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O desenvolvimento tumoral é um processo que compreende diversas etapas e consiste no desenvolvimento progressivo de células normais para um estado neoplásico através de diversas mudanças bioquímicas e fenotípicas. Entre as principais marcas do câncer estão a capacidade de invasão tecidual e metástase. A metástase é responsável por, aproximadamente, 90% das mortes causadas por câncer. Assim, os métodos mais efetivos para a melhoria dos índices de morbidade e mortalidade relacionados ao câncer são a detecção precoce, a prevenção e o tratamento da metástase. O processo de EMT, que ocorre naturalmente durante a embriogênese e reparo tecidual, está envolvido também na progressão e metástase do câncer. A EMT induz alterações celulares e microambientais complexas que resultam na aquisição de um fenótipo mesenquimal pelas células epiteliais, juntamente com um aumento das capacidades migratórias e invasivas celulares. A EMT pode ser induzida por diversos fatores extracelulares, como os fatores de crescimento TGF?, EGF, HGF e PDGF. Além disso, a superexpressão de fatores de transcrição como SNAIL, SLUG, ZEB1 e TWIST1 também é capaz de induzir a EMT in vitro. A fim de ampliar o conhecimento dos mecanismos envolvidos no processo de EMT a nível de proteínas, foram realizadas neste trabalho análises de alterações proteômicas em diferentes modelos de indução da EMT na linhagem de adenocarcinoma de mama MCF7, sendo eles a superexpressão do FT SNAIL, o tratamento com o inibidor de histonas deacetilases SAHA e o tratamento com o fator de crescimento EGF. A análise proteômica detalhada por LC-MS/MS das frações subcelulares de núcleo, citoplasma e membrana das células superexpressando SNAIL gerou uma lista de proteínas reguladas relacionadas com o processo de EMT e que foram avaliadas nos demais modelos de indução. Entre essas, a proteína HDAC1, que teve seus níveis diminuídos pela superexpressão de SNAIL. O tratamento da linhagem MCF7 com o inibidor de histonas deacetilases SAHA demonstrou uma correlação positiva com o aumento dos níveis de SNAIL nas células MCF7, sugerindo um cross-talk entre ambas as proteínas. Além disso, otratamento com SAHA induziu alterações celulares e proteicas que também sugerem a indução do processo de EMT nas células MCF7. Por fim, o tratamento com o fator de crescimento EGF também foi capaz de induzir a EMT nas células MCF7 e apresentou envolvimento na regulação do ciclo celular, alterações de proteínas em comum com os demais tratamentos e regulação diferencial de proteínas entre os subcompartimentos, indicando similaridades entre os processos e potenciais mecanismos de translocação subcelular. Em conclusão, este estudo relevou proteínas alvo relacionadas à EMT, abrindo possibilidades para tentar alterar processos relacionados à progressão tumoral e ao processo metastático. / Tumor development is a process comprising several steps and consists in the progressive development of normal cells into a neoplastic state through various biochemical and phenotypic changes. Among the major marks of cancer are the capacity for tissue invasion and metastasis. Metastasis accounts for approximately 90% of cancer deaths. Thus, the most effective methods for improving cancer-related morbidity and mortality rates are early detection, prevention and treatment of metastasis. The EMT process, which occurs naturally during embryogenesis and tissue repair, is also involved in cancer progression and metastasis. EMT induces complex cellular and microenvironmental changes that result in the acquisition of a mesenchymal phenotype by epithelial cells, together with an increase in migratory and invasive cellular capacities. EMT can be induced by various extracellular factors, such as TGF?, EGF, HGF and PDGF. In addition, overexpression of some transcription factors such as SNAIL, SLUG, ZEB1 and TWIST1 is also capable of inducing EMT in vitro. In order to increase the knowledge of the mechanisms involved in the EMT process, we performed proteomic analysis in different models of EMT induction in the MCF7 breast adenocarcinoma cell line, which were the overexpression of SNAIL, treatment with the histone deacetylase inhibitor SAHA and treatment with the growth factor EGF. The detailed proteomic analysis by LC-MS/MS of the subcellular fractions of nucleus, cytoplasm and membrane of the overexpressing SNAIL cells generated a list of regulated proteins related to the EMT process and that were evaluated in the other models of induction. Among these, the HDAC1 protein, which had its levels decreased by SNAIL overexpression. Treatment of the MCF7 cell line with the histone deacetylase inhibitor SAHA showed a positive correlation with the increase of SNAIL levels, suggesting a cross-talk between both proteins. In addition, SAHA treatment induced cellular and protein alterations that also suggest the induction of the EMT process in MCF7 cells. Finally, the treatment with the growth factor EGF was also able to induce the EMT in MCF7 cells and showed involvement in the regulation of the cell cycle, changes in proteins in common with the other treatments and differential regulation of proteins among thesubcompartiments, indicating similarities between the processes and potential mechanisms of subcellular translocation. In conclusion, this study revealed target proteins related to EMT, opening possibilities to try to alter processes related to tumor progression and metastatic process.

O desenvolvimento tumoral é um processo que compreende diversas etapas e consiste no desenvolvimento progressivo de células normais para um estado neoplásico através de diversas mudanças bioquímicas e fenotípicas. Entre as principais marcas do câncer estão a capacidade de invasão tecidual e metástase. A metástase é responsável por, aproximadamente, 90% das mortes causadas por câncer. Assim, os métodos mais efetivos para a melhoria dos índices de morbidade e mortalidade relacionados ao câncer são a detecção precoce, a prevenção e o tratamento da metástase. O processo de EMT, que ocorre naturalmente durante a embriogênese e reparo tecidual, está envolvido também na progressão e metástase do câncer. A EMT induz alterações celulares e microambientais complexas que resultam na aquisição de um fenótipo mesenquimal pelas células epiteliais, juntamente com um aumento das capacidades migratórias e invasivas celulares. A EMT pode ser induzida por diversos fatores extracelulares, como os fatores de crescimento TGF?, EGF, HGF e PDGF. Além disso, a superexpressão de fatores de transcrição como SNAIL, SLUG, ZEB1 e TWIST1 também é capaz de induzir a EMT in vitro. A fim de ampliar o conhecimento dos mecanismos envolvidos no processo de EMT a nível de proteínas, foram realizadas neste trabalho análises de alterações proteômicas em diferentes modelos de indução da EMT na linhagem de adenocarcinoma de mama MCF7, sendo eles a superexpressão do FT SNAIL, o tratamento com o inibidor de histonas deacetilases SAHA e o tratamento com o fator de crescimento EGF. A análise proteômica detalhada por LC-MS/MS das frações subcelulares de núcleo, citoplasma e membrana das células superexpressando SNAIL gerou uma lista de proteínas reguladas relacionadas com o processo de EMT e que foram avaliadas nos demais modelos de indução. Entre essas, a proteína HDAC1, que teve seus níveis diminuídos pela superexpressão de SNAIL. O tratamento da linhagem MCF7 com o inibidor de histonas deacetilases SAHA demonstrou uma correlação positiva com o aumento dos níveis de SNAIL nas células MCF7, sugerindo um cross-talk entre ambas as proteínas. Além disso, otratamento com SAHA induziu alterações celulares e proteicas que também sugerem a indução do processo de EMT nas células MCF7. Por fim, o tratamento com o fator de crescimento EGF também foi capaz de induzir a EMT nas células MCF7 e apresentou envolvimento na regulação do ciclo celular, alterações de proteínas em comum com os demais tratamentos e regulação diferencial de proteínas entre os subcompartimentos, indicando similaridades entre os processos e potenciais mecanismos de translocação subcelular. Em conclusão, este estudo relevou proteínas alvo relacionadas à EMT, abrindo possibilidades para tentar alterar processos relacionados à progressão tumoral e ao processo metastático. / Tumor development is a process comprising several steps and consists in the progressive development of normal cells into a neoplastic state through various biochemical and phenotypic changes. Among the major marks of cancer are the capacity for tissue invasion and metastasis. Metastasis accounts for approximately 90% of cancer deaths. Thus, the most effective methods for improving cancer-related morbidity and mortality rates are early detection, prevention and treatment of metastasis. The EMT process, which occurs naturally during embryogenesis and tissue repair, is also involved in cancer progression and metastasis. EMT induces complex cellular and microenvironmental changes that result in the acquisition of a mesenchymal phenotype by epithelial cells, together with an increase in migratory and invasive cellular capacities. EMT can be induced by various extracellular factors, such as TGF?, EGF, HGF and PDGF. In addition, overexpression of some transcription factors such as SNAIL, SLUG, ZEB1 and TWIST1 is also capable of inducing EMT in vitro. In order to increase the knowledge of the mechanisms involved in the EMT process, we performed proteomic analysis in different models of EMT induction in the MCF7 breast adenocarcinoma cell line, which were the overexpression of SNAIL, treatment with the histone deacetylase inhibitor SAHA and treatment with the growth factor EGF. The detailed proteomic analysis by LC-MS/MS of the subcellular fractions of nucleus, cytoplasm and membrane of the overexpressing SNAIL cells generated a list of regulated proteins related to the EMT process and that were evaluated in the other models of induction. Among these, the HDAC1 protein, which had its levels decreased by SNAIL overexpression. Treatment of the MCF7 cell line with the histone deacetylase inhibitor SAHA showed a positive correlation with the increase of SNAIL levels, suggesting a cross-talk between both proteins. In addition, SAHA treatment induced cellular and protein alterations that also suggest the induction of the EMT process in MCF7 cells. Finally, the treatment with the growth factor EGF was also able to induce the EMT in MCF7 cells and showed involvement in the regulation of the cell cycle, changes in proteins in common with the other treatments and differential regulation of proteins among thesubcompartiments, indicating similarities between the processes and potential mechanisms of subcellular translocation. In conclusion, this study revealed target proteins related to EMT, opening possibilities to try to alter processes related to tumor progression and metastatic process.

Several parasites that require two or more hosts to complete their life cycles are known to manipulate host behaviour, enhancing their transmission to the next host. The intertidal snail, Littorina sitkana, is host to a diverse assemblage of parasites dominated by trematodes. Trematodes often use snails as first intermediate host and vertebrates as definitive host. Trematode infections can affect host behaviours such as dispersal and foraging. I identified four sites in Barkley Sound that varied in trematode prevalence and species richness. I measured dispersal of snails at these sites and in the laboratory to assess effects of trematode infection on behaviour. I measured feeding rate under laboratory conditions. Trematode effects lowered snail grazing activity at three of the four sites studied, suggesting trematode infection lowers feeding rate of L. sitkana, potentially affecting algal composition of the intertidal zone. Infected male snails travelled longer distances in some sites but shorter distances in others. There was an almost significant effect of trematode infection on vertical displacement of L. sitkana in the field. I estimated survival rates on each site through intensive capture-mark-recapture experiments. There was differential survival among sites, but no negative correlation between survival estimates and trematode prevalence. / Graduate / 0718 / 0329 / 0472 / mayala@uvic.ca

Epithelial-mesenchymal transition (EMT) is an evolutionarily conserved process during which cells lose epithelial characteristics and gain a migratory phenotype. Although downregulation of epithelial cadherins by Snail and other transcriptional repressors is generally considered a prerequisite for EMT, recent studies have challenged this view. Here we investigate the relationship between E-cadherin and P-cadherin expression and localization, Snail function and EMT during gastrulation in chicken embryos. Expression analyses show that while E-cadherin transcripts are detected in the epiblast but not in the primitive streak or mesoderm, P-cadherin mRNA and protein are present in the epiblast, primitive and mesoderm. Antibodies that specifically recognize E-cadherin are not presently available. During EMT, P-cadherin relocalizes from the lateral surfaces of epithelial epiblast cells to a circumferential distribution in emerging mesodermal cells. Cells electroporated with an E-cadherin expression construct undergo EMT and migrate into the mesoderm. An examination of Snail function showed that reduction of Slug (SNAI2) protein levels using a morpholino fails to inhibit EMT, and expression of human or chicken Snail in epiblast cells fails to induce EMT. In contrast, cells expressing the Rho inhibitor peptide C3 rapidly exit the epiblast without activating Slug or the mesoderm marker N-cadherin. Together, these experiments show that epiblast cells undergo EMT while retaining P-cadherin, and raise questions about the mechanisms of EMT regulation during avian gastrulation.

The transforming growth factor beta (TGFβ) is a cytokine that regulates a plethora of cellular processes such as cell proliferation, differentiation, migration and apoptosis. TGFβ signals via serine/threonine kinase receptors and activates the Smads to regulate gene expression. Enigmatically, TGFβ has a dichotomous role as a tumor suppressor and a tumor promoter in cancer. At early stages of tumorigenesis, TGFβ acts as a tumor suppressor by exerting growth inhibitory effects and inducing apoptosis. However, at advanced stages, TGFβ contributes to tumor malignancy by promoting invasion and metastasis. The pro-tumorigenic TGFβ potently triggers an embryonic program known as epithelial-mesenchymal transition (EMT). EMT is a dynamic process whereby polarized epithelial cells adapt a mesenchymal morphology, thereby facilitating migration and invasion. Downregulation of cell-cell adhesion molecules, such as E-cadherin and ZO-1, is an eminent feature of EMT. TGFβ induces EMT by upregulating a non-histone chromatin factor, high mobility group A2 (HMGA2). This thesis focuses on elucidating the molecular mechanisms by which HMGA2 elicits EMT. We found that HMGA2 regulates a network of EMT transcription factors (EMT-TFs), such as members of the Snail, ZEB and Twist families, during TGFβ-induced EMT. HMGA2 can interact with Smad complexes to synergistically induce Snail expression. HMGA2 also directly binds and activates the Twist promoter. We used mouse mammary epithelial cells overexpressing HMGA2, which are mesenchymal in morphology and highly invasive, as a constitutive EMT model. Snail and Twist have complementary roles in HMGA2-mesenchymal cells during EMT, and tight junctions were restored upon silencing of both Snail and Twist in these cells. Finally, we also demonstrate that HMGA2 can epigenetically silence the E-cadherin gene. In summary, HMGA2 modulates multiple reprogramming events to promote EMT and invasion.

Magister en biomedicina celular y molecular / En Chile la incidencia y mortalidad del cáncer de próstata (CaP) está aumentado, siendo actualmente la segunda causa de muerte por cáncer en hombres. Durante la progresión tumoral las células epiteliales disminuyen sus moléculas de adhesión, polaridad, posicionamiento, reordenan su citoesqueleto y aumentan sus capacidades migratorias e invasivas. Todos estos cambios se conocen bajo el concepto de Transición Epitelio-Mesénquima (TEM). La TEM se caracteriza por el aumento de ciertos factores transcripcionales, tales como SNAIL1, que reprime genes característicos de un fenotipo epitelial, como E-cadherina, y aumenta indirectamente la expresión de genes que promueven el fenotipo mesenquimático. Se propone que el factor transcripcional SNAIL1 disminuye la proliferación y aumenta las capacidades migratorias e invasivas en líneas celulares de CaP. Para ello se trabajó con las líneas celulares LNCaP y PC3 con expresión ectópica y silenciamiento de SNAIL1 obtenidas mediante el uso de vectores lentivirales. Se caracterizó la expresión de marcadores de TEM mediante PCR en tiempo real, western blot e inmunofluorescencia. Además, se analizó la sobrevida mediante MTS; proliferación utilizando anticuerpos contra PCNA y Ki-67; migración a través de placas con poros de 8 μm (que permiten el paso de células) e invasividad mediante cámaras de membranas cubiertas con una capa de membrana basal. Complementariamente se determinó la apoptosis mediante un kit que contiene un sustrato que es modificado por las caspasas 3 y 7. Se determinó que ambas líneas celulares de CaP con sobreexpresión y silenciamiento de SNAIL1 la proliferación y sobrevida disminuyen. Sin embargo, la sobreexpresión de SNAIL1 disminuye la apoptosis respecto a las células con silenciamiento de la expresión de éste gen, en las cuales aumenta la muerte celular. Respecto a las capacidades migratorias e invasivas, aumentaron sólo en las células con sobreexpresión de SNAIL1 y disminuyeron en las células con silenciamiento. En conclusión, células de CaP con sobreexpresión de SNAIL1 exhibieron características fenotípicas tipo TEM, mientras que el silenciamiento de éste represor transcripcional condujo a las células a un fenotipo tipo epitelial con la disminución de características mesenquimales como la migración e invasión. / FONDECYT No 1110269

Resistance of various cancers to conventional therapies including radio- and chemo- therapy is one of the most investigated phenomena in the molecular and clinical oncology. Recurrent disease is characterized by the presence of metastases, which are responsible for 90% of cancer-related mortality. Fractionated ionizing radiation (fIR) combined with surgery or hormone therapy represent the first-choice treatment for medium to high risk localized prostate carcinoma (PCa). In PCa, the failure of radiotherapy (RT) is often caused by radioresistance and further dissemination of escaping (surviving) cells. To investigate the radioresistance-associated phenotype, we exposed four metastasis- derived human PCa cell lines (DU145, PC-3, LNCaP, and 22RV1) to clinically relevant daily fractions of ionizing radiation (fIR; 35 doses of 2 Gy) resulting in generation of two surviving populations: adherent senescent-like cells expressing common senescence-associated markers and non-adherent anoikis-resistant stem cell-like cells with active Notch signaling and expression of stem cell markers CD133, Oct-4, Sox2, and Nanog. While the radioresistant adherent cells were capable to resume proliferation shortly after the end of irradiation, the non- adherent cells started to proliferate only after their reattachment...

Submitted by Ricardo Cedraz Duque Moliterno (ricardo.moliterno@uefs.br) on 2015-07-29T22:02:33Z No. of bitstreams: 1 Disserta??o Final_Patr?cia Herc?lia de Almeida.pdf: 2058812 bytes, checksum: f101704775fb70cb9a74a8e794349727 (MD5) / Made available in DSpace on 2015-07-29T22:02:33Z (GMT). No. of bitstreams: 1 Disserta??o Final_Patr?cia Herc?lia de Almeida.pdf: 2058812 bytes, checksum: f101704775fb70cb9a74a8e794349727 (MD5) Previous issue date: 2014-03-27 / Funda??o de Amparo ? Pesquisa do Estado da Bahia - FAPEB / The African snail Achatina fulica Bowdich, 1822, is an invasive species that cause environmental and economic damage, as to human and animal health. The aim of this study was to evaluate the occurrence and parasitological aspects of A. fulica in Feira de Santana (BA). To assess the occurrence of A. fulica, sampling was made from January to June 2013 at four points in the urban perimeter of the city. In the laboratory animals were measured as the total length of the shell and the total mass. From these data the degree of development of the population was estimated, and the relationships between morphometric and climatic variables and the condition factor were calculated. During this period were collected 600 snails showing predominant length in the range of 57.0 to 47.6 mm. Results allowed to infer that the population of A. fulica in these sampling points is in decline, since in these areas were found juvenile and young animals. There was no association between climatic and morphometric variables. The relationship between total length and weight showed that A. fulica presented a negative allometric growth. The condition factor presented variations on time over the six months of collection. In order to evaluate parasitological aspects in A. fulica, animals were collected from may 2012-to june 2013 in eight areas in the urban perimeter of the city.The animals were collected early in the morning and transported to the laboratory of the Zoonoses and Public Health Research Group -UEFS. Each clam was analyzed individually using the artificial digestion technique in hydrochloric acid solution with identification of larvae found. The larvae have been identified as Metastrongylidae were submitted to the polymerase chain reaction for the identification of the genus Angyostrongylus. 220 animals were tested and in 28 (12.7; 95: 8.6 -17.9) were found at least one of four morphotypes of nematodes. Angyostrongylus has not been registered in snails examined. There was no association between climatic variables and the presence of larvae. The location of collection and the size of the snail influenced in the larvae of nematodes. Both parasited and not parasitised animals have negative allometric growth. The relative condition factor did not differ significantly between the groups. Nested-PCR has been used to search Cryptosporidium ssp and Sarcocystidae DNA in fecal samples of Achatina fulica. Amplicons compatibles with Cryptosporidium ssp were found in 2 of 223 stool samples analyzed. Of the 223 stool samples analysed, in 58 samples we found the 290 base pair fragment expected for Sarcocystidae, and then subjected to RFLP. Of these, 41 samples were cleaved by enzymes used in this study, producing a profile compatible with T. gondii. The other 17 samples were not compatible with Sarcocystidae. This is the first record of the occurrence of A. fulica in Feira de Santana (BA), with research of association of climatic variables to the biometric parameters of the animals. These results indicate the presence of animal and humana parasites in Achatina fulica in the municipality of Feira de Santana, demonstrating the potential of these animals in the maintenance and spread of these agents in the environment. / O caramujo africano Achatina fulica Bowdich, 1822, ? uma esp?cie invasora que causa preju?zos ambientais, econ?micos e a sa?de humana e animal. O objetivo desse estudo foi avaliar a ocorr?ncia e os aspectos parasit?rios de Achatina fulica no munic?pio de Feira de Santana (BA). Para avalia??o da ocorr?ncia de A. fulica foram feitas coletas de janeiro a junho de 2013 em quatro pontos no per?metro urbano do munic?pio. No laborat?rio os animais foram mensurados quanto ao comprimento total da concha e a massa total. A partir desses dados o grau de desenvolvimento da popula??o foi estimado, as rela??es entre as vari?veis morfom?tricas e clim?ticas e o fator de condi??o foram calculados. Durante esse per?odo foram coletados 600 caramujos apresentando comprimento predominante no intervalo de 47,6-57,0mm. Os resultados permitiram inferir que a popula??o de A. fulica nos pontos de coleta estudados est? em decl?nio, j? que nessas ?reas foram encontrados animais juvenis e jovens. N?o houve associa??o entre as vari?veis clim?ticas e morfom?tricas. A rela??o entre o comprimento total e a massa corporal mostrou que A. fulica apresentou um crescimento alom?trico negativo. O fator de condi??o apresentou varia??es pontuais ao longo dos seis meses de coleta. A fim de avaliar os aspectos parasit?rios em A. fulica foram realizadas coletas de maio de 2012 a junho de 2013 em oito ?reas no per?metro urbano do munic?pio, com registro de observa??es sobre os locais de coleta. Cada molusco foi analisado individualmente utilizando a t?cnica de digest?o artificial em solu??o de ?cido clor?drico, com identifica??o das larvas encontradas. As larvas identificadas como Metastrongylidae foram submetidas ? rea??o em cadeia pela polimerase para a identifica??o do g?nero Angyostrongylus. Foram analisados 220 animais e em 28 (12,7%; IC95%: 8,6-17,9) foram encontrados pelo menos um de quatro morfotipos de nemat?deos. Angyostrongylus n?o foi registrado nos caramujos examinados. N?o houve associa??o entre vari?veis clim?ticas e a presen?a de larvas. O local de coleta e o tamanho do caramujo influenciaram no encontro de larvas de nemat?deos. Tanto animais parasitados quanto n?o parasitados apresentaram um crescimento alom?trico negativo. O fator de condi??o relativo n?o diferiu significamente entre os grupos. A fim de pesquisar a presen?a de DNA Cryptosporidium ssp. e Sarcocystidae nas amostras fecais de Achatina fulica foi utilizada a t?cnica de nested-PCR (nPCR). Para Cryptosporidium ssp., amplicons compat?veis com este parasito foram encontrados em 2 de 223 amostras de fezes analisadas. Das 223 amostras de fezes de Achatina fulica submetidas ? nPCR para detec??o de Sarcocystidae, 58 amostras amplificaram o fragmento esperado de 290 pares de base, sendo ent?o submetidas ? RFLP. Destas, 41 amostras foram clivadas pelas enzimas utilizadas nesse estudo, produzindo um perfil compat?vel com T. gondii. As outras 17 amostras n?o foram compat?veis com o Sarcocystidae. Este ? o primeiro registro da ocorr?ncia de A. fulica no munic?pio de Feira de Santana (BA), com pesquisa da associa??o das vari?veis clim?ticas aos par?metros biom?tricos dos animais estudados. Estes resultados indicam que h? presen?a de parasitos de interesse em sa?de animal e humana em Achatina fulica no munic?pio de Feira de Santana, Bahia, demonstrando o potencial destes animais na manuten??o e dispers?o destes agentes no ambiente.

No description available.