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Shi, Dan. "Computational analysis of transcriptional responses to the Activin signal." Doctoral thesis, Humboldt-Universität zu Berlin, 2020. http://dx.doi.org/10.18452/21891.
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Die Signalwege des transformierenden Wachstumsfaktors β (TGF-β) spielen eine entscheidende Rolle bei der Zellproliferation, -migration und -apoptose durch die Aktivierung von Smad-Proteinen. Untersuchungen haben gezeigt, dass die biologischen Wirkungen des TGF-β-Signalwegs stark vom Zellkontext abhängen. In dieser Arbeit ging es darum zu verstehen, wie TGF-β-Signale Zielgene unterschiedlich regulieren können, wie unterschiedliche Dynamiken der Genexpression durch TGF-β-Signale induziert werden und auf welche Weise Smad-Proteine zu unterschiedlichen Expressionsmustern von TGF- β-Zielgenen beitragen.
Der Fokus dieser Studie liegt auf den transkriptionsregulatorischen Effekten des Nodal / Activin-Liganden, der zur TGF-β-Superfamilie gehört und ein wichtiger Faktor in der frühen embryonalen Entwicklung ist. Um diese Effekte zu analysieren, habe ich kinetische Modelle entwickelt und mit den Zeitverlaufsdaten von RNA-Polymerase II (Pol II) und Smad2-Chromatin-Bindungsprofilen für die Zielgene kalibriert. Unter Verwendung des Akaike-Informationskriteriums (AIC) zur Bewertung verschiedener kinetischer Modelle stellten wir fest, dass der Nodal / Activin-Signalweg Zielgene über verschiedene Mechanismen reguliert. Im Nodal / Activin-Smad2-Signalweg spielt Smad2 für verschiedene Zielgene unterschiedliche regulatorische Rollen. Wir zeigen, wie Smad2 daran beteiligt ist, die Transkriptions- oder Abbaurate jedes Zielgens separat zu regulieren. Darüber hinaus werden eine Reihe von Merkmalen, die die Transkriptionsdynamik von Zielgenen vorhersagen können, durch logistische Regression ausgewählt.
Der hier vorgestellte Ansatz liefert quantitative Beziehungen zwischen der Dynamik des Transkriptionsfaktors und den Transkriptionsantworten. Diese Arbeit bietet auch einen allgemeinen mathematischen Rahmen für die Untersuchung der Transkriptionsregulation anderer Signalwege.Transforming growth factor-β (TGF-β) signaling pathways play a crucial role in cell proliferation, migration, and apoptosis through the activation of Smad proteins. Research has shown that the biological effects of TGF-β signaling pathway are highly cellular-context-dependent. In this thesis work, I aimed at understanding how TGF-β signaling can regulate target genes differently, how different dynamics of gene expressions are induced by TGF-β signal, and what is the role of Smad proteins in differing the profiles of target gene expression. In this study, I focused on the transcriptional responses to the Nodal/Activin ligand, which is a member of the TGF-β superfamily and a key regulator of early embryonic development. Kinetic models were developed and calibrated with the time course data of RNA polymerase II (Pol II) and Smad2 chromatin binding profiles for the target genes. Using the Akaike information criterion (AIC) to evaluate different kinetic models, we discovered that Nodal/Activin signaling regulates target genes via different mechanisms. In the Nodal/Activin-Smad2 signaling pathway, Smad2 plays different regulatory roles on different target genes. We show how Smad2 participates in regulating the transcription or degradation rate of each target gene separately. Moreover, a series of features that can predict the transcription dynamics of target genes are selected by logistic regression. The approach we present here provides quantitative relationships between transcription factor dynamics and transcriptional responses. This work also provides a general computational framework for studying the transcription regulations of other signaling pathways.
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Ibrahim, Christine. "Exploring the role of the activin A-ActRIIB pathway in sickle cell disease-associated nephropathy and sarcopenia : mechanistic insights and therapeutic potential." Electronic Thesis or Diss., Université Paris Cité, 2024. http://www.theses.fr/2024UNIP5287.
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La drépanocytose est une maladie génétique caractérisée par des crises vaso-occlusives récurrentes et des lésions multi-organes progressives, y compris des atteintes rénales et une perte musculaire, qui aggravent la morbidité et réduisent la qualité de vie des patients. Bien que les mécanismes sous-jacents de la néphropathie associée à la drépanocytose soient bien établis, les facteurs responsables de l'atrophie musculaire restent partiellement compris. Des données récentes suggèrent qu'Activine A, un membre de la superfamille TGF-β, joue un rôle significatif dans la fibrose et la progression des maladies rénales ainsi que dans l'atrophie musculaire. Cependant, son rôle dans les lésions musculaires et rénales associées à la drépanocytose reste à élucider. Cette étude explore le rôle de l'Activine A dans la perte musculaire et la néphropathie associées à la drépanocytose. Nous avons évalué la prévalence de la sarcopénie et les niveaux circulants d'Activine A chez des patients atteints de drépanocytose, et utilisé un modèle murin pour analyser la cinétique des lesions musculaire et rénale de la drépanocytose ainsi que l'implication de la voie Activine dans ces pathologies. Nos résultats confirment une prévalence importante de la sarcopénie chez les patients drépanocytaires, soulignant la nécessité de recherches ciblées sur la pathologie musculaire de la drépanocytose. Les patients et notre modèle murin ont montré des niveaux élevés d'Activine A dans la drépanocytose, soutenant l'hypothèse selon laquelle l'Activine A pourrait contribuer à la maladie rénale et à l'atrophie musculaire dans ce contexte. Chez les souris drépanocytaires, des altérations ultrastructurales, une atrophie des myofibres, une vascularisation réduite et une altération des cellules souches musculaires ont précédé les pathologies rénales détectables. L'inhibition pharmacologique de la voie de l'Activin A atténué les lésions musculaires et a montré des signes précoces d'amélioration rénale, suggérant qu'elle représente une cible thérapeutique prometteuse pour réduire les complications de la drépanocytoseSickle cell disease (SCD) is a genetic disorder marked by recurrent vaso-occlusive crises and progressive multi-organ damage, including kidney disease and muscle wasting, both of which worsen morbidity and reduce quality of life of affected patients. While the mechanisms underlying SCD-related kidney disease are well-established, the drivers of muscle atrophy remain incompletely understood. Emerging evidence suggests that Activin A, a member of the TGF-β superfamily, plays a significant role in both fibrosis and disease progression in kidney disease as well as muscle atrophy. However, its role in SCD-associated muscle and kidney damage has yet to be elucidated. This study investigates the role of Activin A in SCD-associated muscle wasting and kidney disease. We assessed sarcopenia prevalence and circulating Activin A levels in SCD patients and employed a murine model to analyse the temporal changes in muscle and kidney pathology as well as the involvement of Activin pathway in these pathologies. Our findings confirm that sarcopenia is prevalent among SCD patients, emphasizing the need for focused research on SCD muscle pathology. Both patient and murine models showed elevated Activin A levels in SCD, supporting the hypothesis that Activin A may contribute to kidney disease and muscle atrophy in this context. In SCD mice, ultrastructural alterations, myofiber atrophy, reduced vascularization, and impaired muscle stem cells preceded detectable kidney pathology. Pharmacological inhibition of Activin signalling pathway mitigated muscle damage and showed early signs of kidney improvement, suggesting it as a promising therapeutic target for SCD complications and patient outcomes enhancement
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Leon, Florian Luis Anthony. "Role of the Nodal Signaling pathway in amphioxus neural induction." Electronic Thesis or Diss., Sorbonne université, 2018. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2018SORUS151.pdf.
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L'induction neurale (IN) est le processus permettant aux cellules ectodermiques d’avoir un devenir de type neural. Chez les vertébrés, le centre organisateur produit des molécules antagonistes à BMP ainsi que d’autres signaux induisant le devenir neural. Ce processus n'est cependant pas entièrement compris chez les autres chordés. Notre équipe a démontré que l’amphioxus B. lanceolatum possède un centre organisateur fonctionnel ; que l'activation de la voie BMP induit le devenir épidermique de l'ectoderme et que son inhibition conduit à un état indifférencié, suggérant que l'inhibition de la voie BMP n'est pas suffisant à l'IN. De plus, l'inhibition de la voie FGF ne bloque pas l'IN, contrairement à ce qui est démontré chez d’autres chordés. Enfin, Nodal/Activine est capable d'activer l'IN et représente donc une signal clé dans ce processus chez l'amphioxus. Ici, nous avons identifié un groupe de régions régulatrices sensibles à l'activine par la technique d'ATAC-seq ; et trouvé des sites de liaison putatifs de facteurs de transcription dans ces régions. Nos résultats suggèrent donc que des facteurs de type « Zinc Finger », comme Klf1/2/4, peuvent avoir un rôle déterminant dans le développement neural. Ces résultats ont été confirmés par des analyses comparatives d'ARN-seq à plusieurs temps de développement et d’explants ectodermiques après activation de la voie NodalNeural induction (NI) is the process through which pluripotent ectodermal cells are committed to a neural fate. In vertebrates, the dorsal organizer produces BMP antagonists, and other signals that induce neural cell fate. However, not much was known about NI in other chordates. Our team previously shown that the cephalochordate B. lanceolatum presents a functional organizer, and that the acquisition of epidermal fate relies on BMP activation. However, deprivation of BMP signals leads to an undifferentiated state of the ectoderm, indicating that BMP inhibition is not sufficient for NI. Moreover, FGF signal inhibition does not block NI, in the contrary to what is observed in several chordate lineages, suggesting that FGF is not the key signal to induce neural fate in amphioxus. Remarkably, activation of the Nodal/Activin pathway triggers NI and represent an instructive signal in this process in amphioxus. In this work, we have identified a group of putative non-exonic regulatory regions which are Activin-sensitive, through ATAC-seq, and searched for potential transcription factors binding sites. Our results suggest that Zinc Finger-related factors, as Klf1/2/4, might be playing crucial roles in neural development. We have also confirmed these results though comparative RNA-seq analyse at several developmental time points in embryo and ectodermal explants after Nodal activation
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Saharinen, Pipsa. "Signaling through the Jak-Stat pathway : regulation of tyrosine kinase activity." Helsinki : University of Helsinki, 2002. http://ethesis.helsinki.fi/julkaisut/mat/bioti/vk/saharinen/.
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Arngården, Linda. "Analysis of signaling pathway activity in single cells using the in situ Proximity Ligation Assay." Doctoral thesis, Uppsala universitet, Molekylära verktyg, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-281716.
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A cell that senses signals from its environment uses proteins for signal transduction via post translational modifications (PTMs) and protein- protein interactions (PPIs) from cell membrane into the nucleus where genes controlling cell proliferation, differentiation and apoptosis can be turned on or off, i.e. changing the phenotype or fate of the cell. Aberrations within such proteins are prone to cause diseases, such as cancer. Therefore, it is important so study aberrant signaling to be able to understand and treat diseases. In this thesis, signaling aberrations of PTMs and PPIs were analyzed with the use of the in situ proximity ligation assay (in situ PLA), and the thesis also contain method development of rolling circle amplification (RCA), which is the method used for signal amplification of in situ PLA reaction products. Paper I considers the integrity of RCA products. Here, the aim was to generate a smaller and more compact RCA product, for more accurate either visual or automated analysis. This was achieved with the use of an additional so called compaction oligonucleotide that during RCA was able to bind and pull segments of RCA products closer together. The compaction oligonucleotide served to increase the signal to noise ratio and decrease the number of false positive signals. The crosstalk between the Hippo and TGFβ signaling pathways were studied in paper II. Activity of the Hippo signaling pathway is regulated by cell density sensing and tissue control. We found differences in amounts and localization of interactions between the effector proteins of the two pathways depending on cell density and TGFβ stimulation. In paper III the NF-кB signaling pathway constitutively activated in chronic lymphocytic leukemia (CLL) was studied. A 4 base-pair frameshift deletion within the NFKBIE gene, which encodes the negative regulator IкBε, was found among 13 of a total 315 cases by the use of targeted deep sequencing. We found reduced levels of IкBε protein, decreased p65 inhibition, and increased phosphorylation, along with increased nuclear localization of p65 in NFKBIE deleted cases compared to healthy cases. Crosstalk between the Hippo and Wnt signaling pathway are studied within paper IV. Here, we found differences in cellular localization of TAZ/β-catenin interactions depending on colon cancer tumor stage and by further investigate Hippo/WNT crosstalk in cell line model systems we found an increase of complex formations involved in the crosstalk in sparse growing HEK293 cells compared to dense growing cells. Also, active WNT3a signaling was affected by cell density. Since cell density showed to have a big effect on Hippo/WNT crosstalk we continued to investigated the effect of E-cadherin, which has a function in cell junctions and maintenance of epithelial integrity on Hippo/WNT crosstalk. Interestingly, we found that E-cadherin is likely to regulate Hippo/WNT crosstalk.
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Montgomery, Lucy Theresa. "Investigations of ABA signalling pathways in stomatal guard cells." Thesis, Lancaster University, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.242895.
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Grocott, Timothy. "Regulation of Pax6 transcriptional activity by the Smad/TGF-β signalling pathway." Thesis, University of East Anglia, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.436697.
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Gianella-Borradori, Matteo Luca. "The identification & optimisation of endogenous signalling pathway modulators." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:4c87de5d-24a7-4998-8edb-917c3922aae1.
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Chapter 1 Provides an overview of drug discovery with particular emphasis on library selection and hit identification methods using virtual based approaches. Chapter 2 Gives an outline of the bone morphogenetic protein (BMP) signalling pathway and literature BMP pathway modulators. The association between the regulation of BMP pathway and cardiomyogenesis is also described. Chapter 3 Describes the use of ligand based virtual screening to discover small molecule activators of the BMP signalling pathway. A robust cell based BMP responsive gene activity reporter assay was developed to test the libraries of small molecules selected. Hit molecules from the screen were synthesised to validate activity. It was found that a group of known histone deacetylase (HDAC) inhibitors displayed most promising activity. These were evaluated in a secondary assay measuring the expression of two BMP pathway regulated genes, hepcidin and Id1, using reverse transcription polymerase chain reaction (RT-PCR). 188 was discovered to increase expression of both BMP-responsive genes. Chapter 4 Provides an overview of existing cannabinoid receptor (CBR) modulating molecules and their connection to progression of atherosclerosis. Chapter 5 Outlines the identification and optimisation of selective small molecule agonists acting at the cannabinoid 2 receptor (CB2R). Ligand based virtual screen was undertaken and promising hits were synthesised to allow structure activity relationship (SAR) to be developed around the hit molecule providing further information of the functional groups tolerated at the active site. Subsequent studies led to the investigation and optimisation of physicochemical properties around 236 leading to the development of a suitable compound for in vivo testing. Finally, a CB2R selective compound with favourable physicochemical properties was evaluated in vivo in a murine inflammation model and displayed reduced recruitment of monocytes to the site of inflammation.
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Porchet, Nicolas. "Role of signaling pathays in cell-fate specification in the early mouse embryo." Thesis, Université de Paris (2019-....), 2019. http://www.theses.fr/2019UNIP7096.
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Lors du développement précoce de l’embryon de souris, divers évènements de spécification des destins cellulaires induisent la formation du blastocyste pré-implantatoire. Ces évènements sont majoritairement contrôlés par l’action de voies de signalisation activées via la fixation de molécules signal à la membrane de la cellule. L’activité de ces voies de signalisation permet la régulation de la transcription de gènes cible responsable de l’acquisition d’une identité cellulaire et de son arrangement sous forme de tissu. Ici je m’intéresse aux rôles des voies ACTIVINE/NODAL et βCATENIN dans la spécification de ces identités cellulaires lors de la formation du blastocyste de sourisDuring the early mouse embryogenesis, cell-fate specification events result in the formation of the pre-implantation blastocyst. Those events are mainly regulated by the action of signaling cascades activated upon fixation of the signaling molecules at the cell membrane. The activity of these signaling pathways allow the transcriptional regulation of a specific pool of genes responsible for cell-fate decisions and the formation of tissues. Here, I am interested in the roles of both ACTIVIN/NODAL and βCATENIN signaling pathways in the specification of cell identities during the maturation of the mouse blastocyst
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Carlyle, Becky Catherine. "DISC1 & GSK3β modulate PDE4 activity : functional integration of psychiatric associated signalling pathways." Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/4823.
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Following the discovery of the DISC1 gene in 2000, subsequent research has led to DISC1 becoming one of the most promising candidate genes for psychiatric disorders. Acting as a scaffold protein, DISC1 has a large number of interacting proteins and is involved in a series of intracellular signalling pathways. Amongst these binding proteins are two enzymes, PDE4 and GSK3β, that were originally implicated in psychiatric disease by virtue of their inhibition by psychoactive drugs. PDE4 enzymes are inhibited by rolipram, which possesses anti-depressant and anti-psychotic activity, while GSK3β is one of the major targets of lithium, a potent mood stabiliser. Both these enzymes are intricately involved in the PI3K/AKT, cAMP, and MAPK signalling pathways, all of which have a number of downstream outcomes with potential relevance to psychiatric disorders. The Millar and Porteous laboratory had established that DISC1 modulates PDE4 activity, but this predated awareness of GSK3 as another DISC1 interactor whose binding site overlapped with that of PDE4. Since cAMP is a key regulator of signalling pathways in the brain, I hypothesised that not only DISC1, but also GSK3β may be involved in the regulation of PDE4 activity to control local cAMP levels and gradients. To investigate this hypothesis, I characterised SHSY5Y cells as a model for measuring PDE4 activity, and performed a series of genetic and pharmacological manipulations on this system. Inhibition of GSK3β resulted in a decrease of basal PDE4 activity that was amplified by DISC1 overexpression. Wild type cells that were treated with forskolin exhibited a significant increase in PDE4 activity, which was suppressed by GSK3β inhibition and both overexpression and knockdown of DISC1. Further experiments confirmed that none of these changes were a result of differences in PDE4 mRNA or protein expression. Thus I have provided evidence that suggests tonic activation of PDE4 by GSK3β and evidence for modulation of PDE4 activity by DISC1. I provide evidence for the localisation of PDE4B & PDE4D with key psychiatric associated receptors in structures resembling developing dendritic spines; furthermore, agonism of NMDA receptors results in a significant increase in PDE4 activity in primary neurons. These results are a simple demonstration of an emerging principle in psychiatric research: that none of the signalling pathways implicated in psychiatric disease are acting in isolation. There are likely to be multiple points of integration between these pathways, with the demonstrated DISC1-GSK3β-PDE4 interaction forming one of these points. My results add an important new element to the understanding of how the DISC1 complex may regulate intracellular signalling in response to extracellular cues.
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Wright, Lilyan Yi Tian. "The activity of the helix-loop-helix protein, E47, is regulated by the PI3K/Akt signaling pathway." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2007. http://wwwlib.umi.com/cr/ucsd/fullcit?p3258711.
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Thesis (Ph. D.)--University of California, San Diego, 2007.Title from first page of PDF file (viewed June 4, 2007). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references.
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TORRES, N. P. MACHADO. "BIOLOGICAL AND TRANSDUCTIONAL EFFECTS OF ALLOSTERIC ANTAGONISTS ON THE ACTIVITY OF CHEMOATTRACTANT RECEPTORS." Doctoral thesis, Università degli Studi di Milano, 2010. http://hdl.handle.net/2434/150153.
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There are two major chemokine receptors expressed on the surface of neutrophils, CXCR1 and CXCR2.It is thought that targeting both receptors may be necessary for optimal inhibition of PMN influx in several inflammation disorders such as post-ischemia reperfusion injury, pulmonary fibrosis, sepsis, psoriasis among other.
DF 2156A is a potent dual inhibitor of CXCR1 and CXCR2 and was derived from a specific lead optimization program (Moriconi et al., 2007). Because it blocks both CXCR1 and CXCR2, it may overcome the problem of redundancy generated by the ELR+ CXC chemokine-CXCR1/R2 axis. The present study demonstrated, that DF 2156A is a potent blocker of both CXCR1 and CXCR2 expressed on PMNs, lymphocytes, and cells transfectants with a IC50 in the range of 1 to 2 nM. Despite its potency on both CXCL8 receptors, the effect of DF 2156A was highly specific. DF 2156A did not affect the activity of several related chemokine receptor belonging to both CC and CXC subfamilies. Our evidences show that DF 2156A is able to block different signaling pathway activated by CXCL8. Here we describe the molecular mechanism exerted by this compound. In particular it unaffected CXCL8- induced both receptors internalization but also both receptors recycling to the cell membrane after CXCL removal, indicating that it does not interfere more in general with the trafficking of both CXCL8 receptors. By contrats DF 2156A significantly suppressed CXCL8- induced cofilin phosphorylation and subsequent failure of CXCR1/R2 cells to migrate in response to CXCL8. Cofilin is protein that is postulated regulates cell migration and chemotaxis, in chemokine responses.
This model of concerted type of allosteric antagonists raises the possibility to stabilize slightly different active receptor conformations, which might interact preferentially with distinct tranducer molecules or signaling pathway. Moreover, this class of antagonists would offer untapped advantages for drug development programs.
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Kimura, Masahiro. "Homeobox A4 Suppresses Vascular Remodeling as a Novel Regulator of YAP/TEAD Transcriptional Activity." Kyoto University, 2020. http://hdl.handle.net/2433/253486.
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Arató, Krisztina. "Regulation of the stability of the protein kinase DYRK1A: establishing connections with the Wnt signaling pathway." Doctoral thesis, Universitat Pompeu Fabra, 2010. http://hdl.handle.net/10803/38524.
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DYRK1A is the most studied member of the DYRK family of protein kinases, because is one of the human chromosoma 21 proteins for which changes in gene dosage result in neuropathological alterations. DYRKs are activated by autophosphorylation on a tyrosine residue in the activation loop, a one-off event that takes place during translation. Accordingly, DYRK1A would be constitutively active once is synthesized. However, DYRK1A is extremely sensitive to gene dosage, and thus it is predictable that not only its activity but also its actual protein amounts have to be tightly regulated by mechanisms not yet characterized.
In the present study, the protein kinase NLK has been identified as a novel regulator of DYRK1A protein stability. DYRK1A interacts with NLK in physiological conditions. The interaction results in the phosphorylation of DYRK1A at multiple sites, which have been identified by mass spectrometry analysis. These phosphorylation events promote DYRK1A proteasome-dependent degradation. Moreover, DYRK1A degradation is induced by stimulating cells with Wnt1 or Wnt3a, or overexpressing elements of the Wnt signaling cascade such as the Frizzled-1 receptor or NLK activators such as HIPK2. In addition, DYRK1A interacts with and phosphorylates -catenin and TCF-4 and enhances -catenin-dependent transcriptional activity, at least by phosphorylation of -catenin. Thus, these results suggest that DYRK1A acts as a positive factor in the Wnt--catenin signaling pathway and NLK acts as a negative regulator by targeting both DYRK1A and TCF/LEF transcription factors for proteasome-mediated degradation.DYRK1A es el miembro más estudiado de la familia de proteína quinasas DYRK, porque es una de las proteínas de la cromosoma humano 21 para la que cambios en la dosis génica dan lugar a alteraciones neuropatológicas. Las quinasas DYRK se activan por autofosforilación en un residuo tirosina localizado en el lazo de activación, un evento único que ocurre durante la traducción. Como consecuencia, DYRK1A sería constitutivamente activa una vez se ha sintetizado. Sin embargo, DYRK1A es extremadamente sensible a la dosis génica, y por tanto es predecible que no sólo su actividad, pero también los niveles de proteína han de estar estrictamente controlados por mecanismos reguladores que todavía no han sido caracterizados. En este trabajo, la proteína quinasa NLK ha sido identificada como un nuevo regulador de la estabilidad de DYRK1A. DYRK1A interacciona con NLK en condiciones fisiológicas, y la interacción tiene como resultado la fosforilación de DYRK1A en residuos serina/treonina, varios de los cuales han sido identificados por espectrometría de masas. La interacción con NLK y la subsecuente fosforilación promueven la degradación de DYRK1A vía el proteasoma. Además, la degradación de DYRK1A es inducida por estimulación de la células con Wnt1 o Wnt3a, o por sobreexpresión de miembros de la cascada de señalización de Wnt, como el receptor Frizzled-1 o de un activador de NLK como HIPK2. Finalmente, se ha demostrado que DYRK1A se une y fosforila -catenina y TCF-4. La fosforilación de, al menos, -catenina es responsable del incremento de la actividad transcripcional dependiente de esta proteína en presencia de DYRK1A. Todos estos resultados sugieren que DYRK1A actúa como un factor positivo en la vía de señalización Wnt--catenina y NLK actúa como un regulador negativo al inducir la degradación vía proteasoma no sólo de los factores de transcripción TCF/LEF sino también del modulador positivo DYRK1A.
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Hao, Wei. "THE MAP KINASE AND TCF-4 SIGNALING PATHWAYS REGULATE HIF-1A TRANSCRIPTIONAL ACTIVITY IN RESPONSE TO HYPOXIA." Thesis, The University of Arizona, 2009. http://hdl.handle.net/10150/192464.
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Sudheer, Smita [Verfasser]. "Differential modulation of BMP signaling by Activin, Nodal and FGF pathways in lineage specification of human embryonic stem cells / Smita Sudheer." Berlin : Freie Universität Berlin, 2011. http://d-nb.info/1026344670/34.
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Hurst, Samantha. "n-3 Polyunsaturated fatty acid effects on inflammatory mediator activity and intracellular signalling pathways in chondrocyte metabolism." Thesis, Cardiff University, 2005. http://orca.cf.ac.uk/55511/.
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Previous studies have shown that supplementation of n-3 polyunsaturated fatty acids (PUFAs) has a beneficial effect on reducing the expression and activity of degradative enzymes and inflammatory factors known to cause damage and destruction of cartilage in arthritic diseases. The aims of this thesis was to use a well-established in vitro model of cartilage degradation to further these studies and to investigate how n-3 PUFAs effect the expression of inflammatory factors at a proteomic level and to use specific inhibitors to identify possible signalling pathways involved in cartilage metabolism. The results of this thesis research indicate that n-3 PUFAs abrogate IL-1-induced cyclooxygenase-2 (COX-2) mRNA expression, protein levels and activity, measured as PGE2 production, in both normal bovine and human osteoarthritis articular cartilage chondrocytes. These studies were followed by the use of a simple array system to analyse the expression of several marker genes from different signalling pathways after IL-1 exposure, plus or minus n-3 PUFA supplementation. This led us to identify three possible pathways involved in IL-1-induced cartilage catabolism and inflammation. These were analysed further with the use of specific inhibitors to ascertain whether the inhibition profiles were similar to those seen by n-3 PUFAs. Two main pathways, the extracellular signal-regulated kinase (ERK) pathway and NFkappaB pathway were identified. Further analysis using the ERK pathway inhibitor, U0126, showed that it decreased IL-1-induced glycosaminoglycan release from the tissue, endogenous aggrecanase activity, ADAMTS-4 (but not ADAMTS-5) mRNA levels, MMP-3 and MMP-13 mRNA levels, COX-2 message, protein levels and PGE2 production in a manner similar to that seen with n-3 PUFA supplementation. Collectively, these results suggest that n-3 PUFAs may be directing their effects through the ERK pathway.
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Lau, Faye. "Muscular activity regulates the expression of ColQ subunit of acetylcholinesterase : a signaling pathway mediated by Ca2̳+̳/ calmodulin-dependent protein kinase II /." View abstract or full-text, 2007. http://library.ust.hk/cgi/db/thesis.pl?BIOL%202007%20LAU.
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Thongwichian, Rossukon [Verfasser]. "Kinase activity reporters for eukaryotic signaling pathway study : Multiplexed profiling of cellular kinase activities by Nuclear Magnetic Resonance Spectroscopy / Rossukon Thongwichian." Berlin : Freie Universität Berlin, 2013. http://d-nb.info/1033306789/34.
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Tong, Lingying. "The Role of Nitric Oxide Synthase and Carnosol in UVB-induced NF-κB Activity and Skin Damage." Ohio University / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1412768175.
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Minnaar, Estella Lily. "Regional neurochemical characterization of the flinders sensitive line rat with regard to glutamate-nitric oxide and cGMP signalling pathways / Estella Lily Minnaar." Thesis, North-West University, 2008. http://hdl.handle.net/10394/4214.
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The serious nature of MDD has intensified the need to identify and elucidate new neurobiological targets for antidepressant drug action. Depression presents with evidence for degenerative pathology that relates to disturbances in excitatory glutamatergic pathways, particularly the N-methyl-D-aspartate (NMDA) receptormediated release of the pleiotropic molecule, nitric oxide (NO), and cyclic guanosine monophosphate (cGMP). The contribution of the glutamate-NO/cGMP pathway may
realize great importance as a fundamental substrate underlying the pathophysiology
of major depression. In the next generation of antidepressant drugs, the nitric oxide pathway could playa dynamic role in addressing urgent therapeutic needs. In this study, we have used a genetic model of depression, the Flinders Sensitive Line (FSL) rat, to investigate the surrogate markers of the NO/cGMP pathway.
The aim was to determine whether the depressive-like behaviour of the
hypercholinergic FSL rat is accompanied by altered activation of the NO/cGMP
pathway. To this end, the extent to which the FSL and Flinders Resistant Line (FRL)
rats differ neurochemically with regard to basal hippocampal and frontal cortical
NOS-activity, as well as nitric oxide (NO) and cGMP accumulation, were determined.
Additionally, select behavioural assessments were performed to confirm the
anxiogenic phenotype of the FSL strain.
For neurochemical determinations a sensitive fluorometric reversed phase highperformance
liquid chromatographic (HPLC) assay was developed to analyze total
nitrite and nitrate in brain tissue. Nitrate was enzymatically converted to nitrite before
derivatization with 2,3-diaminonaphthalene (DAN). The stable and highly fluorescent
product, 2,3-naphthotriazole (NAT), was quantified. Secondly, the quantity of the
amino acid L-citrulline was measured by HPLC with electrochemical detection after
o-phthalaldehyde (OPA) derivatization. L-citrulline formation was used as an index
for nNOS activity. Finally, a direct, competitive enzyme immunoassay kit was used to
determine the downstream activity of the NO-pathway in brain tissue.
FSL rats were compared to FRL rats with respect to sensitivity to serotonin 5-HT1A .
receptor-mediated hypothermia under our lab-conditions. The Open Field Test (OFT)
behavioural assessment was performed to compare FSL with FRL groups under
baseline conditions according to their level of inherent anxiety. The parameters used
to measure anxiety were number of line crosses (locomotor activity), time spent in
middle blocks and social interaction time between pairs of rats. As an additional
behavioural assessment, the Forced Swim Test (FST) was performed to assess
behavioural restraint measured as time of immobility.
Basal cGMP levels in the frontal cortex were found to be significantly less in FSL
than in FRL rats, whereas the levels in the hippocampus did not differ significantly.
No other significant differences with respect to NO and nNOS activity were apparent
in either of the brain areas. The hypothermia test confirmed a significantly greater
decrease in temperature in the FSL rat than the FRL rat. The FST did not confirm
any differences in immobility time between the two rat strains. In the OFT, FSL rat
groups exhibited behaviour that indicated significantly more anxiety than FRL rats.
Under basal conditions, FSL rats do not present with significant changes in markers
of the NO cascade in the hippocampus and frontal cortex compared to FRL controls,
including NOS activity as well as NO accumUlation. However, cGMP levels were
found to be significantly lower in the frontal cortex of FSL rats versus FRL rats,
although not in the hippocampus. Since the FSL rat is known to be hypercholinergic,
these data support an interaction between the NO/cGMP pathway and the
cholinergIc system in the frontal cortex but not hippocampus of FSL animals. The
mechanisms and implications of such a mutual involvement need further clarification.
Further, this anatomical differentiation may have important implications for
understanding the role of NO in the depressive-like behaviour of the FSL rat and,
indeed, may reveal more on the neurobiology and treatment of depression. Through
the performed behavioural assessments, the FSL and FRL rats were successfully
separated with respect to their anxiety phenotype as well as their heightened
response to serotonergic challenge, thus confirming a contribution of both the
serotonergic and cholinergic systems to the depressogenic nature of these animals.
As concluding remark can be said that under normal basal conditions markers of the
NO/cGMP signalling cascade are not altered in FSL vs FRL rats, although cGMP
levels are reduced in the frontal cortex of FSL rats, supportive of an NO-independent
mechanism of cGMP regulation, possibly involving ACh.Thesis (M.Sc. (Pharmacology)--North-West University, Potchefstroom Campus, 2009.
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ALESIANI, DANIELA. "Melanoma cells differentiation through inhibition of p-Mek activity following treatment with 5,7-dimethoxycoumarin." Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2009. http://hdl.handle.net/2108/906.
Full textAbstract:
In questo lavoro è stata studiata l’attività antineoplastica della 5,7-dimetossicumarina, un metabolita secondario di origine vegetale, su cellule di melanoma murino (B16) e umano (A375). Il composto riduce significativamente la proliferazione cellulare in modo dose e tempo dipendente, bloccando nella fase G0/G1 il ciclo di entrambe le linee cellulari. In seguito a questo blocco, è stato inoltre osservato un processo di differenziamento nelle cellule trattate attraverso l’analisi di markers specifici quali lo sviluppo di proiezioni dendritiche dalla superficie cellulare, l’incremento nella sintesi di melanina e l’accumulo della protoporfirina IX (PpIX).
Considerando le attività antineoplastica e differenziativa della 5,7-dimetossicumarina, è stato esaminato il suo effetto sulla fosforilazione, e quindi sull’attività, delle protein chinasi attivate da mitogeni Ras/Raf/Mek/Erk (MAPK). Questa via chinasica regola la sopravvivenza, la proliferazione, e il differenziamento cellulari ed è costitutivamente attivata, in seguito a mutazioni, nel 70% dei casi di melanoma. Un’inibizione dell’attività chinasica di Mek 1/2 e una conseguente riduzione nell’attivazione di Erk 1/2 è stata osservata in entrambe le linee usate dopo incubazione delle cellule con la 5,7-dimetossicumarina. Inoltre, è stato investigato il processo di melanogenesi indotto dal trattamento andando a monitorare il grado di fosforilazione di CREB (proteina legante CRE), l’espressione di Mitf (fattore di trascrizione associato alla microftalmia) e l’attività della tirosinasi. Mitf è coinvolto nel differenziamento, nella proliferazione e nella sopravvivenza dei melanociti e delle cellule di melanoma, poiché regola la trascrizione di geni codificanti per proteine che controllano la progressione del ciclo cellulare o la melanogenesi, come la tirosinasi che è considerato l’enzima chiave nella sintesi di melanina. L’attività di Mitf è regolata da vari fattori, tra cui CREB che quando è fosforilato induce la trascrizione del gene corrispettivo. Noi abbiamo osservato che la fosforilazione di CREB e l’espressione di Mitf aumentano significativamente in seguito a trattamento con la 5,7-dimetossicumarina nelle cellule di melanoma; di conseguenza, un incremento nell’attività della tirosinasi è stato dimostrato.
Questi risulatati suggeriscono che la 5,7-dimetosicumarina esercita un’attività antineoplastica nelle cellule di melanoma murino e umano, causando un processo di differenziamento con induzione di melanogenesi. Sarà interessante effettuare studi in vitro, per investigare un’eventuale attività antimetastatica del composto in esame, ed in vivo al fine di dimostrare le proprietà antineoplastiche e antimetastatiche della molecola anche in ambito preclinico.In this work the antiproliferative activity of 5,7-dimethoxycoumarin, a plant secondary metabolite, on murine (B16) and human (A375) melanoma cells was analysed. The compound reduced significatively cell proliferation in time and dose-dependent manner blocking cell cycle in G0/G1 phase, in both cell lines. Also a differentiation process, following this block, was detected by monitoring some specific markers such as morphological changes with development of dendrite-like projections from cell surface, melanin synthesis and protoporphyrine IX (PpIX) accumulation. Taking into account the antiproliferative and differentiation activities of this compound, we examined Ras/Raf/Mek/Erk mitogen-activated protein kinase (MAPK) activity following treatment; this kinases pathway regulates cell survival, proliferation and differentiation processes and it is costitutively activated following mutations in 70 % of melanoma. Inhibition of Mek 1/2 kinase activity and subsequent reduction in Erk 1/2 activation were detected in both cell types. Furthermore, we analyzed melanogenesis process by monitoring the phosphorylation of cAMP-response element-binding protein (p-CREB) and the expression of microphthalmia-associated transcription factor (Mitf); also tyrosynase activity was investigated. Mitf is involved in differentiation, proliferation and survival of melanocyte and melanoma cells since it regulates transcription of genes encoding for proteins involved in cell cycle progression or in melanogenesis, such as the enzyme tyrosinase, the key enzyme in melanin synthesis. Transcriptional activity of Mitf is regulated also through the phosphorylation of CREB that upregulates its gene transcription. We detected that CREB phosphorylation and Mitf expression enhanced significantly following 5,7-dimethoxycoumarin treatment in melanoma cells; consequently, an increase of tyrosinase activity was observed. These findings suggest that 5,7-dimethoxycoumarin exerts an antineoplastic activity in murine and human melanoma cells, triggering a differentiation process with stimulation of melanin synthesis. It will be of note to carry out in vitro analyses to investigate the antimetastatic activity of the tested compound; and in vivo studies with the aim to demonstrate cancer antigrowth and antimetastatic properties of compound also in preclinical trials.
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La, Bounty Paul Willoughby Darryn Scott. "The effects of heavy resistance exercise in combination with orally administered branched-chain amino acids or leucine on insulin signaling and Akt/mTOR pathway activity in active males." Waco, Tex. : Baylor University, 2007. http://hdl.handle.net/2104/5069.
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Lu, Hang. "The synthesis and structure-activity relationship study of azo dye related HIV replication inhibitors : Part 2: Plant isolation of signalling pathways inhibitors as anti-cancer agents." Diss., Georgia Institute of Technology, 1994. http://hdl.handle.net/1853/27436.
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Mota, Elia Marilia da Fonte. "Dopamine and non-canonical signaling Phosphodiesterase 1 bridges glutamate inputs with NO- and dopamine-induced cyclic nucleotide signals in the striatum Involvement of phosphodiesterase 2A activity in the pathophysiology of fragile X syndrome." Thesis, Sorbonne université, 2019. http://www.theses.fr/2019SORUS600.
Full textAbstract:
Les neurones épineux striataux de taille moyenne (MSN) intègrent les signaux médiés par la dopamine principalement par la voie de signalisation de l'AMPc. Les récepteurs dopaminergiques D1 ou D2 déclenchent respectivement une augmentation ou une diminution du taux d'AMPc. Ma thèse porte sur la manière dont les phosphodiestérases (PDE), qui dégradent l'AMPc, sont impliquées dans l'intégration des signaux de dopamine dans le striatum. J'ai utilisé des biosenseurs FRET génétiquement codés pour surveiller le niveau d'AMPc en temps réel dans des neurones individuels vivants dans des préparations de tranches de cerveau striatal. J'ai utilisé des inhibiteurs sélectifs pour déterminer la fonction de chaque PDE. La PDE1B, qui est activée par la calcium-calmoduline, apparaît comme un détecteur de la coïncidence des signaux de dopamine et de glutamate, ce qui est essentiel dans la régulation de la plasticité synaptique impliquée dans l’apprentissage par récompense. La PDE10A montre l'activité la plus importante, dégradant efficacement les taux d'AMPc élevés et faibles. L'activité PDE10A est nécessaire pour permettre la désactivation de la PKA, et donc nécessaire pour transduire un signal de dopamine à travers les récepteurs D2 en une diminution de la phosphorylation dépendante de la PKA. PDE2A et PDE4 ont semblé ne dégrader que des niveaux élevés d’AMPc, empêchant de fortes augmentations d’AMPc. La PDE2A, dont l’activité peut être augmentée par le GMPc, apparaît également comme un détecteur de coïncidence dopamine et NO. Comprendre les fonctions des PDE peut mettre en évidence leur potentiel en tant que cibles thérapeutiques dans les pathologies du SNC. A titre d'exemple, nous avons montré une fonction accrue de PDE2A dans un modèle de souris du syndrome du X fragile. En plus de la voie AMPc/PKA, les récepteurs de la dopamine D2 pourraient également activer des voies non canoniques. Les tentatives d'utilisation de biosenseurs pour les voies Akt et ERK n'ont pas fourni de données concluantesStriatal medium-sized spiny neurons (MSNs) integrate dopamine signals mainly through the cAMP signaling pathway. Dopamine D1 or D2 receptors trigger an increase or a decrease in cAMP levels, respectively. My thesis focuses on how phosphodiesterases (PDEs), which degrade cAMP, are involved in the integration of dopamine signals in the striatum. I used genetically-encoded FRET biosensors to monitor cAMP level in real time in individual living neurons in striatal brain slice preparations. I used selective inhibitors to determine the function of each PDE. PDE1B, which is activated by calcium-calmodulin, appears as a detector of the coincidence of dopamine and glutamate signals, which is critical in the regulation of synaptic plasticity involved in reward-based learning. PDE10A shows the most prominent activity, efficiently degrading both high and low cAMP levels. PDE10A activity is required to allow for PKA de-activation, and therefore needed to transduce a dopamine signal through D2 receptors into a decrease in PKA-dependent phosphorylation. PDE2A and PDE4 appeared to degrade only high levels of cAMP, preventing large increases in cAMP. PDE2A, which activity can be increased by cGMP, also appears as a detector of dopamine and NO coincidence. Understanding PDE functions can highlight their potential as therapeutic targets in CNS pathologies. As an example, we showed an increased PDE2A function in the hippocampus of a mouse model of Fragile X syndrome. Besides the cAMP/PKA pathway, dopamine D2 receptors is reported to activate non-canonical pathways. Attempts to use biosensors for Akt and ERK pathways did not provide conclusive data
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Kaladchibachi, Sevag. "Positive Regulation of PKB/Akt Kinase Activity by the Vacuolar-ATPase in the Canonical Insulin Signaling Pathway: Implications for the Targeted Pharmacotherapy of Cancer." Thesis, 2014. http://hdl.handle.net/1807/65674.
Full textAbstract:
The canonical PI3K/Akt pathway is activated downstream of numerous receptor tyrosine kinases, including the insulin and insulin-like growth factor receptors, and is a crucial regulator of growth and survival in metazoans. The deregulation of Akt is implicated in the pathogenesis of numerous diseases including cancer, making the identification of modifiers of its activity of high chemotherapeutic interest. In a transheterozygous genetic screen for modifiers of embryonic Akt function in Drosophila, in which the PI3K/Akt signaling pathway is conserved, we identified the A subunit of the vacuolar ATPase (Vha68-2) as a positive regulator of Dakt function. Our characterization of this genetic interaction in the larval stage of development revealed that Vha68-2 mutant phenotypes stereotypically mimicked the growth defects observed in mutants of the Drosophila insulin signaling pathway (ISP). The loss of Vha68-2 function, like Dakt-deficiency, was found to result in organismal and cell-autonomous growth defects, and consistent with its putative role as a positive regulator of Dakt function, both the mutational and pharmacological inhibition of its activity were found to downregulate Akt
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activation. Genetic epistasis experiments in somatic clones of Vha68-2/dPTEN double mutants demonstrated that the loss of Vha68-2 function suppressed the growth defects associated with dPTEN-deficiency, placing Vha68-2 activity downstream of dPTEN in the ISP, while the examination of PI3K activity and PH domain-dependent membrane recruitment in pharmacologically inhibited larval tissues further placed Vha68-2 function downstream of PI3K. These findings were recapitulated in cultured NIH-3T3 cells, whose treatment with bafilomycin A1, a potent and specific inhibitor of V-ATPase, resulted in the downregulation of Akt phosphorylation, particularly in non-cytoplasmic intracellular compartments. Furthermore, cellular subfractionation of bafilomycin-treated NIH-3T3 cells demonstrated a decrease in the localization of Akt to early endocytic structures, and a downregulation in the localization and activation of Akt in the nuclei of both Drosophila and mammalian cells. Finally, the pharmacotherapeutic relevance of V-ATPase inhibition was addressed in two tumor models – multiple myeloma and glioblastoma – and our preliminary findings in these cancers, which are often associated with ectopic PI3K/Akt signaling, showed significant cytotoxic efficacy in vitro, warranting its consideration as a tractable pharmacological option in the treatment of cancer.
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"Signaling Pathway Deregulation: Identification Through Genomic Aberrations And Verification Through Genomic Activity." Master's thesis, 2011. http://hdl.handle.net/2286/R.I.9422.
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abstract: Given the process of tumorigenesis, biological signaling pathways have become of interest in the field of oncology. Many of the regulatory mechanisms that are altered in cancer are directly related to signal transduction and cellular communication. Thus, identifying signaling pathways that have become deregulated may provide useful information to better understanding altered regulatory mechanisms within cancer. Many methods that have been created to measure the distinct activity of signaling pathways have relied strictly upon transcription profiles. With advancements in comparative genomic hybridization techniques, copy number data has become extremely useful in providing valuable information pertaining to the genomic landscape of cancer. The purpose of this thesis is to develop a methodology that incorporates both gene expression and copy number data to identify signaling pathways that have become deregulated in cancer. The central idea is that copy number data may significantly assist in identifying signaling pathway deregulation by justifying the aberrant activity being measured in gene expression profiles. This method was then applied to four different subtypes of breast cancer resulting in the identification of signaling pathways associated with distinct functionalities for each of the breast cancer subtypes.Dissertation/Thesis
M.S. Computer Science 2011
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"Differential regulation of gonadotropin (FSHb and LHb) transcription: roles of activin/Smad and estrogen/ER signaling pathways." 2005. http://library.cuhk.edu.hk/record=b5896387.
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Lin Sze-Wah.Thesis (M.Phil.)--Chinese University of Hong Kong, 2005.
Includes bibliographical references (leaves 111-127).
Abstracts in English and Chinese.
Abstract (in English) --- p.i
Abstract (in Chinese) --- p.iii
Acknowledgements --- p.iv
Table of Contents --- p.v
Abbreviations --- p.x
Scientific Names --- p.xii
Chapter CHAPTER 1 --- GENERAL INTRODUCTION --- p.1
Chapter 1.1 --- Gonadotropins --- p.1
Chapter 1.1.1 --- Structure --- p.1
Chapter 1.1.2 --- Function --- p.1
Chapter 1.1.3 --- Regulation --- p.2
Chapter 1.1.3.1 --- Gonadotropin-releasing hormone (GnRH) --- p.3
Chapter 1.1.3.2 --- Dopamine --- p.4
Chapter 1.1.3.3 --- Sex steroids --- p.5
Chapter 1.1.3.3.1 --- Functions --- p.5
Chapter 1.1.3.3.2 --- Working mechanism´ؤEstrogen signaling pathway --- p.7
Chapter 1.1.3.4 --- Gonadal peptides --- p.9
Chapter 1.1.3.4.1 --- Functions --- p.9
Chapter 1.1.3.4.2 --- Working mechanism一Activin signaling pathway --- p.11
Chapter 1.2 --- Transcriptional regulation of pituitary gonadotropin subunit genes at the promoter level --- p.13
Chapter 1.2.1 --- Transcriptional regulation of mammalian glycoprotein a subunits --- p.13
Chapter 1.2.1.1 --- GnRH --- p.14
Chapter 1.2.1.2 --- Activin --- p.15
Chapter 1.2.1.3 --- Steroids --- p.15
Chapter 1.2.2 --- Transcriptional regulation of mammalian FSHβ and LHβ subunits --- p.16
Chapter 1.2.2.1 --- Regulation of LHβ expression by GnRH --- p.17
Chapter 1.2.2.1.1 --- Roles of SP-1 binding sites on LHβ promoter --- p.17
Chapter 1.2.2.1.2 --- Effect of SF-1 on LHp expression --- p.17
Chapter 1.2.2.1.3 --- Effect of Egr-1 on LHp expression --- p.18
Chapter 1.2.2.1.4 --- "Synergistic effect ofSP-1, SF-1 and Egr-1 on LHp expression." --- p.18
Chapter 1.2.2.1.5 --- Effect of Pitx-1 on LHβ expression --- p.19
Chapter 1.2.2.1.6 --- "Effect of SF-1, Egr-1 and Pitx-1 on LHβ expression of other mammalian counterparts" --- p.19
Chapter 1.2.2.1.7 --- Effect of other transcription factors on mammalian LHβ expression --- p.19
Chapter 1.2.2.2 --- Regulation of LHβ expression by steroids and activin --- p.20
Chapter 1.2.2.3 --- Regulation of FSHβ expression by activin and GnRH --- p.20
Chapter 1.2.2.4 --- Regulation of FSHβ expression by steroids --- p.21
Chapter 1.2.2.5 --- Regulation of FSHβ expression by other transcription factors --- p.22
Chapter 1.2.3 --- Transcriptional regulation of fish FSHβ and LHβ subunits --- p.22
Chapter 1.3 --- The project objectives and long-term significance --- p.24
Chapter CHAPTER 2 --- CLONING OF ZEBRAFISH FSHB AND LHB PROMOTERS. --- p.26
Chapter 2.1 --- Introduction --- p.26
Chapter 2.2 --- Materials and Methods --- p.27
Chapter 2.2.1 --- Chemicals --- p.27
Chapter 2.2.2 --- Animals --- p.27
Chapter 2.2.3 --- Isolation of genomic DNA --- p.28
Chapter 2.2.4 --- Cloning of promoters of zebrafish FSHβ and LHβ from the genomic DNA --- p.28
Chapter 2.2.5 --- Construction of the reporter plasmids containing zebrafish FSHβ and LHβ promoters --- p.30
Chapter 2.2.6 --- Cell culture and transient transfection --- p.31
Chapter 2.2.7 --- SEAP reporter gene assay --- p.32
Chapter 2.2.8 --- β-galactosidase reporter gene assay --- p.32
Chapter 2.2.9 --- Data analysis --- p.33
Chapter 2.3 --- Results --- p.33
Chapter 2.3.1 --- Cloning of zebrafish FSHβ and LHβ promoters --- p.33
Chapter 2.3.2 --- Sequence characterization of zebrafish FSHβ and LHβ promoters --- p.34
Chapter 2.3.3 --- Basal FSHp and LHβ promoter activities in LβT2 cells --- p.35
Chapter 2.4 --- Discussion --- p.36
Chapter CHAPTER 3 --- ROLES OF ACTIVIN/SMADS AND ESTROGEN/ERS IN THE REGULATION OF ZEBRAFISH FSHB AND LHB PROMOTER ACTIVITY --- p.51
Chapter 3.1 --- Introduction --- p.52
Chapter 3.2 --- Materials and Methods --- p.56
Chapter 3.2.1 --- Chemicals --- p.56
Chapter 3.2.2 --- Animals --- p.56
Chapter 3.2.3 --- Isolation of total RNA --- p.57
Chapter 3.2.4 --- Rapid amplification of full-length cDNA (RACE) --- p.57
Chapter 3.2.5 --- Construction of expression plasmids --- p.57
Chapter 3.2.6 --- cell culture and transient transfection --- p.59
Chapter 3.2.7 --- SEAP reporter gene assay --- p.59
Chapter 3.2.8 --- p-galactosidase reporter gene assay --- p.59
Chapter 3.2.9 --- Data analysis --- p.59
Chapter 3.3 --- Results --- p.60
Chapter 3.3.1 --- Cloning and sequence characterization of zebrafish Smad 4 (zfSmad 4) --- p.60
Chapter 3.3.2 --- Smads regulate FSHβ transcription in LβT2 cells --- p.61
Chapter 3.3.3 --- Smads regulate LHβ transcription in LPβT2 cells --- p.61
Chapter 3.3.4 --- Functionality of the two forms of Smad 4 cloned --- p.62
Chapter 3.3.5 --- Estrogen and ERs regulate zJFSHβ transcription in LβT2 cells --- p.63
Chapter 3.3.6 --- Estrogen and ERs regulate zfLHβ transcription in LβT2 cells --- p.63
Chapter 3.4 --- Discussion --- p.64
Chapter CHAPTER 4 --- PROMOTER ANALYSIS FOR SMAD RESPONSIVE ELEMENT AND ESTROGEN RESPONSIVE ELEMENT IN ZEBRAFISH FSHB AND LHB PROMOTERS --- p.82
Chapter 4.1 --- Introduction --- p.83
Chapter 4.2 --- Materials and Methods --- p.85
Chapter 4.2.1 --- Chemicals and animals --- p.85
Chapter 4.2.2 --- Construction of SEAP reporter plasmids containing different lengths of zfFSHβ promoter --- p.85
Chapter 4.2.3 --- Construction of SEAP reporter plasmids containing different lengths of zfLHβ promoter --- p.85
Chapter 4.2.4 --- Site-directed mutagenesis --- p.86
Chapter 4.2.5 --- cell culture and transient transfection --- p.87
Chapter 4.2.6 --- SEAP reporter gene assay --- p.87
Chapter 4.2.7 --- P-galactosidase reporter gene assay --- p.87
Chapter 4.2.8 --- Data analysis --- p.88
Chapter 4.3 --- Results --- p.88
Chapter 4.3.1 --- Localization of Smad-responsive element (SRE) on zfFSHβ promoter --- p.88
Chapter 4.3.2 --- Localization of estrogen-responsive element (ERE) on zfLHβ promoter --- p.89
Chapter 4.3.3 --- Localization of estrogen-responsive element (ERE) on zfFSHβ promoter --- p.90
Chapter 4.3.4 --- Confirmation of SRE by site-directed mutagenesis --- p.91
Chapter 4.3.5 --- Confirmation of ERE by site-directed mutagenesis --- p.92
Chapter 4.4 --- Discussion --- p.92
Chapter CHAPTER 5 --- GENERAL DISCUSSION --- p.106
Chapter 5.1 --- Overview --- p.106
Chapter 5.2 --- Contribution of the present research --- p.107
Chapter 5.3 --- Future research direction --- p.108
REFERENCE: --- p.111
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Ferris, Heather Anne. "GnRH signaling and LH[B] gene transcription : modulation of promoter activity by signaling pathways and transcription factor occupancy /." 2005. http://wwwlib.umi.com/dissertations/fullcit/3161271.
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Sanchez, Melanie. "Growth factor activation of ErbB2/ErbB3 signaling pathways regulate the activity of Estrogen Receptors (ER)." Thèse, 2010. http://hdl.handle.net/1866/4472.
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La signalisation par l’estrogène a longtemps été considérée comme jouant un rôle critique dans le développement et la progression des cancers hormono-dépendants tel que le cancer du sein. Deux tiers des cancers du sein expriment le récepteur des estrogènes (ER) qui constitue un élément indiscutable dans cette pathologie. L’acquisition d’une résistance endocrinienne est cependant un obstacle majeur au traitement de cette forme de cancer. L’émergence de cancers hormono-indépendants peut est produite par l’activation de ER en absence d’estrogène, l’hypersensibilité du récepteur aux faibles concentrations plasmique d’estrogène ainsi que l’activation de ER par des modulateurs sélectifs. L’activité du ER est fortement influencée par l’environnement cellulaire tel que l’activation de voie de signalisation des facteurs de croissances, la disponibilité de protéines co-régulatrices et des séquences promotrices ciblées. Présentement, les études ont principalement considérées le rôle de ERα, cependant avec la découverte de ERβ, notre compréhension de la diversité des mécanismes potentiels impliquant des réponses ER-dépendantes s’est améliorée. L’activation des voies des kinases par les facteurs de croissance entraîne le développement d’un phénotype tumoral résistant aux traitements actuels. Nos connaissances des voies impliquées dans l’activation de ER sont restreintes. ERα est considéré comme le sous-type dominant et corrèle avec la plupart des facteurs de pronostic dans le cancer du sein. Le rôle de ERβ reste imprécis. Les résultats présentés dans cette thèse ont pour objectif de mieux comprendre l’implication de ERβ dans la prolifération cellulaire par l’étude du comportement de ERβ et ERα suite à l’activation des voies de signalisation par les facteurs de croissance.
Nous démontrons que l’activation des récepteurs de surfaces de la famille ErbB, spécifiquement ErbB2/ErbB3, inhibe l’activité transcriptionnelle de ERβ, malgré la présence du coactivateur CBP, tout en activant ERα. De plus, l’inhibition de ERβ est attribuée à un résidu sérine (Ser-255) situé dans la région charnière, absente dans ERα. Des études supplémentaires de ErbB2/ErbB3 ont révélé qu’ils activent la voie PI3K/Akt ciblant à son tour la Ser-255. En effet, cette phosphorylation de ERβ par PI3K/Akt induit une augmentation de l’ubiquitination du récepteur qui promeut sa dégradation par le système ubiquitine-protéasome. Cette dégradation est spécifique pour ERβ. De façon intéressante, la dégradation par le protéasome requiert la présence du coactivateur CBP normalement requis pour l’activité transcriptionnelle des récepteurs nucléaires. Malgré le fait que l’activation de la voie PI3K/Akt corrèle avec une diminution de l’expression des gènes sous le contrôle de ERβ, on observe une augmentation de la prolifération des cellules cancéreuses. L’inhibition de la dégradation de ERβ réduit cette prolifération excessive causée par le traitement avec Hrgβ1, un ligand de ErbB3. Un nombre croissant d’évidences indique que les voies de signalisations des facteurs de croissance peuvent sélectivement réguler l’activité transcriptionnelle de sous-types de ER. De plus, le ratio ERα/ERβ dans les cancers du sein devient un outil de diagnostique populaire afin de déterminer la sévérité d’une tumeur. En conclusion, la caractérisation moléculaire du couplage entre la signalisation des facteurs de croissance et la fonction des ERs permettra le développement de nouveaux traitements afin de limiter l’apparition de cellules tumorales résistantes aux thérapies endocriniennes actuelles.It has long been appreciated that estrogenic signaling plays a critical role in the development of hormone-dependent cancers such as breast cancer. Two-thirds of breast cancers express estrogen receptor (ER) which has been demonstrated to play an irrefutable role in tumour development and progression. However the acquisition of endocrine resistance has become a major obstacle in the treatment of hormone-dependent cancers that have acquired a hormone-independent state. Hormone-independent cancers emerge from an array of pathways involving ER activation in the absence of estrogen, hypersensitivity of ER to low serum levels of estrogen and activation by estrogen antagonists. The activity of ER is critically influenced by the cellular environment such as growth factor signaling pathways, availability of coregulatory proteins and the promoter sequence of target genes. The mechanisms studied have mostly considered the role of ERα, however with the discovery of the second subtype, ERβ, the understanding on the diversity of potential mechanisms involving ER-dependent responses have improved. Hormonal-independent activation of ER can occur in estrogen-dependent breast tumours, with concomitant rise in kinase signaling pathways, resulting in the acquisition of a therapeutic resistant phenotype in treated women. Our knowledge is relatively limited on which pathways trigger ER signaling and how these phosphorylation-coupled events affect ER activity. ERα is considered the dominant subtype and correlates with most of the prognostic factors in breast cancers. Conversely the role of ERβ remains unclear. The results presented in this thesis were carried out with the objective of gaining a better understanding of ERβ’s role in cellular proliferation by examining the behavior of ERβ and ERα during the activation of growth factor signaling pathways by cell-surface receptor-tyrosine kinases. We demonstrate here that the activation of cell surface receptors of the ErbB family, specifically ErbB2/ErbB3, inhibits the transcriptional activity of ERβ despite the presence of the coactivator CBP, yet activated ERα. Furthermore the inhibition of ERβ was attributed to a specific serine residue located within the hinge region, not present in ERα. Additional studies of ErbB2/ErbB3-initiated signaling revealed that it triggered the activation of the PI3K/Akt pathway which targeted the serine residue within the hinge region of ERβ. In fact, phosphorylation of ERβ by the PI3K/Akt pathway led to an increase in receptor ubiquitination which promoted its degradation by the ubiquitin-proteasome system which was subtype specific. Interestingly, proteasomal degradation required the presence of the coactivator CBP, which is normally involved in assisting nuclear receptor transcriptional activity. Although the activation of the PI3K/Akt pathway correlated with a decrease in the expression of ERβ target genes it led to an increase in the proliferation of breast cancer cells. Inhibiting the degradation of ERβ reduced the enhanced proliferation of breast cancer cells brought about by the treatment of ErbB3’s ligand, Hrgβ1. Increasing evidence indicates that growth factor signaling pathways can selectively regulate the transcriptional activity of ER subtypes, and the ratio of ERα/ERβ expression in breast tumours is becoming a popular prognostic factor to evaluate the severity of the tumour. Therefore the molecular characterization of the coupling between growth factor signaling and ER function should provide improved therapeutical approaches to overcome or delay the onset of resistance to endocrine therapy in hormone-dependent cancers.
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Jia, Tseng Hong, and 曾鴻家. "The sphingomyelinase activity of Helicobacter pylori induces ceramide signaling pathways in AGS gastric epithelial cells." Thesis, 2003. http://ndltd.ncl.edu.tw/handle/49599860304065351026.
Full textAbstract:
碩士長庚大學
基礎醫學研究所
91
Purpose: The TNF-induced sphingomyelinase (SMase) could catalyze the hydrolysis of sphingomyelin in cellular membrane and produce ceramide, which is a secondary messenger modulating several cellular responses, such as proliferation, differentiation, and apoptosis. We found that Helicobacter pylori also possess the activity of SMase. In this study, we investigate whether the exogenous H. pylori SMase can cause the similar effect with the TNF-induced signal transduction pathway in gastric epithelial cells. Methods: SMase was used to treat the gastric cell-line (AGS) in dose and temporal manners for studying the signal mechanism and cellular responses. The amount of cellular hydrolyzed product ceramide was determined by HPLC. The effects of exogenous SMase to AGS cells on MAPK, phosphatase, and PKC pathways were investigated. Results: Exogenous SMase to AGS cells showed both growth inhibition and apoptosis. The cells treated with SMase with concentration range from 0.5 to 10 unit/ml showed increased amount of ceramide in a dose-dependent manner. The treated cells also exhibited elevating activities of ERK (extracellular signal-regulated kinase) and JNK (c-jun-NH2-terminal kinase), as well as protein serine/threonine phosphatase. However, the activity of p38 MAPK was not influenced. The treatment also enhance the translocation of protein kinase Cξ from cytosol to membrane. The inhibitors for JNK, phosphatase, and PKCξ abolished the growth inhibition of treated cells. This study indicates the exogenous SMase could trigger ceramide-mediated signaling pathways and inhibit cell growth.
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Chang, Hui-Wen, and 張慧文. "Effects of areca nut extract on release of prostaglandin E2 , interleukin-8 and signaling pathway activity in neutrophils." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/80736457709712983156.
Full textAbstract:
碩士國立陽明大學
口腔生物研究所
95
Areca quid (AQ) chewing is associated with many oral diseases, such as periodontal disease, oral leukoplakia, and oral cancer. Polymorphonuclear neutrophils (PMNs) can migrate from blood to gingival crevicular fluid (GCF) and execute non-specific defense. Prostaglandins (PGs) are synthesized via the cyclooxygenase (COX) pathway. Interleukin-8 (IL-8) is a cytokine and chemotactic factor secreted by activated PMNs. Areca nut extract (ANE), the main component of AQ, enhances the phosphorylation of p38 MAPK (mitogen-activated protein kinase) and intracellular calcium. The purpose of the study was to investigate the effects of ANE on the production of prostaglandin E2 (PGE2) and IL-8 by PMNs, and to study the potential role of intracellular calcium ions, MAPK and COX-2 pathway involved in the effects of ANE. PMNs isolated from peripheral blood of healthy volunteers were treated with various concentrations of ANE in HBSS/Ca2+ or HBSS for 8 hours. The effects of ANE on granularity, size, and the viability of PMNs were determined by flow cytometer after propidium iodide (PI) staining. In addition, the cell supernatants after ANE treatment were collected. The amounts of PGE2 and IL-8 in culture supernatants were messured using immunoassays. The effects of intracellular calcium chelator (BAPTA-AM), the p38 MAPK inhibitor (SB203580), the ERK MAPK inhibitor (U0126), and the COX-2 inhibitor (NS398) were also examined. The results showed that ANE significantly increased the secretion of PGE2 and IL-8 by PMNs in HBSS/Ca2+. In addition, after pretreated with the intracellular calcium chelator, the p38 MAPK inhibitor, the ERK MAPK inhibitor, and the COX-2 inhibitor, the inductions of PGE2 and IL-8 by ANE were inhibited. In conclusion, the results in this study showed that ANE induced secretion of PGE2 and IL-8 by PMNs, The inducible effects of ANE may be due to the increase of the intracellular calcium. Moreover, p38 and ERK MAPK may play a role in the regulation of ANE-induced PGE2 and IL-8 production. The results might help to explain the influences of AQ chewing on oral health.
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Tsai, Jia Shiuan, and 蔡家玄. "Cadmium activates multiple signaling pathways that coordinately stimulate Akt activity to enhance c-Myc mRNA stability." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/33061166689431248895.
Full textAbstract:
博士國立清華大學
分子與細胞生物研究所
104
Cadmium (Cd) is an environmental contaminant that has been classified as a human carcinogen. Exposure of Cd leads to cell death or malignant transformation. Metallothioneins (MTs) and Glutathione (GSH) are two major branches involved in protecting cells from Cd toxicity. Previous studies showed that c-Myc transcriptionally regulates γ-glutamyl-cysteine synthetase (γ-GCS), the rate-limiting enzyme catalyzing GSH biosynthesis. In addition, c-Myc was reported to associate with MT gene expression. Cd is known to activate c-Myc proto-oncogene. However, the mechanism has not been explored. We investigated here the mechanism of c-Myc expression under Cd treatment in HepG2 cells. The c-Myc protein and mRNA levels increased with dose- and time-dependent manners after Cd treatment. This increase was not associated with promoter activity and protein stability of c-Myc but was due to an increase in c-Myc mRNA stability. To explore the mechanism involved in enhancing the mRNA stability, several cellular signaling factors that evoked by Cd treatment were analyzed. PI3K, p38, ERK and JNK were activated after Cd treatment. However, ERK did not participate in the Cd-induced c-Myc expression. Further analysis revealed that mTORC2 was a downstream factor of p38. PI3K, JNK and p38/mTORC2 coordinately activated Akt. Akt was phosphorylated at Thr450 in the untreated cells. Cd treatment led to additional phosphorylation at Thr308 and Ser473. Blocking any of the three signaling factors resulted in the reduction of phosphorylation level at all three Akt sites. The activated Akt phosphorylated Foxo1 and allowed the modified protein to translocate into the cytoplasm. Overexpression of Foxo1 reduced the Cd-induced c-Myc mRNA and protein levels, and knockdown of Foxo1 increased c-Myc mRNA level. These results suggest that Cd-induced accumulation of c-Myc requires the activation of PI3K、JNK and p38/mTORC2 signaling pathways. The signals act coordinately for Akt activation and drive the Foxo1 from the nucleus to the cytoplasm. Reduction of Foxo1 in the nucleus reduces the transcription of its target genes that may affect c-Myc mRNA stability, resulting in a higher accumulation of the c-Myc proteins. PI3K、JNK、p38 inhibitors and c-Myc knockdown enhanced Cd-induced cell death. These inhibitors and c-Myc knockdown also reduced Cd-induced γ-GCS mRNA level and GSH content, but did not affect MT2A mRNA level. These results suggest that Cd-induced c-Myc level may regulate GSH synthesis and provide protection from Cd-induced toxicity.
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Wu, Po-Huei, and 吳柏慧. "Nomilin exerts anticancer activity through the inhibition of ER-α signaling pathway in ER-α positive breast cancer cells." Thesis, 2018. http://ndltd.ncl.edu.tw/handle/x756g3.
Full textAbstract:
碩士國立中興大學
生物化學研究所
106
Estrogen receptor-α is a ligand-activated transcription factor implicated in breast cell and plays an important role in cancer cell proliferation and metastasis. The binding of estrogen and the estrogen receptor-α in the cytoplasm causes two estrogen receptors to dimerize spontaneously, and translocated into the nucleus. Citrus fruits and its related genera contain about 300 limonoids have been known nowadays. In the present study, we found that limonoids inhibit breast cancer cell proliferation. Interestingly, the effect of limonoids inhibited the growth of ER-α positive breast cancer cells was better than the ER-α negative breast cancer cells. Therefore, we aimed at the relationship between limonoids and ER-α signaling pathway. Our data indicated that among the abundant of the limonoids, the cytotoxic effect of Nomilin was stronger than Limonin in breast cancer cells. Moreover, we demonstrated that Nomilin reduced the expression of ER-α protein levels in ER-α positive breast cancer cells. Nomilin repressed cell proliferation through the inhibition of AKT/mTOR signaling pathway in MCF7 cells. We also found that Nomilin inhibited ER-α positive breast cancer cell metastasis through the regulation of E-cadherin. According to previous studies, Twist was transcription factor that can induced transcription of Y-box binding protein-1. We found that Nomilin reduced the expression of Y-box binding protein-1 through the inhibition of ER-α. This work sheds light on the pathways by which Nomilin inhibits ER-α positive breast cancer cells proliferation and metastasis.
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Alves, Celso Miguel da Maia. "S. Coronopifolius Bromoterpenes : antitumor activity and intracellular signal pathways characterization on in vitro cancer models." Doctoral thesis, 2019. http://hdl.handle.net/10773/29248.
Full textAbstract:
Nowadays, cancer is one of the major threats to human health and, due to distinct factors, it is expected that its incidence will increase in the next decades leading to an urgent need to develop new approaches to fight it, including the development of new anticancer drugs. Marine environment has revealed to harbor a vast diversity of unusual and distinct chemical structures with high potential to be used as scaffolds for the design and development of new drugs with great effectiveness and specificity to human illnesses, such as cancer. Therefore, the main goal of the present thesis was to study the chemical profile of the red alga Sphaerococcus coronopifolius collected in the Berlenga Nature Reserve (Peniche, Portugal) as well as to evaluate the antitumor activities of its major metabolites.
The study of S. coronopifolius chemical profile allowed, through antitumor bioguied screening, to identify seven compounds, including five already known terpenes, alloaromadendrene (1), bromosphaerol (3), sphaerococcenol A (4),12S-hydroxy-bromosphaerol (5), and 12R-hydroxy-bromosphaerol (7), as well as two new natural molecules, a new brominated dactylomelane diterpene, named sphaerodactylomelol (2) and a new brominated 7-epi-eudesmane sesquiterpene, named 6-acetyl-sphaeroeudesmanol (6). Compounds (2-5, 7) exhibited antiproliferative activity on an in vitro model of human hepatocellular carcinoma (HepG2) in an IC50 range between 42.87 to 279.93 μM being the highest activity induced by sphaerococcenol A (4) (IC50: 42.87 μM). The new diterpene, sphaerodactylomelol (2), was the only compound that induced cytotoxicity (IC50: 719.85 μM) on HepG2 cells. Hence, due to the cytotoxic activities exhibited by the compounds 2-5 and 7 on HepG2 cells, their antitumor potential was evaluated on several in vitro human cancer cells derived from distinct tissues (SH-SY5Y; MCF-7; A549; NCI-H226; PC-3; HCT-15; CACO-2; SK-MEL-28; RenG2) to define their selectivity and potency (0.1 - 100 μM; 24 hours). Murine fibroblasts (3T3) were used as non-tumor cells. S. coronopifolius compounds did not displayed selective activity for specific tumor tissue as well as for cancer cells. Concerning the potency of the effects, S. coronopifolius metabolites exhibited an IC50 range between 4.47 to 89.41 μM. Sphaerococcenol A (4) showed the highest cytotoxicity exhibiting a range of IC50 between 4.47 to 16.59 μM and sphaerodactylomelol (2) displayed the lowest cytotoxicity showing a range of IC50 between 33.04 and 89.41 μM. In addition, to understand the intracellular signaling pathways linked to their cytotoxic activities, hallmarks associated to reactive oxygen species production, namely through hydrogen peroxide (H2O2) real-time production, and apoptosis (membrane translocation of phosphatidylserine, mitochondrial membrane potential, Caspase-9 activity, and DNA condensation and/ or fragmentation) were studied on an in vitro breast carcinoma model (MCF-7 cells). Genotoxic activities were evaluated on fibroblasts derived from mouse tissues (L929 cells). The treatment of MCF-7 cells with compounds induced changes in the mitochondrial
membrane potential, increased Caspase-9 activity, and promoted DNA condensation and/or fragmentation. In addition, with the exception of bromosphaerol (3), all compounds promoted the increase of H2O2 levels production. Regarding genotoxic effects, only bromosphaerol (3) mediated DNA damage in L929 cells. Additionally, to evaluate the compounds' effects in a system similar to a tissue, co-cultures of non-malignant human bronchial fibroblasts and malignant human bronchial epithelial cells were implemented. 12R-hydroxy-bromosphaerol (7) revealed to be the compound with highest potential exhibiting cytotoxicity and ability to prevent the formation of malignant stem cells.
Summarizing, Sphaerococcus coronopifolius bromoditerpenes exhibited cytotoxic activities, which seems to be linked to apoptosis, H2O2 generation and induction of DNA damage. Despite the interesting results achieved, this work is an initial approach being of utmost importance to deeply characterize the intracellular signaling pathways associated with antitumor activities mediated by these compounds in order to define their effective pharmacological potential in cancer therapeutics.Atualmente, o cancro representa um dos maiores desafios para a saúde humana e, devido a diversos fatores, é expectável que a sua incidência aumente nas próximas décadas tornando-se de extrema importância desenvolver novas abordagens terapêuticas, incluindo o desenvolvimento de novos fármacos. Neste âmbito, o ambiente marinho tem revelado albergar uma elevada diversidade de estruturas químicas incomuns e distintas com potencial para serem usadas como “scaffolds” no design e desenvolvimento de novos fármacos com grande eficácia e especificidade para o tratamento de doenças humanas, como o cancro. Desta forma, o principal objetivo da presente tese consistiu em estudar o perfil químico da alga vermelha Sphaerococcus coronopifolius recolhida na Reserva Natural da Berlenga (Peniche, Portugal) assim como avaliar as atividades antitumorais dos seus principais metabolitos. O estudo do perfil químico da alga S. coronopifolius, através de screening bioguiado, permitiu identificar sete compostos, incluindo cinco terpenos previamente descritos e denominados como alloaromadendrene (1), bromosphaerol (3), sphaerococcenol A (4), 12S-hydroxy-bromosphaerol (5) e 12R-hydroxy-bromosphaerol (7), assim como duas novas moléculas de origem natural, nomeadamente um diterpeno dactilomelano bromado, designado sphaerodactylomelol (2), e um novo sesquiterpeno 7-epi-eudesmano bromado, designado 6-acetyl-sphaeroeudesmanol (6). Os compostos (2-5 e 7) exibiram atividade antiproliferativa num modelo in vitro de carcinoma hepatocelular humano (HepG2) num intervalo de IC50 entre 42.87 e 279.93 μM, sendo a atividade mais potente induzida pelo sphaerococcenol A (4) (IC50: 42.87 μM). Por sua vez, o novo diterpeno, sphaerodactylomelol (2), foi o único composto que induziu citotoxicidade (IC50: 719.85 μM) nas células HepG2. Consequentemente, devido às atividades exibidas pelos compostos 2-5 e 7 nas células HepG2, o seu potencial antitumoral foi avaliado em diferentes modelos celulares in vitro de cancro humano derivados de diferentes tecidos (SH-SY5Y; MCF-7; A549; NCI-H226; PC-3; HCT-15; CACO-2; SK-MEL-28; RenG2) de modo a definir a sua seletividade e potência (0.1 - 100 μM; 24 h). Fibroblastos derivados de tecidos murinos (3T3) foram usados como modelo não tumoral. Os compostos não demonstraram seletividade nem para células tumorais nem entre os modelos derivados de diferentes tecidos. No que diz respeito à capacidade citotóxica, os compostos exibiram um intervalo de IC50 entre 4.47 e 89.41 μM. O sphaerococcenol A (4) induziu o maior efeito citotóxico exibindo um intervalo de IC50 entre 4.47 e 16.59 μM enquanto o sphaerodactylomelol (2) demonstrou ser o composto menos citotóxico exibindo um intervalo de IC50 entre 33.04 e 89.41 μM. De modo a compreender as vias de sinalização intracelular envolvidas nas atividades citotóxicas observadas, biomarcadores associados à produção de espécies reativas de oxigénio, nomeadamente a produção de peróxido de hidrogénio (H2O2) em tempo real, e apoptose (translocação da fosfatidilserina, potencial mitocondrial membranar, atividade da Caspase-9, condensação de ADN e/ ou fragmentação) foram estudados num modelo in vitro de carcinoma humano da mama (MCF-7). As atividades genotóxicas foram avaliadas em fibroblastos derivados de tecidos murinos (L929). O tratamento realizado nas células MCF-7 com os compostos isolados induziu alterações no potencial mitocondrial membranar, aumentou a atividade da Caspase-9 e promoveu a condensação e/ ou a fragmentação de ADN. Com a exceção do bromosphaerol (3), todos os compostos promoveram um aumento da produção dos níveis de H2O2. No que diz respeito aos ensaios de genotoxicidade, apenas o bromosphaerol (3) mediou danos no ADN das células L929. Tendo em vista avaliar os supracitados compostos num sistema que mais se aproximasse a um tecido foram implementadas co-culturas de fibroblastos bronquiais humanos não malignos e células do epitélio bronquial humano malignizadas tendo o 12R-hydroxy-bromosphaerol (7) induzido citotoxicidade e capacidade de impedir a formação de células estaminais malignas. Resumindo, os bromoterpenos isolados da alga vermelha Sphaerococcus coronopifolius exibiram atividades citotóxicas relevantes, as quais parecem estar associadas aos processos de apoptose, produção de H2O2 e danos de ADN. Por sua vez, o composto 12R-hydroxy-bromosphaerol (7) demonstrou ser o composto mais promissor nos ensaios realizados em sistema de co-cultura impedindo a formação de células tumorais com fenótipo estaminal. Apesar dos resultados obtidos, este trabalho consistiu numa abordagem inicial sendo de extrema importância caraterizar profundamente as vias de sinalização intracelular associadas às atividades antitumorais mediadas por estes compostos de modo a compreender o seu verdadeiro potencial como agentes farmacológicos na terapia do cancro.
Programa Doutoral em Ciência, Tecnologia e Gestão do Mar
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"Modulation of ROR-alpha receptor activity and the calcium/calmodulin signaling pathway by melatonin in MCF-7 human breast cancer cells." Tulane University, 2000.
Find full textAbstract:
The pineal hormone, melatonin has repeatedly been shown to inhibit the proliferation of estrogen receptor alpha (ERalpha)-positive MCF-7 human breast cancer cells. Previous reports have suggested that the actions of melatonin can be mediated either through G protein-coupled membrane receptors, or via retinoid orphan receptors (RORalpha), which constitutively activate gene transcription through their corresponding response elements termed ROREs. Transient transfection assays using an RORE-luciferase reporter construct have shown that the endogenous (RORalpha), receptors expressed in MCF-7 breast cancer cells exhibited a high basal level of transcriptional activity, which was further stimulated by serum. In the presence of serum, both the transcriptional activity and DNA binding ability of (RORalpha), were repressed by melatonin treatment, even though melatonin had no effect on RORalpha protein levels. Although RORalphas were originally proposed as nuclear receptors for melatonin, the direct binding of melatonin to these receptors can not be repeated. Therefore, it is not clear how melatonin can modulate (RORalpha), functions in MCF-7 cells. Consistent with the results from other systems, RORalpha transcriptional activity in MCF-7 cells was found to be responsive to intracellular calcium concentration ([Ca2+]i), calmodulin (CaM), and Ca2+/CaM-dependent protein kinases. Meanwhile, melatonin not only affected CaM subcellular distribution, but also modulated [Ca2+]i change induced by another known calcium stimulator, ATP, indicating that melatonin may regulate RORalpha transcriptional activity via its modulation of the Ca2+/CaM signaling pathway in MCF-7 cells. In exploring the biological functions of (RORalpha), receptors, we found that the decrease in RORalpha protein levels by an (RORalpha), antisense oligonucleotide con-elated with growth inhibition in MCF-7 cells. Our data suggest that RORalpha receptors may play a role in stimulating the proliferation of MCF-7 cells, and thus, the repressive effect of melatonin on these receptors may, at least in part, mediates melatonin's antiproliferative effects on breast cancer cellsacase@tulane.edu
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Tsou, Hsiao-Tung, and 鄒曉彤. "Anti-tumor Activity of AC10 and its major compound Against Murine Melanoma Cells Through the Modulation of Wnt/β-catenin Signaling Pathway." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/56723417987186277666.
Full textAbstract:
碩士中國醫藥大學
營養學系碩士班
99
Melanoma is the most serious form of skin cancer. Aberrant activation of Wnt/β-catenin signaling cascade has been observed in approximately one-third of melanomas, indicating that modulation of Wnt/β-catenin activation might be a novel strategy for melanoma treatment. The downstream targets of Wnt/β-catenin signaling pathway including c-Myc, cyclinD1, MMPs, and survivin, which are regulating number of cellular functions, such as proliferation, differentiation, survival, apoptosis and invasion. Antrodia camphorata, a well known medicinal mushroom in Taiwan that has been used as Chinese folk medicine for many years. Previous studies have shown that A. camphorata (AC) possessed greater anti-tumor activity against a variety of tumor cells. However, the anti-tumor efficacy of AC against melanoma was poorly understood. In addition, the currently employing treatment for melanoma is a tough topic, due to the high resistance to radio-and chemotherapy and most the synthetic chemotherapeutic drugs are volnarable to non-melanoma skin cells.Therefore, the present study, we aimed to investigate the anti-tumor efficacy of fermented culture broth extracts of A. camphorata (AC-10) and it derived pure compound (AC-0) in murine melanoma cells. The first set of experiment, we observed AC-10 treatment significantly decreased murine melanoma B16F1 and B16F10 cell viability with an IC 50 value of 80ug/mL. The reduction of cell viability is directly correlated with the inhibition of β-catenin and its downstream protein expression. Immunofluorescence analysis confirmed that AC-10-treatment markedly reduced β-catenin translocation into the nucleolus, and also downregulates ??-catenin-mediated transcriptional activity. Furthermore, MG132 a proteosomal inhibitor that prevent proteosomal degradation of β-catenin, conversely, GSK3β inhibitor SB216312 also suppressed β-catenin degradation which strongly suggest that β-catenin degradation is GSK3β dependent. In a similar way, AC-10-treatment attenuate GSK3β expression, suggesting that AC-10 may regulate the proteasomal degradation of β-catenin by GSK3β-dependent nmechanism. Furthermore, flow cytometry analysis showed that AC-10-treatment significantly arrest G1 to S-phase transition followed by the suppression of cyclin D1 (wnt/β-catenin target gene), CDK4 expression and increased in p27, p21 levels. Moreover, TUNEL assay revealed that AC-10-treatment induce apoptosis in a dose-dependent manner, followed by the disregulation of BAX/BCL-2 ratio, and the down-regulation of pro-caspase-9, pro-caspase-3 and pro-PARP. Migration and invasion assay shows AC-10 could abate melanoma metastatic ability, through the inhibition of MMP-9, MMP-2, VEGF expression, which are also wnt/β-catenin target genes. In the second part, AC-0 also found to effectively inhibit Wnt/β-catenin pathway cascades, and suppressed β-catenin-mediated transcriptional activity. Similar with AC-10, AC-0 treated cell found to decreased melanoma metastasis and augmented apoptotic induction. MG132 and AC-0 treatment prevents β-catenin expression, however, SB216312 failed to prevent GSK3β expression, which suggesting that AC-0-treatment may regulate the proteasomal degradation of β-catenin via GSK3β-independent mechanism. This phenomenon also demonstrated with immunoprecipition assay that AC-0 treated cells decreased the interaction between β-catenin and GSK3β. In vivo study, AC-0 decreased the growth of B16F10-derived tumors development in the athymic nude mice. The decreased B16F10-derived tumor growth was associated with a down-regulation of Wnt/β-catenin target genes such as c-myc, cyclin D1, MMP-9. AC-0 treatment also induced B16F10-derived tumors apoptosis in nude mice. In conclusion, our data demonstrated that AC-10 and its major compound AC-0 appreciably modulate Wnt/β-catenin pathway in melanoma cells. Therefore, we believe AC-10/AC-0 might be a potential chemo-preventive agent for melanoma treatment.
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Wagner, Toni. "Activity and Crosstalk of STAT3 and BMP Signalling Pathways in Pluripotency Control of Mouse and Medaka Stem Cells." Doctoral thesis, 2007. https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-26495.
Full textAbstract:
- 77/83 allerdings inaktiv in Kulturen und Embryonen von Medaka. Dieser Unterschied wird durch Daten aus humanen ES-Zellkulturen unterstützt. Letztere sind ebenfalls komplett STAT3 unabhängig. Die BMP-Smad Kaskade wiederum ist in Medaka-Stammzellen aktiv, Antidifferenzierungsgene wie id2, die durch BMP direkt kontrolliert werden, sind dementsprechend exprimiert. Diese Daten stimmen wiederum mit dem Maussystem überein, während humane ES-Zellen diesbezüglich bislang nicht untersucht wurden. Die Interaktion zwischen verschiedenen Signalwegen ist ein bisher noch nicht gut verstandenes Gebiet. Die Integration verschiedener Signale ist aber speziell für Stammzellen, die ihr Differenzierungsschicksal von winzigen Abweichungen in der Signalmixtur abhängig machen, von entscheidender Bedeutung. Im zweiten Teil der hier vorgelegten Arbeit konnte eine Interaktion zwischen dem BMP-Rezeptor 1a und STAT3 nachgewiesen werden. Diese Interaktion ist offenbar Teil eines variablen Komplexes. Zum ersten Mal war es auch möglich, funktionale Konsequenzen für STAT3 nach Stimulierung des BMP-Rezeptors 1a zu dokumentieren. Nach Belegung des BMP-Rezeptors 1a mit dem mutierten BMP2-A34D wird STAT3 trotz Aktivierung durch Phosphorylierung an Tyrosin 705 im Zytoplasma von Maus Stammzellen festgehalten. Zusammengenommen konnte hier gezeigt werden, dass eine Interaktion zwischen den bislang als isoliert betrachteten Signalwegen BMP-Smad und STAT3 besteht. Des Weiteren wurde das Medaka-Stammzellkultursystem benutzt, um zu zeigen, dass STAT3 für die Pluripotenz von Stammzellen nur im Maussystem eine Rolle spielt, wohingegen BMPZielgene wie id2 in bislang allen getesteten ES-Zellkultursystemen aktiv sind.
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Wagner, Toni [Verfasser]. "Activity and crosstalk of STAT3 and BMP signalling pathways in pluripotency control of mouse and medaka stem cells / vorgelegt von Toni Wagner." 2008. http://d-nb.info/988085089/34.
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Tzeng, Chung-Yuh, and 曾崇育. "The study of hypoglycemic effect of electroacupuncture by reducing insulin resistance in chronic steroid induced insulin resistance rats and signaling pathways associated with hypoglycemic activity of ST 36 electroacupuncture." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/62714443948862441271.
Full textAbstract:
博士國立清華大學
分子醫學研究所
104
This study is designed to evaluate the treatment effect of electroacupuncture (EA) in chronic steroid induced insulin resistant rat model. An alternative therapy is explored to reduce the chronic steroid induced insulin resistance.The aim of this study is to determine (1) if EA treatment can produce hypoglycemic effect and (2) inhibit the development of glucocorticoid-altered insulin sensitivity in chronic status and to (3) explore the mechanisms of EA by assaying plasma FFAs and proteins of insulin signal pathway. Intravenous glucose tolerance test (IVGTT) and insulin challenge test (ICT) were applied to evaluate the effect of EA on steroid induced insulin resistance (SIIR) rats. Finally, this study evaluates proteins of insulin signaling pathway to investigate the mechanisms by which EA improves the insulin resistance of SIIR rats. We hypothesized that electroacupuncture can produce a hypoglycemic effect in chronic steroid induced insulin resistance diabetes rat model. A diabetes rat model was created by using clinical-like dose dexamethasone, 1 mg/kg, i.p. once a day chronic to induce insulin resistance for 5 days. Then the steroid induced insulin resistant (SIIR) rats were randomly divided into SIIR+EA group and SIIR group. Plasma glucose, insulin challenge test (ICT) and intravenous glucose tolerance test (ivGTT) were used to test the change of plasma glucose levels between SIIR+EA group and SIIR group. The plasma free fatty acids (FFA) and related proteins of the insulin signaling pathway, such as IRS-1 and GLUT4 were also checked to explore the effect of EA on recovering insulin sensitivity of SIIR rats. The results showed that EA could decrease the FFA level and increase insulin sensitivity in SIIR rats. Further clinical studies are needed to determine whether EA can be an alternative and effective treatment for patients for whom chronic usage of dexamethasone is needed by reducing insulin resistance. Data on expression of all genes in a biological sample can be achieved in one experiment using the microarray method, the results of which can be analyzed to determine the potential pathways involved in a given process and identify potential therapeutic targets. Then the microarray analysis experiment was done to explore the possible signaling pathway related to hypoglycemic effect induced by the EA. Previous animal studies have reported a hypoglycemic effect of EA and suggested that the mechanisms are closely related to intracellular signaling pathways. The aim of this study was to screen potential for intracellular signaling pathways that are upregulated by EA at bilateral ST36 in rats with diabetes using microarray analysis. Streptozotocin (STZ) - induced diabetic rats were randomly assigned to experimental (EA, n=8) or control (non-EA, n=8) groups. Plasma glucose levels were measured at baseline, 30 and 60 minutes, and microarray analysis was performed on samples of the gastrocnemius muscle. Relative to baseline values, EA significantly reduced plasma levels of glucose at 30 and 60 minutes. The microarray pathway analysis showed that cell adhesion molecules and type 1 DM gene sets were both upregulated in EA versus non-EA groups (p<0.05). Cell adhesion molecules might be related to the hypoglycemic effect induced by EA in rats with STZ-induced type I diabetes. Future research will be required to examine the involvement of related intracellular signaling pathways.
41
Lu, Chi-Cheng, and 呂啟誠. "Investigations for targeting signaling pathways in the apoptosis induction and anti-metastasis of the novel quinazolinone derivatives in leukemia and oral cancer cells in vitro and anti-tumor activity in vivo." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/69439994355722115199.
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博士國立中興大學
生命科學系所
100
The previous studies have demonstrated that 2-phenyl 6-pyrrolidinyl-4-quinazolinone derivatives exhibited antitumor actions and biological responses. Among them, both of the most potent newly synthesized compounds, 6-pyrrolidinyl-2-(2-hydroxyphenyl)-4-quinazolinone (MJ-29) and 6-pyrrolidinyl- 2-(3-Methoxyphenyl)-4-quinazolinone (HMJ-38) for their mechanisms underlying anticancer activities remain unclear and have not been well clarified. In this dissertation, we attempt to investigate anti-leukemia and anti-oral cancer effects in vitro and in vivo. In the chapter 1, we focused on the in vitro effects of MJ-29 on ER stress and mitochondria-dependent apoptotic death in murine myelomonocytic leukemia WEHI-3 cells, and to hypothesize that MJ-29 might fully impair the orthotopic leukemic mice. In the chapter 2, the objective of current study was explored the effects of the novel compound MJ-29 on anti-metastatic actions of human oral squamous cell carcinoma (OSCC) CAL 27 cells and to verify the underlying related molecular mechanisms in this event. In the chapter 3, this study investigated the newly synthesized and anti-mitotic compound, HMJ-38 addressing its target and precise mechanism of actions. We hypothesized that HMJ-38 might sensitize apoptotic death of CAL 27 cells in vitro and inhibit xenografts tumor growth in vivo. In my Ph.D. study, we found that the induction of cell death in MJ-29-treated WEHI-3 cells has been proven by an assessment of apoptosis in vitro culture through intrinsic apoptotic pathway and ER stress signaling. Strikingly, anti-leukemia responses by MJ-29 were observed in leukemic mice in vivo. We further demonstrated that MJ-29 is capable of inhibiting cell adhesion, invasion and migration through the down-regulation of MMP-2 and MMP-9 in human OSCC CAL 27 cells. Also, the phosphorylation of AKT and MAPK signaling pathway proteins may be coordinately involved in the inhibitory effect on these essential steps of metastasis by MJ-29. Finally, our study provides evidence for G2/M phase arrest and apoptosis caused by HMJ-38 in CAL 27 cells, and HMJ-38 by intraperitoneal administration suppressed CAL 27 xenografts tumor growth in nude mice. Overall, we presented the novel findings that MJ-29 and HMJ-38 possess a potent therapeutic value for use against leukemia and oral cancer in response to cell-fate decision, and the efficacy of both compounds might be sufficient to investigate the potential of treatment in the future.
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Huang, Wei. "Small molecule compounds targeting DNA binding domain of STAT3 for inhibition of tumor growth and metastasis." Thesis, 2014. http://hdl.handle.net/1805/5221.
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Indiana University-Purdue University Indianapolis (IUPUI)Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in malignant tumors, and its activation is associated with high histological grade and advanced cancer stage. STAT3 has been shown to play important roles in multiple aspects of cancer aggressiveness including proliferation, survival, self-renewal, migration, invasion, angiogenesis and immune response by regulating the expression of diverse downstream target genes. Thus, inhibiting STAT3 promises to be an attractive strategy for treatment of advanced tumors with metastatic potential. We firstly identified a STAT3 inhibitor, inS3-54, by targeting the DNA-binding site of STAT3 using an in-silico screening approach; however, inS3-54 was finally found not to be appropriate for further studies because of low specificity on STAT3 and poor absorption in mice. To develop an effective and specific STAT3 inhibitor, we identified 89 analogues for the structure-activity relationship analysis. By using hematopoietic progenitor cells isolated from wild-type and STAT3 conditional knockout mice, further studies showed that three analogues (A18, A26 and A69) only inhibited STAT3-dependent colony formation of hematopoietic progenitor cells, indicating a higher selectivity for STAT3 than their parental compound, inS3-54. These compounds were found to (1) inhibit STAT3-specific DNA binding activity; (2) bind to STAT3 protein; (3) suppress proliferation of cancer cells harboring aberrant STAT3 signaling; (4) inhibit migration and invasion of cancer cells and (5) inhibit STAT3-dependent expression of downstream targets by blocking the binding of STAT3 to the promoter regions of responsive genes in cells. In addition, A18 can reduce tumor growth in a mouse xenograft model of lung cancer with little effect on body weight. Taken together, we conclude that it is feasible to inhibit STAT3 by targeting its DNA-binding domain for discovery of anticancer therapeutics.
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Mathieu, Mélissa. "Étude de la différenciation des lymphocytes T CD8+ effecteurs et mémoires : rôle de la cellule présentatrice d’antigène et de la voie de signalisation Notch." Thèse, 2014. http://hdl.handle.net/1866/11791.
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Lors d’une infection par un pathogène, des lymphocytes T CD8+ naïfs (LTn) spécifiques de l’antigène sont activés, prolifèrent et se différencient en LT effecteurs (LTe). Les LTe produisent différentes cytokines et acquièrent une activité cytotoxique menant à l’élimination du pathogène. Seulement 5 à 10 % des LTe survivront et se différencieront en LT mémoires (LTm), qui sont capables de répondre plus rapidement lors d’une seconde infection par le même pathogène, contribuant au succès de la vaccination. Toutefois, la compréhension de l’ensemble des mécanismes régulant le développement des LTe et des LTm demeure incomplète. Afin de mieux comprendre les signaux requis pour la différenciation des LT CD8+ lors de la réponse immune, nous avons posé deux hypothèses.
Nous avons d’abord proposé que différentes cellules présentatrices d’antigène (CPA) fournissent différents signaux au moment de la reconnaissance antigénique influençant ainsi le devenir des LT CD8+. Vu leur potentiel d’utilisation en immunothérapie, nous avons comparé la capacité d’activation des LT CD8+ par les lymphocytes B activés via le CD40 (CD40-B) et les cellules dendritiques (CD). Nous avons montré que l’immunisation avec des CD40-B induit une réponse effectrice mais, contrairement à l’immunisation avec des CD, pratiquement aucun LTm n’est généré. Les LTe générés sont fonctionnels puisqu’ils sécrètent des cytokines, ont une activité cytotoxique et contrôlent une infection avec Listeria monocytogenes (Lm). Nous proposons qu’une sécrétion plus faible de cytokines par les CD40 B ainsi qu’une interaction plus courte et moins intime avec les LT CD8+ comparativement aux CD contribuent au défaut de différenciation des LTm observé lors de la vaccination avec les CD40-B.
Ensuite, nous posé l’hypothèse que, parmi les signaux fournis par les CPA au moment de la reconnaissance antigénique, la voie de signalisation Notch influence le développement des LTe, mais aussi des LTm CD8+ en instaurant un programme génétique particulier. D’abord, grâce à un système in vitro, le rôle de la signalisation Notch dans les moments précoces suivant l’activation du LT CD8+ a été étudié. Ce système nous a permis de démontrer que la voie de signalisation Notch régule directement l’expression de la molécule PD-1. Ensuite, grâce à des souris où il y a délétion des récepteurs Notch1 et Notch2 seulement chez les LT CD8+ matures, un rôle de la voie de signalisation Notch dans la réponse immune des LT CD8+ a été démontré. Nos résultats démontrent que suite à une infection avec Lm ou à une immunisation avec des CD, la signalisation Notch favorise le développement de LTe, exprimant fortement KLRG1 et faiblement CD127, destinés à mourir par apoptose. Toutefois, la signalisation Notch n’a pas influencé la génération de LTm. De façon très intéressante, l’expression des récepteurs Notch influence la production d’IFN- en fonction du contexte d’activation. En effet, suite à une infection avec Lm, l’absence des récepteurs Notch n’affecte pas la production d’IFN- par les LTe, alors qu’elle est diminuée suite à une immunisation avec des CD suggérant un rôle dépendant du contexte pour la voie de signalisation Notch.
Nos résultats permettent une meilleure compréhension des signaux fournis par les différentes CPA et de la voie de signalisation Notch, donc des mécanismes moléculaires régulant la différenciation des LT CD8+ lors de la réponse immunitaire, ce qui pourrait ultimement permettre d’améliorer les stratégies de vaccination.Following an infection with a pathogen, antigen-specific naive CD8+ T lymphocytes (Tn) will proliferate and differentiate into effector (Te) cells. Those Te cells will produce different cytokines and acquire a cytotoxic activity, leading to pathogen clearance. Only 5 to 10 % of Te cells will survive and differentiate into memory CD8+ T lymphocytes (Tm) able to respond rapidly following a second encounter with the same pathogen, contributing to the success of vaccination. However, the mechanisms regulating Te and Tm cells development remain incompletely understood. To better understand the signals required for CD8+ T lymphocytes during an immune response, we proposed two hypotheses. First, we propose that different antigen presenting cells (APCs) can deliver different signals to CD8+ T lymphocytes at the time of priming leading to different outcome. Given their potential for use in immunotherapy, we compared the ability of CD40 activated B lymphocytes (CD40-B) and dendritic cells (DCs) to activate CD8+ T lymphocytes. We have shown that CD40-B cell immunisation leads to an effector response but very few Tm cells are generated compared to DC immunisation. The Te cells generated following CD40-B cell immunisation are functional because they secrete cytokine, are cytotoxic and control a Listeria monocytogenes (Lm) infection. We propose that CD40-B cells secrete less cytokines and interact during shorter period of time with the CD8+ T lymphocytes, without engulfment, contributing to the decreased Tm generation observed following immunisation with CD40-B cells. Second, among the signals provided by APC at the time of CD8+ T lymphocyte priming, we have hypothesised that the Notch signalling pathway influences Te and Tm cell differentiation by inducing a particular genetic program. Using an in vitro system, we first studied the role of the Notch signalling pathway in the hours following CD8+ T lymphocyte priming. We demonstrated that Notch signalling directly regulates PD-1 expression. Then, studying mice where Notch1 and Notch2 receptor genes are deleted only in mature CD8+ T lymphocytes, we characterised the role of the Notch signalling pathway on Te and Tm differentiation during an immune response. Our results show that following Lm infection or a DC immunisation, the Notch signalling pathway promotes the differentiation of short lived effector cells Te cells (KLRG1highCD127low) meant to die by apoptosis. However, the Notch signalling pathway did not influence the generation of CD8+ Tm cells. Most interestingly, IFN- regulation by the Notch signalling pathway depends on the activation context. Indeed, following Lm infection, lack of Notch receptors does not impact IFN- secretion by Te cells while it is significantly decreased following a DC immunisation suggesting a context dependant role for the Notch signalling pathway. Our findings provide a better understanding of the key signals provided by APC as well as the Notch signalling pathway, and thus the molecular mechanisms leading to CD8+ lymphocyte effector and memory generation which is crucial as this knowledge may ultimately lead to improved vaccination.