Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-04-27T18:46:26Z No. of bitstreams: 1 Thiago Almeida Pereira. Participação...pdf: 128345460 bytes, checksum: 463e735ff04d1040b79e06171b01301e (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-04-27T18:46:42Z (GMT) No. of bitstreams: 1 Thiago Almeida Pereira. Participação...pdf: 128345460 bytes, checksum: 463e735ff04d1040b79e06171b01301e (MD5) / Made available in DSpace on 2016-04-27T18:46:42Z (GMT). No. of bitstreams: 1 Thiago Almeida Pereira. Participação...pdf: 128345460 bytes, checksum: 463e735ff04d1040b79e06171b01301e (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / INTRODUÇÃO/OBJETIVO: A esquistossomose mansonica é causa importante de fibrose hepática e hipertensão porta em regiões tropicais, e a patogênese da fibrose não está bem esclarecida. Como a via do hedgehog e um dos seus genes alvos, a osteopontina, estão envolvidos em fibroses hepáticas de outras etiologias o objetivo foi investigar a ativação destas vias na esquIsitossomose humana e murina experimental, no intuito de verificar o seu envolvimento no desenvolvimento da forma hepatoesplênica da esquistossomose mansonica (FHE). MATERIAL E MÉTODOS: 87 biópsias em cunha de fígados de pacientes com FHE submetidos a cirurgia e fragmentos de fígado de camundongos suiços infectados com Schistosoma mansoni foram submetidos a métodos imunohistoquímicos e de biologia molecular para avaliar a expressão de ligantes hedgehog (Ihh, Shh), receptor Patched, fatores de transcrição Gli 1 e 2 e osteopontina. Osteopontina sérica e ligante Shh do hedgehog foram avaliados em camundongos suíços infectados e os de osteopontina em camundongos CBA/J infectados e em pacientes com FHE e forma hepatointestinal da esquistossomose. In vitro foi avaliado o efeito de antígeno solúvel do ovo (SEA) em células de Kuppfer, células estreladas, macrófagos, colangiócitos e células endoteliais sinusoidais hepáticas. A relação com a via da IL-13 foi avaliada em camundongos geneticamente deficientes ou hiperexpressando a citocina. Foi avaliado in vitro se a IL-13 induz ligantes hedghog ou ativação da via em células de Kuppfer. RESULTADOS: Os resultados mostraram: (a) aumento expressão de ligantes Ihh, de fatores de transcrição Gli2 e de osteopontina no fígado de camundongos suíços infectados com Schistosoma mansoni, aumento de shh e osteopontina no plasma de camundongos suíços e de osteopontina no plasma de camundongos CBA/J infectados com S. mansoni; (b) aumento na expressão de Ihh, Shh, Gli1 e 2, receptor Patched e de osteopontina no fígado de pacientes com esquistossomose e aumento da osteopontina sérica em pacientes com a FHE; (c) A expressão de ligantes hedgehog e de Gli2 foi observada em macrófagos, células estreladas, ductos biliares e células endoteliais, e a de osteoponina em ductos biliares, macrófagos e células estreladas/miofibroblastos; (d) correlação positiva entre ativação do hedgehog (Gli2 e osteopontina) e fibrose, no modelo murino experimental e nos pacientes; nestes a correlação também foi observada com o grau de fibrose classificada pelo ultrassom e com a hipertensão porta; (e) Inibição in vitro do hedgehog com ciclopamina e vismodegib ou por nocauteamento condicional de receptor Smoothened bloqueou a ativação alternativa de macrófagos e inibiu a angiogênese a partir de células endoteliais sinusoidais hepáticas; (f) que o bloqueio da via da IL-13 reduziu e a hiperexpressão aumentou a ativação da via do hedgehog e IL-13 diretamente induziu, in vitro, produção de ihh em células de Kupffer de camundongos e de humanos, demonstrando a inter-relação das duas vias. CONCLUSÃO: Pode-se concluir que a via do hedgehog tem participação importante na patogênese da fibrose hepática esquistossomótica, atuando através de estímulos à fibrogênese e à angiogênese e que a osteopontina é candidata a ser um biomarcador de intensidade da fibrose e da hipertensão porta na doença. / BACKGROUND AND AIMS: Schistosomiasis is a major cause of liver fibrosis and portal hypertension in tropical regions, and the pathogenesis of fibrosis is not well established. As hedgehog pathway and one of its target genes, osteopontin, are involved in liver fibrosis of other etiologies our aims were to investigate the activation of these pathways in human and experimental murine schistosomiasis, in an attempt to verify their involvement in the development of hepatosplenic schistosomiasis mansoni (HS). METHODS: 87 wedge liver biopsies of patients with HS submitted to surgery and liver fragments Swiss mice infected with Schistosoma mansoni were submitted to immunohistochemistry and molecular biology methods to evaluate the expression of hedgehog ligands (Ihh, Shh), patched receptor , Gli transcription factors and osteopontin. Serum osteopontin and Shh were evaluated in infected Swiss mice and osteopontin was evaluated in serum of infected CBA/J mice and plasma from patients with hepatointestinal and HS forms of schistosomiasis. The effect of soluble egg antigen (SEA) on Kuppfer cells, stellate cells, macrophages, cholangiocytes and liver sinusoidal endothelial cells was evaluated in vitro. Relationship with IL-13 pathway was evaluated in mice genetically deficient or with hyperexpression of this cytokine. Whether IL-13 induces production of ligands and/or activation of the hedgehog pathway in Kuppfer cells was evaluated in vitro. RESULTS: Results demonstrated: (a) increased expression of Ihh, transcription factor Gli2 and osteopontin in the livers of Swiss mice infected with S. mansoni, increased plasma levels of shh and osteopontin in infected Swiss mice and increased osteopontin in plasma of S. mansoni infected CBA/J mice; (b) increased expression of ihh, shh, Gli1 and 2, patched and osteopontin receptor in the liver of patients with schistosomiasis and increased serum osteopontin in patients with HS; (c) expression of hedgehog ligands and Gli2 was observed in macrophages, stellate cells, endothelial cells and bile duct and expression of osteopontin was detected in macrophages and stellate/myofibroblast cells; (d) positive correlation between activation of the hedgehog (Gli2 and osteopontin) and fibrosis in experimental murine model and in patients; these correlation was also observed with the degree of fibrosis classified by ultrasound and with portal hypertension; (e) in vitro inhibition of hedgehog pathway with cyclopamine or vismogedib or by conditional knockout of Smoothened co-receptor blocked the alternative activation of macrophage and inhibited angiogenesis in liver sinusoidal endothelial cells; (f) reduction of IL-13 pathway or IL-13 over-expression respectively reduced or increased the activation of the hedgehog pathway and IL-13 directly induced in vitro ihh production in Kupffer cells from mice and human, demonstrating a cross-talk between the two pathways. CONCLUSION: In conclusion the hedgehog pathway plays an important role in the pathogenesis of liver fibrosis in schistosomiasis mansoni, acting through stimulation of angiogenesis and fibrogenesis and osteopontin is a putative candidate to be a biomarker of intensity of fibrosis and portal hypertension in the disease.

Les récepteurs à dépendance (RDs) ont la particularité d’induire deux types de signalisation selon la disponibilité de leur ligand. En présence de leur ligand, ils induisent une signalisation positive favorisant la survie, la prolifération ou encore la différentiation cellulaire, tandis qu’en son absence, ils induisent la mort cellulaire par apoptose. Ptc et CDON, tous deux récepteurs du morphogène Shh, appartiennent à la famille des RDs. Ces deux récepteurs sont impliqués dans le développement embryonnaire, mais aussi dans le contrôle de la tumorigenèse et de l’homéostasie. La plupart des mécanismes moléculaires de la signalisation proapoptotique de ces récepteurs sont encore inconnus. Nous avons donc étudié les voies de signalisations proapoptotiques de ces récepteurs grâce à un crible d’ARN interférents. Parmi les partenaires potentiels identifiés, nous nous sommes intéressés à l’IGFBP7, caractérisé comme gène suppresseur de tumeurs dans de nombreux cancers. Nous avons mis en évidence que la mort induite par Ptc implique un contrôle transcriptionnel d’IGFBP7 de façon p53 dépendante. Dans un second temps, nous nous sommes intéressés au rôle des voies apoptotiques des récepteurs Ptc et CDON dans des cancers colorectaux associés à des maladies inflammatoires chroniques de l’intestin, pour lesquels il a été montré que le ligand Shh est surexprimé. La surexpression du ligand est l’un des mécanismes d’échappement au contrôle apoptotique exercé par les RDs acquit par les cellules tumorales. Notre étude vise à démontrer que le ciblage de Shh dans ces cancers pourrait induire une réactivation des voies apoptotiques des RDs Ptc et CDON, et ainsi constituer une nouvelle stratégie anti-tumorale / Dependence receptors (DRs) are able to inducing two types of signaling according to their ligand availability. In the presence of their ligand, they induce a positive signaling promoting survival, proliferation or cell differentiation, whereas in its absence, they induce cell death by apoptosis. Ptc and CDON, both receptors of the morphogen Shh, belong to the DRs family. Both receptors are involved in embryonic development, but also in the control of tumorigenesis and homeostasis. Most of the molecular mechanisms of the pro-apoptotic signaling of these receptors remain unknown. We therefore studied both receptors pro-apoptotic pathways through a shRNA screen. Among the identified potential partners, we have been interested in IGFBP7 shown as a tumor suppressor gene in many cancers. Secondly, we studied the role of Ptc and CDON pro-apoptotic pathways in colorectal cancers associated with chronic inflammatory bowel diseases, in which it has been shown that Shh is overexpressed. Overexpression of the ligand is one mechanism of pro-apoptotic control escapement by DRs acquired by tumor cells. Our study aims to demonstrate that targeting Shh in these cancers could induce reactivation of DRs pro-apoptotic pathways and thus constitute a new anti-tumor strategy

Der Hedgehog Signaltransduktionsweg (HhP) ist in der Embryologie und für die Tumor-entstehung bedeutsam und kann durch den spezifischen Antagonisten Cyclopamin (Cyc) inhibiert werden. Simvastatin (Sim) kann die für den HhP essentielle Cholesterolsynthese blockieren. Die therapeutische Unterdrückung des HhP in Kopf-Hals-Plattenepithel-karzinomen (HNSCC) zu untersuchen erschien nach verschiedenen Literaturhinweisen lohnend. In den Experimenten, deren Ergebnisse bereits in Artikeln publiziert wurden, konnten antineoplastische Effekte von Cyc bzw. Sim allein und in Kombination mit den Leitlinientherapeutika Cisplatin (Cis) oder Docetaxel (DTX) an der epithelialen Zelllinie KB, den Kopf-Hals-Zelllinien FaDu und HN-5, sowie an primären HNSCC ex vivo nachgewiesen werden. Biopsien von 49 HNSCC wurden im FLAVINO-Assay mit Cyc bzw. Sim in steigenden Konzentrationen allein und kombiniert mit Cis oder DTX untersucht. In die Auswertung konnten gemäß den Einschlusskriterien (histopathologisch bestätigtes HNSCC und suffiziente Koloniebildung im FLAVINO-Assay) 18 HNSCC einbezogen werden. Bei den Voruntersuchungen führten sowohl Cyc als auch Sim zu einer signifikanten Zeit- und Dosis-abhängigen Reduktion der Lebensfähigkeit von KB, FaDu und HN-5. Ebenso unterdrückten sowohl Cyc als auch Sim die Koloniebildung epithelialer Zellen im FLAVINO-Assay hochsignifikant. Auch tolerierbare Cis- und DTX-Konzentrationen zeigten eine signifikante Wachstumshemmung. In der Analyse des Interaktionsmodus wurde in den untersuchten Kombinationen (Sim+Cis, Sim+DTX, Cyc+Cis und Cyc+DTX) in allen Fällen Additivität als prädominanter Interaktionstyp ermittelt. Die Ergebnisse dieser Arbeit weisen den HhP als potentielles Target in HNSCC aus. Potentere und human besser verträgliche HhP-Blocker sollten unsere Ergebnisse bestätigen und in klinischen Studien getestet werden. Auch die Wirksamkeit von Sim auf HNSCC sollte in prospektiven klinischen Studien weiter analysiert und bestätigt werden. Möglicherweise vermag Sim bzw. die HhP-Blockade zukünftig einen Beitrag zur Therapie von HNSCC im Rahmen multimodaler Therapiekonzepte zu leisten.

The interplay between biochemical signals and mechanical processes during animal development is key for the formation of tissues and organs with distinct shapes and functions. An important step during the formation of many tissues is the formation of compartment boundaries which separate cells of different fates and functions. Compartment boundaries are lineage restrictions that are characterized by a straight morphology. Biochemical signaling across compartment boundaries induce the expression of morphogens in the cells along the boundaries. These morphogens then act at long-range to direct growth and patterning of the whole tissue. Compartment boundaries stabilize the position of morphogens and thereby contribute to proper tissue development. The straight morphology of compartment boundaries is challenged by cell rearrangements caused by cell division and tissue reshaping. Physical mechanisms are therefore required to maintain the straight morphology of compartment boundaries. The anteroposterior (A/P) compartment boundary in the developing Drosophila melanogaster wing is established by biochemical signals. Furthermore, mechanical processes are required to maintain the straight shape of the A/P boundary. Recent studies show that mechanical tension mediated by actomyosin motor proteins is increased along the A/P boundary. However, it was not understood how biochemical signals interact with mechanical processes to maintain the A/P boundary. Here I provide the first evidence that Hedgehog signaling regulates mechanical tension along the A/P boundary. I was able to show that differences in Hedgehog (Hh) signal transduction activity between the anterior and posterior compartments are necessary and sufficient to maintain the straight shape of the A/P boundary, which is crucial for patterning and growth of the adult wing. Moreover, differences in Hh signal transduction activity are necessary and sufficient for the increase in mechanical tension along the A/P boundary. In addition, differences in Hh signal transduction activity are sufficient to generate smooth borders and to increase mechanical tension along ectopic interfaces. Furthermore, the differential expression of the transmembrane protein Capricious is sufficient to increase mechanical tension along ectopic interfaces. It was previously suggested that mechanical tension is generated by an actomyosin-cable through which the increase in mechanical tension is transmitted between the junctions along the A/P boundary. Here I show that mechanical tension is generated locally at each cell bond and not transmitted between junctions by an actomyosin cable. My results provide new insights for our understanding of the interplay between biochemical signals and mechanical processes during animal development.

Hedgehog is a lipid-linked morphogen that is carried on lipoprotein particles and that regulates both patterning and proliferation in a wide variety of vertebrate and invertebrate tissues. Hyperactivity of Hedgehog signaling causes numerous forms of cancer. Hedgehog acts by binding to its receptor Patched, relieving the suppression of Smoothened and initiating Smoothened signaling. The mechanism by which Patched represses Smoothened has been unclear, but correlates with reduced Smoothened levels on the basolateral membrane. The structural homology of Patched with the Niemann-Pick-Type C1 protein and bacterial transmembrane transporters suggests that Patched might regulate lipid trafficking to repress Smoothened. However, no endogenous lipid regulators of Smoothened have yet been identified, nor has it ever been shown that Patched actually controls lipid trafficking. This work shows that, in Drosophila melanogaster, the Sterol-Sensing Domain of Patched regulates Smoothened trafficking from Patched-positive endosomes. Furthermore, it demonstrates that Patched recruits internalized lipoproteins to Patched-positive endosomes. Thereby, Patched regulates the efflux of specific lipoprotein-derived lipids from this compartment via its Sterol-Sensing Domain and utilizes these lipids to destabilize Smoothened on the basolateral membrane. We propose that Patched normally promotes Smoothened degradation and subsequently downregulates its activity by changing the lipid composition of endosomes through which Smoothened passes. For this purpose, Patched utilizes a specific lipid – possibly a modified sterol or sphingolipid – derived from lipoproteins. Further, we suggest that the presence of Hedgehog on lipoprotein particles inhibits utilization of their lipids by Patched.

The presented doctoral thesis is focused on the role of the Hedgehog (HH) signaling pathway in cancer pathogenesis. HH signaling pathway is an evolutionarily conserved signaling pathway that plays an essential role in embryonic development. Its activity is strictly limited to stem and progenitor cells for example in brain, lung, skin or prostate. HH pathway also plays a key role in tissue homeostasis and regeneration. Aberrantly activated HH pathway is essential in cancer progression. The aim of the presented thesis was to elucidate new details about the HH signaling pathway. We identified a new target gene of the HH pathway - the anti-apoptotic protein survivin. Survivin is considered to be an important tumor marker associated with a poor prognosis of patients. We showed that the inhibitor of HH pathway effectors GLI1 and GLI2 GANT61 reduced the survivin level in cancer cells. Subsequently, we used GANT61 and the inhibitor of the anti-apoptotic BCL2 protein family obatoclax to inhibit melanoma cells growth. We showed that the combination of these inhibitors was very effective in the eradication of melanoma cells in vitro. We also proved that GANT61 triggers the process of apoptosis in melanoma cells. We found out that the HH signaling pathway is canonically activated in many cell lines of various...

The liver is the central metabolic hub in organisms and a complex, intertwining regulatory network guarantees efficient liver processes. The morphogenic Hedgehog pathway was recently shown to play a role in regulating the underlying genetic program. Transgenic mouse models with hepatocyte-specific inactivation of Hedgehog signaling showed alterations in insulin-like growth factor homeostasis and in energy metabolism associated with increased lipid accumulation in the liver. In this thesis, it was possible to connect the observed infertility of female knockout mice with an unexpected activation of sex steroid synthesis in the liver. Associated with increased steroidogenic gene expression exclusively in hepatocytes, the plasma testosterone level was significantly elevated, which led to androgenization and an anovulatory phenotype. With these characteristics, the mouse model mimicked the human polycystic ovarian syndrome and suggested an influence of liver and hepatic Hedgehog signaling on reproduction under disease conditions. Further, murine liver metabolism was challenged with starvation starting at different times of day. The transcriptomic results were analyzed with a self-organizing map approach, allowing an intuitive interpretation of data and a thus far unknown diurnally different response of hepatic regulatory mechanisms due to starvation was revealed. In contrast to the manifoldly published and observed switch from energy-consuming to energy-providing processes due to starvation started in the morning, evening starvation led to a novel hepatic expression signature with decreased gluconeogenic gene expression and increased levels of lipid and steroid metabolism-related genes. These differences can be explained by the equally diurnally regulated expression of the corresponding regulatory transcription factors and hormones. Additionally, lipidome analysis confirmed the diurnal differences after starvation. Thus, this study emphasized the immense impact of circadian regulation on liver metabolism and suggests high accuracy when starvation is the focus of research to avoid varying results.:BIBLIOGRAPHISCHE DARSTELLUNG ................................................................................ II LIST OF ABBREVIATIONS .................................................................................................. III TABLE OF CONTENTS ....................................................................................................... IV SUMMARY ............................................................................................................................ 1 ZUSAMMENFASSUNG ......................................................................................................... 5 INTRODUCTION ................................................................................................................... 9 Liver architecture and metabolism ..................................................................................... 9 Diverse possibilities of liver metabolism regulation .......................................................... 10 Connection of Hedgehog signaling to hepatic metabolism ............................................... 10 Impact of feeding schemes on hepatic metabolism .......................................................... 13 Aims of the thesis ............................................................................................................ 14 References ...................................................................................................................... 15 CHAPTER 1 ........................................................................................................................ 18 CHAPTER 2 ........................................................................................................................ 39 PERSPECTIVE ................................................................................................................... 64 CURRICULUM VITAE ........................................................................................................... V PUBLICATIONS AND PRESENTATIONS ............................................................................ VI Publications ...................................................................................................................... VI Oral presentations ............................................................................................................ VI Poster presentations ........................................................................................................ VII AUTHOR CONTRIBUTION STATEMENT .......................................................................... VIII SELBSTSTÄNDIGKEITSERKLÄRUNG .............................................................................. XII DANKSAGUNG .................................................................................................................. XIII

The correct patterning of embryos requires the exchange of information between cells. This is in part achieved by the proper distribution of signaling molecules, many of which exert their function by establishing gradients of concentration. Because of this property they were named "morphogens", or "form giving" substances. Among these, proteins belonging to the Hedgehog (Hh) family have received much attention, owing to their unusual double lipid modification and their involvement in human disease, causing congenital birth defects and cancer. Great efforts have been made in order to elucidate the mechanisms by which Hh molecules are propagated in the embryo. However, no conclusive evidence exists to date to which structures these molecules localize and how they, despite their membrane association, establish a gradient of concentration. Therefore, I decided to study the distribution of the vertebrate Hh homolog, Sonic Hedgehog (Shh) in developing zebrafish embryos. By fluorescently tagging Shh proteins, I found that these localize to discrete punctate structures at the membranes of expressing cells. These were often regions from which filopodial protrusions emanated from the cells. Puctate deposits of Shh were also located outside of expressing cells. In dividing cells, Shh accumulated at the cleavage plane. Furthermore, by making use of confocal microscopy and time lapse analysis, I visualized Shh proteins moving in filopodial extensions present between cells. This suggests a novel mechanism of Shh distribution, which relies on the direct contact of cells by filopodia for the exchange of signaling proteins. In a second part of my thesis, I characterized genes implicated in regulating Shh protein distribution and signaling function. I cloned three zebrafish genes belonging to the Ext1 (exostosin) family of glycosyltransferases required for the synthesis of Heparan Sulfate Proteoglycans and established a tentative link of these genes to somitic Hh signaling. In addition, I characterized the developmental expression and function of zebrafish Rab23, a small GTPase, which acts as a negative regulator of the Shh signaling pathway. Performing knock-down experiments of zebrafish Rab23, I found that Rab23 functions in left-right axis specification, a process previously shown to depend on proper Shh signaling.

The correct patterning of embryos requires the exchange of information between cells. This is in part achieved by the proper distribution of signaling molecules, many of which exert their function by establishing gradients of concentration. Because of this property they were named "morphogens", or "form giving" substances. Among these, proteins belonging to the Hedgehog (Hh) family have received much attention, owing to their unusual double lipid modification and their involvement in human disease, causing congenital birth defects and cancer. Great efforts have been made in order to elucidate the mechanisms by which Hh molecules are propagated in the embryo. However, no conclusive evidence exists to date to which structures these molecules localize and how they, despite their membrane association, establish a gradient of concentration. Therefore, I decided to study the distribution of the vertebrate Hh homolog, Sonic Hedgehog (Shh) in developing zebrafish embryos. By fluorescently tagging Shh proteins, I found that these localize to discrete punctate structures at the membranes of expressing cells. These were often regions from which filopodial protrusions emanated from the cells. Puctate deposits of Shh were also located outside of expressing cells. In dividing cells, Shh accumulated at the cleavage plane. Furthermore, by making use of confocal microscopy and time lapse analysis, I visualized Shh proteins moving in filopodial extensions present between cells. This suggests a novel mechanism of Shh distribution, which relies on the direct contact of cells by filopodia for the exchange of signaling proteins. In a second part of my thesis, I characterized genes implicated in regulating Shh protein distribution and signaling function. I cloned three zebrafish genes belonging to the Ext1 (exostosin) family of glycosyltransferases required for the synthesis of Heparan Sulfate Proteoglycans and established a tentative link of these genes to somitic Hh signaling. In addition, I characterized the developmental expression and function of zebrafish Rab23, a small GTPase, which acts as a negative regulator of the Shh signaling pathway. Performing knock-down experiments of zebrafish Rab23, I found that Rab23 functions in left-right axis specification, a process previously shown to depend on proper Shh signaling.

No description available.