus control cells following serum stimulation for 9 hours. This decrease was even greater for p21 in allylamine cells when grown on collagen relative to control cells. Alterations in peak and temporal activation of ERK1/2 were observed in allylamine cells seeded on a plastic substrate as compared to control cells, following serum stimulation. Seeding on collagen decreased the enhanced peak phosphorylation of ERK1/2 and increased the sustained activity in allylamine cells compared with control counterparts. Inhibition of ERK1/2 activity resulted in reduced p21 expression in both cells types, but the response was markedly enhanced in allylamine cells, and preferentially observed on a restrictive collagen substrate. We conclude that induction of proliferative (i.e. atherogenic) phenotypes following repeated cycles of oxidative injury involves ERK1/2 activity and modulation of the cyclin dependent kinase inhibitors, p21 and p27, in a matrix-dependent manner.

Thesis (Ph. D.)--Cornell University, May, 2008. / Vita. Includes bibliographical references (leaves 140-170).

Made available in DSpace on 2015-07-22T20:49:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-08-26. Added 1 bitstream(s) on 2015-08-11T03:25:28Z : No. of bitstreams: 1 Publico-00291a.pdf: 2012251 bytes, checksum: 21e4927ff9907a0e614761f475e1bc26 (MD5). Added 1 bitstream(s) on 2015-08-11T03:25:28Z : No. of bitstreams: 2 Publico-00291a.pdf: 2012251 bytes, checksum: 21e4927ff9907a0e614761f475e1bc26 (MD5) Publico-00291b.pdf: 1285004 bytes, checksum: b092509fe084d14584b2356d3e9d3722 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Introdução: Endometriose é uma afecção ginecológica benigna e, em sua fisiopatologia, encontramos os distúrbios do ciclo celular e apoptose. Os genes TP53, P27 e FAS, que participam da regulação do ciclo celular e apoptose, apresentam polimorfismos que podem ter influência funcional. Objetivo: Avaliar se há associação entre a presença dos polimorfismos nos genes TP53 (códons 11, 72, 248), P27 (V109G) e FAS (A670G) com a endometriose. Método: Estudo transversal, casocontrole onde foram pesquisados polimorfismos nos genes TP53 (códons 11, 72 e 248), P27 e FAS por PCR-RFLP em DNA extraídos de swab bucal de mulheres com e sem endometriose. Resultados: Os códons 11 e 248 do gene TP53 apresentaram raros alelos mutados, levando-se a crer que os polimorfismos destes códons não apresentam relevância clínica para a endometriose. O polimorfismo do gene FAS não apresentou diferença estatisticamente significativa entre os grupos endometriose e controle. Já no estudo do polimorfismo do gene TP53 (códon 72) e do gene P27, houve diferença estatisticamente significativa entre os grupos endometriose e controle (p= 0,01; OR= 2,19; IC de 95%= 1,196-4,039 e p= 0,017; OR= 1,93; IC de 95%= 1,12-3,34; respectivamente). Conclusões: Os polimorfismos dos genes TP53*72 e P27 parecem estar associados à endometriose. Os polimorfismos dos genes TP53*11, TP53*248 e FAS não parecem estar associados à endometriose. / Introduction: Endometriosis is a benign gynaecologycal disease, defined as the presence of functional endometrial implants out of uterus, mainly in pelvis. Cell cycle and apoptosis disturbs could be associated with this disease as well as alterations in the genes that control. Aim: To investigate the possible association between the presence of the polymorphisms in the genes TP53: codons 11; 72; 248, P27 (V109G) and FAS –A670G with endometriosis. Methods: Cross sectional case–control study was performed. Genomic DNA was extracted from cells collected from buccal swabs. The TP53: codons 11; 72; 248, P27 (V109G) and FAS –A670G polymorphisms were investigated using the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method in a hospital-based Brazilian population. Results: The P53 gene, codon 11 (P53*11) polymorphism study showed that mostly of women did not present the allelic variance (p=0,255). The P53 gene, codon 72 (P53*72) polymorphism study presented significant differences between endometriosis and control groups (P=0,01; OR=2,19; 95% IC=1,196-4,039). The P53 gene, codon 248 polymorphism study showed that only one woman from endometriosis group presented heterozygosis to variate allele. The distribution of genotype and allele frequencies of p27 V109G polymorphism was significantly different between the endometriosis cases and healthy women (p = 0.016 and 0.002). Finally, FAS gene polymorphism study showed any significant difference between groups (p=0,97). Conclusions: P53*72 and P27 polymorphisms studies could be associated with endometriosis. / TEDE / BV UNIFESP: Teses e dissertações

ANDRÉ, Angela Rosa. Associação da presença de Helicobacter pylori e dos genótipos caga e vaca com as alterações moleculares dos supressores tumorais P53 e P27 nos adenocarcinomas gástricos. 2008. 146 f. Dissertação (Mestrado em Patologia) - Universidade Federal do Ceará, Faculdade de Medicina, Fortaleza, 2008. / Submitted by denise santos (denise.santos@ufc.br) on 2011-12-20T13:56:04Z No. of bitstreams: 1 2008_dis_arandré.pdf: 5783302 bytes, checksum: 36586c0859cef2aa2be2b5835b3602c4 (MD5) / Approved for entry into archive by Eliene Nascimento(elienegvn@hotmail.com) on 2012-02-01T16:04:09Z (GMT) No. of bitstreams: 1 2008_dis_arandré.pdf: 5783302 bytes, checksum: 36586c0859cef2aa2be2b5835b3602c4 (MD5) / Made available in DSpace on 2012-02-01T16:04:09Z (GMT). No. of bitstreams: 1 2008_dis_arandré.pdf: 5783302 bytes, checksum: 36586c0859cef2aa2be2b5835b3602c4 (MD5) Previous issue date: 2008 / Gastric carcinoma is the second cause of death by cancer in the world. On State of Ceara-Brazil is the second most frequent type of cancer in men and third in women. Adenocarcinomas account for approximately 95% of all malignant gastric neoplasms. It has been associated to genetic and environmental factors and a intimate relationship between the infection by the bacteria Helicobacter pylori and the gastric carcinoma have been related. The presence of the cagA gene and specific genotypes (s1m1) of the gene vacA have been detected in more pathogenic strains. Although the precise molecular mechanisms by which H. pylori could promote the process of gastric carcinogenesis are under investigation, one hypothesized mechanism involves the tumor supressor genes inactivation. The aim of the present study was to verify if the presence of Helicobacter pylori, cagA and vacA genes is related to mutations in the tumor supressor gene p53 and altered expression of p53 and p27 proteins in gastric adenocarcinomas. Seventy-four (74) samples were analyzed to detect the presence of H. pylori, cagA and genotypes of vacA by Polymerization Chain Reaction (PCR). The mutational analysis of p53 gene was performed by PCR-SSCP (Polymerization Chain Reaction for analysis of the Single-strand Conformation Polymorphism). Analysis of mutation or overexpression of p53 protein and p27 expression was detected by the immunohistochemical method. The bacteria was detected in 95% of the samples, 63% was cagA(+). Among the vacA allele it was observed prevalence of s1 (74%) and m1 (82%), associated in 69% of the cases. Mutation analysis of p53 demonstrated 72% of the cases with altered electrophoretic mobility; The alterations were significatively more frequent in the presence of the cagA gene. Immunohistochemical analysis detected only 29% of cases with the expression of p53 protein. The protein p27 showed accentuated reduction in its expression (detected in only 19% of the cases), it has not demonstrated compensatory activity in relation to the p53 altered protein, neither association to H. pylori presence. Finally, these data suggest that simultaneous inactivation of these tumor suppressors genes may be the key point of deregulation of the cellular cycle that, associated to the other factors, favor the development and progression of the gastric cancer. There is some evidence that the bacterial presence, cagA and vacA/s1m1 genes, may influence, in a not understood way, the alterations observed in the tumor suppressors p53 and p27. / O carcinoma gástrico é a segunda causa de morte por câncer no mundo. No Ceará é o segundo mais freqüente entre os homens e o terceiro entre as mulheres. Dos cânceres gástricos os adenocarcinomas representam em torno de 95%. A doença tem sido associada a fatores genéticos e ambientais sendo demonstrada íntima relação com a infecção por Helicobacter pylori, principalmente associada à presença do gene cagA e genótipos vacAs1m1. Entretanto, apesar dos mecanismos pelos quais a bactéria promove a carcinogênese gástrica ainda não estarem esclarecidos, uma das hipóteses seria através da inativação de supressores tumorais. O objetivo do presente trabalho foi verificar, em adenocarcinomas gástricos, se a presença de H. pylori, e de seus genes cagA e vacA, está relacionada com a mutação e/ou alteração na expressão protéica dos supressores tumorais p53 e p27. Neste estudo, 74 amostras de pacientes foram analisadas quanto à presença de H. pylori, cagA+ e os genótipos de vacA, pela reação em cadeia da polimerase (PCR). A análise mutacional do gene p53 foi realizada por PCR-SSCP e a detecção da mutação/superexpressão do p53 e expressão da proteína p27 pelo método imunohistoquímico. A bactéria foi detectada em 95% das amostras, das quais 63% eram cagA(+). Dentre os alelos de vacA, observou-se predomínio de s1 (74%) e m1 (82%), associados em 69% dos casos. Na análise mutacional do p53 verificou-se que 72% dos casos exibiram alteração no padrão de mobilidade eletroforética, sendo esta associada significativamente à presença do gene cagA. Por outro lado, apenas 29% dos casos apresentaram detecção pelo método imunohistoquímico, não sendo encontrada associação com a H. pylori. A proteína p27 demonstrou acentuada redução em sua expressão (detectada em apenas 19% dos casos), não demonstrando atividade compensatória em relação à proteína p53 mutada e sem associação estatística dos casos negativos com a presença da H. pylori. Finalmente, os resultados sugerem que estes supressores simultaneamente inativados podem ser o ponto chave da desregulação do ciclo celular que, associados a outros fatores, favoreçam o desenvolvimento e progressão dos adenocarcinomas gástricos. Há indícios de que a presença bacteriana, e dos seus genes cagA(+) e vacA/s1m1, possam influenciar, de forma não esclarecida, as alterações moleculares ocorridas nos supressores tumorais p53 e p27.

A major problem with breast cancer treatment is the prevalence of antiestrogen resistance, be it de novo or acquired after continued use. Many of the underlying mechanisms of antiestrogen resistance are not clear, although estrogen receptor-mediated actions have been identified as a pathway that is blocked by antiestrogens. Selective estrogen receptor modulators (SERMs), such as tamoxifen, are capable of producing reactive oxygen species (ROS) through metabolic activation, and these ROS, at high levels, can induce irreversible growth arrest that is similar to the growth arrest incurred by SERMs. This suggests that SERM-mediated growth arrest may also be through ROS accumulation. Breast cancer receiving long-term antiestrogen treatment appears to adapt to this increased, persistent level of ROS. This, in turn, leads to the disruption of reversible redox signaling that involves redox-sensitive phosphatases and protein kinases and transcription factors. This has downstream consequences for apoptosis, cell cycle progression, and cell metabolism. For this dissertation, we explored if altering the ROS formed by tamoxifen also alters sensitivity of the drug in resistant cells. We explored an association with a thioredoxin/Jab1/p27 pathway, and a possible role of dysregulation of thioredoxin-mediated redox regulation contributing to the development of antiestrogen resistance in breast cancer. We used standard laboratory techniques to perform proteomic assays that showed cell proliferation, protein concentrations, redox states, and protein-protein interactions. We found that increasing thioredoxin reductase levels, and thus increasing the amount of reduced thioredoxin, increased tamoxifen sensitivity in previously resistant cells, as well as altered estrogen and tamoxifen-induced ROS. We also found that decreasing levels of Jab1 protein also increased tamoxifen sensitivity, and that the downstream effects showed a decrease p27 phosphorylation in both cases. We conclude that the chronic use of tamoxifen can lead to an increase in ROS that alters cell signaling and causing cell growth in the presence of tamoxifen, and that this resistant cell growth can be reversed with an alteration to the thioredoxin/Jab1 pathway.

A proliferação celular mediada pela progesterona (P4) é essencial para a funcão uterina. Dessa forma, alterações nesse processo podem comprometer a reprodução. O diabetes do tipo 1 (DM1) está associado a diversos distúrbios reprodutivos. No entanto, o impacto do DM1 sobre a ação da P4 no ambiente uterino ainda não é conhecido. Para isso, utilizamos fêmeas de camundongo DM1 induzidas por aloxana, submetidas à ovarectomia (OVX) e reposição por P4. Verificamos por meio de histomorfometria e imunohistoquímica (PCNA) uma diminuição da área de estroma uterino e do índice de proliferação. As quantificações proteícas por Western blot monstraram um aumento do PR-A nas fêmeas diabéticas OVX e nas tratadas pela P4. Ressalta-se que as fêmeas DM1 tratados pela P4 não apresentaram a mesma expressão do RNAm para o fator de crescimento Hoxa-10. Houve também um aumento do RNAm da p27 nas fêmeas DM1 não tratadas, visto por qPCR. Nossos resultados demonstraram que o DM1 interfere negativamente na resposta proliferativa promovida pela P4. Contribuindo para compreensão dos mecanismos biológicos pelos quais o diabetes compromete as funções reprodutivas. / Progesterone (P4)-mediated cell proliferation is essential for uterine function. Therefore, alteration in this process could compromise reproduction. The type 1 diabetes (DM1) relates to several reproductive disturbs. However, the impact of DM1 on the P4 function is still not elucidated. Thus, we used alloxan-induced diabetic mice females subjected to ovariectomy and hormonal replacement therapy with P4. Histomorphometrical and immunohistochemistry to PCNA approaches showed a decrease of the uterine stromal area and the cell proliferation index. Protein quantification by Western blot showed increased levels of PR-A in both ovariectomized and P4-treated diabetic females. Importantly, P4 did not recovered the mRNA expression to the Hoxa-10 transcription factor in diabetic females. Additionally, qPCR analysis revealed increased level of p27 mRNA in diabetic females non-treated with P4. Together these results show that DM1 has a negative action on the P4-mediated cell proliferative response. These are new and important results to a better understand of the biological mechanisms by which diabetes affects the reproductive functions.

A proliferação celular mediada pela progesterona (P4) é essencial para a funcão uterina. Dessa forma, alterações nesse processo podem comprometer a reprodução. O diabetes do tipo 1 (DM1) está associado a diversos distúrbios reprodutivos. No entanto, o impacto do DM1 sobre a ação da P4 no ambiente uterino ainda não é conhecido. Para isso, utilizamos fêmeas de camundongo DM1 induzidas por aloxana, submetidas à ovarectomia (OVX) e reposição por P4. Verificamos por meio de histomorfometria e imunohistoquímica (PCNA) uma diminuição da área de estroma uterino e do índice de proliferação. As quantificações proteícas por Western blot monstraram um aumento do PR-A nas fêmeas diabéticas OVX e nas tratadas pela P4. Ressalta-se que as fêmeas DM1 tratados pela P4 não apresentaram a mesma expressão do RNAm para o fator de crescimento Hoxa-10. Houve também um aumento do RNAm da p27 nas fêmeas DM1 não tratadas, visto por qPCR. Nossos resultados demonstraram que o DM1 interfere negativamente na resposta proliferativa promovida pela P4. Contribuindo para compreensão dos mecanismos biológicos pelos quais o diabetes compromete as funções reprodutivas. / Progesterone (P4)-mediated cell proliferation is essential for uterine function. Therefore, alteration in this process could compromise reproduction. The type 1 diabetes (DM1) relates to several reproductive disturbs. However, the impact of DM1 on the P4 function is still not elucidated. Thus, we used alloxan-induced diabetic mice females subjected to ovariectomy and hormonal replacement therapy with P4. Histomorphometrical and immunohistochemistry to PCNA approaches showed a decrease of the uterine stromal area and the cell proliferation index. Protein quantification by Western blot showed increased levels of PR-A in both ovariectomized and P4-treated diabetic females. Importantly, P4 did not recovered the mRNA expression to the Hoxa-10 transcription factor in diabetic females. Additionally, qPCR analysis revealed increased level of p27 mRNA in diabetic females non-treated with P4. Together these results show that DM1 has a negative action on the P4-mediated cell proliferative response. These are new and important results to a better understand of the biological mechanisms by which diabetes affects the reproductive functions.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O carcinoma gÃstrico à a segunda causa de morte por cÃncer no mundo. No Cearà à o segundo mais freqÃente entre os homens e o terceiro entre as mulheres. Dos cÃnceres gÃstricos os adenocarcinomas representam em torno de 95%. A doenÃa tem sido associada a fatores genÃticos e ambientais sendo demonstrada Ãntima relaÃÃo com a infecÃÃo por Helicobacter pylori, principalmente associada à presenÃa do gene cagA e genÃtipos vacAs1m1. Entretanto, apesar dos mecanismos pelos quais a bactÃria promove a carcinogÃnese gÃstrica ainda nÃo estarem esclarecidos, uma das hipÃteses seria atravÃs da inativaÃÃo de supressores tumorais. O objetivo do presente trabalho foi verificar, em adenocarcinomas gÃstricos, se a presenÃa de H. pylori, e de seus genes cagA e vacA, està relacionada com a mutaÃÃo e/ou alteraÃÃo na expressÃo protÃica dos supressores tumorais p53 e p27. Neste estudo, 74 amostras de pacientes foram analisadas quanto à presenÃa de H. pylori, cagA+ e os genÃtipos de vacA, pela reaÃÃo em cadeia da polimerase (PCR). A anÃlise mutacional do gene p53 foi realizada por PCR-SSCP e a detecÃÃo da mutaÃÃo/superexpressÃo do p53 e expressÃo da proteÃna p27 pelo mÃtodo imunohistoquÃmico. A bactÃria foi detectada em 95% das amostras, das quais 63% eram cagA(+). Dentre os alelos de vacA, observou-se predomÃnio de s1 (74%) e m1 (82%), associados em 69% dos casos. Na anÃlise mutacional do p53 verificou-se que 72% dos casos exibiram alteraÃÃo no padrÃo de mobilidade eletroforÃtica, sendo esta associada significativamente à presenÃa do gene cagA. Por outro lado, apenas 29% dos casos apresentaram detecÃÃo pelo mÃtodo imunohistoquÃmico, nÃo sendo encontrada associaÃÃo com a H. pylori. A proteÃna p27 demonstrou acentuada reduÃÃo em sua expressÃo (detectada em apenas 19% dos casos), nÃo demonstrando atividade compensatÃria em relaÃÃo à proteÃna p53 mutada e sem associaÃÃo estatÃstica dos casos negativos com a presenÃa da H. pylori. Finalmente, os resultados sugerem que estes supressores simultaneamente inativados podem ser o ponto chave da desregulaÃÃo do ciclo celular que, associados a outros fatores, favoreÃam o desenvolvimento e progressÃo dos adenocarcinomas gÃstricos. Hà indÃcios de que a presenÃa bacteriana, e dos seus genes cagA(+) e vacA/s1m1, possam influenciar, de forma nÃo esclarecida, as alteraÃÃes moleculares ocorridas nos supressores tumorais p53 e p27. / Gastric carcinoma is the second cause of death by cancer in the world. On State of Ceara-Brazil is the second most frequent type of cancer in men and third in women. Adenocarcinomas account for approximately 95% of all malignant gastric neoplasms. It has been associated to genetic and environmental factors and a intimate relationship between the infection by the bacteria Helicobacter pylori and the gastric carcinoma have been related. The presence of the cagA gene and specific genotypes (s1m1) of the gene vacA have been detected in more pathogenic strains. Although the precise molecular mechanisms by which H. pylori could promote the process of gastric carcinogenesis are under investigation, one hypothesized mechanism involves the tumor supressor genes inactivation. The aim of the present study was to verify if the presence of Helicobacter pylori, cagA and vacA genes is related to mutations in the tumor supressor gene p53 and altered expression of p53 and p27 proteins in gastric adenocarcinomas. Seventy-four (74) samples were analyzed to detect the presence of H. pylori, cagA and genotypes of vacA by Polymerization Chain Reaction (PCR). The mutational analysis of p53 gene was performed by PCR-SSCP (Polymerization Chain Reaction for analysis of the Single-strand Conformation Polymorphism). Analysis of mutation or overexpression of p53 protein and p27 expression was detected by the immunohistochemical method. The bacteria was detected in 95% of the samples, 63% was cagA(+). Among the vacA allele it was observed prevalence of s1 (74%) and m1 (82%), associated in 69% of the cases. Mutation analysis of p53 demonstrated 72% of the cases with altered electrophoretic mobility; The alterations were significatively more frequent in the presence of the cagA gene. Immunohistochemical analysis detected only 29% of cases with the expression of p53 protein. The protein p27 showed accentuated reduction in its expression (detected in only 19% of the cases), it has not demonstrated compensatory activity in relation to the p53 altered protein, neither association to H. pylori presence. Finally, these data suggest that simultaneous inactivation of these tumor suppressors genes may be the key point of deregulation of the cellular cycle that, associated to the other factors, favor the development and progression of the gastric cancer. There is some evidence that the bacterial presence, cagA and vacA/s1m1 genes, may influence, in a not understood way, the alterations observed in the tumor suppressors p53 and p27.