Made available in DSpace on 2016-12-23T13:56:07Z (GMT). No. of bitstreams: 1 Dissertacao de Thiago de Almeida Pereira.pdf: 7083594 bytes, checksum: b087de1012807ea82b96d6e7ada7b3cc (MD5) Previous issue date: 2010-03-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Hedgehog (Hh) pathway activation promotes many processes that occur during fibrogenic liver repair. Whether the Hh pathway modulates the outcomes of virally-mediated liver injury has never been examined. Gene-profiling studies of human hepatocellular carcinomas (HCC) demonstrate Hh pathway activation in HCCs related to chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV). Because most HCC develop in cirrhotic livers, we hypothesized that Hh pathway activation occurs during fibrogenic repair of liver damage due to chronic viral hepatitis, and that Hh-responsive cells mediate disease progression and hepatocarcinogenesis in chronic viral hepatitis. Methods Immunohistochemistry and qRT-PCR analysis were used to analyze Hh pathway activation and identify Hhresponsive cell types in archival liver biopsies from 45 patients with chronic HBV or HCV with different stages of fibrosis (F0-4), some of which also presented HCC. Hh signaling was manipulated with cyclopamine in primary human hepatic stellate cells (HSC) and sinusoidal endothelial cells (SEC). Angiogenesis assay was used to investigate if the pathway plays a role in tube formation. SECs were incubated with recombinant Shh, conditioned media from HSC, cyclopamine, short hairpin RNA against smoothened or their respective controls and the length of vascular tubes were measured by morphometry. Hedgehog production was investigated in Huh7 cells infected with JFH-1 and in a rat hepatoma cell line that express the HBV X gene by qRT-PCR. Results We found increased hepatic expression of Hh ligands (Shh and Ihh) in all patients with chronic viral hepatitis (p<0.005), and demonstrated that infection with JFH-1 or ectopic expression of the HBV X gene stimulated cultured hepatocytes to produce Shh (p<0.005). The major cell populations that expanded during cirrhosis and HCC (i.e., liver myofibroblasts, activated endothelial cells, and progenitors expressing markers of tumor stem/initiating cells Krt7, CD133, Epcam and survivin) were Hh-responsive (Patched and Gli-2 positive) and higher levels of Hh pathway activity associated with cirrhosis and HCC (p<0.005). Inhibiting pathway activity in Hh-responsive target cells (HSC and SEC) reduced fibrogenesis (p<0.005 for &#945;SMA and p<0.05 for col1&#945;1 expression) and angiogenesis (p<0.05). Conclusions HBV/HCV infection increases hepatocyte production of Hh ligands in hepatocytes and expands types of Hh-responsive cells, including myofibroblasts and xiv sinusoidal endothelial cells that associate with fibrosis progression to cirrhosis and hepatocellular carcinoma. Moreover, hedgehog-responsive progenitors that are undergoing epithelial-to-mesenchymal transition accumulate during the progression of viral hepatitis and may play a role in the development or progression of hepatocellular carcinoma. In vitro inhibition of Hh-signaling in primary human hepatic stellate cells and sinusoidal endothelial cells suggest the possibility of using Hh inhibitors as potential tools to prevent cirrhosis and hepatocellular carcinoma in patients with chronic hepatitis B and C / Ativação da via do Hedgehog (Hh) promove vários processos que ocorrem durante o reparo fibrogênico hepático. O papel da via do Hh na lesão causada pela hepatite crônica B e C ainda não foi investigado. Estudos de expressão global em carcinomas hepatocelulares (CHC) demostraram a ativação da via do Hh em pacientes com CHC relacionados à infecção crônica com o vírus da hepatite B (VHB) ou vírus da hepatite C (VHC). Como a maioria dos CHCs desenvolve em fígados cirróticos, levantamos a hipótese de que a ativação da via do Hh ocorre durante o reparo fibrogênico relacionado à hepatite viral crônica B e C e que células Hh-reatoras poderiam orquestrar a progressão da doença e a hepatocarcinogênese na hepatite viral crônica. Métodos Immunohistoquímica e análise por PCR quantitativo em tempo real (qRTPCR) foram utilizados para investigar a ativação da via do Hh e identificar os tipos celulares que respondem aos ligantes dessa via em biópsias arquivadas de 45 pacientes com hepatite crônica B ou C, de diferentes graus de fibrose (F0-4), sendo que 7 também apresentavam CHC. A via do Hh foi manipulada com Ciclopamina em células estreladas hepáticas (HSC) e em células endoteliais sinusoidais hepáticas (SEC) primárias humanas. O ensaio da angiogênese foi usado para investigar o papel da via do Hh na formação de tubos. SECs foram incubadas com Shh recombinante, meio condicionado de HSC, ciclopamina, short hairpin RNA (shRNA) contra o Smoothened ou os seus controles respectivos e o comprimento dos tubos vasculares foram quantificados por morfometria. A produção de ligantes Hh foi investigada por qRT-PCR em células Huh7 infectadas com o virus JFH-1 e em uma linhagem de hepatoma de rato que expressa o gene da proteína X do VHB. Resultados Observamos um aumento dos níveis de expressão de ligantes Hh (Shh e Ihh) em todos os pacientes com hepatite crônica viral (p<0,005) e demonstramos que a infecção pelo vírus da hepatite C JFH-1 ou a expressão ectópica do gene X do VHB estimulam culturas de hepatoma a produzirem Shh (p<0,005). Os principais tipos celulares que proliferam durante a cirrose e CHC (miofibroblastos, células endoteliais ativadas e progenitores que expressam marcadores de células tronco/iniciadoras de tumor CD133, Krt7, EpCAM e survivina) são reatores aos ligantes Hh e os níveis mais altos de expressão de componentes da via do Hh (Shh, Ihh, Gli-2 e Ptch) estão associados xii com cirrose e CHC (p<0,005). Inibição da via do Hh em células reatoras (HSC e SEC) reduziu fibrogênese (p<0,005 para expressão de &#945;SMA e p<0,05 para expressão de col1&#945;1) e angiogênese (p<0,05). Conclusões A Infecção pelo VHB/VHC aumenta a produção de ligantes hedgehog em hepatócitos e expande o número de células estreladas hepáticas e células endoteliais sinusoidais primárias humanas reatoras a esses ligantes, em relação direta com a progressão da fibrose para a cirrose e o carcinoma hepatocelular. Também induz o acúmulo progressivo de células progenitoras reatoras a via do Hh e que estão em processo de transição epitélio mesenquimal, o que pode estar relacionado com o desenvolvimento ou a progressão do carcinoma hepatocelular. A inibição in vitro da ativação da via do Hedgehog em células estreladas e em células sinusoidais endoteliais hepáticas primárias humanas sugere a possibilidade de investigar inibidores dessa via como potenciais ferramentas para reduzir a progressão da fibrose hepática para cirrose e carcinoma hepatocelular nas hepatites crônicas produzidas pelos vírus B e C

Le glioblastome (GBM), tumeur de plus haut grade du système nerveux central (OMS grade 4) a un pronostic très sombre, quelque soit le traitement, lié à une résistance à l’apoptose. L’erlotinib (Tarceva®, OSI 774) est un inhibiteur de la tyrosine kinase du récepteur au facteur de croissance épithélial (EGFR). L’hyper-expression et l’amplification du gène de l’EGFR dans 40 à 60% des GBM, fourni un rationnel pour utiliser l’erlotinib. Nous avons montré sur U87-MG et DBTRG-05MG, deux lignées de GBM, l’absence d’apoptose avec l’erlotinib, liée soit à un déficit en pro-caspase 3, soit à une accumulation d’αB-crystalline bloquant l’activation de la caspase-3. L’absence d’apoptose dévie alors la cellule vers l’autophagie. L’inhibition de l’autophagie par ARN interférents ou par la chloroquine permet d’obtenir une synergie avec l’erlotinib en induisant la mort des cellules tumorales à des doses acceptables.Les GBM ont composition cellulaire hétérogène, avec un petit nombre d’éléments appelés cellules souches cancéreuses (CSC). Douées d’auto-renouvellement, elles participent à la propagation tumorale et à la résistance aux traitements. Nous avons testé l’erlotinib sur trois lignées issues de GBM humains, ayant deux modes de croissance distincts selon les conditions de milieu: en neurosphères (NS) et de type adhérent. Erlotinib a un effet inhibiteur minime sur les trois lignées adhérentes, alors que l’effet est significatif sur les lignées NS, traduisant l’importance de la voie d’EGFR pour les NS. Dans les lignées en NS, l’erlotinib est efficace sur les cellules progénitrices, mais n’a pas d’action ni sur les cellules initiatrices de NS, ni sur les cellules différenciées. L’auto-renouvellement des NS n’est pas non plus altéré. L’association cyclopamine, inhibiteur pharmacologique de la voie de Hedgehog, -erlotinib est synergique en bloquant la croissance et l’initiation des NS, laissant présager une efficacité sur les CSC. Les résultats obtenus sur ces différents modèles permettent d’une part de préciser certains mécanismes de résistance des cellules de GBM, et aussi d’orienter les indications et le choix des traitements susceptibles d’être les plus efficaces. / Glioblastoma (GBM) is the most common primary central nervous system tumor in adults and the prognosis remains dismal, any treatment used. Epidermal Growth Factor Receptor (EGFR) is amplified, overexpressed, and/or mutated in GBM, making it a rational for therapy. Erlotinib, an EGFR kinase inhibitor is strongly associated with clinical response in several cancers. We showed for U87-MG and DBTRG-05MG, two human GBM cell lines, that erlotinib can’t trigger apoptosis, related either to accumulation of αB-crystallin capable to impair caspase 3 cleavage, or to constitutive deficit for procaspase 3 in DBTRG-05MG. Apoptosis deficit switches the cell to autophagic process. Inhibition of autophagy with RNA interference or chloroquine resulted in sensitization of U87 and allowed a synergistic effect with erlotinib at therapeutic doses.Moreover, GBM showed a heterogeneous cell composition with cancer stem cells, progenitors and more differentiated cells. In this study, we test erlotinib in vitro on other GBM models: three cell lines established from surgically resected GBM specimens, grown along two features adherent and neurospheres. On the three differentiated adhering cell lines, erlotinib had only a moderate activity. Conversely, on neurosphere forming cell lines, erlotinib induced a strong inhibition of cell growth related to the EGFR amplification and EGFR expression. A short erlotinib exposure induced cell death primarily in nestin-positive cells; however it was found without effect on neurosphere initiating activity and self renewal. These results suggest that EGFR pathway activation is essential for the proliferation of GBM progenitor cells but dispensable for stem-like cancer cells self–renewal. As Hedgehog pathway is known to be activated in neural stem cells, we assayed the Hedgehog pathway inhibitor cyclopamine in association with erlotinib. While each drug separately was without effect on sphere initiation, their combination led to a 25 fold decrease in the sphere number (p=0.0004).These in vitro models are convenient to investigate resistance mechanisms in GBM. Furthermore, they focus on the necessity to exploit drug combinations for greatest efficiency.

Nous avons récemment démontré que le récepteur du morphogène Hedgehog, Patched, qui est exprimé dans de nombreux cancers, est un transporteur de multiples drogues qui contribue à la résistance des cellules cancéreuses à la chimiothérapie. Le criblage d'une banque de molécules nous a permis d'identifier deux molécules qui inhibent l'activité d'efflux de doxorubicine de Patched. Nous avons montré que ces molécules renforcent les effets cytotoxiques, proapoptotiques, antiprolifératifs et anticlonogéniques de la doxorubicine sur les cellules de cancer de la glande surrénale (surrénalome) qui expriment de façon endogène Patched. De plus, nous avons observé que l’ajout de la molécule P375 au traitement à la doxorubicine inhibe le développement des tumeurs chez des souris ayant reçu une xénogreffe de cellules de surrénalome de façon plus significative que la doxorubicine seule. Nos résultats suggèrent que l'utilisation d'un inhibiteur de l'activité d'efflux de drogues de Patched en association avec la doxorubicine est une option thérapeutique prometteuse pour le surrénalome, et très probablement pour d'autres cancers exprimant Patched. Nous avons découvert qu'une petite fraction seulement des cellules de la lignée de surrénalome exprime Patched au niveau de la membrane plasmique (cellules PM-Patched). Les cellules PM-Patched sont plus résistantes à la doxorubicine, et présentent une expression plus élevée de Patched mais aussi de la protéine ABCG2. ABCG2 étant un marqueur de cellules souches cancéreuses (CSC), nous pensons que les cellules PM-Patched pourraient être des CSC.D'autres expériences sont nécessaires pour valider cette hypothèse. / We recently demonstrated that the Hedgehog receptor Patched, which is expressed in many recurrent and metastatic cancers, is a multidrug transporter contributing to chemotherapy resistance. The screening of a chemical library allowed us identifying two molecules which inhibit the doxorubicin efflux activity of Patched. We showed that these molecules enhance the cytotoxic, proapoptotic, antiproliferative and anticlonogenic effects of doxorubicin on adrenocortical carcinoma (ACC) cells which endogenously express Patched. Moreover, we reported that the addition of the drug-like molecule P375 to doxorubicin treatment prevents the development of xenograft ACC tumours in mice much more significantly than the doxorubicin alone. Our results suggest that the use of an inhibitor of Patched drug efflux activity in combination with doxorubicin is a promising therapeutic option for ACC and most likely for other Patched-expressing cancers. We discovered that only a small fraction of the ACC cell line expressed Patched at the plasma membrane (PMPatched cells). We observed that these cells are more resistant to doxorubicin treatment than ACC cells that express Patched only in intracellular compartments. Moreover, we estimated that PMPatched cells have higher expression of Patched but also of ABCG2/BCRP proteins. Based on the fact ABCG2/BCRP is a cancer stem cell (CSC) marker and that Hedgehog signaling is involved in maintenance of CSC, we think that PM-Patched cells could be CSCs. More experiments are needed to confirm this hypothesis.

Morphogens of the Wnt and Hedgehog families are secreted signaling molecules that coordinate growth and patterning of many different tissues. Both, Wingless and Hedgehog spread across long distances in developing wing of Drosophila melanogaster. However, both proteins are covalently modified with lipid moieties. The mechanisms that allow long-range movement of such hydrophobic molecules are unclear. Like Wingles and Hedgehog, glycosylphosphatidylinositol (gpi)-linked proteins also transfer between cells with their lipid anchor intact. It has been speculated that gpi-linked proteins and lipid-linked morphogens travel together on a membranous particle, which was termed an argosome. As yet however, no functional link between argosome production and dispersal of lipid-linked proteins has been established. The topic of this thesis is to understand the cell biological nature of the argosome and thus contribute to understanding of morphogen gradient formation. To address the question of argosome biosynthesis, at least two models have been proposed. One possibility is that argosomes are membranous exovesicles with a complete membrane bilayer. Alternatively, argosomes might resemble lipoprotein particles that comprise on of a family of apolipoproteins scaffolded around a phospholipid monolayer that surrounds a core of esterified cholesterol and triglyceride. Lipid-modified proteins of the exoplasmic face of the membrane (like GFPgpi, Wingless or Hedgehog) might fit well into the outer phospholipid monolayer of such a particle. Here, I utilize biochemical fractionation to determine the sort of particle that lipid-linked proteins associate with. I show that Wingless, Hedgehog and gpi-linked proteins bind Drosophila lipoprotein particles in vitro, and colocalize with them in wing imaginal discs. Next, I use genetic means to address the functional importance of this association. I demonstrate that reducing Lipophorin levels in Drosophila larvae perturbs long-range but not shor-range Wingless and Hedgehog signaling, and increases the sequestration of Hedgehog by Patched. I propose that Lipophorin particles are vehicles for the long-range movement of lipid-linked morphogens and gpi-linked proteins.

No description available.

Einleitung: Das Darmmikrobiom, ein hochkomplexes Ökosystem an Mikroorganismen, geht eine lebenslange wechselseitige Beziehung mit seinem Wirt ein und beeinflusst wesentlich dessen Darmreifung post partum. Dazu interagieren Darmbakterien direkt und indirekt mit den intestinalen Stammzellen in den Krypten und regulieren Zellteilung und -differenzierung, wobei die Mechanismen nicht abschließend geklärt sind. Das Darmmikrobiom stellt eine wichtige Quelle für Mikroben-assoziierte molekulare Muster (MAMPs) dar, die von Mustererkennungsrezeptoren wie den Toll-like-Rezeptoren (TLR) auf den intestinalen Epithelzellen erkannt werden können. Somit können TLR auf den intestinalen Epithelzellen eine mögliche Verbindung zwischen Darmmikrobiom und Anpassungsreaktionen der Dünndarmmorphologie darstellen. Ziele der Untersuchung: Folgende Hypothesen sollten untersucht werden: 1. Die Anwesenheit von Darmbakterien beeinflusst die Morphologie der Dünndarmschleimhaut. 2. Diese Wirkung wird über Toll-like-Rezeptoren und den Hedgehog-Signalweg in Epithelzellen vermittelt. 3. Über diese Signalwege werden auch funktionelle Eigenschaften wie die Durchlässigkeit der Darmbarriere verändert. Tiere, Material und Methoden: Zur Untersuchung des Einflusses des Mikrobioms wurden in einem gnotobiotischen Mausmodell keimfreie Tiere mit dem minimalen mikrobiellen Konsortium Altered Schaedler Flora (ASF) besiedelt. Die ASF, welche sich aus acht definierten Bakterienarten zusammensetzt, wurde aus dem Caecum ASF-besiedelter C3H/HeNTac-Mäuse entnommen und männlichen und weiblichen keimfreien C57BL/6J-Mäusen appliziert. Der Nachweis der Bakterienspezies erfolgte aus bakterieller DNA, die aus frischen Kotpellets isoliert wurde. Der Vergleich wurde zwischen Dünndärmen ASF-kolonisierter Mäuse (n = 13), keimfreien Tieren (n = 8), konventionell gehaltenen (n = 7) und einer Gruppe Antibiotika-behandelter Mäuse (n = 8) durchgeführt. Zur Untersuchung des Einflusses der Toll-like-Rezeptoren TLR2 und TLR4 wurden epithelspezifische TLR2-defiziente (TLR2ΔIEC) bzw. TLR4-defiziente Mäuse (TLR4ΔIEC) verwendet und mit ihren jeweiligen Wildtyp-Geschwistertieren (WT) verglichen. Die morphometrische Eigenschaften des Dünndarms wurden anhand von histologischen Schnitten untersucht, die mit Hämatoxylin-Eosin oder Periodic-Acid-Schiff gefärbt waren. Hierbei wurden u.a. Mukosahöhe und Villuslänge gemessen sowie die Anzahl der Epithelzellen und Anzahl der Becherzellen pro Villus gezählt. Weiterhin wurden sowohl in Dünndarmgewebe als auch in isolierten intestinalen Epithelzellen mittels qPCR die mRNAExpression der Liganden des Hedgehog (Hh)-Signalwegs Sonic Hedgehog (Shh) und Indian Hedgehog (Ihh) sowie Hedgehog-Zielgene wie Glioma-associated oncogene transcription factor 1 (Gli-1), Patched-1 (Ptch-1) und Hedgehog Interacting Protein (Hhip) untersucht. In einem Teil der Tiere wurde der Hedgehog-Signalweg durch Applikation des Inhibitors Vismodegib gehemmt. Ein möglicher Einfluss auf die Permeabilität des Dünndarms wurde über die mRNA-Expression diverser Tight Junction-Proteine wie Occludin oder Claudin-4 sowie durch einen Permeabilitätsassay mit FITC-Dextran untersucht. Gruppenvergleiche wurden mit einer einfaktoriellen Varianzanalyse (ANOVA) und Holm-Šídák post-hoc Test bzw. zwischen den TLR2ΔIEC oder TLR4ΔIEC und WT-Mäusen mittels zweiseitigem ungepaarten t-Test durchgeführt. Unterschiede wurden bei P < 0,05 als statistisch signifikant betrachtet. Ergebnisse: Durch den Nachweis aller acht Bakterienarten in den Kotproben der behandelten Mäuse konnte die erfolgreiche Übertragung der ASF auf die Empfängertiere nachgewiesen werden. Hinsichtlich Mukosahöhe, Villuslänge, Anzahl der Epithelzellen und der Becherzellen wiesen keimfreie gegenüber konventionell gehaltenen Mäusen signifikant höhere Werte auf. Die Kolonisierung mit ASF führte zu einer signifikanten Verringerung dieser Merkmale, so dass sie sich denen der konventionell gehaltenen Tiere annäherten. Die mRNA-Expression der Hh-Liganden Shh und Ihh war sowohl im Dünndarm als auch in isolierten intestinalen Epithelzellen ASF-besiedelter Mäuse signifikant erhöht. Die Hh-Zielgene Gli-1, Ptch-1 und Hhip waren auf mRNA-Ebene im Dünndarm ASF-besiedelter Mäuse ebenfalls signifikant höher exprimiert. Mukosahöhe, Epithelzellzahl und Villuslänge waren im Jejunum von TLR2ΔIEC- und TLR4ΔIEC-Mäusen gegenüber ihren WT-Geschwistern signifikant höher. Während bei TLR2ΔIEC-Mäusen außer Shh alle untersuchten Hh-Zielgene vermehrt exprimiert waren, waren bei TLR4ΔIEC-Mäusen die gleichen Gene signifikant weniger exprimiert. Wurde hingegen der Hh-Signalweg in konventionell gehaltenen C57BL/6-Mäusen mit Vismodegib inhibiert, waren der Expression der Hh-Zielgene, des TLR2 und des TLR4 signifikant vermindert. Sowohl die Hemmung des Hh-Signalwegs mit Vismodegib als auch das Fehlen der epithelialen TLR2 oder TLR4 bedingte eine verminderte Expression von Tight Junction-Proteinen, die mit einer erhöhten Darmpermeabilität einherging. Konventionell gehaltene Mäuse hatten sowohl gegenüber keimfreien als auch gegenüber mit ASF besiedelten Tieren eine signifikant niedrigere Expression von Tight Junction-Proteinen, was mit einer signifikant höheren Darmpermeabilität verbunden war. Schlussfolgerungen: Zusammenfassend lassen die Ergebnisse dieser Studie darauf schließen, dass Darmbakterien über intestinale TLR2 und TLR4 den Hh-Signalweg regulieren und darüber die Morphologie der Dünndarmmukosa als auch deren funktionelle Eigenschaften wie die Durchlässigkeit der Darmbarriere beeinflussen. / Introduction: The intestinal microbiome, a highly complex ecosystem of microorganisms, enters into a lifelong reciprocal relationship with its host and significantly influences its intestinal maturation postpartum. To this end, intestinal bacteria interact directly and indirectly with intestinal stem cells in the crypts and regulate cell division and differentiation, although the mechanisms are not conclusively understood. The intestinal microbiome represents an important source of microbeassociated molecular patterns (MAMPs) that can be recognized by pattern recognition receptors such as Toll-like-Receptors (TLRs) on intestinal epithelial cells. Thus, TLRs on intestinal epithelial cells may represent a potential link between gut microbiome and adaptation responses of small intestinal morphology. Aims: The following hypotheses should be investigated: 1. The presence of intestinal bacteria affects the morphology of the small intestinal mucosa. 2. This effect is mediated via Toll-like-Receptors and the Hedgehog signaling pathway in epithelial cells. 3. Functional properties such as the permeability of the intestinal barrier are also altered via these signaling pathways. Animals, materials and methods: To investigate the influence of the microbiome, germ-free animals were colonized with the minimal microbial consortium Altered Schaedler Flora (ASF) in a gnotobiotic mouse model. The ASF, which is composed of eight defined bacterial species, was harvested from the caecum of ASF-colonized C3H/HeNTac mice and applied to male and female germ-free C57BL/6J mice. Bacterial species were detected from bacterial DNA isolated from fresh fecal pellets. Comparison was made between small intestines of ASF-colonized mice (n = 13), germ-free animals (n = 8), conventionally housed (n = 7), and a group of antibiotic-treated mice (n = 8). To investigate the influence of Toll-like-Receptors TLR2 and TLR4, epithelial-specific TLR2-deficient (TLR2ΔIEC) or TLR4-deficient mice (TLR4ΔIEC) were used and compared with their respective wild-type (WT) littermates. Morphometric characteristics of the small intestine were examined using histological sections stained with hematoxylin-eosin or periodic-acid-Schiff. Here, mucosa height and villus length were measured, and the number of epithelial cells and number of goblet cells per villus were counted, among other measurements. Furthermore, mRNA expression of the ligands of the Hedgehog (Hh) signaling pathway Sonic Hedgehog (Shh) and Indian Hedgehog (Ihh) as well as Hedgehog target genes such as Gliomaassociated oncogene transcription factor 1 (Gli-1), Patched-1 (Ptch-1) and Hedgehog Interacting Protein (Hhip) were examined in small intestinal tissue as well as in isolated intestinal epithelial cells by qPCR. In a subset of animals, Hedgehog signaling was inhibited by application of the inhibitor Vismodegib. A possible influence on small intestinal permeability was investigated by mRNA expression of various tight junction proteins such as Occludin or Claudin-4 and by permeability assay with FITC-dextran. Group comparisons were performed with a one-way analysis of variance (ANOVA) and Holm-Šídák post-hoc test or between the TLR2ΔIEC or TLR4ΔIEC and WT mice by two-tailed unpaired t test. Differences were considered statistically significant at P < 0.05. Results: Detection of all eight bacterial species in the fecal samples of treated mice demonstrated successful transfer of ASF to recipient animals. In terms of mucosa height, villus length, number of epithelial cells and goblet cells, germ-free mice had significantly higher values compared to conventionally housed mice. Colonization with ASF significantly decreased these characteristics to approach those of conventionally housed animals. The mRNA expression of the Hh ligands Shh and Ihh was significantly increased in the small intestine as well as in isolated intestinal epithelial cells of ASF colonized mice. The Hh target genes Gli-1, Ptch-1, and Hhip were also significantly higher expressed at the mRNA level in the small intestine of ASF-colonized mice. Mucosa height, epithelial cell number, and villus length were significantly higher in the jejunum of TLR2ΔIEC and TLR4ΔIEC mice compared with their WT littermates. Whereas in TLR2ΔIEC mice, except for Shh, all Hh target genes examined were increased in expression, the same genes were significantly less expressed in TLR4ΔIEC mice. In contrast, when the Hh pathway was inhibited with Vismodegib in conventionally maintained C57BL/6J mice, the expression of Hh target genes, TLR2, and TLR4 were significantly decreased. Both inhibition of the Hh pathway with Vismodegib and absence of epithelial TLR2 or TLR4 caused decreased expression of tight junction proteins, which was associated with increased intestinal permeability. Conventionally housed mice had significantly lower expression of tight junction proteins compared with both germ-free and ASF-populated animals, which was associated with significantly higher intestinal permeability. Conclusions: In summary, the results of this study suggest that intestinal bacteria regulate the Hh signaling pathway via intestinal TLR2 and TLR4 and thereby influence the morphology of the small intestinal mucosa as well as its functional properties such as intestinal barrier permeability.

Purpose: The Hedgehog-signalling pathway (Hh) is frequently active in head and neck squamous cell carcinoma (HNSCC). Overexpressed Hh associates with poor prognosis. The Hh inhibitor vismodegib targets smoothened (SMO) and, based on molecular data, may prevent resistance to EGFR targeting. Methods: To elucidate potential roles of vismodegib in HNSCC therapy, its sole effects and those combined with cisplatin, docetaxel, and cetuximab on HNSCC cell lines were assessed by MTT metabolisation and BrdU incorporation. Colony formation (CF) of primary HNSCC cells was studied utilizing the FLAVINO-protocol. Combinatory effects were analysed regarding antagonism, additivity or synergism. Associations between the ex vivo detected mode of action of vismodegib with other treatments related to patient characteristics were assessed and progression-free survival (PFS) in patient groups compared using Kaplan-Meier curves. Results: Vismodegib suppressed BrdU incorporation significantly stronger than MTT turnover; CF was significantly inhibited at ≥20 µM vismodegib while concentrations <20 µM acted hormetic. Combining 20 µM vismodegib plus docetaxel (T), cisplatin (P), and cetuximab (E), additively enhanced antitumoral activity in HNSCC samples from patients with superior PFS highlighting a potential role for ex-vivo testing of this combination for use as a prognostic classifier. Conclusion: We provide ex-vivo evidence for vismodegib’s potential in HNSCC therapies especially if combined with cetuximab, cisplatin and docetaxel.:Abkürzungsverzeichnis 1 Einleitung 1.1 Kopf-Hals-Tumore 1.1.1 Therapie von Kopf-Hals-TumoreN 1.1.2 Limitationen der etablierten Therapien 1.2 Eingesetzte Chemotherapeutika 1.2.1 Cisplatin 1.2.2 Docetaxel 1.2.3 Cetuximab 1.3 Hedgehog-Signaltransduktionsweg 1.3.1 Hedgehog-Signalweg und Karzinogenese 1.3.2 Vermittlung von Tumortherapieresistenz durch den Hedgehog-Signalweg 1.3.3 Zielgerichtete Tumortherapie durch Blockade des Hedgehog-Signalweges 1.4 Vismodegib 1.5 Ex-Vivo-Chemoresponse-Testung mittels FLAVINO-Assay 1.6 Zusammenfassung der Rationale der Untersuchung 1.7 Aufgabenstellung der Promotionsarbeit 2 Publikation 2.1 Reduzierte Proliferation und Koloniebildung von Plattenepithelkarzinomen der Kopf Hals Region unter dualer Inhibition des EGFR- und Hedgehog-Signalweges 3 Zusammenfassung der Arbeit 4 Literaturverzeichnis 5 Anlagen 5.1 Darstellung des Eigenanteils 5.2 Erklärung über die eigenständige Abfassung der Arbeit 5.3 Lebenslauf 5.4 Publikationen 5.5 Danksagung

Рассматриваются два топологических пространства: квази-метрический ёж и фактор-ёж, у которых находятся следующие кардинальные инварианты: вес, характер, плотность, спред, экстент, клеточность, теснота, число открытых множеств и число Линделефа; наследственные кардинальные инварианты: наследственный вес, наследственный характер, наследственная плотность, наследственный спред, наследственный экстент, наследственная клеточность, наследственная теснота и наследственное число Линделефа. / We consider two topological spaces, the quasi-metric hedgehog and the factor-hedgehog, which have the following cardinal invariants: weight, character, density, spread, extent, cellularity, tightness, the number of open sets, and the Lindelöf number; hereditary cardinal invariants: hereditary weight, hereditary character, hereditary density, hereditary spread, hereditary extent, hereditary cellularity, hereditary tightness, and the hereditary Lindelöf number.

The development of the vertebrate eye is tightly controlled by precise genetic regulations. From a single ocular primordium to bilateral eyes with complex structures and cell types, it requires intensive proliferation and migration for cells in both the ectoderm and mesoderm to accomplish ocular morphogenesis, and during this process cell differentiation and interaction takes place to establish the complex composition of ocular cell types and cellular connections. Genetic defects can lead to severe abnormalities in eye morphogenesis and cell differentiation during ocular development. A tremendous amount of work has been done to identify both intrinsic and extrinsic factors that regulate ocular development. However, much more work is needed to fully understand this complex process. Sox4 is known as a transcription activator that regulates cell survival and differentiation in multiple embryonic tissues during development. Evidence of its requirement during ocular development has recently emerged, but the mechanism by which Sox4 regulates ocular development is far from elucidated. Chapter 1 of this dissertation provides an overview of different stages in embryonic eye development and known genetic interactions during each stage. It also reviews recent knowledge about SoxC proteins and their roles in ocular development. Chapter 2 presents data characterizing the expression profile of the zebrafish sox4 co-orthologs, sox4a and sox4b, in the developing eye. Additionally, it presents data from morpholino-mediated sox4 knockdown in zebrafish, which indicate that Sox4 deficiency leads to defects in choroid fissure closure through elevation in the Hedgehog (Hh) signaling pathway. Sox4 knockdown causes upregulation of the Hh ligand indian hedgehog b (ihhb), which alters the proximal-distal boundary of the optic vesicle and inhibits choroid fissure closure. Chapter 3 presents data reporting the generation of sox4 mutant zebrafish lines using the CRISPR/Cas9 genome editing system. Characterization of one sox4a maternal zygotic (MZ) mutant line confirms Sox4’s role in negative regulation of Hh signaling and reveals new evidence that maternal and zygotic sox4 are both critical for ocular development. Chapter 4 presents data demonstrating that sox4 is required for rod photoreceptor neurogenesis. Rod photoreceptor terminal differentiation is delayed in both sox4 morphants and sox4 CRISPR mutants, while rod progenitor and precursor cells are properly specified. In Chapter 5, the roles of Sox4 in regulating ocular development are summarized based on the results, and implications of the results are discussed to expand our understanding of the genetic regulation of ocular morphogenesis and retinal neurogenesis.

Introduction. The inflammatory bowel diseases (IBD), Crohn’s disease (CD) and ulcerative colitis (UC), are common in Western Europe (200-400 cases /100,000) and associated with substantial morbidity, although mortality is now low. There is presently a great unmet need for novel therapeutics in IBD as present agents are limited by lack of efficacy, toxicity and poor patient acceptance. Recent findings from genome-wide association studies (GWAS) have characterised the genetic architecture of CD and UC. Defects in innate and adaptive immunity have been clearly established, and substantial novel insights into disease pathogenesis have been gained. Over 30 genes / loci are now associated with CD; a number of these, along with a few specific loci, are also associated with UC. The hedgehog (HH) signalling pathway is critical to gastrointestinal development and plays key roles in intestinal and immune homeostasis. Furthermore, in addition to well described roles in tumorigenesis, it is evident that recapitulation of embryonic HH signals play critical roles in response to acute and chronic inflammatory challenge in diverse tissues. Aims. The main aims of the work presented in this thesis were to characterise the expression of key HH signalling components in the healthy and inflamed human intestine, establish whether germline variation in HH genes is associated with IBD and describe the in vitro responses of intestinal epithelial cells to pathogen associated molecular patterns. The WNT pathway, antagonised by HH in the intestine, and two HH target genes (NKX2.3 and CCL20) were also analysed for evidence of association with IBD. Methods. Expression of HH and WNT signalling components was described by immunohistochemistry and microarray analysis in healthy controls (HC), CD, UC, and non- IBD inflamed terminal ileal and colonic samples. Gene-wide haplotype-tagging studies were performed for GLI1 in Scottish, English and Swedish CD and UC, and Scottish early-onset colo-rectal cancer, IHH in Scottish IBD, NKX2.3 in Scottish and UK IBD, and CCL20 in Scottish, Swedish and Japanese IBD. Evidence for association of all HH (n=13) and WNT (n=27) signalling genes in CD was established by analysis of UK GWAS data and metaanalysis from UK, French/Belgium and N American studies. The effect of lipopolysaccharide (LPS) and muramyl dipeptide (MDP) on HH signalling was assessed in colonic epithelial cells (SW480). The effect of HH pathway agonists and antagonists on NFκB activity and cytokine expression was analysed in SW480 cells and peripheral blood mononuclear cells (HC and IBD patients) in vitro. Results. The expression of HH pathway ligand is present in the intestinal epithelium and the pathway response network in the lamina propria demonstrating the paracrine nature of HH signalling in the intestine. Immunohistochemical studies and microarray analysis demonstrates that HH pathway activity is decreased in all forms of colonic inflammation studied in man. Variation in Glioma-associated oncogene homolog 1 (GLI1), a key HH transcription factor located at 12q13 (IBD2), was associated with IBD (p<0.0001), UC (p<0.0001) and to a lesser extent CD (p=0.03) in Scotland, a finding replicated in English IBD and UC. This association was attributed to a non-synonymous SNP (rs2228226C→G) with pools odds ratio of 1.194 in meta-analysis of over 5000 individuals from Scotland, England and Sweden (p=0.0002). There was association of this SNP with early-onset colorectal cancer, but of borderline significance (p=0.05). The variant protein (Q1100E) is 50% less active than wild-type protein in vitro. IHH was not associated with CD or UC. Preliminary evidence was produced for association at SUFU (10q24; p=0.005), a GLI1- binding protein, and at the WNT3 / WNT9B locus (17q21; p=0.0005). MDP stimulation of colonic epithelial cells decreased HH pathway activity. Exogenous HH increased expression of CCL20. CCL20 promoter polymorphisms were associated with UC in Japanese patients (p=0.018) but not in Scotland or Sweden. NKX2.3 was associated with IBD in Scotland (UC>CD), but there was insufficient power for fine-mapping of causative variants. Conclusions. Multiple lines of evidence presented here demonstrate that the HH signalling pathway is involved in IBD pathogenesis. In key complementary in vivo studies (conceived by CWL; conducted in collaboration with the Gumucio lab in Ann Arbor) we have demonstrated that Gli1+/- mice develop early, severe colitis with high mortality in response to acute inflammatory challenge. Furthermore, lamina propria antigen presenting cells are identified as the key HH target cells. With HH agonists and antagonists in extensive preclinical and early clinical testing, these studies have real potential to translate into novel therapeutics for patients with IBD.