In der vorliegenden Arbeit wurde das Expressionsverhalten fünf zellulärer Marker beim metastasierenden Plattenepithelkarzinom des Kopf- und Halsbereiches untersucht. Bei den getesteten Markern handelte es sich um einen MAGE-A, zwei verschiedenen VEGF, einen EGFR und einen C-Src-Tyrosinkinase Antikörper. Im Einzelnen sollte hinterfragt werden, ob ein Zusammenhang zwischen der Antikörperexpression und verschiedenen, klinischen und histopathologischen Parametern (pT-Stadium, pN-Stadium, histologisches Grading, Tumorverhornung, Patientenalter, Geschlecht des Patienten) besteht. Weiterhin war von Interesse, ob Parallelen zwischen dem Expressionsverhalten der verschiedenen Antikörper untereinander zu erkennen sind. Die Ergebnisse wurden anschließend mit Erkenntnissen aus anderen Studien und Literaturangaben verglichen. / This study aimed to evaluate the expression profiles of cellular marker in metastatic head and neck cancer in the primary tumor and in the metastases. The expression profiles of MAGE-A, VEGF-A, VEGF-C, EGFR and c-Src in 50 squamous cell carcinoma were characterised by immunhistochemical stainig.

No description available.

Background: Pancreatic cancer is one of the leading causes of cancer-related death. Using DNA gene expression analysis based on a custom made Affymetrix cancer array, we investigated the expression pattern of both primary and established pancreatic carcinoma cell lines. Methods: We analyzed the gene expression of 5 established pancreatic cancer cell lines (AsPC-1, BxPC-3, Capan-1, Capan-2 and HPAF II) and 5 primary isolates, 1 of them derived from benign pancreatic duct cells. Results: Out of 1,540 genes which were expressed in at least 3 experiments, we found 122 genes upregulated and 18 downregulated in tumor cell lines compared to benign cells with a fold change > 3. Several of the upregulated genes (like Prefoldin 5, ADAM9 and E-cadherin) have been associated with pancreatic cancer before. The other differentially regulated genes, however, play a so far unknown role in the course of human pancreatic carcinoma. By means of immunohistochemistry we could show that thymosin [β-10 (TMSB10), upregulated in tumor cell lines, is expressed in human pancreatic carcinoma, but not in non-neoplastic pancreatic tissue, suggesting a role for TMSB10 in the carcinogenesis of pancreatic carcinoma. Conclusion: Using gene expression profiling of pancreatic cell lines we were able to identify genes differentially expressed in pancreatic adenocarcinoma, which might contribute to pancreatic cancer development. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Zugl.: Kiel, Univ., Diss., 2008

Epigenetic control of gene expression by histone post-translational modifications (PTMs) is a complex process regulated by proteins that can 'read', 'write' or 'erase' these PTMs. The histone lysine demethylase (KDM) family of epigenetic enzymes remove methyl modifications from lysines on histone tails. The Jumonji C domain (JmjC) family is the largest family of KDMs. Investigating the scope and mechanisms of the JmjC KDMs is of interest for understanding the diverse functions of the JmjC KDMs in vivo, as well as for the application of the basic science to medicinal chemistry design. The work described in this thesis aimed to biochemically investigate the inhibitor and substrate diversity of the JmjC KDMs, it led to the identification of new inhibitors and substrates and revealed a potential combinatorial dependence between adjacent histone PTMs. Structure-activity relationship studies gave rise to an n-octyl ester form of IOX1 with improved cellular potency and selectivity towards the KDM4 subfamily. This compound should find utility as a basis for the development of JmjC inhibitors and as a tool compound for biological studies. The rest of this thesis focused on the biochemical investigations of potential substrates and inhibitors for KDM3A, a JmjC demethylase with varied physiological functions. Kinetic characterisation of reported KDM3A substrates was used as the basis for evaluations of novel substrates and inhibitors. Further studies found TCA cycle intermediates to be moderate co-substrate competitive inhibitors of KDM3A. Biochemical investigations were carried out to study potential protein-protein interactions of KDM3A with intraflagellar transport proteins (IFTs), non-histone proteins involved in the formation of sperm flagellum. Work then addressed the exploration of novel in vitro substrates for KDM3 (KDM3A and JMJD1C) mediated catalysis, including: methylated arginines, lysine analogues, acetylated and formylated lysines. KDM3A, and other JmjC KDMs, were found to catalyse novel arginine demethylation reaction in vitro. Knowledge gained from studies with unnatural lysine analogues was utilised to search for additional novel PTM substrates for KDM3A. These results constitute the first evidence of JmjC KDM catalysed hydroxylation of an Nε-acetyllysine residue. The H3 K4me3 position seems to be required for acetyllysine substrate recognition, implying a combinatorial effect between PTMs. Preliminary results provide evidence that JMJD1C, a KDM3 protein previously reported to be inactive, may catalyse deacetylation in vitro. An additional novel reaction, observed with both KDM3A and JMJD1C, is deformylation of N^ε-formyllysine residues on histone H3 fragment peptides. Interestingly, H3 K4 methylation was also observed to enhance the apparent deformylation of both KDM3A and JMJD1C catalysed reactions. Overall, findings in this thesis suggest that the catalytic activity of JmjC KDMs extends beyond lysine demethylation. In a cellular context, members of the KDM3 subfamily might provide a regulatory link between methylation and acylation marks. Such a link will further highlight the complex relationships between histone PTMs and the epigenetic enzymes that regulate them. The observed dependency of H3 K9 catalysis on H3 K4 methylation adds another layer of complexity to the epigenetic regulation by histone PTMs.

Este trabalho descreve o planejamento, desenvolvimento e validação de um modelo de software educacional. O aplicativo é um ambiente multimídia de ensino e aprendizagem do Metabolismo dos Glicídios e o Ciclo de Krebs, denominado e-Metabolismo: Glicídios e contém um jogo de seqüência para o ensino de Bioquímica, denominado Diagrama Metabólico Dinâmico Virtual. O estudo de teorias pedagógicas e a experiência em aulas com os alunos do curso de medicina da Fundação Faculdade Federal de Ciências Médicas de Porto Alegre apontou a necessidade de mudanças no ensino de Bioquímica com uso das novas tecnologias de informação e comunicação. A justificativa do uso de um jogo virtual como método de ensino tem por base os resultados obtidos com o uso de um jogo de seqüência lógica em tabuleiro, na Disciplina de Bioquímica. O desenvolvimento do e-Metabolismo: Glicídios, tendo como referência a prática pedagógica baseada na epistemologia genética Jean Piaget, incluiu no seu planejamento a escolha de ferramenta de programação para permitir a interação do usuário (aluno) com o ambiente. O produto utiliza amplamente recursos de multimídia e pode ser disponibilizado num servidor ou em forma de CD-ROM. O ambiente virtual possibilita a interação do aluno com o ambiente e com colegas e professores através de ferramentas como, por exemplo, acesso a e-mails, chats, fóruns, mapas conceituais e diário de bordo. Instrumentos de avaliação de software foram estudados e aplicados com alunos de Disciplinas de Bioquímica no sentido de validar o software e-Metabolismo tanto no que se refere aos aspectos técnicos como a aprendizagem do conteúdo pelos alunos. Experiências com o uso do software foram, primeiramente, realizadas com alunos do curso de Medicina da FFFCMPA e depois com alunos de outros cursos. O primeiro grupo de alunos que avaliaram o e-Metabolismo foi formado pelos monitores da Disciplina. Mapas conceituais, testes escritos e avaliação dos registros deixados pelos usuários no próprio software foram utilizados como instrumentos de avaliação do conhecimento dos alunos. O grau de satisfação com o uso do método de estudo, foi avaliado por um questionário, cujas respostas foram analisadas e categorizadas. Os resultados obtidos indicam que o ambiente apresenta interface de fácil acesso, desperta o interesse, possibilita ao aluno escolher de que maneira quer fazer o seu estudo sem prejuízos no seu desempenho e facilita o estudo, sendo, portanto, considerado válido como instrumento educacional. Por se tratar de um ambiente dinâmico, deve ser constantemente atualizado, e a versão atual contém as modificações sugeridas por professores e alunos, facilitando o uso na Internet e o acompanhamento do aluno. / This work describes the planing, the development and the validation of a game-like educational software. This multimedia ambient was designed for the study of carbohydrates metabolic pathways and the Krebs's Cycle, called e-Metabolism: carbohydrates, and contains the sequential game, called Virtual Dynamic Metabolic Diagram. The study of pedagogical theories and experiments in classroom with medicine students of the “Fundação Faculdade Federal de Ciências Médicas de Porto Alegre”, pointed the necessity of changes in Biochemistry courses, involving new technologies of information and communication. The use of a game-like software as a tool for teaching is based on experiments related to the use of tray games at Biochemistry courses. The development of the e-Metabolism took as a reference the integrationists’ pedagogical practice, based on Jean Piaget's concepts, related to genetic epistemology and constructivism, yet allowing the professors to choose the teaching method they wish to use. This product integrates multimedia resources extensively, and can be used in computer networks or in the format of a CD-ROM. In the virtual environment students will be able to interact with the environment as well as with classmates and professors through such tools as chats, forums, concept maps and notepads. Software ’s evaluation Instruments were studied and applied with undergraduate students of Biochemistry classes in the way to value the eMetabolism software in its technical aspects and student’s content learning aspects. Conceptual maps, written tests and evaluation of user’s registers realized with this software where used as evaluation instruments of students knowledge. The level of satisfaction was evaluated by a questionnaire, which answers had been analyzed and categorized. The results show that the e-Metabolism is easy to use, awakes the interest and facilitates the study, improving the student performance and can be considered a valid educational instrument. Since this is a dynamic ambient and is constantly actualized, the current version contains the changes suggested by teachers and students, making easier to use it at the Internet and to do a better analysis of the student’s learning.

Este trabalho descreve o planejamento, desenvolvimento e validação de um modelo de software educacional. O aplicativo é um ambiente multimídia de ensino e aprendizagem do Metabolismo dos Glicídios e o Ciclo de Krebs, denominado e-Metabolismo: Glicídios e contém um jogo de seqüência para o ensino de Bioquímica, denominado Diagrama Metabólico Dinâmico Virtual. O estudo de teorias pedagógicas e a experiência em aulas com os alunos do curso de medicina da Fundação Faculdade Federal de Ciências Médicas de Porto Alegre apontou a necessidade de mudanças no ensino de Bioquímica com uso das novas tecnologias de informação e comunicação. A justificativa do uso de um jogo virtual como método de ensino tem por base os resultados obtidos com o uso de um jogo de seqüência lógica em tabuleiro, na Disciplina de Bioquímica. O desenvolvimento do e-Metabolismo: Glicídios, tendo como referência a prática pedagógica baseada na epistemologia genética Jean Piaget, incluiu no seu planejamento a escolha de ferramenta de programação para permitir a interação do usuário (aluno) com o ambiente. O produto utiliza amplamente recursos de multimídia e pode ser disponibilizado num servidor ou em forma de CD-ROM. O ambiente virtual possibilita a interação do aluno com o ambiente e com colegas e professores através de ferramentas como, por exemplo, acesso a e-mails, chats, fóruns, mapas conceituais e diário de bordo. Instrumentos de avaliação de software foram estudados e aplicados com alunos de Disciplinas de Bioquímica no sentido de validar o software e-Metabolismo tanto no que se refere aos aspectos técnicos como a aprendizagem do conteúdo pelos alunos. Experiências com o uso do software foram, primeiramente, realizadas com alunos do curso de Medicina da FFFCMPA e depois com alunos de outros cursos. O primeiro grupo de alunos que avaliaram o e-Metabolismo foi formado pelos monitores da Disciplina. Mapas conceituais, testes escritos e avaliação dos registros deixados pelos usuários no próprio software foram utilizados como instrumentos de avaliação do conhecimento dos alunos. O grau de satisfação com o uso do método de estudo, foi avaliado por um questionário, cujas respostas foram analisadas e categorizadas. Os resultados obtidos indicam que o ambiente apresenta interface de fácil acesso, desperta o interesse, possibilita ao aluno escolher de que maneira quer fazer o seu estudo sem prejuízos no seu desempenho e facilita o estudo, sendo, portanto, considerado válido como instrumento educacional. Por se tratar de um ambiente dinâmico, deve ser constantemente atualizado, e a versão atual contém as modificações sugeridas por professores e alunos, facilitando o uso na Internet e o acompanhamento do aluno. / This work describes the planing, the development and the validation of a game-like educational software. This multimedia ambient was designed for the study of carbohydrates metabolic pathways and the Krebs's Cycle, called e-Metabolism: carbohydrates, and contains the sequential game, called Virtual Dynamic Metabolic Diagram. The study of pedagogical theories and experiments in classroom with medicine students of the “Fundação Faculdade Federal de Ciências Médicas de Porto Alegre”, pointed the necessity of changes in Biochemistry courses, involving new technologies of information and communication. The use of a game-like software as a tool for teaching is based on experiments related to the use of tray games at Biochemistry courses. The development of the e-Metabolism took as a reference the integrationists’ pedagogical practice, based on Jean Piaget's concepts, related to genetic epistemology and constructivism, yet allowing the professors to choose the teaching method they wish to use. This product integrates multimedia resources extensively, and can be used in computer networks or in the format of a CD-ROM. In the virtual environment students will be able to interact with the environment as well as with classmates and professors through such tools as chats, forums, concept maps and notepads. Software ’s evaluation Instruments were studied and applied with undergraduate students of Biochemistry classes in the way to value the eMetabolism software in its technical aspects and student’s content learning aspects. Conceptual maps, written tests and evaluation of user’s registers realized with this software where used as evaluation instruments of students knowledge. The level of satisfaction was evaluated by a questionnaire, which answers had been analyzed and categorized. The results show that the e-Metabolism is easy to use, awakes the interest and facilitates the study, improving the student performance and can be considered a valid educational instrument. Since this is a dynamic ambient and is constantly actualized, the current version contains the changes suggested by teachers and students, making easier to use it at the Internet and to do a better analysis of the student’s learning.

Este trabalho descreve o planejamento, desenvolvimento e validação de um modelo de software educacional. O aplicativo é um ambiente multimídia de ensino e aprendizagem do Metabolismo dos Glicídios e o Ciclo de Krebs, denominado e-Metabolismo: Glicídios e contém um jogo de seqüência para o ensino de Bioquímica, denominado Diagrama Metabólico Dinâmico Virtual. O estudo de teorias pedagógicas e a experiência em aulas com os alunos do curso de medicina da Fundação Faculdade Federal de Ciências Médicas de Porto Alegre apontou a necessidade de mudanças no ensino de Bioquímica com uso das novas tecnologias de informação e comunicação. A justificativa do uso de um jogo virtual como método de ensino tem por base os resultados obtidos com o uso de um jogo de seqüência lógica em tabuleiro, na Disciplina de Bioquímica. O desenvolvimento do e-Metabolismo: Glicídios, tendo como referência a prática pedagógica baseada na epistemologia genética Jean Piaget, incluiu no seu planejamento a escolha de ferramenta de programação para permitir a interação do usuário (aluno) com o ambiente. O produto utiliza amplamente recursos de multimídia e pode ser disponibilizado num servidor ou em forma de CD-ROM. O ambiente virtual possibilita a interação do aluno com o ambiente e com colegas e professores através de ferramentas como, por exemplo, acesso a e-mails, chats, fóruns, mapas conceituais e diário de bordo. Instrumentos de avaliação de software foram estudados e aplicados com alunos de Disciplinas de Bioquímica no sentido de validar o software e-Metabolismo tanto no que se refere aos aspectos técnicos como a aprendizagem do conteúdo pelos alunos. Experiências com o uso do software foram, primeiramente, realizadas com alunos do curso de Medicina da FFFCMPA e depois com alunos de outros cursos. O primeiro grupo de alunos que avaliaram o e-Metabolismo foi formado pelos monitores da Disciplina. Mapas conceituais, testes escritos e avaliação dos registros deixados pelos usuários no próprio software foram utilizados como instrumentos de avaliação do conhecimento dos alunos. O grau de satisfação com o uso do método de estudo, foi avaliado por um questionário, cujas respostas foram analisadas e categorizadas. Os resultados obtidos indicam que o ambiente apresenta interface de fácil acesso, desperta o interesse, possibilita ao aluno escolher de que maneira quer fazer o seu estudo sem prejuízos no seu desempenho e facilita o estudo, sendo, portanto, considerado válido como instrumento educacional. Por se tratar de um ambiente dinâmico, deve ser constantemente atualizado, e a versão atual contém as modificações sugeridas por professores e alunos, facilitando o uso na Internet e o acompanhamento do aluno. / This work describes the planing, the development and the validation of a game-like educational software. This multimedia ambient was designed for the study of carbohydrates metabolic pathways and the Krebs's Cycle, called e-Metabolism: carbohydrates, and contains the sequential game, called Virtual Dynamic Metabolic Diagram. The study of pedagogical theories and experiments in classroom with medicine students of the “Fundação Faculdade Federal de Ciências Médicas de Porto Alegre”, pointed the necessity of changes in Biochemistry courses, involving new technologies of information and communication. The use of a game-like software as a tool for teaching is based on experiments related to the use of tray games at Biochemistry courses. The development of the e-Metabolism took as a reference the integrationists’ pedagogical practice, based on Jean Piaget's concepts, related to genetic epistemology and constructivism, yet allowing the professors to choose the teaching method they wish to use. This product integrates multimedia resources extensively, and can be used in computer networks or in the format of a CD-ROM. In the virtual environment students will be able to interact with the environment as well as with classmates and professors through such tools as chats, forums, concept maps and notepads. Software ’s evaluation Instruments were studied and applied with undergraduate students of Biochemistry classes in the way to value the eMetabolism software in its technical aspects and student’s content learning aspects. Conceptual maps, written tests and evaluation of user’s registers realized with this software where used as evaluation instruments of students knowledge. The level of satisfaction was evaluated by a questionnaire, which answers had been analyzed and categorized. The results show that the e-Metabolism is easy to use, awakes the interest and facilitates the study, improving the student performance and can be considered a valid educational instrument. Since this is a dynamic ambient and is constantly actualized, the current version contains the changes suggested by teachers and students, making easier to use it at the Internet and to do a better analysis of the student’s learning.

Weltweit hat das duktale Adenokarzinom des Pankreas (PDAC) unter den Krebserkrankungen die schlechteste Prognose. Aufgrund der unspezifischen Symptome wird die Erkrankung meist erst in einem lokal fortgeschrittenem Stadium diagnostiziert, wenn eine kurative Tumorresektion nicht mehr möglich ist und es bereits zur Metastasierung gekommen ist. Trotz intensiver klinischer Forschung wird mit bisherigen Therapieansätzen wie zum Beispiel Gemcitabin (auch in Kombination mit nab-Paclitaxel) nur eine geringfügige Verbesserung des medianen Überlebens erzielt. Diese Therapieresistenz gegenüber Gemcitabin wurde in in vitro Versuchen mit verschiedenen humanen PDAC-Zelllinien bestätigt. Die zusätzlich behandelte nichttumorigene Pankreasdukt-Zelllinie HDPE-E6E7 zeigte hingegen eine starke Sensitivität gegenüber dem Chemotherapeutikum. Ursächlich beteiligt an der generellen Resistenz dieser Tumore gegenüber Chemo- und Strahlentherapie sind verschiedene Apoptose-Evasionsmechanismen. Diese sind vor allem durch ein Ungleichgewicht pro- und antiapoptotischer Faktoren bedingt (Lowe 2004). Zusätzlich fördern KRAS-Mutationen, die nahezu universell in allen PDACs vorliegen, die Proliferation der Tumorzellen und unterstützen die Apoptose-Dysregulation durch eine verstärkte Expression antiapoptotischer Proteine. Nach umfangreichen Literaturrecherchen (Werner 2011a, Werner 2011b) wurden verschiedenste PDAC-Zelllinien hinsichtlich ihrer Expression von Apoptose-assoziierten Genen analysiert. Untersucht wurden diesbezüglich verschiedene humane PDAC-Zelllinien, aber auch Primärzellkulturen (PaCaDD-Zellen), die in unserem Labor per Outgrowth-Methode aus Patiententumorgewebe etabliert wurden. Ergänzend wurden murine Zelllinien analysiert, die aus einem genetischen PDAC-Mausmodell (KRASG12D; P53R172H; PDX1 CRE) stammen. Normiert bezüglich der Expression der HDPE-E6E7-Zellen wurde eine vielfältige, sehr heterogene Dysregulation von verschiedenen, mit der Apoptose assoziierten Proteinen festgestellt. Per siRNA-basiertem Knockdown wurden verschiedene Kandidatengene hinsichtlich ihrer funktionellen Bedeutung für die Zellproliferation und Apoptose-Induktion näher charakterisiert. In einem dynamischen Prozess wurde eine multimodale Therapie entwickelt, bei der fünf antiapoptotische Zielgene (BCLXL, FLIP, MCL1L, SURVIVIN und XIAP) kombiniert mit KRAS als zentralem Onkogen simultan in ihrer Expression inhibiert wurden. Ziel war es, hierdurch sowohl die extrinsische als auch intrinsische Apoptose zu normalisieren und eine möglicherweise durch Caspase-Inhibitoren blockierte Signaltransduktion zu ermöglichen. Ein zusätzlicher Knockdown von KRAS sollte einer Gegenregulierung dieser Therapie vorbeugen und die gesteigerte Proliferation der Tumorzellen unterbinden. Dieser so genannte SGS6-Therapieansatz zeigte in vitro in allen fünf getesteten humanen sowie in zwei murinen Zelllinien eine starke Apoptose-Induktion und Verminderung der Zellzahl. Durch Zweit-siRNAs wurde die spezifische Wirksamkeit der Knockdowns bestätigt und zelluläre off-target-Effekte wurden ausgeschlossen. Auch in vivo wurde in einem subkutanen Allograftmodell mit dem SGS6-Therapiekonzept eine starke Tumorreduktion erzielt. Die analoge Untersuchung der nichttumorösen Pankreasdukt-Zelllinie HDPE-E6E7 zeigte, dass die SGS6-Therapie deutlich spezifischer für die Krebszellen war verglichen zur Behandlung mit Gemcitabin.

Photodynamic therapy (PDT) has shown to be a promising technique to treat various forms of malignant neoplasia. The photodynamic eradication of the tumor cells is achieved by applying a photosensitizer either locally or systemically and following local activation through irradiation of the tumor mass with light of a specific wavelength after a certain time of incubation. Due to preferential accumulation of the photosensitizer in tumor cells, this procedure allows a selective inactivation of the malignant tumor while sparing the surrounding tissue to the greatest extent. These features and requirements make the PDT an attractive therapeutic option for the treatment of retinoblastoma, especially when surgical enucleation is a curative option. This extreme solution is still in use in case of tumours that are resistant to conventional chemotherapy or handled too late due to poor access to medical care in less advanced country. In this study we initially conducted in-vitro investigations of the new cationic water-soluble photo sensitizer tetrahydroporphyrin-tetratosylat (THPTS) regarding its photodynamic effect on human Rb-1 and Y79 retinoblastoma cells. We were able to show, that neither the incubation with THPTS without following illumination, nor the sole illumination showed a considerable effect on the proliferation of the retinoblastoma cells, whereas the incubation with THPTS combined with following illumination led to a maximal cytotoxic effect on the tumor cells. Moreover the phototoxicity was lower in normal primary cells from retinal pigmented epithelium demonstrating a higher phototoxic effect of THPTS in cancer cells than in this normal retinal cell type. The results at hand form an encouraging foundation for further in-vivo studies on the therapeutic potential of this promising photosensitizer for the eyeball and vision preserving as well as potentially curative therapy of retinoblastoma.