Brain tumours comprise about 25% of all cancers in children. Medulloblastoma – which arise in the cerebellum – are the most common and severe malignant pediatric brain tumour and the leading cause of cancer-related deaths in children under the age of 9. Treatment of medulloblastoma remains conventional, with surgery followed by chemotherapy and radiation. These measures are successful in about 60-80% of cases but treatment results in severe side effects due to its toxicity to the central nervous system. Therefore it is of utmost importance to define the signalling pathways and genetic changes involved in the formation of medulloblastoma in order to allow for better diagnosis and treatments with higher efficiency and decreased toxicity. The cell of origin for medulloblastoma is thought to be the granule neuron progenitor, a cell type arising from cerebellar stem cells of the ventricular zone. After birth granule neuron progenitors differentiate into mature granule neurons which populate the majority of the cerebellum and are crucial for its cognitive functions and motor coordination. The Hedgehog signalling pathway plays an important role in medulloblastoma generation and murine models with activated Hedgehog signalling develop medulloblastoma at high frequencies. In addition, the Notch pathway has been implicated in the generation of medulloblastoma, and interaction between the two pathways has been suggested. Inhibitors of both Hedgehog and Notch are currently in clinical trials however knowledge of possible interactions between them could lead to more effective treatment strategies. The aim of this project was to investigate the interaction of Hedgehog and Notch signalling in normal brain development and medulloblastoma. Two mouse models allowed activation of Hedgehog and inactivation of Notch signalling in granule neuron progenitors and cerebellar ventricular zone stem cells. In granule neuron progenitors canonical Notch signalling is not required and the layering and cell types of RBP-Jlox/lox;Math1-Cre cerebella appear identical to control brains. In contrast, Notch inactivation in ventricular zone stem cells with GFAP-Cre resulted in increased differentiation of stem cells into progenitor cells accompanied by an overall developmental delay in neuronal differentiation. Medulloblastoma generated by Hedgehog activation (through inactivation of the negative Hedgehog regulator Ptc1) in both cell types cannot be blocked by Notch inactivation. Furthermore, medulloblastoma of Ptc1lox/lox;RBP-Jlox/lox;GFAP-Cre and those of Ptc1lox/lox;RBP-Jlox/lox;Math1-Cre mice are identical in incidence as well as histology to the tumours in which only Hedgehog signalling is activated. This implies that even though Notch signalling plays an important role in cerebellar stem cells it is not required for the initiation and development of Hedgehog induced medulloblastoma. Therefore it may be crucial to consider the Hedgehog status of patients in order to interpret clinical data of Notch pathway inhibitors and even more importantly these results suggest that determining the Hedgehog status might be crucial before treatment of medulloblastoma patients with Notch pathway inhibitors.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 92-100).

Pancreatic neuroendocrine tumours (PNETs) are the most common neuroendocrine tumours diagnosed in humans and dogs. Due to the highly heterogeneous nature of these tumours, definitive data are still lacking over the molecular mechanisms involved in their cancerous behaviour. This study focused on insulinoma (INS), as it is the most commonly diagnosed PNET in human and veterinary oncology. INS is an insulin-producing tumour that causes a hypoglycaemic syndrome related to the excessive insulin production. In humans, it is often a small benign neoplasm readily curable by surgical resection whereas, in dogs, INS is often malignant. Despite current treatment modalities, malignant canine and human INS have a poor prognosis as patients tend to develop metastases in liver and lymph nodes that do not respond to current therapies. From a comparative oncology perspective, the close resemblance of canine and human malignant INS makes canine INS an interesting study model for human INS. Cancer stem cells (CSCs) are critical for the engraftment and chemoresistance of many tumours. Although CSCs have been isolated from a range of solid tumours, a comprehensive characterisation of INS CSCs has not yet been reported. In this study, it was confirmed that INS CSCs can be enriched and are potential targets for novel INS therapies. Highly invasive and tumourigenic human and canine INS CSCs were successfully isolated and exhibited greater resistance to chemotherapy, which may play a significant role in the poor prognosis of this disease. To date, the mechanisms by which tumours spread and the clinical causes of chemoresistance remain only partially understood. Here, RNA-sequencing analysis was performed over a small set of canine INS tumour samples in order to identify mechanisms involved in INS carcinogenesis through different stages of the disease. Preliminary data showed that distinct gene profiles characterised early and late stage of canine INS. Interestingly, differential gene expression and gene pathways analysis, highlighted that sets of genes involved in pancreatic embryogenesis and insulin secretion were overexpressed in canine primary INS lesions compared with normal pancreas. The Notch pathway is fundamental in pancreatic embryogenesis and it has been previously associated with carcinogenesis of neuroendocrine tumours and with the CSC phenotype. Protein analysis showed that the Notch pathway is activated in both human and canine INS CSCs, particularly when treated with chemotherapy, indicating that the Notch pathway may be involved in chemoresistance. Additionally, it was demonstrated that inhibition of the Notch pathway decreased INS CSCs' survival and chemoresistance, both in vitro and in vivo. These findings provide preclinical evidence that anti-Notch therapy may improve outcomes for patients with malignant INS.

Background: Intrahepatic cholangiocarcinoma (CCA) is a highly treatment-resistant malignancy of biliary epithelium with increasing global mortality. Histologically, CCA is characterised by a pronounced inflammatory stroma of tumour-associated myofibroblasts, macrophages, immune cells and a modified extracellular matrix (ECM). In other solid cancers, the stroma plays a tumour promoting role. The functional role of the stroma in CCA remains unclear. The origin and the proportional contribution to the stroma by haematopoietic and mesenchymal bone marrow (BM) -derived cells is not known in CCA. Intriguingly, reports suggest that mesenchymal stem cells (MSCs) may contribute to the epithelial compartment of malignant tumours. Furthermore, the Notch signalling pathway is known to play oncogenic and tumour suppressive roles in diverse neoplasms but its role in CCA remains unclear. Aims and Methods: The functional role of myofibroblasts and macrophages in the tumour stroma of CCA was investigated together with an analysis of the origin and contribution of BM-derived cells to the stromal and epithelial compartments of CCA. The Notch signalling pathway was studied as a potential signalling mechanism through which the stroma and malignant epithelial compartments of CCA may interact. Results: The thioacetamide rat model of CCA was optimised and found to display excellent histological congruence with human lesions. The tumour cellular microenvironment comprised of myofibroblasts, migratory macrophages and immune cells. During cholangiocarcinogenesis, progressive intrahepatic accumulation of inflammatory cells and proliferation of bipotential progenitor cells preceded the development of invasive CCA. In vitro, CCA lines were identified to contain a side population of stem cells. Adoptive transfer of BM from Enhanced Green Fluorescent Protein (EGFP) transgenic rats to wild type rats to establish chimeras was undertaken. In transplanted rats, persistent EGFP+ chimerism of both haematopoietic and mesenchymal stem cell compartments was established. In tumours, macrophages and neutrophils were overwhelmingly EGFP+ve, whereas myofibroblasts, fibroblasts and benign and malignant bile ducts were EGFP-ve. There was no evidence of cell fusion or EGFP silencing. These findings were confirmed in spontaneous breast, skin and colon tumours in EGFP+ chimeric rats not treated with TAA. In vitro studies to recapitulate the cellular and extracellular elements of the tumour niche identified that ECM components induce characteristic cell proliferation patterns dependent on the matrix component but do not appear to affect chemosensitivity. Bidirectional interaction between CCA cells and hepatic stellate cells (mediated by soluble factors) was identified. Furthermore, in direct co-culture, M2 polarised macrophages appear to enhance CCA cell proliferation compared to M1 macrophages. In considering the Notch pathway, Notch signalling components (particularly Notch3) and target genes were upregulated in human CCA specimens. Immunohistochemical analysis identified apparent distribution of Notch ligand on tumour stroma and Notch receptor subtypes on malignant epithelia. Although direct co-culture of CCA cells with myofibroblasts and M1/M2 polarised macrophages did not clearly demonstrate stromal:epithelial Notch pathway activation, this may have been a function of in vitro experimental limitations. Gamma-secretase inhibition downregulated the Notch pathway, reduced proliferation and appeared to enhance chemosensitivity of CCA cells in vitro. Conclusions: A stereotypical niche forms around CCA in developing and malignant lesions. There was no evidence of a BM-derived stem cell contribution to the epithelial component of CCA, breast, colon or skin malignancies. Haematopoietic but not mesenchymal components of the tumour stroma were of BM origin. Notch signalling is upregulated in CCA and appears to play a tumour promoting role in CCA; pathway inhibition represents a potential therapeutic target.

The aim of this thesis was to examine role of Sirt1 in the Notch signalling pathway, using Drosophila as a model organism. Based on in vivo and in vitro studies, we conclude that Sirt1 plays a positive role in Notch signalling. In embryonic S2N cells, Sirt1 is responsible for the protection from metabolic stress-induced down-regulation of subset of E(Spl) genes. During development, Sirt1 is responsible for proper Notch-dependent specification of SOPs and wing development. Sirt1 can regulate the Notch signalling on multiple levels via deacetylation of various substrates involved in the Notch signalling revealed by our proteomic survey.

O aumento de demanda por combustíveis menos poluentes e de fontes renováveis faz com que o Etanol se destaque como uma alternativa as fontes convencionais de energia. O crescente aumento no consumo deste produto, para os próximos anos, exigirá meios de transporte e estocagem eficientes e seguros, desde os diversos produtores até os terminais de distribuição. O transporte por dutos é, sem dúvida, a forma mais econômica de transporte deste produto, porém, há evidências de que esses dutos de transporte e fundo de tanques de armazenamento de etanol poderiam ser susceptíveis ao fenômeno de corrosão sob tensão. Este fenômeno pode ser avaliado, por exemplo, por ensaios de baixa taxa de deformação (BTD) com corpos de prova com ou sem entalhe. Este trabalho tem por objetivo avaliar a influência da severidade do entalhe em corpos de prova ensaiados a baixa taxa de deformação no meio etanol combustível simulado (PRCI SFGE). Foram preparados quatro tipos de corpos de prova de aço API X70: sem entalhe, com entalhe da norma NACE TM 0111, com entalhe “intermediário” (severidade 18,9% menor que o da norma) e com entalhe “menos severo” (redução de 37,2% na severidade do entalhe, segundo concentração de tensões, quando comparado com o da norma). Os resultados mostram que os quatro tipos de corpos de prova apresentaram susceptibilidade ao meio (etanol PRCI SFGE). Nos corpos de prova sem entalhe esta susceptibilidade foi evidenciada somente nas imagens fractográficas. Já nos CPs com entalhe, mesmo com redução de concentrações de tensões no entalhe, observou-se resultados bem semelhantes, ficando evidenciada a susceptibilidade ao meio tanto nas imagens fractográficas (fratura frágil), quanto pela diminuição da ductilidade. / The increasing demand for cleaner fuels and renewable sources makes the ethanol rise as an alternative to conventional energy sources. The increasing consumption rate of this product, predicted for the upcoming years, will require safer and more efficient methods for transportation and storage through the entire transport cycle. Pipelines are the most economical way to transport it. However, there is evidence that pipeline transportation and the bottom of the storage tanks for ethanol might be susceptible to the stress corrosion cracking phenomenon. This phenomenon can be assessed by slow strain rate tests (SSRT) using smooth or notched specimen. This work aims to evaluate the influence of notch severity in specimens submitted to SSRT technique in simulated fuelgrade ethanol (PRCI SFGE). Four types of specimens of steel API X70 were prepared: unnotched, notched according to standard NACE TM 0111, notched with “intermediate” severity (severity 18.9% lower than the standard) and “less severe” notched (reduction of 37.2% on severity of the notched when compared with the standard). The results show that both sample types presented susceptibility in the solution (ethanol PRCI SFGE). For the unnotched specimens this susceptibility was observed only in fractographic images. In the notched specimens, despite the reduction of stress concentrations in the notch, the results were very similar. In this case the susceptibility can be observed in fractographic images (brittle fracture) as well as by decrease in the ductility.

Made available in DSpace on 2015-07-22T20:50:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-07-30. Added 1 bitstream(s) on 2015-08-11T03:25:48Z : No. of bitstreams: 1 Publico-10890a.pdf: 1984275 bytes, checksum: 9c806a399d111fa36900927053424e9e (MD5). Added 1 bitstream(s) on 2015-08-11T03:25:49Z : No. of bitstreams: 2 Publico-10890a.pdf: 1984275 bytes, checksum: 9c806a399d111fa36900927053424e9e (MD5) Publico-10890b.pdf: 1607433 bytes, checksum: 8c8860e6bda38169d779192272e62a56 (MD5). Added 1 bitstream(s) on 2015-08-11T03:25:49Z : No. of bitstreams: 3 Publico-10890a.pdf: 1984275 bytes, checksum: 9c806a399d111fa36900927053424e9e (MD5) Publico-10890b.pdf: 1607433 bytes, checksum: 8c8860e6bda38169d779192272e62a56 (MD5) Publico-10890c.pdf: 1673660 bytes, checksum: a0d2f4bc84915ed191f7e86a2fa11945 (MD5). Added 1 bitstream(s) on 2015-08-11T03:25:49Z : No. of bitstreams: 4 Publico-10890a.pdf: 1984275 bytes, checksum: 9c806a399d111fa36900927053424e9e (MD5) Publico-10890b.pdf: 1607433 bytes, checksum: 8c8860e6bda38169d779192272e62a56 (MD5) Publico-10890c.pdf: 1673660 bytes, checksum: a0d2f4bc84915ed191f7e86a2fa11945 (MD5) Publico-10890d.pdf: 1518283 bytes, checksum: 5bd6b7757a463a7b6c64cd275e3e0014 (MD5) / É observado que a cascata de sinalização de Notch tem ativação desregulada em diversos tipos de cânceres. Em câncer, é descrito que a função específica da via de sinalização de Notch e dependente do contexto celular, dos tipos específicos expressos de receptores homólogos e do cruzamento de informação (crosstalk) entre as diferentes vias de sinalização. Nosso laboratório descreveu previamente, que a via de sinalização de Notch apresenta-se desregulada em meningiomas de todos os graus. Entretanto, a conseqüência funcional desta sinalização anormal de Notch e desconhecida. Neste trabalho, descrevemos que, em linhagens celulares de meningiomas, a expressão exógena do gene Hes-1, o componente efetor da via de Notch, esta associada à formação de células tetraplóides. De forma similar, a expressão exógena das formas ativas dos receptores Notch 1 e Notch2, induz ao aumento da expressão de Hes-l, que contribui para a formação de células tetraplóides. Células tetraplóides de meningioma exibem indícios de instabilidade genética, caracterizados pelo aumento da freqüência de figuras atípicas nucleares, como a presença de mitoses com fusos multipolares e de células com núcleos de tamanho aumentado. As células tetraplóides isoladas por citometria de fluxo demonstram ser viáveis em cultura, embora apresentem uma maior taxa de apoptose espontânea, quando comparadas as células diplóides. A técnica de cariotipagem espectral foi usada para demonstrar que, em comparação as células diplóides, as células tetraplóides desenvolvem uma taxa mais elevada de aberrações cromossômicas estruturais e numéricas. Nossos resultados identificam uma nova função para a via de sinalização de Notch na geração tetraplóidia e conseqüentemente de instabilidade cromossômica. Portanto, a via desregulada da sinalização de Notch pode ser um mecanismo genético inicial em meningiomas, contribuindo potencialmente para a tumorigênese.. / TEDE / BV UNIFESP: Teses e dissertações

Submitted by Tatiana Silva (tsilva@icict.fiocruz.br) on 2012-11-01T14:31:00Z No. of bitstreams: 1 karis_maria_p_rodrigues_ioc_bp_0027_2011.pdf: 37180086 bytes, checksum: 5edafa91606122063b098c48eb81a834 (MD5) / Made available in DSpace on 2012-11-01T14:31:00Z (GMT). No. of bitstreams: 1 karis_maria_p_rodrigues_ioc_bp_0027_2011.pdf: 37180086 bytes, checksum: 5edafa91606122063b098c48eb81a834 (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz.Instituto Oswaldo Cruz. Rio de janeiro, RJ, Brasil / A Leishmaniose Tegumentar Americana (LTA) é uma doença que se caracteriza pela presença de lesões cultâneas (LCL) e, em um número pequeno de pacientes, pelo desenvolvimento de lesão mucosa (ML). A evolução clínica e a resposta ao tratamento dependem de múltiplos fatores, incluindo o equilíbrio entre as citocinas Th1 e Th2, influenciando no controle parasitário e na extensão da lesão. Nesse equilíbrio complexo é possível que outras citocinas inflamatórias como IL-17 e IL-18, bem como fatores que influenciem na diferenciação de queratinócitos e nos processos inflamatórios da pele, como o sistema Notch, também possam se relacionar ao prognóstico das lesões. IL-18 é uma potente citocina indutora de IFN-γ, já tendo sido observado efeito protetor em doenças por protozoários. IL-17 é produzida por uma linhagem de células T descritas mais recentemente, que são consideradas essenciais em processos inflamatórios de vários tecidos e em doenças auto-imunes. O sistema de sinalização Notch é composto por um grupo de proteínas transmembrana que regulam processos decisórios em diversos tecidos, tanto no período embrionário como na vida adulta, incluindo a diferenciação de queratinócitos e de linfócitos T, na dependência do ligante envolvido em sua ativação (Delta-like – DLL1/DLL3/DLL4 ou Jagged 1 e 2). Estudamos a expressão das proteínas do sistema Notch e das citocinas IL-17 e IL-18 em lesões e sangue de pacientes com LTA causada por Leishmania (Viannia) braz possa estar associado com reação inflamatória mais intensa e retardo na resolução das lesões. Nas lesões mucosas foi observada maior expressão do receptor Notch3 e dos ligantes Delta-like (DLL1 e DLL4). O sistema Notch está associado à homeostase da pele regulando a diferenciação de queratinócitos e a cicratização. Nas células do sistema imune, quando linfócitos T CD2+ expressando Notch são ativados por células apresentadoras de antígenos expressando ligantes Delta-like, uma resposta Th1 ou Th17 é gerada. Nesse sentido, o papel do sistema Notch nas lesões de LTA pode ser associado tanto à homeostase do tecido cutâneo quanto ao sistema imune ou a ambos, além de poder estar correlacionado ao aumento de IL-17 observado no mesmo grupo de pacientes. A alta expressão de receptores Notch, ligantes Delta-like e das citocinas inflamatórias IL-17 e IL-18 sugere que o aumento desses fatores pode indicar pacientes para os quais esteja indicado um esquema terapêutico mais agressivo, ou pelo menos, um acompanhamento mais rigoroso

O aumento de demanda por combustíveis menos poluentes e de fontes renováveis faz com que o Etanol se destaque como uma alternativa as fontes convencionais de energia. O crescente aumento no consumo deste produto, para os próximos anos, exigirá meios de transporte e estocagem eficientes e seguros, desde os diversos produtores até os terminais de distribuição. O transporte por dutos é, sem dúvida, a forma mais econômica de transporte deste produto, porém, há evidências de que esses dutos de transporte e fundo de tanques de armazenamento de etanol poderiam ser susceptíveis ao fenômeno de corrosão sob tensão. Este fenômeno pode ser avaliado, por exemplo, por ensaios de baixa taxa de deformação (BTD) com corpos de prova com ou sem entalhe. Este trabalho tem por objetivo avaliar a influência da severidade do entalhe em corpos de prova ensaiados a baixa taxa de deformação no meio etanol combustível simulado (PRCI SFGE). Foram preparados quatro tipos de corpos de prova de aço API X70: sem entalhe, com entalhe da norma NACE TM 0111, com entalhe “intermediário” (severidade 18,9% menor que o da norma) e com entalhe “menos severo” (redução de 37,2% na severidade do entalhe, segundo concentração de tensões, quando comparado com o da norma). Os resultados mostram que os quatro tipos de corpos de prova apresentaram susceptibilidade ao meio (etanol PRCI SFGE). Nos corpos de prova sem entalhe esta susceptibilidade foi evidenciada somente nas imagens fractográficas. Já nos CPs com entalhe, mesmo com redução de concentrações de tensões no entalhe, observou-se resultados bem semelhantes, ficando evidenciada a susceptibilidade ao meio tanto nas imagens fractográficas (fratura frágil), quanto pela diminuição da ductilidade. / The increasing demand for cleaner fuels and renewable sources makes the ethanol rise as an alternative to conventional energy sources. The increasing consumption rate of this product, predicted for the upcoming years, will require safer and more efficient methods for transportation and storage through the entire transport cycle. Pipelines are the most economical way to transport it. However, there is evidence that pipeline transportation and the bottom of the storage tanks for ethanol might be susceptible to the stress corrosion cracking phenomenon. This phenomenon can be assessed by slow strain rate tests (SSRT) using smooth or notched specimen. This work aims to evaluate the influence of notch severity in specimens submitted to SSRT technique in simulated fuelgrade ethanol (PRCI SFGE). Four types of specimens of steel API X70 were prepared: unnotched, notched according to standard NACE TM 0111, notched with “intermediate” severity (severity 18.9% lower than the standard) and “less severe” notched (reduction of 37.2% on severity of the notched when compared with the standard). The results show that both sample types presented susceptibility in the solution (ethanol PRCI SFGE). For the unnotched specimens this susceptibility was observed only in fractographic images. In the notched specimens, despite the reduction of stress concentrations in the notch, the results were very similar. In this case the susceptibility can be observed in fractographic images (brittle fracture) as well as by decrease in the ductility.

O aumento de demanda por combustíveis menos poluentes e de fontes renováveis faz com que o Etanol se destaque como uma alternativa as fontes convencionais de energia. O crescente aumento no consumo deste produto, para os próximos anos, exigirá meios de transporte e estocagem eficientes e seguros, desde os diversos produtores até os terminais de distribuição. O transporte por dutos é, sem dúvida, a forma mais econômica de transporte deste produto, porém, há evidências de que esses dutos de transporte e fundo de tanques de armazenamento de etanol poderiam ser susceptíveis ao fenômeno de corrosão sob tensão. Este fenômeno pode ser avaliado, por exemplo, por ensaios de baixa taxa de deformação (BTD) com corpos de prova com ou sem entalhe. Este trabalho tem por objetivo avaliar a influência da severidade do entalhe em corpos de prova ensaiados a baixa taxa de deformação no meio etanol combustível simulado (PRCI SFGE). Foram preparados quatro tipos de corpos de prova de aço API X70: sem entalhe, com entalhe da norma NACE TM 0111, com entalhe “intermediário” (severidade 18,9% menor que o da norma) e com entalhe “menos severo” (redução de 37,2% na severidade do entalhe, segundo concentração de tensões, quando comparado com o da norma). Os resultados mostram que os quatro tipos de corpos de prova apresentaram susceptibilidade ao meio (etanol PRCI SFGE). Nos corpos de prova sem entalhe esta susceptibilidade foi evidenciada somente nas imagens fractográficas. Já nos CPs com entalhe, mesmo com redução de concentrações de tensões no entalhe, observou-se resultados bem semelhantes, ficando evidenciada a susceptibilidade ao meio tanto nas imagens fractográficas (fratura frágil), quanto pela diminuição da ductilidade. / The increasing demand for cleaner fuels and renewable sources makes the ethanol rise as an alternative to conventional energy sources. The increasing consumption rate of this product, predicted for the upcoming years, will require safer and more efficient methods for transportation and storage through the entire transport cycle. Pipelines are the most economical way to transport it. However, there is evidence that pipeline transportation and the bottom of the storage tanks for ethanol might be susceptible to the stress corrosion cracking phenomenon. This phenomenon can be assessed by slow strain rate tests (SSRT) using smooth or notched specimen. This work aims to evaluate the influence of notch severity in specimens submitted to SSRT technique in simulated fuelgrade ethanol (PRCI SFGE). Four types of specimens of steel API X70 were prepared: unnotched, notched according to standard NACE TM 0111, notched with “intermediate” severity (severity 18.9% lower than the standard) and “less severe” notched (reduction of 37.2% on severity of the notched when compared with the standard). The results show that both sample types presented susceptibility in the solution (ethanol PRCI SFGE). For the unnotched specimens this susceptibility was observed only in fractographic images. In the notched specimens, despite the reduction of stress concentrations in the notch, the results were very similar. In this case the susceptibility can be observed in fractographic images (brittle fracture) as well as by decrease in the ductility.