Pax3 and Pax7 belong to a family of conserved transcription factors that play important and diverse roles in development. In the embryo, they carry out similar roles in neural and somite development, but Pax7 fails to compensate for critical functions of Pax3 in the development of limb musculature. Conversely, in the adult, Pax7 is necessary for the maintenance and survival of muscle satellite cells, whereas Pax3 cannot effectively fulfill these roles in the absence of Pax7. To identify the unique roles of Pax7 in adult muscle cells, we have analyzed global binding of Pax3 and Pax7 by ChIP-Seq. Here, we show that despite highly homologous DNA-binding domains, the majority of binding sites are uniquely recognized by Pax7 and are enriched for homeobox motifs. Genes proximal to conserved, unique Pax7 binding sites cluster into specific functional groups which may reflect the unique biological roles of Pax7. Combining Pax7 binding sites with gene expression data, we describe the regulatory networks directed by Pax7 and show that Pax7 binding is associated with positive gene regulation. Moreover, we show Myf5 is a direct target of Pax7 and identify a novel binding site in the satellite cell control region upstream of Myf5.

Pax3 and Pax7 belong to a family of conserved transcription factors that play important and diverse roles in development. In the embryo, they carry out similar roles in neural and somite development, but Pax7 fails to compensate for critical functions of Pax3 in the development of limb musculature. Conversely, in the adult, Pax7 is necessary for the maintenance and survival of muscle satellite cells, whereas Pax3 cannot effectively fulfill these roles in the absence of Pax7. To identify the unique roles of Pax7 in adult muscle cells, we have analyzed global binding of Pax3 and Pax7 by ChIP-Seq. Here, we show that despite highly homologous DNA-binding domains, the majority of binding sites are uniquely recognized by Pax7 and are enriched for homeobox motifs. Genes proximal to conserved, unique Pax7 binding sites cluster into specific functional groups which may reflect the unique biological roles of Pax7. Combining Pax7 binding sites with gene expression data, we describe the regulatory networks directed by Pax7 and show that Pax7 binding is associated with positive gene regulation. Moreover, we show Myf5 is a direct target of Pax7 and identify a novel binding site in the satellite cell control region upstream of Myf5.

Pax3 and Pax7 belong to a family of conserved transcription factors that play important and diverse roles in development. In the embryo, they carry out similar roles in neural and somite development, but Pax7 fails to compensate for critical functions of Pax3 in the development of limb musculature. Conversely, in the adult, Pax7 is necessary for the maintenance and survival of muscle satellite cells, whereas Pax3 cannot effectively fulfill these roles in the absence of Pax7. To identify the unique roles of Pax7 in adult muscle cells, we have analyzed global binding of Pax3 and Pax7 by ChIP-Seq. Here, we show that despite highly homologous DNA-binding domains, the majority of binding sites are uniquely recognized by Pax7 and are enriched for homeobox motifs. Genes proximal to conserved, unique Pax7 binding sites cluster into specific functional groups which may reflect the unique biological roles of Pax7. Combining Pax7 binding sites with gene expression data, we describe the regulatory networks directed by Pax7 and show that Pax7 binding is associated with positive gene regulation. Moreover, we show Myf5 is a direct target of Pax7 and identify a novel binding site in the satellite cell control region upstream of Myf5.

Pax3 and Pax7 belong to a family of conserved transcription factors that play important and diverse roles in development. In the embryo, they carry out similar roles in neural and somite development, but Pax7 fails to compensate for critical functions of Pax3 in the development of limb musculature. Conversely, in the adult, Pax7 is necessary for the maintenance and survival of muscle satellite cells, whereas Pax3 cannot effectively fulfill these roles in the absence of Pax7. To identify the unique roles of Pax7 in adult muscle cells, we have analyzed global binding of Pax3 and Pax7 by ChIP-Seq. Here, we show that despite highly homologous DNA-binding domains, the majority of binding sites are uniquely recognized by Pax7 and are enriched for homeobox motifs. Genes proximal to conserved, unique Pax7 binding sites cluster into specific functional groups which may reflect the unique biological roles of Pax7. Combining Pax7 binding sites with gene expression data, we describe the regulatory networks directed by Pax7 and show that Pax7 binding is associated with positive gene regulation. Moreover, we show Myf5 is a direct target of Pax7 and identify a novel binding site in the satellite cell control region upstream of Myf5.

<p>This thesis reports on the development of a novel comprehensive database (Dragon Database of Genes Implicated in Esophageal Cancer, DDEC) as an integrated knowledge database aimed at representing a gateway to esophageal cancer related data. More importantly, it illustrates how the biocurated genes in the database may represent a reliable starting point for divulging transcriptional regulation, diagnostic markers and the biology related to esophageal cancer.</p>

<p>This thesis reports on the development of a novel comprehensive database (Dragon Database of Genes Implicated in Esophageal Cancer, DDEC) as an integrated knowledge database aimed at representing a gateway to esophageal cancer related data. More importantly, it illustrates how the biocurated genes in the database may represent a reliable starting point for divulging transcriptional regulation, diagnostic markers and the biology related to esophageal cancer.</p>

INTRODUÇÃO: A doença de Parkinson é causada pela perda significativa de neurônios dopaminérgicos na substância nigra e perda celular no locus cerúleo, com perda do neurotransmissor dopamina e continuada deposição de inclusões proteicas nos tecidos cerebrais. A doença tem progressão caudo-rostral, iniciando-se no núcleo dorsal do nervo vago e, em grau menor, nos sistema olfativo, com evolução para o mesencéfalo e posteriormente para o prosencéfalo e neocórtex. Cerca de 90% dos casos são idiopáticos e o principal fator de risco é o envelhecimento. Para se compreender a interação genoma-ambiente e o mecanismo molecular nessa doença têm sido conduzidas investigações dos perfis de expressão gênica global em diversos tecidos-alvo. Essa abordagem de genômica funcional utiliza a tecnologia de DNA microarrays para estudo da expressão gênica e ferramentas de bioinformática para análise dos dados gerados. Neste trabalho foi feita uma análise das redes de interação transcricional em tecidos-alvo da doença de Parkinson utilizando-se amostras post mortem de tecidos cerebrais obtidas de pacientes e controles livres da doença. MÉTODOS: Estudo comparativo das redes de interação transcricional no núcleo dorsal do nervo vago, locus cerúleo e substância nigra entre pacientes com doença de Parkinson idiopática nos estágios Braak 4-5 e controles livres da doença utilizando material de necropsia. Foram utilizados DNA microarrays Agilent de 44 K e a análise estatística comparativa de dos transcritos válidos (TMEV) foi feita no vetor paciente X controle para cada região anatômica sob estudo. Para a análise das redes de interação transcricional dos grupos de pacientes e controles em cada região anatômica utilizou-se o software FunNet e as anotações genômicas do Gene Ontology Consortium. RESULTADOS: Os genes com maior número de ligações gene-gene em cada rede transcricional, ou hubs, foram identificados e correlacionados com sua função biológica e possível papel na doença de Parkinson. A análise comparativa entre o perfil de hubs (número de ligações, posição na rede) em cada região anatômica para pacientes e controles revelou que: i) no núcleo dorsal do nervo vago os hubs principais nas redes de controles e pacientes estão relacionados a funções de manutenção da homeostase cerebral e organização neuronal, ii) no locus cerúleo os hubs principais dos controles são genes ligados à manutenção das funções cerebrais, mobilização de células progenitoras (pericitos) e controle de vias inflamatórias, enquanto que na rede de pacientes esses hubs estão ligados aos processos de endo e exocitose, desenvolvimento neuronal e controle do estresse oxidativo e degradação de proteínas; iii) finalmente, na substância nigra os principais hubs da rede de controles estão envolvidos na proteção contra estresse oxidativo e proteínas não dobradas e na manutenção do sistema dopaminérgico mesodiencefálico, enquanto que na rede de pacientes predominam hubs ligados a processos epigenéticos de envelhecimento mitocondrial, transporte vesicular, neurogênese, inflamação e morte neuronal. CONCLUSÕES: Os resultados da análise de redes de interação transcricional de pacientes e controles em diferentes regiões anatômicas são compatíveis com o modelo de progressão caudo-rostral da doença de Parkinson e apontam para mecanismos compensatórios no núcleo dorsal do nervo vago e locus cerúleo / INTRODUCTION: Parkinson\'s disease is caused by a substantial loss of dopaminergic neurons in the substantia nigra and cell loss in locus coeruleus, concomitant loss of dopamine neurotransmitter and continuing deposition of protein within the brain as intracellular inclusions. The disease has a caudal-rostral progression, beginning in the dorsal nucleus of vagus nerve and, in a less extent, in the olfactory system, progressing to the midbrain and finally to the basal forebrain and the neocortex. About 90% of the cases are idiopathic and the main risk factor is ageing. In order to have a better understanding of the genome-environment interactions and of the molecular mechanisms involved in this disease, the investigation of global gene expression in different target tissues has been conducted. This functional genomic approach is based on DNA microarray technology and on the use of bioinformatics for analyzing the data. In the present work an analysis of transcriptional interaction networks in Parkinson\'s disease target tissues was conducted in post mortem cerebral tissue samples obtained from patients and disease-free controls. METHODS: Comparative study of transcriptional interaction networks in the dorsal nucleus of vagus nerve, locus coeruleus, and substantia nigra of idiopathic Parkinson\'s disease patients in Braak stages 4-5 and disease-free controls using post mortem tissue samples. Agilent 44 K DNA microarrays were used and the statistical comparative analysis of valid transcripts was accomplished (TMEV) in the vector patient X control for each anatomic region under study. In order to analyze the transcriptional interaction networks for patient and control groups in each anatomic region the FunNet software and the Gene Ontology genomic annotations were used. RESULTS: The genes with high number of gene-gene connections in each transcriptional network, or hubs, were identified and related to their biological function and putative role in Parkinson\'s disease. The comparative analysis between hub profiles (number of connections, position in the network) in each anatomic region for patients and controls revealed that: i) in the dorsal nucleus of vagus nerve the main hubs in patient and control networks are related to the maintenance of brain homeostasis and to neuronal organization; ii) in the locus coeruleus the main hubs of control network are related to the maintenance of brain functions, mobilization of progenitor cells (pericytes) and control of inflammatory pathways, whereas in the patient network the main hubs are linked to exo and endocytosis processes, neuronal development and control of oxidative stress and protein degradation; iii) finally, in the substantia nigra the main hubs in the control network are related to protection against oxidative stress and unfolded/misfolded proteins and in the maintenance of the midbrain dopaminergic system, whereas in the patient network the main hubs are related to epigenetic processes of mitochondrial ageing, vesicular transport, neurogenesis and inflammation and neuronal death. DISCUSSION: The results of transcriptional interaction networks analyses performed for patient and control groups in different anatomic regions are compatible with the caudal-rostral model of Parkinson\'s disease progression and point out to compensatory mechanisms acting in vagus nerve and locus coeruleus

INTRODUÇÃO: A doença de Parkinson é causada pela perda significativa de neurônios dopaminérgicos na substância nigra e perda celular no locus cerúleo, com perda do neurotransmissor dopamina e continuada deposição de inclusões proteicas nos tecidos cerebrais. A doença tem progressão caudo-rostral, iniciando-se no núcleo dorsal do nervo vago e, em grau menor, nos sistema olfativo, com evolução para o mesencéfalo e posteriormente para o prosencéfalo e neocórtex. Cerca de 90% dos casos são idiopáticos e o principal fator de risco é o envelhecimento. Para se compreender a interação genoma-ambiente e o mecanismo molecular nessa doença têm sido conduzidas investigações dos perfis de expressão gênica global em diversos tecidos-alvo. Essa abordagem de genômica funcional utiliza a tecnologia de DNA microarrays para estudo da expressão gênica e ferramentas de bioinformática para análise dos dados gerados. Neste trabalho foi feita uma análise das redes de interação transcricional em tecidos-alvo da doença de Parkinson utilizando-se amostras post mortem de tecidos cerebrais obtidas de pacientes e controles livres da doença. MÉTODOS: Estudo comparativo das redes de interação transcricional no núcleo dorsal do nervo vago, locus cerúleo e substância nigra entre pacientes com doença de Parkinson idiopática nos estágios Braak 4-5 e controles livres da doença utilizando material de necropsia. Foram utilizados DNA microarrays Agilent de 44 K e a análise estatística comparativa de dos transcritos válidos (TMEV) foi feita no vetor paciente X controle para cada região anatômica sob estudo. Para a análise das redes de interação transcricional dos grupos de pacientes e controles em cada região anatômica utilizou-se o software FunNet e as anotações genômicas do Gene Ontology Consortium. RESULTADOS: Os genes com maior número de ligações gene-gene em cada rede transcricional, ou hubs, foram identificados e correlacionados com sua função biológica e possível papel na doença de Parkinson. A análise comparativa entre o perfil de hubs (número de ligações, posição na rede) em cada região anatômica para pacientes e controles revelou que: i) no núcleo dorsal do nervo vago os hubs principais nas redes de controles e pacientes estão relacionados a funções de manutenção da homeostase cerebral e organização neuronal, ii) no locus cerúleo os hubs principais dos controles são genes ligados à manutenção das funções cerebrais, mobilização de células progenitoras (pericitos) e controle de vias inflamatórias, enquanto que na rede de pacientes esses hubs estão ligados aos processos de endo e exocitose, desenvolvimento neuronal e controle do estresse oxidativo e degradação de proteínas; iii) finalmente, na substância nigra os principais hubs da rede de controles estão envolvidos na proteção contra estresse oxidativo e proteínas não dobradas e na manutenção do sistema dopaminérgico mesodiencefálico, enquanto que na rede de pacientes predominam hubs ligados a processos epigenéticos de envelhecimento mitocondrial, transporte vesicular, neurogênese, inflamação e morte neuronal. CONCLUSÕES: Os resultados da análise de redes de interação transcricional de pacientes e controles em diferentes regiões anatômicas são compatíveis com o modelo de progressão caudo-rostral da doença de Parkinson e apontam para mecanismos compensatórios no núcleo dorsal do nervo vago e locus cerúleo / INTRODUCTION: Parkinson\'s disease is caused by a substantial loss of dopaminergic neurons in the substantia nigra and cell loss in locus coeruleus, concomitant loss of dopamine neurotransmitter and continuing deposition of protein within the brain as intracellular inclusions. The disease has a caudal-rostral progression, beginning in the dorsal nucleus of vagus nerve and, in a less extent, in the olfactory system, progressing to the midbrain and finally to the basal forebrain and the neocortex. About 90% of the cases are idiopathic and the main risk factor is ageing. In order to have a better understanding of the genome-environment interactions and of the molecular mechanisms involved in this disease, the investigation of global gene expression in different target tissues has been conducted. This functional genomic approach is based on DNA microarray technology and on the use of bioinformatics for analyzing the data. In the present work an analysis of transcriptional interaction networks in Parkinson\'s disease target tissues was conducted in post mortem cerebral tissue samples obtained from patients and disease-free controls. METHODS: Comparative study of transcriptional interaction networks in the dorsal nucleus of vagus nerve, locus coeruleus, and substantia nigra of idiopathic Parkinson\'s disease patients in Braak stages 4-5 and disease-free controls using post mortem tissue samples. Agilent 44 K DNA microarrays were used and the statistical comparative analysis of valid transcripts was accomplished (TMEV) in the vector patient X control for each anatomic region under study. In order to analyze the transcriptional interaction networks for patient and control groups in each anatomic region the FunNet software and the Gene Ontology genomic annotations were used. RESULTS: The genes with high number of gene-gene connections in each transcriptional network, or hubs, were identified and related to their biological function and putative role in Parkinson\'s disease. The comparative analysis between hub profiles (number of connections, position in the network) in each anatomic region for patients and controls revealed that: i) in the dorsal nucleus of vagus nerve the main hubs in patient and control networks are related to the maintenance of brain homeostasis and to neuronal organization; ii) in the locus coeruleus the main hubs of control network are related to the maintenance of brain functions, mobilization of progenitor cells (pericytes) and control of inflammatory pathways, whereas in the patient network the main hubs are linked to exo and endocytosis processes, neuronal development and control of oxidative stress and protein degradation; iii) finally, in the substantia nigra the main hubs in the control network are related to protection against oxidative stress and unfolded/misfolded proteins and in the maintenance of the midbrain dopaminergic system, whereas in the patient network the main hubs are related to epigenetic processes of mitochondrial ageing, vesicular transport, neurogenesis and inflammation and neuronal death. DISCUSSION: The results of transcriptional interaction networks analyses performed for patient and control groups in different anatomic regions are compatible with the caudal-rostral model of Parkinson\'s disease progression and point out to compensatory mechanisms acting in vagus nerve and locus coeruleus

No description available.

Tumormodelle, in denen die maligne Transformation durch definierte Onkogene experimentell ausgelöst und unterhalten wird, bieten vielfältige Möglichkeiten, die komplexen Mechanismen der Tumorentstehung und Therapieresistenz zu untersuchen und neue Ansätze für Diagnostik und Therapie auszuarbeiten. KRAS-Onkogen-„getriebene“ Transformationsmodelle spiegeln neben anderen tumorspezifischen Veränderungen insbesondere die charakteristischen Änderungen des Transkriptoms wider. In der vorliegenden Arbeit wird ein Modell für Ovarialtumore auf Grundlage von Rose Zellen („Rat ovarian surface epithelium“) verwendet, um die Rolle von Transkriptionsfaktoren, welche durch die KRAS-vermittelte Signaltransduktion hoch reguliert werden, zu untersuchen. Die KRAS-transfomierten Derivate der normalen Rose Zellen zeigen die typischen Merkmale von ankerunabhängigen und invasiven Tumorzellen. Aufgrund der hohen Komplexität sind die Interaktionen zwischen der zytoplasmatischen Signaltransduktion und dem durch sie regulierten Transkriptionsfaktornetzwerk noch weitgehend unverstanden. Die Transkriptionsfaktoren Fosl1, Hmga2, Klf6, JunB, Otx1, Gfi1 und RelA wurden systematisch mittels RNA-Interferenz in KRAS-transformierten Rose Zellen transient ausgeschaltet. Danach wurden Proliferation, Morphologie (epithelial-mesenchymale Transition, EMT) und Ankerunabhängigkeit der Zellen bestimmt. Alle untersuchten Transkriptionsfaktoren beeinflussten die KRAS-induzierten morphologischen Veränderungen teilweise, belegt durch die Abnahme der EMT-Merkmale nach siRNA-vermittelter Ausschaltung. Der Knock-down der Transkriptionsfaktoren Otx1, Gfi1 und RelA hemmte die Proliferation, während Fosl1, Hmga2, Klf6 und JunB die generelle Proliferationsfähigkeit nicht beeinflussten, jedoch spezifisch die ankerunabhängige Proliferation blockierten. Diesen Faktoren kommt daher eine spezifische Funktion in der neoplastischen Transformation zu, da die Ankerunabhängigkeit sehr gut mit der Tumorigenität korreliert ist. Um die Beteiligung der Transkriptionsfaktoren an der Deregulation von Zielgenen zu erfassen, wurden Genexpressionsmuster aller Zellen, in denen jeweils ein Faktor durch siRNA ausgeschaltet war, mittels Microarray-Analyse identifiziert. Auf dieser Grundlage wurde ein Netzwerk-Modell der regulatorischen Interaktionen zwischen den Transkriptionsfaktoren berechnet. Die Existenz der beiden funktionellen Gruppen wurde im Modell bestätigt. Darüber hinaus zeigte sich eine gegenseitige Abhängigkeit des transkriptionellen Netzwerks und der zytoplasmatischen Signaltransduktion, gemessen mittels Proteinanalyse der mitogenabhängigen Signalkinasen (MAPK). Diese wird als kompensatorische Regulation interpretiert, welche trotz Pertubation, experimentell durch siRNA, das effiziente Überleben der transformierten Zellen sicherstellt. Die vorliegende Studie schafft somit die Voraussetzung und Motivation, das reduzierte Netzwerk aus sieben Komponenten auf alle differentiell exprimierten Transkriptionsfaktoren zu erweitern. Möglicherweise behindern solche Regulationskreise in der klinischen Situation die effektive Wirkung zielgerichteter Therapien. / Tumor models, in which malignant transformation was experimentally triggered and maintained through defined oncogenes, offer manifold opportunities to determine the complex mechanisms of tumor progression and resistance to therapies, and to develop new strategies for diagnosis and therapy. Particularly, KRAS oncogene driven models of transformation reflect the characteristic alterations of the transcriptome, among other tumor specific changes. In the present work a model for ovarian cancer based on Rose („Rat ovarian surface epithelium“) cells has been used to evaluate the role of transcription factors, which are up-regulated through KRAS dependent signaling. The KRAS transformed derivates of normal ROSE cells exhibit typical characteristics of anchorage-independent and invasive tumor cells. Due to the high complexity of cellular networks, the interactions between cytoplasmic signalling and their regulated transcription factors are not well understood. The transcription factors Fosl1, Hmga2, Klf6, JunB, Otx1, Gfi1 and RelA were systematically eliminated by transient RNA interference in KRAS transformed ROSE cells. The proliferation, morphology (epithelial-mesenchymal transition, EMT) and anchorage-independence of the cells were determined. All of the selected transcription factors had partial effect on the KRAS induced morphologic changes, documented by reduction of EMT-properties after siRNA treatment. The knock-down of the transcription factors Otx1, Gfi1 and RelA blocked proliferation in general, whereas Fosl1, Hmga2, Klf6 and JunB had no influence on proliferation but specifically blocked the anchorage-independence. Thus, these factors exhibited essential functions in the process of neoplastic transformation, because the anchorage-independence correlates very well with tumorigenicity. In order to elucidate the involvement of the transcription factors in the genetic deregulation of their target genes, microarray based gene expression profiles were determined from all cells in which one factor was eliminated by siRNA. Based on these data, a network model of regulatory interactions among these transcription factors was calculated. The existence of both functional groups was confirmed by the model. Furthermore, an interdependence of the transcriptional networks and cytoplasmatic signaling was observed by protein analysis of the mitogen dependent signal kinases (MAPK). This was interpreted as compensatory regulation, which in spite of experimental perturbation by siRNA, permitted efficient survival of the transformed cells. Thus, the present work provides the basis and motivation to extend the reduced network composed of seven components to all regulated transcription factors. Potentially, such regulatory networks diminish the efficacy of targeted therapies in clinical situations.