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Swift, Nathan Butler IV. "HEDGEMON: A HEDGEHOG-INSPIRED HELMET LINER." Case Western Reserve University School of Graduate Studies / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=case1459380535.
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Jovanovic, Biljana. "The role of Hedgehog acyltransferase in Sonic hedgehog signalling." Thesis, Imperial College London, 2012. http://hdl.handle.net/10044/1/14486.
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Protein acyltransferases (PATs), a group of enzymes catalysing fatty acylation of a variety of proteins, play an important role in protein sorting and signalling in the cell. In the Hedgehog (Hh) signalling pathway the PAT Hedgehog acyltransferase (Hhat) is responsible for addition of a palmitic acid to the N-terminal cysteine of Hh proteins. This modification is necessary for proper packaging and release of Hh ligands from the signalling cell and for transmitting a potent signal to the receiving cell. Hh signalling regulates cell growth and differentiation. Improper activation of the Hh signalling pathway in cancer arises either through an upregulation of ligand-dependent autocrine or paracrine signalling, or oncogenic mutations in the signal receiving cell. To date, drug therapies targeting the pathway in cancer have been directed at pathway regulators downstream of ligand binding. In this thesis, I investigate the potential of Hhat as a therapeutic target in lung cancer cell lines that express Sonic hedgehog (Shh). Studies of lung cancer cell lines A549, H209 and H82 treated with Shh pathway inhibitors showed no autocrine dependence on Shh signalling. Nonetheless, A549 cells were capable of inducing a Shh signal response in C3H10T½ cells in co-culture in a juxtacrine/paracrine fashion indicating that Shh in lung cancer cells may be involved in juxtacrine/paracrine signalling to the surrounding stroma. This effect was effectively attenuated by siRNA-mediated Hhat knock-down confirming the potential of Hhat as therapeutic target in cancer. I further provide a membrane topology model of Hhat which will aid in understanding the molecular mechanism of this enzyme. Membrane topology determination resulted in a topology model where Hhat consists of 11 transmembrane regions with the signal peptide preserved in the mature protein. Surprisingly, the suggested catalytic site, a conserved His, is localised in a transmembrane region near the cytosol away from the site of Hh palmitoylation which occurs in the lumen of the endoplasmic reticulum. Taken together, my results provide a firm basis for further investigation of the mechanism of action of Hhat and its role as a potential drug target in multiple human cancers.
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Lau, C. I. "Hedgehog signalling in haematopoiesis." Thesis, University College London (University of London), 2014. http://discovery.ucl.ac.uk/1428443/.
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The Hedgehog (Hh) family proteins and their signalling pathway are key mediators of, and important in, many mammalian developmental processes. Malfunction of the Hh signalling pathway contributes to developmental disorders and birth defects. This project aims to investigate the role of the Hh signalling pathway in murine haematopoiesis. In our study, we found that Dhh plays a negative regulatory role in normal erythropoiesis and under stress conditions. However, it is not required for regulating erythropoiesis in the fetal liver during embryo development. In contrast, analysis of conditional deletion of Smo in haematopoietic cells revealed that Smo controls early haematopoietic differentiation in the fetal liver but is dispensable for regulating haematopoiesis in adult bone marrow and spleen. Furthermore, pervious studies have demonstrated that Hh signalling is involved in T-cell development throughout maturation. We tested the hypothesis that Foxa2, a downstream target gene of Hh during pre-TCR signalling, is also required for late T-cell development and activation. Analysis of mice conditionally Foxa2-deficient in mature T-cells revealed that Foxa2 is important in the process of maturation in late thymocyte development. In addition, Foxa2 is also involved in regulation of T-cell activation, and the differentiation of T helper cells. Gene expression experiments confirmed that Foxa2 is also a Hh target gene in the thymus. Taken together, our findings revealed that the Hh signalling pathway and its target genes play critical roles in haematopoiesis during embryogenesis and in adult mice.
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Siegwarth, Mark D. "The Arizona Hedgehog Project." University of Arizona (Tucson, AZ), 2014. http://hdl.handle.net/10150/622042.
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An intergovernmental agreement was signed on August 26, 2008 between the Arizona Department of Transportation (ADOT) and Boyce Thompson Arboretum (BTA) for the Arizona Hedgehog Project. The project was to transplant individuals of the Arizona hedgehog cactus (Echinocereus triglochidiatus var. arizonicus or AHC) from the US Highway 60 ADOT project area to Boyce Thompson Arboretum and conduct a 5-year research study on the AHC to learn more about how to increase success of future salvage efforts. In addition, the project included the development of interpretive/educational materials including printed materials and signage to explain the project to the more than 75,000 annual visitors who visit the Arboretum. The transplant sites at the Arboretum offer an excellent opportunity for informing the general public, adults and children alike, about the importance of conserving the Arizona hedgehog and other endangered species. As stated above, the Arizona Hedgehog project is comprised of two distinct but overlapping parts: the physical movement of the plants to Boyce Thompson Arboretum and an extended study of transplantation success. In brief, we first evaluated the
plants in situ at the US Highway 60 ADOT project location, then removed the plants and transported them to BTA. Although there were to be two different plantings, fall and spring, delays and fewer than the expected number of AHC needing salvage mandated that all plantings be done in the fall. Finally, we evaluated the success of the transplants over a 5-year period.
This was essentially an observational study. Germplasm was shared with other researchers, which will provide additional information.
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Chang, Shu-Chun. "The role of hedgehog acyltransferase & heparan sulphate proteoglycans in human sonic hedgehog signalling." Thesis, Imperial College London, 2011. http://hdl.handle.net/10044/1/6837.
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Hedgehog proteins (Hh) are morphogens and major mediators in many developmental processes. Hh signalling is significant for many aspects of embryonic development, whereas dysregulation of this pathway is associated with several types of cancers. Hh proteins require dual lipidation and Heparan Sulfate Proteoglycans (HSPGs) for their proper distribution and signalling activity. My first aim was to study the role of HSPGs in human (h) Sonic Hedgehog (Shh) signalling and clarify the biological function of hShh/HSPGs complexes in hShh signalling, by investigating the interaction between human hShh and HSPGs. I used DNA mutagenesis and heparin affinity chromatography to determine key residues in hShh involved in heparin binding (K37/38 and K178). The activity of these mutants was tested by detecting induced Alkaline Phosphatase activity in C3H10T1/2 cells and hShh-inducible gene expression in PANC1 human pancreatic carcinoma cells. I examined the biological function of mutated hShhs (K37/38S, K178S and K37/38/178S) that cannot interact with heparin efficiently and showed that they had reduced signalling activity compared to wild type hShh and a control mutation (K74S). Also, I showed that mutant hShh proteins mediate reduced proliferation and invasion of PANC1 cells following hShh RNAi knockdown (KD), and this correlated with reduced Shh multimeric complex formation. Structurally, Shh proteins are unusual in being dually lipid-modified to be fully active. During the post-translational modifications of Shh, N-terminal palmitoylation is facilitated by the product (Hhat) of the hedgehog acyltransferase gene. I have carried out a thorough analysis of Hhat in PANC1 cells. First, I characterised an antibody prepared in the lab to hHhat. I confirmed the specificity of the antibody by immunoblotting using a self-constructed hHhat-EGFP clone, and a control mGup1-EGFP clone. By subcellular fractionation and Western blotting I found Hhat to be a membrane protein. In addition, I used the hHhat antibody to determine the intracellular localisation of hHhat in PANC1 cells by confocal microscopy and showed that hHhat localised in ER mainly but not in Golgi apparatus. I confirmed this using the hHhat-EGFP clone for fluorescence microscopy in transfected cells. To illuminate the biological function of palmitoylation of hShh in production of active hShh and in the formation of hShh multimeric complex I optimised hHhat RNAi knockdown (KD) in PANC1 cells and confirmed this by a cell-based palmitoylation assay. Using semi-quantitative RT-PCR and immunoblot analyses, I showed that hHhat KD caused decreased signalling through the Shh pathway due to reduced production of active hShh. In addition, I investigated the effect of the addition of palmitate to hShh on its association with cells by comparing hHhat KD cells with control cells. Immunoblotting suggests that palmitoylation of Shh improves its ability to associate to cell membranes. Using hHhat KD, gel filtration of high molecular weight complexes of hShh and immunoblotting of hShh I characterised the role of palmitoylation of hShh in multimeric complex formation. Lastly, I investigated the effect of hHhat KD on PANC1 proliferation and invasion, showing that it represses PANC1 proliferation and invasion. These studies provide a firm basis for understanding the functional roles of hShh palmitoylation and its interactions with HSPGs, and provide proof-of-principle for targeting these aspects of hShh biology in tumour cell therapeutics, specifically in the pancreatic carcinoma context.
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Haines, Nicola. "Mutational analysis of hedgehog signalling." Thesis, University of Oxford, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365823.
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Gorka, Oliver. "Hedgehog-Signale in rheumatoider Arthritis." [S.l. : s.n.], 2008. http://nbn-resolving.de/urn:nbn:de:bsz:25-opus-43288.
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Lau, Janet. "Hedgehog signaling in the pancreas epithelium." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2010. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3398879.
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Koziel, Lydia. "Indian hedgehog signaling during endochondral ossification." [S.l. : s.n.], 2004. http://www.diss.fu-berlin.de/2005/68/index.html.
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Tukachinsky, Hanna. "Mechanistic Studies of Vertebrate Hedgehog Signaling." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10691.
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Metazoans use Hedgehog signaling to direct many stages of embryonic development, and deregulation of this pathway is implicated in many types of cancer. I investigated several steps of Hedgehog pathway transduction that were poorly understood in mechanistic terms. The mature Hedgehog ligand is produced by a self-proteolysis reaction that covalently attaches a cholesterol molecule to the signaling half of the protein. I showed that the catalytic cysteine forms a disulfide bridge that is essential for the folding and function of the C-terminal tail of Hedgehog, and identified two protein disulfide isomerases that remodel this bridge to free the catalytic thiol group after folding is complete. Using pulse chase assays to follow Hedgehog processing, I demonstrated that the self-proteolysis reaction takes place in the endoplasmic reticulum, that the cleaved C-terminal tail of Hedgehog is degraded before moving to the Golgi, and that Hedgehog mutants defective in processing get degraded in their entirety by the same route. Lipidated Hedgehog ligand requires the transmembrane protein Dispatched for secretion. I devised a system to test Dispatched function in cultured cells, and showed that some inactive Dispatched mutants fail to bind Hedgehog, while others bind more tightly than the wild type protein. Scube2 was implicated as a Hedgehog pathway component in zebrafish genetic studies. I showed that Scube2 is a secreted protein that binds Hedgehog via its cholesterol adduct and solubilizes it in aqueous media. Dispatched and Scube2 bind Hedgehog on opposing faces, and they function synergistically to release it from the membrane. Vertebrate Hedgehog signaling relies on intraflagellar transport through an antenna-like organelle called the primary cilium. The Hedgehog receptor Patched and transducer protein Smoothened localize to primary cilia in a mutually exclusive pattern, depending on Hedgehog ligand presence. I showed that cytoplasmic components of the pathway Suppressor of Fused (SuFu, a pathway inhibitor) and Glioma-associated oncogene transcription factors (the Gli family, the effectors of the pathway) localize to primary cilia and accumulate there when Smoothened is activated. SuFu and Gli form a complex that dissociates when the pathway is turned on, and this dissociation depends on trafficking through the cilium.
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Moshiri, Ala. "Sonic hedgehog in the vertebrate retina /." Thesis, Connect to this title online; UW restricted, 2004. http://hdl.handle.net/1773/10669.
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Palm, Wilhelm, Marta M. Swierczynska, Veena Kumari, Monika Ehrhart-Bornstein, Stefan R. Bornstein, and Suzanne Eaton. "Secretion and Signaling Activities of Lipoprotein-Associated Hedgehog and Non-Sterol-Modified Hedgehog in Flies and Mammals." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-180911.
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Hedgehog (Hh) proteins control animal development and tissue homeostasis. They activate gene expression by regulating processing, stability, and activation of Gli/Cubitus interruptus (Ci) transcription factors. Hh proteins are secreted and spread through tissue, despite becoming covalently linked to sterol during processing. Multiple mechanisms have been proposed to release Hh proteins in distinct forms; in Drosophila, lipoproteins facilitate long-range Hh mobilization but also contain lipids that repress the pathway. Here, we show that mammalian lipoproteins have conserved roles in Sonic Hedgehog (Shh) release and pathway repression. We demonstrate that lipoprotein-associated forms of Hh and Shh specifically block lipoprotein-mediated pathway inhibition. We also identify a second conserved release form that is not sterol-modified and can be released independently of lipoproteins (Hh-N*/Shh-N*). Lipoprotein-associated Hh/Shh and Hh-N*/Shh-N* have complementary and synergistic functions. In Drosophila wing imaginal discs, lipoprotein-associated Hh increases the amount of full-length Ci, but is insufficient for target gene activation. However, small amounts of non-sterol-modified Hh synergize with lipoprotein-associated Hh to fully activate the pathway and allow target gene expression. The existence of Hh secretion forms with distinct signaling activities suggests a novel mechanism for generating a diversity of Hh responses.
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Wilson, Christopher William. "Mechanism and evolution of mammalian hedgehog signaling." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3378515.
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Wenzel, Hans Markus. "Identifizierung neuer Zielgene im Indian-Hedgehog-Signalweg." [S.l.] : [s.n.], 2003. http://www.diss.fu-berlin.de/2003/196/index.html.
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Zhu, Yanhua. "MBD genes and Hedgehog signalling in cancer." Thesis, University of Edinburgh, 2004. http://hdl.handle.net/1842/27742.
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In this study, two sets of candidate genes in colon and lung cancer tumourigenesis were studied. The first set comprised members of a family of genes whose proteins are important in the recognition of the methylation/epigenetic status of other genes. The second set were members of a pathway that normally regulate tissue development but whose abnormal, epigenetic loss of activity could lead to tissue dysregulation and tumourigenesis. MBD3 and MBD2 are two members of the MBD family of proteins with a methyl-CpG-binding domain (MBD) involved in transcriptional silencing of methylated genes. Both genes are located in chromosomal regions that suffer loss of heterozygosity in colon and lung cancers. By SSCP analysis and methylation sensitive restriction followed by PCR, 2 mutations were found in 28 cell lines and in no cases was there evidence of gene silencing by hypermethylation of putative promoter regions. RT-PCR and northern hybridisation showed expression of MBD3 in all cancer cell lines examined. The results indicate that neither MBD2 nor MBD3 are major targets of genetic and epigenetic alteration in colon and lung cancers. The Hedgehog (Hh) pathway is a highly conserved signalling cascade involved in many developmental processes. In this study, two genes of this pathway, SMO and GLI3 were investigated for expression and epigenetic alterations in colon and lung cancers. In three cell lines expression of SMO was absent, the putative SMO promoter was fully methylated and GLI3 was not expressed. Two other cell lines had a methylated wild-type SMO allele and expressed mutant SMO, and also did not express GLI3. The results indicate that SMO is silenced by CpG island hypermethylation in colon and lung cancer cell lines, that GLI3 is also silenced in colon and lung cancer cell lines by an as yet unrevealed mechanism and that GLI3 is possibly regulated by SMO in a manner outside the normal sequence of steps currently thought to comprise the Hh pathway.
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Nedelcu, Daniel. "Smoothened regulation in the Hedgehog signaling pathway." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:11080.
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Hedgehog signaling is a pathway essential in embryonic development, adult stem cell maintenance, and is implicated in the formation and progression of cancer. Signaling in this pathway is triggered when the secreted protein Hedgehog binds to its membrane receptor, Patched. Patched normally inhibits the seven-spanner transmembrane protein Smoothened (Smo). Binding of Hedgehog inhibits Patched resulting in Smo derepression. Active Smo then triggers the activation of the cytoplasmic steps of the signaling pathway.
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Hartmann, David [Verfasser], and Christine [Akademischer Betreuer] Dierks. "Der Hedgehog-Signalweg in Non-Hodgkin-Lymphomen." Freiburg : Universität, 2017. http://d-nb.info/1135572119/34.
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Hsieh, David. "Hedgehog signaling in glioblastoma multiforme stem cells." Thesis, The University of Arizona, 2009. http://hdl.handle.net/10150/192485.
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Braun, Stefanie Anett. "Analyse des Hedgehog-Signalweges in Zellkulturen maligner Gliome." Doctoral thesis, Universitätsbibliothek Leipzig, 2013. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-102026.
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Hedgehog-signalling in malignant gliomas The Hedgehog signalling pathway is important for the development of the central nervous system. On the other hand, aberrant induction is observed in different tumors. Immunofluorescence and real-time qRT-PCR confirmed that in some gliomas, specifically in Glioblastoma multiforme (GBM), Gli1, a transcription factor activated by signalling, is present. In general, the hedgehog pathway is initiated by binding of extracellular ligands to the transmembrane receptor Patched and leads finally to the activation of the transcription factors Gli1, Gli2, Gli3 and Gli4. Whereas Gli1 acts as an activator, Gli2 appears to be an activator but retains some repressor activities and Gli3 and Gli4 are believed to act only as inhibitors. Therefore, the determination of hedgehog activity at the level of transcription requires additional experiments measuring gene activation. For that reason, cells isolated from 13 tumors of patients with glioblastoma (WHO Grade IV) and cells from two different glioma cell lines were transfected with reporter genes. These reporter genes carried the luciferase gene from Gaussia princeps under the control of two promoters (pT109 and pT81) conjugated to Gli binding sites. The activity of the reporter genes was compared to a control plasmid with mutant Gli-binding sites. In addition reporter gene activity was analysed in the absence and presence of the hedgehog signalling inhibitor cyclopamine and the effect of cyclopamine on cellular metabolism was studied. The analysis revealed that the two cell lines and cells from 6 glioblastomas exhibited enhanced reporter gene activity compared to the activity mutant control. This points towards an enhanced expression of Gli1. In three cultures a repression was detected suggesting that Gli3 may be active in these cells. Four cultures did neither show activation nor repression. This could provide evidence that Gli1 and Gli3 effects cancel each other out or that there is no effect at all. Enhanced luciferase activity in cells from the line T98G and in cells from four primary cultures was not influenced by the hedgehog inhibitor cyclopamine, whereas one cell line significantly responded to its presence with a decreased activity. Interestingly, ATP level was suppressed by cyclopamine in cells from the line T98G and also in cells from one primary culture that responded to the inhibitor. This may point towards an effect of cyclopamine independent of smo. Since cyclopamine is a potential new substance for the treatment of tumors, the observed effect of this inhibitor even in cells without an indication of hedgehog signalling activity should be investigated in further experiments in more detailDer Hedgehog (Hh) -Signalweg spielt während der Embryonalentwicklung eine wichtige Rolle, so auch bei der Entstehung des zentralen Nervensystems (Varjosalo & Taipale 2008). Andererseits führt seine unregulierte Aktivität zur Ausbildung verschiedenster Tumore (Bailey et al. 2009; Fiaschi et al. 2009; Shaw et al. 2009; Velcheti & Govindan 2007). Vorausgegangene Studien wiesen durch Immunfluoreszenz und real-time qRT-PCR nach, dass auch in Gliomen, speziell in Glioblastoma multiforme, dem agressivsten Hirntumor des Menschen, Effektoren des Signalweges (Gli1) überexprimiert werden (Wang et al. 2010). Die Aktivierung des Signalweges geschieht über Bindung des Hh-Liganden an den Rezeptor Ptch und endet mit der Aktiverung der Transkriptionsfaktoren der Gli Familie (Kinzler & Vogelstein 1990; Stone et al. 1996). Die aktuell bekannten Vertreter dieser Familie sind der Aktivator der Transkription Gli1, Gli2, der als Aktivator und Repressor agieren kann sowie Gli3 und Gli4, die die Transkription inhibieren (Marine et al. 1997; Ruppert et al. 1988). Ziel dieser Arbeit war es, herauszufinden, inwieweit die Transkriptionsfaktoren der Gli-Familie in Zellen von Glioblastoma multiforme aktiv sind. Dafür wurden Zellen aus Tumormaterial isoliert und daraus Primärkulturen hergestellt. In diese 13 Primärkulturen, wie auch in zwei Gliom-Zelllinien, wurden mittels transienter Transfektion Reporterplasmide eingebracht. Diese enthielten ein Gen der Gaussia-Luciferase, das unter der Kontrolle zweier verschiedener Promotoren (pT109 und pT81) mit Bindungsmotiven für die Transkriptionsfaktoren der Gli-Familie stand. Weiterhin wurde der Einfluss des Inhibitors des Hh-Signalweges Cyclopamin auf die Gli-Aktivität und die Metabolische Aktivität der Zellen untersucht. Die Beobachtungen ergaben, dass die zwei Zelllinien und sechs der primären Kulturen eine erhöhte Luciferaseaktivität und damit gesteigerte Aktivität von Gli1 zeigten. Weiterhin wiesen vier Kulturen eine verminderte Luciferaseaktivität auf. Dies ließ darauf schließen, dass in diesen Zellen Gli3 aktiv war. In den restlichen vier Kulturen zeigte sich keine Veränderung der Luciferaseaktiviät, was für einen Aufhebungseffekt von Gli1 und Gli3 oder gar keinen Effekt spricht. Weiterhin konnte gezeigt werden, dass die Luciferaseaktivität und damit die Aktivität von Gli1 in Zellen der Zelllinie T98G und von vier Primärkulturen nicht durch Cyclopamin beeinflusst wird. Lediglich eine Probe der Primärkulturen reagierte mit einer Abnahme der Luciferaseaktivität. Außerdem konnte Cyclopamin die ATP-Produktion sowohl in Zellen von T98G als auch in Zellen der Zelllinie, deren Gli-Aktivität durch Cyclopamin vermindert wurde, senken. Dies sprach für eine Smo unabhängige Wirkung des Cyclopamins. Da Cyclopamin ein potenzielles Pharmakon für die Antitumortherapie ist, bedarf dieser Umstand näherer Untersuchungen
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Laubner, Daniela. "Regulation der Wirkung von Sonic Hedgehog durch Cholesterin." [S.l.] : [s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=964183757.
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Hagl, Beate. "Epigenetische Veränderungen des Hedgehog-Signalwegs in embryonalen Tumoren." Diss., lmu, 2011. http://nbn-resolving.de/urn:nbn:de:bvb:19-126012.
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Steg, Adam. "Analysis of the hedgehog pathway in pancreatic adenocarinoma." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2008. https://www.mhsl.uab.edu/dt/2008d/steg.pdf.
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Jacinto, Antonio Alfred Coelho. "Analysis of hedgehog signalling in Drosophila melanogaster development." Thesis, Imperial College London, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.313805.
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Blewitt, Alex. "HEDGEHOG : automatic verification of design patterns in Java." Thesis, University of Edinburgh, 2006. http://hdl.handle.net/1842/1459.
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Design patterns are widely used by designers and developers for building complex systems in object-oriented programming languages such as Java. However, systems evolve over time, increasing the chance that the pattern in its original form will be broken. To verify that a design pattern has not been broken involves specifying the original intent of the design pattern. Whilst informal descriptions of patterns exist, no formal specifications are available due to differences in implementations between programming languages. This thesis shows that many patterns (implemented in Java) can be verified automatically. Patterns are defined in terms of variants, mini-patterns, and artefacts in a pattern description language called SPINE. These specifications are then processed by HEDGEHOG, an automated proof tool that attempts to prove that Java source code meets these specifications.
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Hardcastle, Zoe. "The Sonic Hedgehog signalling pathway in tooth development." Thesis, King's College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.368160.
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Papaioannou, Eleftheria. "The role of Hedgehog signalling in atopic dermatitis." Thesis, University College London (University of London), 2018. http://discovery.ucl.ac.uk/10057775/.
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Hedgehog (Hh) proteins are morphogens which regulate embryonic development and adult tissue homeostasis, with distinct outcomes dependent on the strength and duration of their signals. In skin, aberrant Hh pathway activation is linked to cancer and malformations, but the role of the pathway in skin inflammation remains largely unknown. Here I show that the Hh signalling pathway modulates the induction and pathology of mouse atopic dermatitis. Sonic hedgehog (Shh) and Hh pathway target genes were upregulated on induction of atopic dermatitis, and the Hh pathway was activated in skin T cells, showing that they respond in vivo to Hh signals secreted from the skin. Higher Shh upregulation reduced skin inflammation in mice, whereas pharmacological Smoothened-inhibition exacerbated skin pathology and reduced Shh upregulation. Hh-signalling to T cells prevented skin inflammation on induction of dermatitis, while inhibition of Hh-mediated transcription in T cells substantially exacerbated the disease. RNA-sequencing analysis of skin CD4+ T cells from mice with chronic atopic dermatitis revealed decreased expression of immune regulatory genes in mice with conditional inhibition of Hh-mediated transcription in T cells, and increased expression of inflammatory and chemokine genes. In contrast, constitutive Hh mediated transcription in T cells led to increased expression of immune regulatory genes in skin CD4+ T cells from mice with chronic atopic dermatitis and protected against inflammation. Hh-mediated transcription in T cells resulted in increased regulatory T (Treg) cells in the periphery and skin of dermatitis-induced mice, and increased TGF-β expression, supporting their immunoregulatory phenotype, whereas, inhibition of T cell specific Hh-mediated transcription, resulted in impaired Treg function, which permitted progression of skin inflammation. Thus, my data demonstrated a critical role for the Hh pathway through Shh upregulation, in the prevention of skin inflammation through upregulation of Shh signalling to T cells to increase Treg populations and promote their immunoregulatory function. This opens the possibility of novel therapeutic strategies for chronic inflammatory skin diseases and conversely suggests that Hh inhibitors could be used to enhance T cell immunity to Hh-secreting tumours.
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Landwerlin, Klemens [Verfasser], and Christine [Akademischer Betreuer] Dierks. "Die Rolle des Hedgehog - Signalweges in der AML." Freiburg : Universität, 2015. http://d-nb.info/1122830521/34.
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Nasr, Talia S. "Identification of Hedgehog/Gli Targets during Tracheoesophageal Development." University of Cincinnati / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1593273349807685.
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Moran, Carlos M. "Role of Hedgehog Signaling on Endothelial Vascular Patterning." Diss., The University of Arizona, 2010. http://hdl.handle.net/10150/194116.
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During embryonic vasculogenesis, endothelial cells form in the mesoderm , assemble into cord-like structures and then undergo tube formation. Previous studies have shown that signaling by members of the hedgehog family of secreted growth factors is essential for normal development of embryonic blood vessels. Embryos lacking hedgehog function show the presence of abundant endothelial cells but the cells fail to assemble into vascular cords and lumenized endothelial tubes do not form. At present it is not known whether active hedgehog signaling is required for both cord and tube formation or only for the initial step. To address this question, we have used small molecule inhibitors and agonists to the alter activity of the hedgehog signaling pathway in the chick embryo. If development is allowed to proceed until endothelial cells of the future dorsal aortae have assembled into cords, subsequent inhibition of hedgehog signaling, using cyclopamine, does not prevent aortal cells from forming endothelial tubes, however, it does lead to a reduction in cross sectional area of the aorta and to a loss of density of the adjacent vascular plexus. In contrast, activation of the hedgehog pathway with SAG leads to formation of enlarged aortae and increased density of the plexus. Very little, if any, of the observed effects are due to differences in number of endothelial cells in the treated embryos. Examination of endothelial cells during vascular plexus formation shows that inhibition of hedgehog signaling with cyclopamine inhibits formation of filopodia while treatment with SAG increases the number of filopodial extensions. These studies show that hedgehog signaling levels must be tightly regulated for normal vascular patterning to be achieved.
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Sales, Caroline Brandi Schlaepfer. "Identificação dos transcritos e proteínas glipicans 1, 3 e 5 em carcinoma escamocelular de boca: associação com moléculas Hedgehog e Vegfa." Centro de Pesquisas Gonçalo Moniz, 2015. https://www.arca.fiocruz.br/handle/icict/9849.
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Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil
INTRODUÇÃO: A Via Hedgehog (HH) está ativada em algumas neoplasias humanas, incluindo o Carcinoma Escamocelular de Boca (CEB), o qual corresponde a mais de 95% dos casos diagnosticados na cavidade bucal. Os glipicans (GPC) participam como reguladores desta cascata, atenuando (GPC1 e GPC3) ou regulando positivamente (GPC5) a via HH. OBJETIVO: O objetivo deste trabalho foi avaliar o perfil de expressão dos genes GPC1, 3 e 5, associando-os com genes da via HH (SHH, PTCH1 e SMO) e VEGFA, bem como caracterizar a imunoexpressão das proteínas GPC, em CEB. MATERIAL E MÉTODOS: Trinta e um casos de CEB foram submetidas a reações de qPCR para os genes SHH, PTCH1, SMO, VEGFA, GPC1, 3 e 5. O RNA total foi extraído utilizando uma coluna composta por membrana de silica (Rneasy Mini Kit). O DNA complementar foi obtido com auxílio da enzima Superscript Vilo™. As reações de qPCR foram conduzidas no aparelho ViiA™ 7 Real-Time PCR System utilizando o sistema Taqman, sendo a quantificação relativa avaliada pelo método comparativo de Cq (ΔΔCQ). Vinte e seis CEBs, 9 casos de margens tumorais (MAT) e 4 casos de mucosa bucal não neoplásica (MNN) foram submetidos à reação imuno-histoquímica para as proteínas GPC1, GPC3, GPC5, CD105 e MCM3 utilizando o sistema polimérico AdvanceTM ou LSABTM. As análises das proteínas GPC1, 3 e 5 foram realizadas de acordo com os parâmetros semi-quantitativos descritos por Gurgel et al. (2008). O número de células MCM3 positivas e de vasos/mm² (microdensidade vascular- MDV) foram avaliados em 5 campos, sendo a mediana de e intervalo de confiança utilizados para agrupar os CEBs em alto e baixo perfil proliferativo (AP e BP) e alta e baixa MDV, respectivamente. A análise estatística foi realizada utilizando GraphPad Prism versão 6.03. RESULTADOS: Transcritos do gene GPC1 (26; 83,87%); GPC3 (n=22; 70,97%) e GPC5 (n=15; 48,38%) foram observados em CEBs. SHH RNAm foi detectado em 5 CEBs (16,13%). A maioria dos CEBS apresentou expressão gênica de PTCH1 (n=25; 80.6%), SMO (n=26; 83,87%) e VEGFA (n=28; 90,32%). Correlação positiva forte e estatisticamente significante foi demonstrada para GPC5 e PTCH1 (rs=0,60; p=0,02) e entre PTCH1 e VEGFA (rs=0,69; p=0,0003). Imunomarcação citoplasmática e membranar de GPC1 foi observada principalmente em epitélio de MNN (n=4;100%) e MAT (n=9; 100%), enquanto que uma perda de imunomarcação desta proteína foi detectada no parênquima do CEB. A imunoexpressão da proteína GPC3 estava ausente em MNN (n= 4; 100%) e MAT (n=9; 100%). O GPC3 ocorreu na membrana e citoplasma de células do parênquima, observadas principalmente na periferia das ilhas tumorais, predominando o escore 3+ (n=5; 19.23%) entre os CEBs positivos (n=23; 88,46%). Ausência de imunomarcação de GPC5 foi observada em MNN (n=4; 0%) e apenas 2 espécimes de MAT (n=2; 22,22%) apresentaram baixa imunoexpressão, escore 1+. GPC5 citoplasmático em células tumorais positivas predominou o escore 1+ (n=5; 38.46%). Ao mesmo tempo, GPC5 foi detectado em estroma de 13 (50%) CEBs, especialmente em células endoteliais e semelhantes a fibroblastos. A expressão dos genes avaliados foi similar em tumores com AP e BP, assim como foi independente da MDV. CONCLUSÕES: A correlação entre os transcritos GPC5 e PTCH1, bem como a superexpressão das proteínas GPC5 e GPC3 e perda de imunopositividade de GPC1 são consistentes com a participação destas proteoglicanas como reguladoras da via HH em CEB. O perfil de expressão do gene e proteína GPC1 sugere que este glipican pode participa da biologia tumoral como uma proteína supressora tumoral, enquanto GPC3 e GPC5 participariam oncoproteínas. A presença de GPC5 em estroma tumoral (células endoteliais e fibroblastos) pode estar associada a regulação da via HH neste compartimento do microambiente tumoral.
INTRODUCTION: The Hedgehog pathway is activated in some human neoplasms, including Oral Squamous Cell Carcinoma (OSCC), which account for more than 95% of all oral cancers diagnosed. Glypicans are involved in the regulation of HH pathway through GPC3 e GPC1 downregulation or/and GPC5 upregulation. AIM: The aim of this study was to evaluate the expression profile of GPC1, 3 and 5 genes, correlating to HH and VEGFA gene, even as to characterize the immunoexpression of these proteins at OSCC. MATERIAL AND METHODS: A total of 31 cases of OSCC were assessed by qPCR for the SHH, PTCH1, SMO, VEGFA, GPC1, GPC3 and GPC5 genes. The total RNA were extracted using silica membrane column (Rneasy Mini Kit). Complementary DNA was obtained using of Superscript ™ Vilo enzyme. The qPCR reactions were performed in VIIA™ 7 Real-Time PCR System using the Taqman enzime, and relative quantification (RQ) was evaluated by the comparative method of Cq (ΔΔCQ). Immunohistochemical reactions for GPC1, GPC3, GPC5, MCM3 and CD105 proteins was performed on twenty-six OSCC, 9 cases of tumor margins (TM) and 4 cases of non-neoplastic oral mucosa (NNM) using AdvanceTM or LSABTM system. The analysis of GPC1, 3 and 5 proteins were conducted according to the semi-quantitative parameters described by Gurgel et al. (2008). The number of MCM3 positive cells and vessels//mm² (microvessel density -MVD) were evaluated in 5-matching areas, and the median and confidence interval being used to group the OSCC in high and low proliferative profile (HP and LP) and high and low MDV, respectively. Statistical analysis were carried out with GraphPad Prism v.6.03. RESULTS: Transcripts of GPC1 (26; 83.87%), GPC3 (n=22; 70.97%) and GPC5 (n=15; 48.38%) genes were observed in OSCC. SHH mRNA was detected in 5 OSCC (16:13%), PTCH1 gene in 25 CEBs (80.6%), SMO in 26 (83.87%) and VEGFA in 28 (90.32%). Strong and statistically significant positive correlation was demonstrated for GPC5 and PTCH1 genes (rs=0.60; p= 0.02) and PTCH1 and VEGFA transcripts (rs = 0.69; p = 0.0003). Cytoplasmic and membrane immunostaining of GPC1 was mainly observed in epithelial MNN (n = 5; 100%) and MAT (n=9; 100%), while a reduction of this protein was detected in parenchymal cells. GPC3 protein were absent in MNN (n = 4; 0%) and MAT (n=9; 0%). The GPC3 occurred in the membrane and cytoplasm of parenchymal cells, mainly observed in the periphery of the tumor islands and the 3+ score was predominat (n=3; 11:56%) in positive OSCC. GPC5 positive tumor cells occurred in the cytoplasm, scored 1+ (n = 5; 38.46%). In addition, GPC5 was detected in the stroma of 13 (50%) OSCC, especially in endothelial and fibroblast cells. The gene expression was similar in tumors with HP and LP, and was independent of MDV. CONCLUSIONS: The correlation between the GPC5 and PTCH1 transcripts, as well the overexpression of GPC5 and GPC3 protein and the loss of GPC1 positive cells are consistent with the participation of these proteoglycans as regulators of HH pathway in OSCC. The gene and protein expression profile of GPC3 indicate that this proteins participates in tumor biology as a tumor suppressor protein, while GPC5 and GPC3 function as oncoproteins. The presence of GPC5 in tumor stroma (endothelial cells and fibroblasts) could be associated with the regulation of the HH pathway in this compartment of the tumor microenvironment.
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Douglas, Adam Thomas. "Chromatin dynamics at the Sonic Hedgehog locus : a study using limb derived Sonic Hedgehog inducible cell lines to investigate chromatin architecture." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/23587.
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Enhancers are cis-regulatory sequences which promote the expression of target genes in a spatial and temporal fashion. They can be located within genes or between them and can act at distances of over 1 Mb. There are several different mechanisms by which enhancers regulate gene expression. Some, such as those regulating the Hox genes, are located close to each other in the genome in a structure referred to as a regulatory archipelago. These come together and act in combination to regulate gene expression, with different enhancer combinations resulting in different patterns of expression. On the other hand, enhancers can act individually, with designated enhancers responsible for regulating the expression of the same gene in different tissues or at different stages of development. Indeed, this is the case for the Sonic Hedgehog gene (Shh) where several different enhancers located within a gene sparse region referred to as a gene desert, act separately leading to Shh expression in areas such as the brain, the lungs, the notochord and neural tube and the limbs. Within the developing mouse embryo, Shh is expressed over roughly a two day period from E10 to E12 in a posterior distal region referred to as the Zone of Polarising Activity (ZPA). Ectopic expression in anterior regions has been observed in some common congenital diseases which affect the limbs, sometimes resulting in the formation of extra digits. The reason for this mis-expression is largely due to defects in the Shh limb enhancer commonly referred to as the Zone of Polarising Activity Regulatory Sequence (ZRS). Mutations within this highly conserved sequence create additional protein binding sites thus activating the enhancer in the wrong locations. The associated diseases are known collectively as the ZRS associated syndromes and can range from the less severe phenotype of preaxial polydactyly type II (characterised by an extra digit near the thumb) to the more severe Werner Mesomelic Syndrome (WMS), where patients present with a clear displacement of their tibia. The mechanism by which the ZRS functions is yet to be fully elucidated, with current studies producing conflicting data. What is known, is that the region encapsulating the Shh gene is highly compact, with both the gene and its enhancers located in a highly conserved Toplogical Associated Domain (TAD) as proven by Hi-C experiments. The boundaries of this domain are likely created by the binding of the protein CTCF to specified binding sites located at the either end of the locus. This restricts the ability of the enhancers to regulate the expression of genes outside the TAD. To study the exact mechanism by which the ZRS is activated and regulates Shh expression, the Hill laboratory has used cultured cell lines derived from the posterior regions of an E11.5 limb bud. Gene expression in these cells is highly reflective of the posterior limb bud, with the key exception being Shh, which is not expressed. However, using different drug treatments or biological manipulations Shh can be activated thereby making this the perfect system to analyse the mechanisms leading to Shh activation. In this investigation the cell lines were used to determine how the position of the ZRS changes upon activation. Using techniques such as Fluorescent in situ hybridisation (FISH) with either fosmid probes or directly labelled probes called MYtags, it was confirmed that the Shh locus is indeed highly compact in both Shh expressing and non-expressing cells. However, no differences were observed in terms of the distance between the ZRS and Shh between these two conditions in our cell lines. Next, both carbon copy chromosome conformation capture (5C) and circular chromosome conformation capture (4C) were used to look at changes to the Shh locus in different conditions. This confirmed Hi-C experiments and other recent publications suggesting that Shh is located within a TAD, the position of which is highly conserved between different conditions and cell lines. Furthermore, treatments activating the Shh gene resulted in significant deviations to the chromatin interactions within the locus suggesting a repositioning of structures when the gene is active. It is believed that the use of Shh inducible limb derived cell lines will prove extremely useful in future scientific endeavours to study the mechanisms of mammalian limb development. These provide a quick and easy means of accessing large numbers of Shh expressing cells, a feature which is increasingly important in an era where large cell numbers are needed for conducting chromosome conformation capture experiments such as Hi-C, 5C and 4C.
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Novelli, Caterina. "Rôle des cytonèmes dans la sécrétion de Hedgehog chez Drosophila melanogaster." Electronic Thesis or Diss., Université Côte d'Azur (ComUE), 2019. http://www.theses.fr/2019AZUR4015.
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Notre laboratoire étudie une molécule de morphogène appelée Hedgehog (Hh) utilisant Drosophila melanogaster comme modèle animal. La voie de signalisation Hh est conservée au cours de l'évolution, des invertébrés aux vertébrés, et joue un rôle régulateur dans divers aspects du développement animal et de l'homéostasie tissulaire, tels que le renouvellement cellulaire, la réparation tissulaire et la régénération d'organes. Hh est une molécule bi-lipidique modifiée par le cholestérol au niveau de son extrémité C-terminale et par l'acide palmitique au niveau de son extrémité N-terminale, et se lie donc étroitement à la membrane plasmique. Bien que la molécule soit hydrophobe, elle exerce sa fonction sur une longue distance. Un moyen particulier utilisé par les cellules pour communiquer repose sur un mécanisme basé sur des contacts directs entre cellules via de longues extensions de filopodes à base d'actine, appelées cytonèmes. Cette nouvelle modalité de transfert d’informations est au coeur de mon projet actuel. Dans ce travail, nous avons étudié le rôle des cytonèmes dans un tissu épithélial polarisé, appelé disque imaginal de l'aile, tissu précurseur de la larve à partir duquel les ailes adultes se développent. Les cytonèmes ont été largement étudiés dans ce tissu avec l'utilisation de la surexpression d'une protéine marquée par fluorescence appelée Interference of Hedgehog (Ihog). L'expression d'Ihog est une protéine qui peut être utilisée pour stabiliser ces longues extensions membranaires, sans quoi elles sont trop fragiles et détruites par les agents de fixation classiques. Nous présentons ici Ihog, Hh et Dispatched (Disp) comme des acteurs importants de la croissance des cytonèmes. En l'absence de différents domaines Ihog, nous avons constaté une réduction significative de la longueur et du nombre de cytonèmes. En outre, la perte totale de fonction Ihog et de son homologue Boi, réduit le nombre et la longueur des cytonèmes, sans pour autant affecter l’activité à longue distance de Hh. En absence de Hh, la longueur des cytonèmes est réduite, sans modification du nombre de cytonèmes. Avec ce travail, nous proposons une nouvelle fonction non canonique de Hh sur la croissance du cytonème. Pour comprendre le rôle des cytonèmes dans la sécrétion de Hh, nous avons également analysé des disques entièrement mutants pour disp. Chez les mutants disp, l’activité longue distance de Hh est fortement restreinte aux cellules qui juxtapose la source de Hh. En augmentant le nombre et la longueur des cytonèmes contenant Hh dans le mutant disp, nous montrons que la présence de Hh sur ces structures n’est pas suffisante pour activer la voie à longue distance. En conclusion, bien que les protéines susmentionnées contribuent à la structure des cytonèmes, nous n'avons pu mesurer de corrélation directe entre la longueur des cytonèmes et l’activité à longue distance de Hh. Enfin, nous avons voulu corréler l’apparition des cytonèmes avec l’établissement du gradient de Hh in vivo. Pour ce faire, nous avons développé un deuxième modèle alternatif permettant d’étudier la formation de cytonèmes: le modèle des histoblastes abdominaux, que nous pouvons utiliser pour analyser la formation et la dynamique des extensions de membrane chez les animaux vivants. En particulier, nous avons examiné deux nids d'histoblastes dorsaux, où le groupe de cellules postérieures produit Hh et le groupe de cellules antérieures répond au signal Hh. Nos résultats indiquent que l'établissement du gradient de Hh a lieu avant la juxtaposition des deux nids et en l'absence de cytonèmes. Nous avons donc proposé que les cytonèmes qui dépendent de Ihog ne sont pas impliqués dans les premières étapes de l’établissement du gradient de Hh. En conclusion, nos travaux montrent que Hh, bien qu’il soit nécessaire à la croissance du cytonème, n’utilise pas cette structure pour un transport à longue distance, mais certainement pour permettre une communication à courte distanceOur laboratory studies a morphogen molecule called Hedgehog (Hh) using Drosophila melanogaster as an animal model. The Hh signaling pathway is evolutionarily conserved from invertebrates to vertebrates, and plays regulatory roles in various aspects of animal development and tissue homeostasis, such as stem cell renewal, tissue repair, and organ regeneration. Hh is a dually lipidated molecule modified by cholesterol at its C-terminus and palmitic acid at its N-terminus, and therefore tightly binds the plasma membrane. Although the hydrophobic nature of the molecule, Hh exerts its function over a long-range of distance. One particular way cells adopted to communicate over long distances is through a new mechanism based on direct cell-cell contacts via long actin-based filopodia extensions, called cytonemes. This new modality for information transfer is at the core of my present project. In this work, we studied this Hh transport mechanism in a polarized epithelial tissue, called the wing imaginal disc, a larval precursor tissue from which the adult wings develop. Cytonemes have been extensively studied in this tissue with the use of the overexpression of a fluorescently tagged protein called Interference of hedgehog (Ihog). The expression of Ihog protein in the wing disc is necessary and sufficient to stabilize these long plasma membrane extensions, otherwise they would be too fragile and easy to be disrupted by conventional fixatives. Here we present Ihog, Hh and Disp as important players in cytoneme growth. In absence of different Ihog domains, we found a significant reduction of cytoneme length and numbers. In addition, Ihog/Boi loss of function was able to reduce the number and length of wild type cytonemes, marked with mCD8GFP. Further, we saw that in loss of function and gain of function genotype for Hh, cytoneme length was reduced and increased respectively, without any change at the cytoneme numbers. With this work, we suggest that Hh has a novel, non-canonical function in the cytoneme growth. To understand the role of cytonemes in Hh secretion we also analyzed discs entirely mutant for Dispatched (Disp). In disp mutants, the Hh gradient is strongly restrained, with most of Hh targets expressed only in anterior cells juxtaposing the A/P border. This phenotype could not be rescued by Ihog overexpression, despite the fact that Hh was very abundant on cytonemes in the disp mutant. Additionally, in the absence of Disp, we observed a reduction of cytoneme length, which suggests a role for Disp in the formation of these filaments, which is likely independent from its function in Hh secretion. In conclusion, although the aforementioned proteins contribute to the structure of cytonemes, we could not measure any direct correlation between the expression of Hh target genes and the simultaneous manipulation of cytoneme length in any mutant condition checked. Finally, we wanted to correlate the time of cytoneme initiation with the establishment of the Hh gradient. In order to do that, we have introduced in the laboratory an alternative system to study cytoneme formation: the abdominal histoblast model, which allowed us to directly analyze the formation and dynamics of membrane extensions in live animals. In particular, we looked at two separated dorsal histoblast nests of the pupal stage (distanced by 30 microns at the onset of pupariation) where the posterior group of cells produces Hh and the anterior cells show expression of various Hh targets. Approximately 15 hours after the onset of the metamorphosis, the abdominal histoblast cells begin to divide and migrate while simultaneously inducing apoptosis in the surrounding larval cells. Our results indicate that the establishment of the Hh gradient occurs before the juxtaposition of the two nests. Nevertheless, we could only observe cytoneme formation after the juxtaposition of the anterior and posterior histoblast cells and not when the two nests were still separated
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Mercier, Audrey. "Impact des mutations d'un modificateur chromatinien dans le développement du cervelet et le médulloblastome de groupe Sonic Hedgehog." Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS466.
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Le médulloblastome (MB), une tumeur formée à partir du cervelet en développement, est l’un des cancers pédiatriques malins les plus fréquents. Des profils d’expression géniques ont montré l’existence de quatre groupes distincts de MB qui présentent des profils moléculaires et des pronostics différents. Parmi ces groupes, l’un d’entre eux est caractérisé par une activation de la voie de signalisation Sonic Hedgehog (SHH). Ce groupe de MB provient des précurseurs de neurones en grain lors du développement cérébelleux. Les traitements actuels comprennent la chirurgie, la chimiothérapie ainsi que la radiothérapie, ce qui a pour effet d’altérer les capacités cognitives et sociales des survivants. Ainsi, des efforts considérables ont été mis en œuvre dans le but de trouver des cibles thérapeutiques afin de bloquer spécifiquement les mécanismes tumorigéniques sans affecter le développement normal. De récentes analyses à grande échelle ont révélé le rôle crucial de mécanismes épigénétiques, et en particulier dans le groupe SHH, dans lequel la perte de fonction d’un certain nombre de modificateurs chromatiniens a été identifiée. Ainsi, l'objectif principal de ma thèse est d'étudier l'implication de potentiels candidats modificateur chromatinien, à la fois au cours du développement cérébelleux et lors du MB SHH. Nous avons concentré notre étude sur plusieurs modificateurs de la chromatine qui ont été trouvés mutés dans les MB SHH humains. Nous avons commencé l’étude avec trois modificateurs chromatiniens sélectionnés selon (i) leur impact sur la survie, (ii) leur expression au cours du développement du cervelet, (iii) leur expression dans les MB SHH humains et nous sommes finalement concentré sur un.Afin d'étudier ce candidat, un objectif important de ma thèse a été de développer des outils fiables. Dans ce contexte, nous avons développé des modèles de souris knock-out conditionnelles et le système CRISPR-cas9 dans le développement cérébelleux postnatal afin d'étudier l'impact de la perte du candidat à la fois dans le développement du cervelet et dans le MB SHH. Ensuite, nous nous sommes intéressés aux mécanismes moléculaires contrôlés par ce modificateur de la chromatine. Plus précisément, nous avons défini (i) l’interactome, et (ii) des cibles transcriptionnelles spécifiques qui nous ont aidé à comprendre comment une protéine impliquée dans la modification de la chromatine peut favoriser l’état tumoral. En conclusion, ces travaux permettent de mettre en évidence comment la perte de la fonction de modificateur chromatinien spécifique peut différemment affecter le destin cellulaire dans le développement normal cérébelleux et dans le MB SHH et soulève la question d’une prise en charge plus personnalisée des patients atteints de MB SHHMedulloblastoma (MB), a tumor arising from the developing cerebellum, is one of the most common malignant pediatric brain tumors. Gene expression profiling showed the existence of four groups of MB with distinct molecular profiles and patient outcomes. Among these groups, one of them is associated with an activation of the Sonic Hedgehog (SHH) pathway.This specific group is thought to arise from cerebellar Granule Neuron Progenitors (GNPs) during cerebellar development. The actual treatment is heavy and consists of surgery, chemotherapy as well as radiotherapy impairing social and cognitive ability of survivors. Thus, considerable effort has been made in order to find drug targets that would specifically block tumorigenic mechanisms without affecting normal development.Recent large scale analysis revealed the crucial role of epigenetic mechanisms, and especially in the SHH group of MB in which loss of function mutation of several chromatin modifiers has been identified. Thus, the main goal of my PhD is to study the involvement of potential candidate chromatin modifiers both during cerebellar development and in SHH MB.We focused our study on several chromatin modifiers that were found mutated in human SHH MB. We began to study three chromatin modifiers that were selected according to (i) their impact on survival, (ii) their expression during cerebellar development, (iii) their expression in human SHH MB and finally we selected one for further functional validation.In order to study this candidate, one important goal of my PhD has been to develop reliable tools. In that context, we developed conditional knock-out mice models and the CRISPR-Cas9 system in postnatal cerebellar development in order to study the impact of the loss of this chromatin modifier both in cerebellar development and SHH MB initiation. Then, we investigated the molecular mechanisms controlled by this chromatin modifier. In particular, we defined (i) the interactome, and (ii) specific target genes that helped us understanding how a protein implicated in chromatin modification can favor tumors. In conclusion, this work provides insights into how the loss of function of a specific chromatin modifier can differentially affect cell fate in the context of normal cerebellar development and in SHH MB, stressing the question of a more personalized patient care
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Kumari, Veena. "Discovery of a novel form of Hedgehog that systemically circulates, and its signaling implications in Drosophila." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2011. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-64956.
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Hedgehog (Hh) shape up development by playing important role in signaling, and thereby controlling growth and pattern formation. It is for this reason that their spatial distribution is tightly regulated. The 19kDa active form of Hh is modified with a palmitate at its N-terminal and with cholesterol at its C-terminal. This dually lipid modified form of Hh act as a morphogen, and is also referred to as HhNp (Mann and Beachy, 2004). In most cases, they are released from producing cells and spread into adjacent non-expressing cells within the tissue, where it activates target gene expression in a concentration-dependent manner. In Drosophila, Lipophorin (Lpp) particles carry these lipid-modified forms of Hh and play a role in long range signaling in the developing wing disc. Further, these particles circulate throughout the larvae in the hemolymph to distribute nutrients mostly in the form of lipids to different tissues of the animal. Thus, Lpp plays important role in metabolism and development.
Hh as a morphogen plays a very important role in development and patterning of embryo and imaginal discs in Drosophila. We wanted to understand the role of Hh in overall development of Drosophila. In my thesis work, I discovered a new form of Hh that is systemically circulating in the 3rd instar larva of Drosophila. I show that imaginal tissues do not produce this form of circulating Hh. Our experiments strongly suggest that systemic Hh can travel from one tissue to another, a feature that was previously unknown. I also show that it could rescue the growth of the imaginal disc, implying its ability to influence cell proliferation. Since the concentration of systemic Hh is low it fails to up regulate the target genes. I characterized fat body as a target of systemically circulating Hh. I clearly demonstrate that fat body transcribes most of the components of Hh signaling pathway except Hh. Further, Hh accumulates in the fat body during late 3rd instar larvae. That makes the fat body an ideal target of systemic Hh. This could shed light in understanding the role of Hh in overall development of Drosophila melanogaster that includes tissue-based interaction.
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Vila, Greisa. "Sonic hedgehog signaling pathway in normal and adenomatous pituitary." Diss., lmu, 2004. http://nbn-resolving.de/urn:nbn:de:bvb:19-20853.
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Saldanha, Gerald Stephen. "The Hedgehog signalling pathway and its role in cancer." Thesis, University of Leicester, 2001. http://hdl.handle.net/2381/29393.
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Mutations in Hedgehog (Hh) pathway genes such as Patched1 are of established importance in Basal cell carcinoma (BCC). However, BCC is unusual because it has indolent behaviour and rarely metastasises. Hh pathway gene mutations are rare in cancers with more typical malignant behaviour, leading to the hypothesis that the pathway plays an important role in BCC but has limited importance in cancers with typical malignant behaviour. This has been addressed in three ways. First, evidence that superficial BCC is a clonal proliferation driven by alterations at the Patched1 locus was sought. Second, evidence of Wnt pathway activation in BCC was sought by looking for nuclear accumulation of -catenin, because Wnt is a putative Hedgehog pathway target gene. Third, to determine whether the Hh pathway was important in a cancer with typical malignant behaviour, breast cancer was studied. In superficial BCC, microdissection of individual tumour nests and analysis for LOH in six cases using three microsatellite markers at the Patched1 locus was performed. Only one of the microsatellites was successfully employed. In four of the cases, all nests showed no LOH and in two cases, all nests lost the same allele. Concordant patterns of allelic loss/retention within cases represented strong evidence of clonality. In addition, the two cases with LOH suggested that clonal proliferation in superficial BCCs might be driven by Patched1 alternations. Accumulation of nuclear -catenin was found in 20 out of 86 BCCs and had a significant relationship with proliferation, indicating that the Hh pathway may act through its target genes. Alternative factors influencing -catenin distribution were sought: no relationship between nuclear -catenin and expression of E-cadherin was seen and no -catenin mutations in eight cases were found. Four breast cancer cell lines and HBL100, derived from normal mammary epithelium, showed substantially lower expression of Hh pathway target genes than BCC and were unresponsive to the pathway ligand, Sonic Hh, suggesting that the pathway is not important in this tumour. However, a relationship was found between the expression of Patched1 and in vitro invasion, the importance of which is uncertain. In conclusion, these results support the hypothesis that the Hh pathway is important in BCC but not in cancers with typical malignant behaviour.
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Peris, Celda María. "Influencia de la ruta Hedgehog-Gli en tumores gliales." Doctoral thesis, Universitat de València, 2011. http://hdl.handle.net/10803/78804.
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Introducción
Los astrocitomas de alto grado son los tumores intrínsecos más frecuentes en el cerebro adulto con una supervivencia media alrededor de 14,2 meses.
Desde el aislamiento de las células madre tumorales (CMT) de glioblastomas (GBM) como población minoritaria implicada en el origen y mantenimiento de la masa tumoral, hay un interés creciente en el estudio de la ruta clásicamente implicada en desarrollo Hedgehog-Gli (Hh-Gli) con gran controversia en la literatura en cuanto a su papel. Hh-Gli se ha visto alterada en distintos tumores malignos y algunos trabajos apuntan a una reducción tumoral in vitro e in vivo en GBM con la aplicación de distintos inhibidores de esta ruta.
En este trabajo se analiza el papel de intermediarios de la ruta Hh-Gli en una serie de tumores gliales y CMT procedentes de pacientes intervenidos en el Hospital Universitario La Fe de Valencia a partir de enero de 2008.
Material y métodos
Se estudiaron 43 tumores gliales (32 GBM, 7 astrocitomas anaplásicos, 4 gliomas de bajo grado) y 12 controles (4 de sustancia blanca, 4 de sustancia gris, 4 de hipocampo) procedentes de pacientes adultos con esclerosis mesial del lóbulo temporal a los que se les realizó lobectomía temporal y amigdalohipocampectomía.
Tras comprobar la anatomía patológica tumoral se aislaron y crecieron CMT a partir de muestras frescas de GBM. Se confirmó que las características de estas células era consistente con la definición de CMT mediante ensayos de multipotencialidad y evaluación de la capacidad para formar tumores cerebrales en ratones nude. Se diseñaron primers de los intermediarios de la ruta Hh-Gli y se cuantificó la expresión de los mismos mediante Real Time PCR tanto en tumores y controles como en líneas de CMT y células madre neurales.
Posteriormente se realizó un estudio estadístico sobre diferentes parámetros clínicos (demográficos, tumorales, pronósticos y de seguimiento) y se compararon con los análisis de expresión de intermediarios de la ruta Hh-Gli.
Resultados
26 de 43 tumores mostraron disregulación Hh-Gli. Gli1, Gli2 y SMO se encuentran significativamente elevados en tumores.
El análisis de las 4 líneas de CMT ha mostrado un incremento de los efectores de Hh-Gli con los sucesivos pases in vitro.
Importantes características clínicas se han relacionado con Hh-Gli: Sufu, supresor tumoral, se encuentra sobreexpresado en tumores diseminados a través de los tractos de sustancia blanca al diagnóstico y Smo con un mayor tamaño tumoral, ambos factores implicados en el pronóstico.
Conclusiones
La ruta Hh-Gli está sobreexpresada en la mayoría de tumores gliales. Variables clínicas estudiadas relacionadas con la ruta y correlaciones entre los diferentes intermediarios, permiten establecer una nueva hipótesis de disregulación de la ruta que podría ayudar en el desarrollo de nuevas estrategias terapéuticas.Introduction Glioblastoma(GBM) is the most common primary malignant brain tumor in adults accounting for 12-15% of all intracranial neoplasms. Since the isolation of tumor stem cells(TSC) from GBM, numerous works have been focusing on this subpopulation of cells instead of the tumor bulk. Certain signals involved classically in embryonic development like Notch, BMP, Noggin, Eph/ephrins and Hedgehog-Gli(Hh-Gli) seem to be important in maintaining neural stem cell(NSC) niches and might play an important role in brain tumors. The aim of the present study is to analyze the expression of Hh-Gli intermediates in a series of human glioma and TSC derived from some of them. Materials and methods We studied 43 astrocytomas(39 high-grade and 4 low-grade) and 12 controls: 4 white matter, 4 grey matter and 4 hippocampus (from adult epileptic patients with mesial temporal sclerosis), one culture of NSC from WM and NSC from hippocampus. After the histological diagnosis, TSC were cultured. We designed primers of the intermediates of the Hh-Gli pathway: Patch1, Smoothened(Smo), Gli1, Gli2, Sufu and the stem cell marker CD133. The transcription of these genes in tumors, TSC and controls was quantified with Real-Time PCR. We demonstrated the multipotentiality of the TSC with differentiation assays in vitro and oncogenicity in athymic mice. Demographic, clinical and radiologic characteristics were obtained from the patient’s clinical record. All the data were statistically analyzed. Results 26 out of 43 tumours showed dysregulation of some of the intermediates of Hh-Gli. Gli1, Gli2 and Smo were significantly up-regulated in tumors compared to controls. The analysis of 4 lineages of TSC has shown an increase of effectors of Hh-Gli with serial passages in vitro. Several important clinical characteristics were related to Hh-Gli: Sufu, a tumor suppressor, was up-regulated in disseminated tumors across white matter fibers at diagnosis and Smo with an increased tumor size, both factors involved in patient’s prognosis. Conclusions An up-regulation in transcription of Hh-Gli intermediates was demonstrated in 60,46% of tumors. More studies in vitro and in vivo have to confirm if Sufu has a direct influence in tumor dissemination or growth. Data obtained from TSCs culture also reveal that Hh-Gli intermediates are not only dysregulated in tumor bulk, which supports the hypothesis of the influence of developmental pathways in TSC. These data open an interesting investigation line towards drug discovery with Hh-Gli inhibitors.
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Rodenfels, Jonathan Konstantin. "The Role of Systemically Circulating Hedgehog in Drosophila melanogaster." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2013. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-128624.
Full textAbstract:
The physiological response to environmental cues involves complex interorgan communication via endocrine factors and hormones, but the underlying mechanisms are poorly understood. In particular, little is known about how animals coordinate systemic growth and developmental timing in response to environmental changes. The morphogen Hedgehog (Hh), which is well studied in tissue patterning and homeostasis, has only recently been implicated in the regulation of lipid and sugar metabolism. Interestingly, Hh is present in systemic circulation in both, ies and mammals.
Here, we demonstrate that systemic Hh is produced in the midgut and secreted in association with the lipoprotein particle lipophorin (Lpp) into the hemolymph to mediate the interorgan communication between the midgut and two tissues, the fat body and the prothoracic gland (PG). We show that midgut hh expression is regulated by dietary sugar and amino acid levels, and RNAi-mediated knock-down of circulating Hh leads to starvation sensitivity. We demonstrate that circulating Hh is required to inhibit systemic growth and developmental progression. In insects, developmental transitions are regulated by steroid hormones, which are produced by the PG. Nutritional regulation of growth is, in part, mediated by the Drosophila fat body. Strikingly, canonical Hh pathway components are present in both tissues, the fat body and the PG. To understand the Hh-mediated function during nutritional stress, we ectopically activated or inhibited the Hh signaling pathway specifically in the fat body and the PG. Our results show that systemic Hh exerts its function through these two target tissues. Hh signaling in the fat body is required for survival during periods of nutrient deprivation, and ectopic activation of fat body Hh signaling causes an inhibition of systemic growth. Hh signaling in the PG slows down developmental progression by inhibiting steroid hormone biosynthesis.
In conclusion, we propose that the midgut senses the uptake of dietary sugar and amino acids and secrets Hh in association with Lpp particles into circulation to relay information about the feeding status to the developing animal. Therefore, circulating Hh functions as a hormone and signals in an endocrine manner to the fat body and the prothoracic gland to coordinate systemic growth and developmental timing in response to changes in nutrient availability.
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Shah, Divya Kantilal. "The role of hedgehog signalling in T-cell development." Thesis, Imperial College London, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.416933.
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Becher, Suzanne Anette. "Population genetics of the European hedgehog Erinaceus europeaeus L." Thesis, University of Oxford, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.308461.
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Bishop, Benjamin F. "Structural and functional characterisation of hedgehog ligand-receptor complexes." Thesis, University of Oxford, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.642625.
Full textAbstract:
Members of the Hedgehog (HH) family of morphogenic signalling molecules are key mediators of many fundamental processes in embryonic development. A relatively small change in HH concentration results in the specification of distinct cell types. Such fine-tuning necessitates a range of regulatory cell-surface proteins to control the concentration of HH to which responding cells are exposed. This thesis focuses on the structural and functional characterisation of three human extracellular modulators of the HH pathway, namely the hedgehog-interacting protein HHIP, the glypican GPC3 and the Growth Arrest Specific 1 (GAS1). Cell culture robot protocols were developed to allow large-scale expression of these targets in mammalian cells (Chapter 3). In Chapter 4, the structure of the HH antagonist HHIP alone and in complex with HH ligands is described. These studies combined with functional experiments reveal a binding mode distinct from previously defined ligand interaction sites, with the HH metal-binding sites playing key roles. The structural and functional analysis of GPC3, another negative HH regulator, is described in Chapter 5. Binding studies suggest that the GPC3 core domain does not bind directly to SHH and that the GPC3-attched heparan sulphate chains play an important role in HH regulation. Chapter 6 reveals crystal structures of the GAS 1 ectodomain, a HH agonist, allowing comparison to glial cell line-derived neurotrophic factor receptors, the identification of an unexpected ligand and mapping of the HH binding site. In summary, this work provides insights into the extracellular modulation of HH signalling and extends our current knowledge of this fundamental signalling pathway. It also offers a model to explain how both agonists and antagonists can adopt similar mechanisms of HH binding.
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Li, Jun. "Indian hedgehog stimulates chondrocyte hypertrophic differentiation inendochondral bone formation." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2007. http://hub.hku.hk/bib/B39558009.
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Lopez, Lyle Villamater. "Regulation of Gli proteins by the Hedgehog Signaling Pathway." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:11095.
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Blagden, Christopher Simon. "The role of sonic hedgehog in slow muscle formation." Thesis, King's College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312763.
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Whalen, Daniel M. "Structural and functional studies of the hedgehog signalling pathway." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:ce0e765c-04f1-4a64-a67b-89204ecaa155.
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Hedgehog (Hh) morphogens play fundamental roles in development whilst dysregulation of Hh signalling leads to disease. Multiple receptors are involved in the modulation of Hh morphogens at the cell surface. Among these, the interactions of Hh ligands with glycosaminoglycan (GAG) (for example heparan or chondroitin sulphate) chains of proteoglycans in the extracellular matrix play a key role in shaping morphogen gradients and fulfil important functions in signal transduction. Several high resolution crystal structures of Sonic Hh (Shh)-GAG complexes have been determined. The interaction determinants, confirmed by binding studies and mutagenesis reveal a novel Hh site for GAG interactions, which appears to be common to all Hh proteins. This novel site is supported by a wealth of published functional data, and resides in a hot spot region previously found to be crucial for Hh receptor binding. Crystal packing analysis combined with analytical ultracentrifugation on Hh-GAG complexes suggest a potential mechanism for GAG-dependent multimerisation. A key step in the Hh pathway is the transduction of the Hh signal into the receiving cell. The Hh signal transducer, Smoothened, is a key target drug target in the pathway with several modulators in clinical trials, despite an absence of structural data. Smoothened is required to activate all levels of Hh signalling. Recent evidence points to the conserved N-terminal ectodomain (ECD) in regulating Smo activity, from vertebrates to invertebrates. Despite the central importance of the ECD, its precise function remains elusive. A crystal structure of the ECD at 2.2 Å resolution is reported here. Structural analysis and biophysical experiments are discussed with reference to the potential function of this intriguing domain.
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Vanderlaan, Gary. "Differential roles for hedgehog signaling in motor neuron development." Diss., Columbia, Mo. : University of Missouri-Columbia, 2006. http://hdl.handle.net/10355/4461.
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Thesis (Ph.D.)--University of Missouri-Columbia, 2006.The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on April 29, 2009) Vita. Includes bibliographical references.
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Patten, Iain. "Mechanisms of floor plate formation in the developing chick embryo." Thesis, University of Sheffield, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.251319.
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Anichini. "Function and targeting of the Hedgehog signaling in human cholangiocarcinoma and melanoma." Doctoral thesis, Università di Siena, 2021. http://hdl.handle.net/11365/1133254.
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ABSTRACT
Hedgehog (HH) signaling is a conserved pathway that plays a pivotal role during embryonic development, tissue homeostasis and regeneration. Aberrant activation of the HH pathway has been reported to drive tumor progression in numerous cancers, including those of the skin, brain, lung and breast. The key signaling components of the HH signaling are the ligands, the cell membrane receptor Smoothened and the three transcription factors GLI1, GLI2 and GLI3, which upon activation of the HH signaling translocate into the nucleus where they activate the transcription of specific target genes. In the last few years, the Smoothened receptor has become an interesting target for the development of anticancer
agents.
In the first part of this work, we tested the requirement of Hedgehog signaling in intrahepatic cholangiocarcinoma (iCCA), a group of aggressive hepatobiliary malignancies with still poorly understood molecular background. Due to the lack of effective therapeutic strategies, CCA is mostly considered an intractable disease. We show that the Hedgehog signaling pathway is upregulated in intrahepatic cholangiocarcinoma (iCCA) cell lines compared to normal cholangiocytes, and it drives and regulates iCCA cell proliferation and survival. Furthermore, we present evidence that pharmacological inhibition of the HH signal transducer Smoothened with novel acylguanidine derivative compounds negatively affects iCCA in vitro growth and viability, leading to cellular apoptosis as a result of the induction of double strand break (DSB) DNA damage. Altogether, these data report new findings in cholangiocarcinoma biology and point to a possible therapeutic potential of targeting the Hedgehog signaling in iCCA.
In the second part of this thesis, we identified a novel axis downstream of Hedgehog signaling, that drives in vitro and in vivo melanoma cell invasiveness and metastatic behavior through the induction of a group of enzymes belonging to the O-glycan biosynthesis pathway. In particular, we find that GLI1 regulates transcription of the sialyltransferase ST3GAL1, through direct binding to regulatory regions in the enhancer II of ST3GAL1. Functional assays show that ST3GAL1 is a critical mediator of GLI1-induced melanoma cell invasiveness. In addition, our biochemical data suggest that the receptor tyrosine kinase AXL is a substrate of ST3GAL1, which is required to induce dimerization and activation of AXL in melanoma cells. Taken together, our results describe a novel HH-GLI1-ST3GAL1-AXL axis as crucial driver of melanoma invasiveness, proposing new potential therapeutic targets for management of metastatic melanoma.
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Li, Jun. "Indian hedgehog stimulates chondrocyte hypertrophic differentiation in endochondral bone formation." Click to view the E-thesis via HKUTO, 2007. http://sunzi.lib.hku.hk/HKUTO/record/B39558009.
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Lees, Charles William. "Role of the hedgehog signalling pathway in inflammatory bowel disease." Thesis, University of Edinburgh, 2009. http://hdl.handle.net/1842/4233.
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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.