A síndrome de Knobloch (SK) é uma doença autossômica recessiva rara, caracterizada por problemas oculares e presença de encefalocele occipital, porém o quadro clínico é variável. Os pacientes apresentam principalmente miopia de grau elevado, degeneração vítreo-retiniana e descolamento de retina; o grau de comprometimento da alteração no occipital também é variável. Nossos estudos mostraram que a SK é causada por mutações no gene COL18A1, que codifica o colágeno tipo XVIII. Esse colágeno, uma proteoglicana da matriz extracelular, tem sido estudado principalmente por liberar a endostatina, um fragmento de 20 kDa clivado proteoliticamente de sua porção C-terminal e que possui atividade inibidora da angiogênese. O colágeno XVIII possui três isoformas conhecidas, as quais diferem entre si apenas na porção N-terminal e apresentam padrões de expressão distintos nos tecidos, mesmo estando ubiquamente presentes nas membranas basais epiteliais e endoteliais. Além da endostatina, o colágeno XVIII apresenta outros motivos com funções ainda desconhecidas: um domínio trombospondina, presente em todas as isoformas e um domínio frizzled, encontrado apenas na forma mais longa da proteína. O espectro de variação clinica na SK ainda é incerto, assim como os mecanismos moleculares que levam ao fenótipo. Nosso trabalho teve como objetivos principais a identificação de mutações no COL18A1 em um número maior de famílias com a SK, estabelecimento de novos protocolos que possam auxiliar no diagnóstico clínico e a avaliação do efeito funcional de variações encontradas na endostatina, domínio de maior conservação do colágeno XVIII. Propusemo-nos ainda a identificar proteínas que interagem com o domínio trombospondina. Apresentamos aqui a caracterização de sete novas mutações no colágeno XVIII em pacientes com SK, permitindo assim uma melhor determinação do espectro de variação fenotípica da SK. Com base na identificação dessas mutações pudemos incluir problemas neurológicos nos possíveis sinais clínicos presentes na SK ao apresentar pela primeira vez alterações de migração neuronal em pacientes com a síndrome. A ausência de mutações detectadas em três famílias sugere ainda a existência de heterogeneidade genética na SK. Também propomos neste trabalho a utilização da imunohistoquímica em biópsias de pele como teste diagnóstico para essa doença. Nossos resultados mostram também que a variação A48T da endostatina leva a alterações em sua interação com proteínas da matriz extracelular, enquanto a variação polimórfica D104N, previamente associada ao desenvolvimento de câncer de próstata, não leva a um efeito sobre a interação com as proteínas testadas. E, por último, o método de duplo híbrido não foi eficaz para a identificação de proteínas que possam interagir com o domínio trombospondina do colágeno XVIII. / Knobloch syndrome (KS) is an autosomal recessive disorder characterized by ophthalmological defects and presence of an occipital encephalocele. Clinical variability is present, however, all patients present high grade myopia, vitreoretinal degeneration and in most cases, retinal detachment; the occipital defect is also variable. Studies show that the KS is caused by mutations in COL18A1, the gene that codes for type XVIII collagen. This collagen is an extracellular matrix proteoglycan and has been the focus of a great number of studies due to its C-terminal domain, endostatin. Endostatin is a 20 kDa fragment that is proteolytically cleaved and possesses a high antiangiogenic activity. Type XVIII collagen is known to be expressed in three isoforms, different among themselves in the N-terminal region. These isoforms have distinct expression patterns, but are present in most basement membranes. Besides endostatin, type XVIII collagen also presents other domains with unknown functions: a thrombospondin domain, found in all isoforms; a frizzled domain, present in the longest isoform. The clinical variability spectrum in KS and the molecular mechanisms that lead to the phenotype are still uncertain. The aim of this study was to identify novel mutations in COL18A1 in additional KS families, to develop biochemical diagnostic tests that could allow the screening of a larger number of patients and to evaluate the effect of naturally found variants in the function of endostatin. We also performed a two-hybrid screening in order to identify proteins that can interact with the thrombospondin domain. The characterization of seven novel mutations in KS patients allowed us to better determine the clinical variability of KS. This work shows for the first time the presence of neuronal migration defects in some KS patients. The lack of detected pathogenic mutations in three families led us to propose the genetic heterogeneity of this syndrome. We demonstrate the possibility to use immunohistochemistry in skin biopsies as a diagnosis method. Our results also show the altered properties of T48 endostatin in its interaction with some extracellular matrix proteins. The N104 variant, that has been previously associated with prostate cancer, do not present any change in its interaction to the tested molecules. Finally, the two-hybrid system was not a good method to detect interacting proteins with the thrombospodin domain of collagen XVIII.

A síndrome de Knobloch (SK) é uma doença autossômica recessiva rara, caracterizada por problemas oculares e presença de encefalocele occipital, porém o quadro clínico é variável. Os pacientes apresentam principalmente miopia de grau elevado, degeneração vítreo-retiniana e descolamento de retina; o grau de comprometimento da alteração no occipital também é variável. Nossos estudos mostraram que a SK é causada por mutações no gene COL18A1, que codifica o colágeno tipo XVIII. Esse colágeno, uma proteoglicana da matriz extracelular, tem sido estudado principalmente por liberar a endostatina, um fragmento de 20 kDa clivado proteoliticamente de sua porção C-terminal e que possui atividade inibidora da angiogênese. O colágeno XVIII possui três isoformas conhecidas, as quais diferem entre si apenas na porção N-terminal e apresentam padrões de expressão distintos nos tecidos, mesmo estando ubiquamente presentes nas membranas basais epiteliais e endoteliais. Além da endostatina, o colágeno XVIII apresenta outros motivos com funções ainda desconhecidas: um domínio trombospondina, presente em todas as isoformas e um domínio frizzled, encontrado apenas na forma mais longa da proteína. O espectro de variação clinica na SK ainda é incerto, assim como os mecanismos moleculares que levam ao fenótipo. Nosso trabalho teve como objetivos principais a identificação de mutações no COL18A1 em um número maior de famílias com a SK, estabelecimento de novos protocolos que possam auxiliar no diagnóstico clínico e a avaliação do efeito funcional de variações encontradas na endostatina, domínio de maior conservação do colágeno XVIII. Propusemo-nos ainda a identificar proteínas que interagem com o domínio trombospondina. Apresentamos aqui a caracterização de sete novas mutações no colágeno XVIII em pacientes com SK, permitindo assim uma melhor determinação do espectro de variação fenotípica da SK. Com base na identificação dessas mutações pudemos incluir problemas neurológicos nos possíveis sinais clínicos presentes na SK ao apresentar pela primeira vez alterações de migração neuronal em pacientes com a síndrome. A ausência de mutações detectadas em três famílias sugere ainda a existência de heterogeneidade genética na SK. Também propomos neste trabalho a utilização da imunohistoquímica em biópsias de pele como teste diagnóstico para essa doença. Nossos resultados mostram também que a variação A48T da endostatina leva a alterações em sua interação com proteínas da matriz extracelular, enquanto a variação polimórfica D104N, previamente associada ao desenvolvimento de câncer de próstata, não leva a um efeito sobre a interação com as proteínas testadas. E, por último, o método de duplo híbrido não foi eficaz para a identificação de proteínas que possam interagir com o domínio trombospondina do colágeno XVIII. / Knobloch syndrome (KS) is an autosomal recessive disorder characterized by ophthalmological defects and presence of an occipital encephalocele. Clinical variability is present, however, all patients present high grade myopia, vitreoretinal degeneration and in most cases, retinal detachment; the occipital defect is also variable. Studies show that the KS is caused by mutations in COL18A1, the gene that codes for type XVIII collagen. This collagen is an extracellular matrix proteoglycan and has been the focus of a great number of studies due to its C-terminal domain, endostatin. Endostatin is a 20 kDa fragment that is proteolytically cleaved and possesses a high antiangiogenic activity. Type XVIII collagen is known to be expressed in three isoforms, different among themselves in the N-terminal region. These isoforms have distinct expression patterns, but are present in most basement membranes. Besides endostatin, type XVIII collagen also presents other domains with unknown functions: a thrombospondin domain, found in all isoforms; a frizzled domain, present in the longest isoform. The clinical variability spectrum in KS and the molecular mechanisms that lead to the phenotype are still uncertain. The aim of this study was to identify novel mutations in COL18A1 in additional KS families, to develop biochemical diagnostic tests that could allow the screening of a larger number of patients and to evaluate the effect of naturally found variants in the function of endostatin. We also performed a two-hybrid screening in order to identify proteins that can interact with the thrombospondin domain. The characterization of seven novel mutations in KS patients allowed us to better determine the clinical variability of KS. This work shows for the first time the presence of neuronal migration defects in some KS patients. The lack of detected pathogenic mutations in three families led us to propose the genetic heterogeneity of this syndrome. We demonstrate the possibility to use immunohistochemistry in skin biopsies as a diagnosis method. Our results also show the altered properties of T48 endostatin in its interaction with some extracellular matrix proteins. The N104 variant, that has been previously associated with prostate cancer, do not present any change in its interaction to the tested molecules. Finally, the two-hybrid system was not a good method to detect interacting proteins with the thrombospodin domain of collagen XVIII.

Background: Among the common malignancies, pancreatic cancer has the shortest long-term survival. The aggressive, rapid, and infiltrative growth pattern of pancreatic cancer, together with the lack of specific symptoms, often leads to late diagnosis. Metastases are frequently found at the time of diagnosis, which prevents curative surgical treatment. Good tumor markers would enable early detection, thus improving the prognosis. Unfortunately, no such markers are available in the clinic. The tumor stroma is defined as the non-malignant cells and the extracellular matrix (ECM) of a cancer. Pancreatic cancer is characterized by an abundant tumor stroma, rich in ECM proteins such as collagens, which have been shown to play important roles in tumor progression. Furthermore, pancreatic cancer cells produce large quantities of ECM proteins, especially the basement membrane (BM) protein type IV collagen. All epithelial cells are anchored to a BM, which must be degraded in order for an in situ cancer to become invasive. Matrix metalloproteinases (MMPs) are enzymes involved in BM degradation. In this thesis, the tumor stroma of pancreatic cancer is studied, focusing on the BM proteins type IV and type XVIII collagen, with the aim to clarify if the stroma could be a source of novel tumor markers for this form of cancer. Additionally, the role of type IV collagen produced by the cancer cells is studied. Methods: Expression patterns of type IV and type XVIII collagen, MMPs involved in collagen degradation, and collagen receptors (integrins) were studied by immunoflourescence in both normal and pancreatic cancer tissue, and in pancreatic cancer cell lines. Circulating plasma levels of type IV and type XVIII collagen and conventional tumor markers (TPS, Ca 19-9, CEA and Ca 125) were measured in controls and pancreatic cancer patients at the time of diagnosis and after treatment. The role of cancer cell produced type IV collagen was studied in human pancreatic cancer cell lines by functional blocking of integrin receptors (integrin a1, a2 and b1) and integrin-binding sites on type IV collagen, and by siRNA-induced down-regulation of type IV collagen synthesis. Proliferation was analyzed by a luminescence based cell viability assay, migration by time-lapse microscopy, and apoptosis by M30-neoepitope detection. Results: MMPs involved in BM degradation were upregulated in pancreatic cancer tissue. The expression of type XVIII collagen shifted from a general BM expression pattern in normal tissue, to mainly being found in the tumor vasculature in pancreatic cancer. Type IV collagen, on the other hand, remained highly expressed in the vicinity of the cancer cells. The a1, a2, and b1 integrin receptors were highly expressed at the cancer cell surface. Both down-regulation of type IV collagen synthesis and blocking the integrin/type IV collagen interaction decreased cell proliferation and migration. The proliferative capacity was rescued by the addition of exogenous type IV collagen. Furthermore, the circulating levels of both type IV and type XVIII collagen were increased in pancreatic cancer patients at the time of diagnosis compared to controls. After treatment, the levels were normalized for type XVIII collagen, whereas the levels of type IV collagen remained high after surgery. High postoperative levels of type IV collagen were associated with short overall survival. A similar association to short survival was found for preoperative type XVIII collagen levels. No such associations to survival could be detected for the conventional markers.   Conclusion: The results of this thesis show that type IV and type XVIII collagens can serve as tumor markers for pancreatic cancer with advantages compared to conventionally used markers. Additionally, evidence is provided of an autocrine loop, involving type IV collagen and its integrin receptors, with importance for retaining a proliferative and migratory phenotype in pancreatic cancer cells.