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Falha, Layal. "Implication du facteur de transcription dans Nkx2.2 gliomagenesis." Thesis, Montpellier 2, 2014. http://www.theses.fr/2014MON20065.
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Glioblastome représente la tumeur la plus courante du cerveau primaire avec une survie de moins de 2 ans. Ces tumeurs sont très infiltrantes et angiogéniques et contiennent une sous population de cellules souches cancéreuses. Nkx2.2 est un homéodomaine facteur de transcription, impliqué dans la formation d'oligodendrocytes au cours du développement. Nkx2.2 joue un rôle central dans la tumorogenèse de Ewing'sarcoma. L'utilisation de la QPCR et de la matrice du tissu de gliome, nous a permis de mettre en évidence la forte expression de Nkx2 dans le glioblastome. Nkx2.2 a également été détecté dans 3 cultures de cellules de gliomes où il est co-exprimé avec des marqueurs de cellules souches tels que CD133 et CD15. Il a été récemment proposé que la surexpression de Nkx2.2 pourrait induire à la différenciation oligodendrocytaire de la cellule du gliome tige-comme et au blocage de la formation des tumeurs dans la xénotransplantation (Cancer Res fév 2011 1; 71 (3): 1135-1145). Pour explorer cette possibilité, nous avons utilisé des rétrovirus pour surexprimer Nkx2.2 dans nos cultures cellulaires. De manière surprenante, nous avons trouvé que Nkx2.2, induit la prolifération des cellules souches du gliome et n'a en conséquent aucun effet de différenciation. Microarray analyses a confirmé que la surexpression de Nkx2.2 n'a en effet aucune influence sur la différenciation des oligodendrocytes. Cette analyse a également révélé que Nkx2.2 était capable d'induire une forte expression de YKL-40 40 dans le surnageant des cellules souches du gliome. YKL-40 est en fait, une glycoprotéine sécrétée et impliquée dans l'inflammation, l'angiogenèse et la prolifération. Elle est souvent associée à un mauvais pronostic dans plusieurs types de cancers. En outre, nous avons effectué une transplantation orthotopique afin d'explorer le rôle de Nkx2.2 dans la gliomagenèse in vivo et avons constaté que Nkx2.2 ne réduit pas l'agressivité du glioblastome.Dans l'autre partie de ma thèse, nous avons utilisé la gamme Taqman à basse densité et la validation des miRNA par la Qpcr afin de chercher ces derniers dans la culture cellulaire du glioblastome humain. Nous avons ensuite étudié le rôle des miARN dans la transcription de 3'UTR de Nkx2.2. Les résultats d'analyse de la mutagénèse dirigée (SDM) et de la double-luciférase ont montré que l'expression de Nkx2.2 est régulée par la diminution de mir-133b ainsi que celle de mir-202Glioblastoma represent the most common primary brain tumor with an overall survival of less than 2 years. These tumors are highly infiltrative and angiogenic and contain a sub population of cancer stem cells. Nkx2.2 is a homeodomain transcription factor which is implicated in the formation of oligodendrocytes during development. Nkx2.2 is central in tumorogenesis of Ewing'sarcoma. Using QPCR and glioma tissue array, we found that Nkx2.2 is highly expressed in glioblastoma. Nkx2.2 was also detected in 3 glioma stem-like cell cultures (neurospheres) where it is co-expressed with stem cell markers such as CD133 and CD15. It was recently proposed that overexpression of Nkx2.2 could induce terminal oligodendrocytic differentiation of glioma stem-like cell and inhibit tumor formation in xenotransplantation (Cancer Res. 2011 Feb 1;71(3):1135-45).To explore this possibility further, we used retroviruses to overexpress Nkx2.2 in our cell cultures. Surprisingly, we found that Nkx2.2, induce glioma stem cell proliferation and had no oligodendrocyte differentiating effect. Microarray analyses confirmed that Nkx2.2 overexpression had no influence in oligodendrocyte differentiation. This analysis further revealed that Nkx2.2 was able to induce a strong expression of YKL40 protein in the supernatant of glioma stem cells and increase YKL-40 promoter activity. YKL-40 is a secreted glycoprotein which is involved in inflammation, angiogenesis and proliferation and which is often associated with a bad prognosis in several cancers. In addition, we performed orthotopic transplantation to explore the role of Nkx2.2 in gliomagenesis in vivo and found that Nkx2.2 did not reduce the aggressiveness of glioblastoma. In the other part of my thesis we used Taqman low-density arrays (TLDA) and individual miRNA QPCR validation to find the microRNA (miRNA) signature in human glioblastoma cell cultures. Then we investigated the role of miRNA in the 3'UTR of Nkx2.2 transcript. Site directed mutagenesis (SDM) and dual-Luciferase reporter assay results showed that the Nkx2.2 expression is downregulated by mir-133b and mir-202
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Pravemann, Jana Verfasser], and Hans-Henning [Akademischer Betreuer] [Arnold. "Die Rolle der Transkriptionsfaktoren Nkx2.2 und Nkx2.9 in der Entwicklung des Neuralrohrs: Herstellung einer Nkx2.9 Cre knock-in Mausmutante / Jana Pravemann ; Betreuer: Hans-Henning Arnold." Braunschweig : Technische Universität Braunschweig, 2014. http://d-nb.info/1175820709/34.
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Guichet, Pierre-Olivier. "Rôle de NKX2-2, NGN2 et DCX dans la prolifération, différenciation et migration des cellules tumorales de glioblastomes." Thesis, Montpellier 2, 2011. http://www.theses.fr/2011MON20143.
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Les Glioblastomes (Gb) sont des tumeurs primaires du SNC les plus fréquentes et sont particulièrement agressives car résistantes à la radio/chimiothérapie. Elles présentent généralement une composante solide et infiltrante. Cette dernière étant difficile à éliminer par la chirurgie sera en partie responsable de la récurrence de la tumeur. Une des avancées majeures du domaine est la mise en évidence dans les Gb de sous populations présentant des caractéristiques de précurseurs neuraux. Ces cellules cancéreuses utilisent des réseaux de gènes spécifiques pour maintenir leur prolifération et leur état indifférencié. Une approche possible pour éliminer ces cellules cancéreuses serait de cibler les facteurs de transcription impliqués dans la prolifération ou encore de forcer leur différenciation. Dans ce but, j'ai étudié le rôle de NKX2.2 et NGN2 à partir de 3 cultures primaires multipotentes. Les résultats montrent que l'expression de NKX2.2 dans ces cultures est nécessaire pour la survie, la prolifération et la capacité à former des neurosphères. A l'inverse, la surexpression de NGN2 conduit à une apoptose massive, à un arrêt de la prolifération avec formation de neurones dont certains sont électrophysiologiquement actifs. Une approche différente consisterait à cibler une des protéines impliquées dans la migration pour limiter la composante infiltrante. Des études antérieures ont montrées un rôle clef de DCX dans la migration des jeunes neurones au cours du développement. La forte expression de DCX dans certains Gb m'a conduit à étudier la régulation et le rôle de ce gène. In vitro, les résultats obtenus montrent que DCX est exprimé par une sous population de cellules. La purification des cellules Dcx+ ainsi qu'une étude clonale a permis de montrer qu'elles se comportent comme des progéniteurs multipotents avec une capacité d'autorenouvellement restreinte. Par ailleurs, j'ai montré que les cellules Dcx+ peuvent réverter vers un état Dcx- et que le gène Dcx est régulé par les voies NOTCH et SHHGlioblastomas (GB) are the most common primary tumors of the CNS and are particularly resistant to radio/chemotherapy. They generally have a solid and infiltrative component. The latter being difficult to remove by surgery will be partly responsible for tumor recurrence. One of the major advances in the field is highlighted in the Gb of subpopulations with features of neural precursors. Cancer cells use specific gene networks to maintain their proliferation and undifferentiated state. One approach to eliminate these cancer cells would be to target transcription factors involved in the proliferation or to force their differentiation. To this end, I studied the role of NKX2.2 and NGN2 from 3 primary multipotent cultures. The results show that NKX2.2 expression in these cultures is necessary for survival, proliferation and ability to form neurospheres. Conversely, overexpression of NGN2 led to massive apoptosis, proliferation arrest with formation of neurons, some of which are electrophysiologically active. A different approach would be to target proteins involved in migration to limit the invasive component. Previous studies have shown a key role of DCX in the migration of young neurons during development. The strong expression of DCX in some Gb led me to study the regulation and the role of this gene. In vitro, the results show that DCX is expressed by a subpopulation of cells. Purification of Dcx+ cells and clonal study has shown that they behave as multipotent progenitors with limited self-renewal capacity. I also found that Dcx+ cells can revert back to a Dcx- state and that DCX is regulated by SHH and NOTCH pathways
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岡原, 京平. "糖脂質ガラクトシルセラミドのオリゴデンドロサイト特異的な発現調節機構に関する研究." 京都大学 (Kyoto University), 2014. http://hdl.handle.net/2433/192153.
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Jarrar, Wassan [Verfasser], and Hans-Henning [Akademischer Betreuer] Arnold. "Genetic analysis of Nkx2.2 and Nkx2.9 transcription factors in mouse brain development: specific functions in the hindbrain / Wassan Jarrar ; Betreuer: Hans-Henning Arnold." Braunschweig : Technische Universität Braunschweig, 2014. http://d-nb.info/1175821233/34.
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Doyle, Michelle Joanne. "Multiple transcriptional activities of NKX2.2 in the embryonic and adult pancreas /." Connect to full text via ProQuest. Limited to UCD Anschutz Medical Campus, 2006.
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Thesis (Ph.D. in Molecular Biology) -- University of Colorado at Denver and Health Sciences Center, 2006.Typescript. Includes bibliographical references (leaves 137-151). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;
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Chao, Christina Seng. "The roles of Nkx2.2 in determination of mouse islet cell fates /." Connect to full text via ProQuest. Limited to UCD Anschutz Medical Campus, 2007.
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Thesis (Ph.D. in Cell & Developmental Biology) -- University of Colorado Denver, 2007.Typescript. Includes bibliographical references (leaves 144-158). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;
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Hill, Katy Victoria. "Regulation of Nkx2.2 gene expression in the vertebrate neural tube : a target of graded Sonic hedgehog signalling." Thesis, University College London (University of London), 2007. http://discovery.ucl.ac.uk/1444785/.
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Sonic hedgehog (Shh) is a morphogen implicated in the developmental patterning of many vertebrate tissues. One such tissue is the neural tube (NT). In ventral regions of the NT distinct neuronal subtypes emerge in precise spatial order from progenitor cells arrayed along its dorsal-ventral axis. Shh regulates this process by controlling the expression patterns of transcription factors in progenitor cells. In addition, cross- repressive interactions between pairs of transcription factors, expressed in adjacent regions, ensures the generation of defined domains. The regulation of Nkx2.2 and Nkx2.9 expression represents an example of this mechanism. The expression of these genes is restricted to a ventral (p3) domain, comprising neural progenitors dorsal to the floor plate. Induction of Nkx2.2 and Nkx2.9 requires high levels of Shh signalling. In part this appears to be because the homeodomain protein Pax6 must be repressed to allow Nkx2.2/2.9 induction. We have analysed the regulatory regions of the Nkx2 genes in order to understand the molecular mechanisms underpinning their expression pattern. The 5' flanking region of Nkx2.2 and Nkx2.9 contains a 250bp block of highly conserved DNA (CNCR) that is found in human, mouse, Fugu and zebrafish. This region includes a binding site for the transcriptional regulators of the Shh pathway: Gli (GBS). Using a BAC homologous recombination system and assays in zebrafish, we provide evidence that the CNCR is required to direct Nkx2.2a-like gene expression. Mouse in vivo reporter assays using fragments containing the CNCR of zNkx2.2a, indicate the CNCR is sufficient to direct reporter gene expression in the p3 domain of the NT. Mutational analysis indicates that the GBS is necessary but not sufficient to account for this expression profile. In vivo assays further suggest correct Nkx2.2 expression requires input from additional transcriptional activators as well as a floor plate repressor. All these factors appear to act through regulatory elements within the CNCR.
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Wankerl, Ludwig Verfasser], and Gunter [Akademischer Betreuer] [Meister. "Characterization of CAMTA1 and Nkx2.2 in the context of glioblastoma cancer stem cell biology / Ludwig Wankerl. Betreuer: Gunter Meister." Regensburg : Universitätsbibliothek Regensburg, 2016. http://d-nb.info/1083251341/34.
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Kordowich, Simon Verfasser], Ernst A. [Akademischer Betreuer] Wimmer, Ahmed [Akademischer Betreuer] Mansouri, Detlef [Akademischer Betreuer] [Doenecke, and Annette [Akademischer Betreuer] Borchers. "Funktionelle Charakterisierung der Transkriptionsfaktoren Nkx2.2 und Arx in der Entwicklung der endokrinen Zellen im murinen Pankreas / Simon Kordowich. Gutachter: Ahmed Mansouri ; Detlef Doenecke ; Annette Borchers. Betreuer: Ernst A. Wimmer." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2011. http://d-nb.info/1042641218/34.
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Schnittert-Hübener, Sarah [Verfasser]. "Analyse des phänotypischen Spektrums von NKX2.1 Genmutationen / Sarah Schnittert-Hübener." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2009. http://d-nb.info/1027814115/34.
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Lemmer, Julia. "Differentielle Regulation der alternativen Promotoren des Endothelin-Konvertierungsenzyms-1 durch den kardialen Transkriptionsfaktor Nkx2.5." [S.l.] : [s.n.], 2002. http://www.diss.fu-berlin.de/2002/255/index.html.
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Nguyen, Thi-Hong-Minh. "Functional defects and molecular mechanisms of Left Ventricular Noncompaction (LVNC) in Nkx2.5 mutant mice." Thesis, Aix-Marseille, 2016. http://www.theses.fr/2016AIXM4039.
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La LVNC est une cardiomyopathie rare, caractérisée par une hypertrabéculation et de profonds replis du ventricule gauche. A ce jour, nous ne savons toujours pas si la LVNC résulte d'un défaut se produisant durant le développement cardiaque et si sa gravité dépend du stade embryonnaire auquel l'arrêt de la compaction se produit. Notre objectif a été d'étudier l'évolution pathologique de la LVNC en caractérisant les défauts fonctionnels et en identifiant les mécanismes moléculaires dans des modèles de souris présentant un développement anormal des trabécules ventriculaires. Pour établir un modèle de LVNC, nous avons généré des souris KO conditionnel pour Nkx2.5 grâce au système Flox/loxP inductible par injection de tamoxifène qui active la recombinaison Cre. Nous avons ainsi supprimé l'allèle Nkx2.5 dans l'oreillette et les cardiomyocytes dérivant des trabécules. Nous avons choisi de sipprimé Nkx2.5 au stade embryonnaire E10 quand le trabécule s'accroît, au stade E14 quand il se compacte, ou juste après la naissance quand le cœur a terminé son processus de compaction. En résumé, nous avons réussi à générer différents modèles de LVNC, dans lesquelles nous avons pu étudier cette pathologie, en supprimant le facteur de transcription Nkx2.5 dans les oreillettes et les cardiomyocytes dérivés des trabécules. Nous avons également confirmé que la sévérité de la LVNC dépend du stade de développement du trabécule auquel le défaut se produit. Peu de publications décrivent à ce jour les mécanismes responsables de l'état inflammatoire observé dans la LVNC, nos résultats sont donc prometteurs pour de futures recherches dans cette voieLVNC is a rare cardiomyopathy, characterized by hypertrabeculation and deep trabecular recesses in the left ventricle. It is still unclear whether LVNC results from a defect occurring during cardiac development. One hypothesis to consider is that the severity of LVNC depends on which embryonic stage the arrest of myocardial compaction occurs. Our aim was to study the pathological evolution of LVNC by characterizing functional defects and identifying molecular mechanisms in mouse models with abnormal ventricular trabeculae development. To establish a LVNC mouse model, we generated specific Nkx2.5 conditional knockout mice to delete Nkx2.5 allele in atria and trabecular derived cardiomyocytes at embryonic stages when trabeculae arise (at around E10), or start to compact (at around E14), or at neonatal stages (after birth) when the heart is almost finish compaction step. After all, we were successful in generating several LVNC mouse models by the conditional deletion of Nkx2.5 transcription factor in atria and trabecular derived cardiomyocytes. These mouse models are suitable for studying LVNC pathology. We also confirmed the hypothesis that the severity of LVNC depends on stages when disturbances in the trabecular development occur. Hypertrabeculation, cardiac conduction defects, decreased ejection fraction, and existence of fibrosis are robustly observed following deletion at E10.5/11.5 meaning that the deletion at early stage of trabecular development causes the most severe pathological phenotype of LVNC. There had been just a few publications showing inflammation in LVNC heart, which could be a very good finding for future researches
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Jabs, Normund. "Expressionsanalyse des Transkriptionsfaktors Nkx6.1 in Mus musculus (Linneaus, 1758)." [S.l.] : [s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=975901818.
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Patel, Ruchi. "The role of homeobox gene NKX3.1 in prostate cancer." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:ca3d2321-3296-4c61-a5fb-57f1d99f9bb1.
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NKX3.1, a prostate specific homeobox gene is a known marker of prostate epithelium during embryogenesis and is also expressed subsequently through different stages of prostate differentiation. However, all studies on NKX3.1 are focused on its regulation by androgen receptor (AR). The aim of this project is to establish the role of NKX3.1 in differentiation in prostate cancer, independent of AR regulation. In this thesis, I characterize the cell lines in terms of their differentiation capabilities in 3D, expression levels of NKX3.1 and the mismatch repair status. The genes potentially involved in differentiation and regulators of NKX3.1 are also identified using microarray data of the cell lines (Chapter 3). Although NKX3.1 plays a key role in prostate development no studies have been conducted on the effect of NKX3.1 expression on differentiation capabilities of prostate cell lines. In Chapter 4, this was investigated by siRNA mediated knockdown of NKX3.1 in 22Rv1 cell line and overexpression of NKX3.1 in PC3 (designated PC3-Nkx3.1) and PNT1a cells followed by growth in 3D. These functional studies show that the expression of NKX3.1 is vital for lumen formation in 3D, which is used as a measure of differentiation. The microarray data and overexpression of NKX3.1 studies suggest that this gene may also be involved in inhibiting epithelial to mesenchymal transition (EMT). Homeobox B13 (HOXB13) was identified as one of the downstream targets of NKX3.1. NKX3.1 and HOXB13 expression levels are positively correlated not only in the panel of prostate cell lines but also in the NKX3.1 overexpression and knockdown studies (Chapter 5). The results of the work presented in this thesis demonstrate that there is a striking parallel between the function of NKX3.1 in prostate and Caudal-type homeobox 1 (CDX1) in the colon and rectum. In conclusion, NKX3.1 plays a key role as a tumour suppressor in prostate cancer by controlling differentiation of prostate cancer cells.
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Thorwarth, Anne [Verfasser]. "Molekulargenetische und molekularzytogenetische Analyse des NKX2.1 Gens bei Patienten mit kongenitaler Hypothyreose und assoziierter Choreoathetose / Anne Thorwarth." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2014. http://d-nb.info/1062949552/34.
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Zakariyah, Abeer. "The Characterization of a Human Disease-Associated Mutation Nkx2.5 R142C Using In vitro and In vivo Models." Thesis, Université d'Ottawa / University of Ottawa, 2017. http://hdl.handle.net/10393/35817.
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Nkx2.5 is a cardiac transcription factor that plays a critical role in heart development. In humans, heterozygous mutations in the NKX2.5 gene result in congenital heart defects (CHDs), but the molecular mechanisms by which these mutations cause the defects are still unknown. NKX2.5 R142C is a mutation that is found to be associated with atrial septal defect and atrioventricular block in 13 patients from one family. The R142C mutation is located within both the DNA-binding domain and the nuclear localization sequence of NKX2.5 protein. The pathogenesis of CHDs in humans with R142C point mutation is not well understood. Also, a previous study in our laboratory has identified Mypt1/PP1 as a novel interacting partner of Nkx2.5 in stem cells during cardiomyogenesis. Nkx2.5 has a PP1-binding consensus sequence RVxF located in the N-terminus of the homeodomain. Notably, the PP1-binding sequence, RVxF, is mutated from arginine to cysteine in patients with the R142C heterozygous mutation. However, the ability of the R142C mutation to bind to the Mypt1/PP1 complex has not been investigated yet. The following thesis addresses the functional deficit associated with R142C by utilizing a combination of in vitro, and in vivo models. It also addresses the interaction of Mypt1/PP1 with the R142C mutation. We have generated a heterozygous mouse embryonic stem cell (mESC) line, harboring the murine homologue (R141C) of the human mutation R142C in Nkx2.5 gene. We show reduced cardiomyogenesis and impaired subcellular localization of Nkx2.5 protein in Nkx2.5R141C/+ mESCs. Gene expression profiling of Nkx2.5R141C/+ mESCs revealed a global misregulation of genes important for heart development and identified putative direct target genes of Nkx2.5 that are affected by the R141C heterozygous mutation. We also generated a mouse model harboring the human mutation R142C. We show that the Nkx2.5R141C/R141C homozygous embryos are developmentally arrested around E10.5 with delayed heart morphogenesis. Moreover, Nkx2.5R141C/+ newborn mice are grossly normal but show variable cardiac defects and downregulation of ion channel genes that later cause AV block in adult mice. Finally, we show that the R141C mutant binds to the Mypt1/PP1 complex but is not inhibited or translocated to the perinuclear region in the presence of Mypt1/PP1 as the WT Nkx2.5 is.
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Schisler, Jonathan Cummings. "New roles of the transcription factor NKX6.1 in beta cell biology." Access to abstract only; dissertation is embargoed until after 5/15/2007, 2006. http://www4.utsouthwestern.edu/library/ETD/etdDetails.cfm?etdID=164.
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Markowski, Mark Christopher. "Inflammatory cytokines induce ubiquitination and loss of the prostate suppressor protein NKX3.1." Connect to Electronic Thesis (CONTENTdm), 2008. http://worldcat.org/oclc/454182234/viewonline.
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Ramos, Helton Estrela [UNIFESP]. "Disgenesias tiroidianas: estudo clínico e pesquisa molecular dos genes candidatos PAX8, receptor de TSH e NKX2.5 em pacientes com hipotiroidismo congênito." Universidade Federal de São Paulo (UNIFESP), 2007. http://repositorio.unifesp.br/handle/11600/23674.
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Decker, Josua [Verfasser]. "Analyse des signalinduzierten Verlusts des Tumorsuppressors NKX3.1 in Prostatitis und Prostatakarzinomzellen / Josua Decker." Ulm : Universität Ulm, 2016. http://d-nb.info/1112603093/34.
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Borglund, Kajsa. "Utvärdering och implementering av antikroppen anti-NKX3.1 för diagnostik av metastaserande prostata-adenocarcinom." Thesis, Jönköping University, HHJ, Avd. för naturvetenskap och biomedicin, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:hj:diva-53076.
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Prostata-adenocarcinom är den vanligaste cancerformen hos män. Anti-PSA och anti-P501S är frekvent använda antikroppar för diagnostisering av prostata-adenocarcinom i vävnadsbiopsier, men kan ha svag eller negativ infärgning av antigenen. Antikroppen anti-NKX3.1 har visat sig ha en högre sensitivitet och starkare infärgning än anti-PSA och anti-P501S vid prostata-adenocarcinom. Det rekommenderas att använda anti-NKX3.1 tillsammans med anti-PSA och/eller anti-P501S för att diagnostisera metastaserande prostata-adenocarcinom. Studiens syfte var att utvärdera och implementera antikroppen anti-NKX3.1 för diagnostisering av metastaserande prostata-adenocarcinom. Positiva vävnadskontroller (prostatavävnad och testisvävnad) och negativ vävnadskontroll (appendix) färgades med anti-NKX3.1 i Roche Ventana Benchmark ULTRA med immunohistokemisk färgning. Förbehandlingarna CC1 mild, CC1 standard och Proteas 1 samt visualiseringskiten OptiView samt UltraView jämfördes. Spädningen optimerades från 1:50-1:200. Vävnadssnitten graderades sedan från 1 (ingen infärgning)-6 (starkast infärgning). Visualiseringskitet OptiView samt förbehandlingen CC1 standard gav den starkaste infärgningen med anti-NKX3.1 och valdes som det optimala visualiseringskitet respektive förbehandlingen. Med tanke på materialkostnad och smidighet valdes spädningen 1:100 som den optimala spädningen.Prostate adenocarcinoma is the most common form of cancer in men. Anti-PSA and anti-P501S are frequently used antibodies for diagnosing of prostate adenocarcinoma in tissue biopsies but may show weak or negative staining of the antigens. Antibody anti-NKX3.1 has been shown to have a higher sensitivity and stronger staining then anti-PSA and anti-P501S in prostate adenocarcinoma. It is recommended to use anti-NKX3.1 along with anti-PSA and/or anti-P501S to diagnose metastases of prostate adenocarcinoma. The purpose of the study was to evaluate and implement the antibody anti-NKX3.1 for diagnosis of metastatic prostate adenocarcinoma. Positive tissue controls (prostate and testis) and negative tissue control (appendix) were stained with anti-NKX3.1 immunohistochemical staining in Roche Ventana Benchmark ULTRA machine. The pre-treatments CC1 mild, CC1 standard and Protease 1 and the visualization kits OptiView and UltraView were compared. The dilution was optimized from 1:50-1:200. Immunohistochemical staining of the tissue was graded from 1 (no staining) - 6 (strongest staining). Visualization kit OptiView and pre-treatment CC1 standard gave the strongest staining with anti-NKX3.1 and were chosen as the optimal visualization kit and pre-treatment. Considering the material cost and flexibility, the 1:100 dilution was chosen as the optimal dilution.
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Krude, Heiko. "Beschreibung drei neuer endokrinologischer Syndrome." Doctoral thesis, [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=972715754.
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Muhlbradt, Erin Elizabeth. "IGFBP-3 mediates the effect of tumor suppressor NKX3.1 on prostate cancer cell proliferation." Connect to Electronic Thesis (CONTENTdm) Connect to Electronic Thesis (ProQuest), 2008. http://worldcat.org/oclc/642191611/viewonline.
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Alves, Carlos Eduardo Fonseca. "Avaliação epigenética dos genes NKX3.1 E CDH1 e expressão do C-MYC, NKX3.1 e E-Caderina por imuno-histoquímica em microarranjo de tecido (TMA) de lesões pré-neoplásicas e neoplásicas na próstata de cães." Botucatu, 2016. http://hdl.handle.net/11449/143108.
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Orientador: Renée Laufer AmorimResumo: A próstata canina é um bom modelo para estudos comparados entre o cão e o homem, uma vez que essas duas espécies desenvolvem espontaneamente carcinoma de próstata (CP). Para melhor caracterização do CP canino, a presente pesquisa foi dividia em quatro capítulos que avaliam diferentes aspectos dos CPs em cães. A atrofia inflamatória proliferativa (PIA) é uma lesão pré-neoplásica descritas em humanos e pouco estuda em cães. Nós caracterizamos essa lesão em cães e identificamos uma forte relação entre a localização topográfica da PIA com os CPs. Além disso, foi identificada a perda de expressão gênica e proteica de PTEN e AR na PIA. Esses fatores associados corroboram com o potencial pré-neoplásico desta lesão em cães. Um achado interessante foi a alta expressão de P63 na PIA e em um grupo de CP caninos. Para melhor caracterizar este grupo, foi avaliada a expressão imuno-histoquímica de diferentes citoqueratinas e outras proteínas relacionadas ao desenvolvimento do CP em humanos. Os carcinomas que apresentam expressão de P63 apresentaram padrões morfológicos com escore de Gleason alto e um fenótipo mais agressivo quando comparado à tumores que não apresentação expressão de P63. Posteriormente, a expressão gênica e proteica de E-caderina, NKX3.1 e C-MYC foi avaliada em CP como marcadores nas diferentes lesões. Além disso, nós avaliamos a metilação como mecanismo regulatórios dos genes CDH1 e NKX3.1. Foi possível identificar a perda de E-caderina e NKX3.1 nos tumores, comparado à ... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The canine prostate gland can be used as a model to human prostatic disease since dogs and men are the only species that spontaneously develop prostate carcinoma (PC). To better characterize the canine PC, this research was divided into four chapters that evaluated different aspects of the PC in dogs. The proliferative inflammatory atrophy (PIA) is a pre-neoplastic lesion described in humans and few studies in dogs describe it as a preneoplastic lesion. This study characterized PIA in dogs and identified a strong relationship between the PIA topography with PC. In addition, we identified the loss of PTEN and AR expression in PIA. These findings demonstrated the potential of PIA as a preneoplastic lesion in dogs. An interesting finding in this research was the high expression of P63 in PIA and a group of PC. This study found a group of PC showing P63 positive expression in neoplastic epithelial cells. Thus, these tumors were selected to better characterize them using immunohistochemistry. These tumors had an aggressive phenotype and presented high expression of AKT and C-MYC and loss of NKX3.1. Further, we selected a usual group of PC and evaluate the expression of E-cadherin, NKX3.1 and C-MYC. In addition, we evaluated the methylation as a regulatory mechanism of CDH1 and NKX3.1 genes. We have identified loss of E-cadherin and NKX3.1 in PC compared to normal prostate and C-MYC overexpression. The expression of E-cadherin was related to overall survival and Gleason score. The ... (Complete abstract click electronic access below)
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Dritsoula, A. "Regulation of NKX2-5 in blood vessels." Thesis, University College London (University of London), 2017. http://discovery.ucl.ac.uk/1551692/.
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NKX2-5 is a transcription factor required for the formation of the heart and vessels during development. Postnatal expression is significantly downregulated, and then re-activated in diseased conditions characterised by vascular remodelling. However, the mechanisms regulating NKX2-5 activation in diseased vessels remain unknown. The aim of this thesis is to identify these mechanisms and provide information on how the gene contributes to cardiovascular pathologies, such as sclerodermaassociated pulmonary hypertension. A case-control genetic association study was performed in two independent cohorts of scleroderma patients. Associated SNPs located in the NKX2-5 genomic region were cloned into reporter vectors, and transcriptional activity was assessed by reporter-gene assays. Associated SNPs were further evaluated through proteinDNA binding assays, chromatin immunoprecipitation and RNA silencing. Signalling mechanisms activating NKX2-5 expression were investigated in vascular endothelial and smooth muscle cells using a panel of selective inhibitors. Meta-analysis across the two independent cohorts revealed that rs3131917 was associated with scleroderma. Rs3132139, downstream of NKX2-5, was significantly associated with pulmonary hypertension in both cohorts. The region containing rs3132139 and rs3131917 was shown to be a novel functional enhancer, which increased NKX2-5 transcriptional activity through the binding of GATA6, c-JUN, and MEF-2c. An activator TEAD/YAP1 complex was shown to bind at rs3095870, another functional SNP upstream of NKX2-5 transcription start site, which showed marginal association with scleroderma. Signalling mechanisms, involving TGF-β, ERK5, AKT and hypoxia, stimulated NKX2-5 expression during phenotypic modulation of vascular endothelial and smooth muscle cells. Overall, the data showed that NKX2-5 is genetically associated with scleroderma and pulmonary hypertension. Functional evidence revealed a regulatory mechanism, activated by TGF-β, which results in NKX2-5 transcription in human vascular smooth muscle cells through the interaction of an upstream promoter and a novel downstream enhancer. These regulatory mechanisms can act as a model for NKX2-5 activation in cardiovascular disease characterised by vascular remodelling.
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Nakashima, Yasuhiro. "The Search for Nkx2-5-regulated Genes Using Purified Embryonic Stem Cell-derived Cardiomyocytes with Nkx2-5 Gene Targeting." Kyoto University, 2010. http://hdl.handle.net/2433/120558.
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Mittelhaus, Sebastian [Verfasser]. "Mutationsanalysen in den Genen NKX2.5 und BMP4 bei Patienten mit Ostium secundum Defekt (ASD II) sowie Evaluierung angewendeter Mutationsdetektionstechniken im Vergleich zur DHPLC-Methode / Sebastian Mittelhaus." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2012. http://d-nb.info/1030290768/34.
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Müller, Myriam. "Die Funktion des Transkriptionsfaktors Nkx6.1 bei der Entwicklung von Motoneuronen im Hirnstamm von Mus musculus (Linneaus, 1758)." [S.l.] : [s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=969421397.
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Li, Wenzhao. "A homeobox protein, NKX6.1, up-regulates interleukin-6 expression for cell growth in basal-like breast cancer cells." Kyoto University, 2016. http://hdl.handle.net/2433/216184.
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Goedel, Alexander Rudolf Peter [Verfasser], Karl-Ludwig [Akademischer Betreuer] Laugwitz, Martin [Akademischer Betreuer] Halle, and Georg [Akademischer Betreuer] Schmidt. "Die Rolle von Isl1 und Nkx2.5 im transkriptionellen Netzwerk der kardialen Progenitorzelle / Alexander Rudolf Peter Goedel. Gutachter: Martin Halle ; Georg Schmidt ; Karl-Ludwig Laugwitz. Betreuer: Karl-Ludwig Laugwitz." München : Universitätsbibliothek der TU München, 2012. http://d-nb.info/1025538625/34.
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Goedel, Alexander [Verfasser], Karl-Ludwig [Akademischer Betreuer] Laugwitz, Martin [Akademischer Betreuer] Halle, and Georg [Akademischer Betreuer] Schmidt. "Die Rolle von Isl1 und Nkx2.5 im transkriptionellen Netzwerk der kardialen Progenitorzelle / Alexander Rudolf Peter Goedel. Gutachter: Martin Halle ; Georg Schmidt ; Karl-Ludwig Laugwitz. Betreuer: Karl-Ludwig Laugwitz." München : Universitätsbibliothek der TU München, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:91-diss-20120628-1081655-1-4.
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日高, 京子, Kyoko Hidaka, 佳子 三輪, Keiko Miwa, 豊明 室原, Toyoaki Murohara, 謙次 笠井, et al. "Paracrine factors of vascular endothelial cells facilitate cardiomyocyte differentiation of mouse embryonic stem cells." Elsevier, 2008. http://hdl.handle.net/2237/10608.
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Alves, Carlos Eduardo Fonseca [UNESP]. "Expressão de c-MYC, NKX3.1 e E-Caderina por Imuno-Histoquímica em Microarranjo de Tecido (TMA) de Lesões Pré- Neoplásicas e Neoplásicas na Próstata De Cães." Universidade Estadual Paulista (UNESP), 2013. http://hdl.handle.net/11449/89317.
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000735057.pdf: 1377720 bytes, checksum: 27a124629e14b0c3deac7a29c3f1f0e7 (MD5)A próstata canina pode utilizada como modelo para estudo comparativo de afecções desta glândula no homem, constituindo as únicas espécies em que o carcinoma de próstata (CaP) se desenvolve espontaneamente. A próstata canina é sede de diversos processos patológicos que comumente acometem cães adultos e idosos. O papel do oncogene c-MYC e supressores tumorais NKX3.1 e E-caderina são bem estabelecidos no processo de carcinogênese da próstata humana, no entanto, não há pesquisas que relatem a expressão proteica dessas proteínas na progressão do CaP canino. Neste contexto, este estudo teve por objetivo avaliar a expressão proteica de c-MYC, NKX3.1 e Ecaderina na próstata normal, com Hiperplasia Prostática Benigna (HPB), Atrofia Inflamatória Proliferativa (PIA) e CaP e avaliar o papel dessas proteínas no processo de gênese tumoral. O presente estudo foi realizado em uma plataforma de Microarranjo de Tecido (TMA) contendo diferentes lesões prostáticas. Foi realizada Imuno-histoquímica com os anticorpos c-MYC, NKX3.1 e E-caderina pelo método da peroxidase e DAB. A análise estatística foi realizada pelo teste de Qui-quadrado ou teste de Fischer para determinar associação entre as variáveis. A proteína c-MYC apresentou maior expressão no CaP e na PIA quando comparada com a próstata normal e com HPB (p= 0,0068). Observou-se maior número de amostras com ausência da expressão do NKX3.1 no CaP (94.75% ) e PIA (100%) quando comparando com a próstata normal e HPB (p=0,0022). Em relação a E-caderina houve maior número de amostras positivas na HPB e próstata normal quando comparado CaP e a PIA, no entanto sem diferença estatística. O processo de gênese tumoral na próstata canina esta associado com o aumento de expressão proteica do c-MYC e a perda da expressão proteica do NKX3.1
The dog is the only species besides humans that develop spontaneous and naturally prostatic carcinoma (PCa) with high frequency. The canine model is primarily used for studies of molecular mechanisms of PCa and provides a natural animal model for studies of potential therapies. PCa in men presents CMYC oncogene mutation and reduced E-cadherin and NKX3.1 protein expression. In this context our objective was to evaluate NKX3.1, C-MYC and E-cadherin expression in benign prostatic hyperplasia (BPH), prostatic inflammatory atrophy (PIA) and PCa in dogs and verify the role of these proteins in the progression to prostate cancer. A tissue microarray (TMA) slide was constructed and immunohistochemistry with antibodies C-MYC, NKX3.1 and E-cadherin was performed with peroxidase method and DAB. Chi-square or Fisher exact test was used to determine the association between the categorical variables. The evaluation of the C-MYC oncogene protein showed higher cytoplasmic positivity in canine PCa and PIA compared to BPH (p= 0.0068). We observed a decrease of NKX3.1 expression in 94.75% of PCa, 100% of PIA in comparison to BPH (p=0.0022). E-cadherin expression was higher in BPH and PIA in comparison to PCa, with no statistical difference. The carcinogenesis process of canine prostatic lesion may be related to gain of CMYC and loss of NKX3.1
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Alves, Carlos Eduardo Fonseca. "Expressão de c-MYC, NKX3.1 e E-Caderina por Imuno-Histoquímica em Microarranjo de Tecido (TMA) de Lesões Pré- Neoplásicas e Neoplásicas na Próstata De Cães /." Botucatu, 2013. http://hdl.handle.net/11449/89317.
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Orientador: Renée Laufer AmorimBanca: Veridiana Maria Brianezi Dignani de Moura
Banca: Marcela Marcondes Pintos Rodrigues
Resumo: A próstata canina pode utilizada como modelo para estudo comparativo de afecções desta glândula no homem, constituindo as únicas espécies em que o carcinoma de próstata (CaP) se desenvolve espontaneamente. A próstata canina é sede de diversos processos patológicos que comumente acometem cães adultos e idosos. O papel do oncogene c-MYC e supressores tumorais NKX3.1 e E-caderina são bem estabelecidos no processo de carcinogênese da próstata humana, no entanto, não há pesquisas que relatem a expressão proteica dessas proteínas na progressão do CaP canino. Neste contexto, este estudo teve por objetivo avaliar a expressão proteica de c-MYC, NKX3.1 e Ecaderina na próstata normal, com Hiperplasia Prostática Benigna (HPB), Atrofia Inflamatória Proliferativa (PIA) e CaP e avaliar o papel dessas proteínas no processo de gênese tumoral. O presente estudo foi realizado em uma plataforma de Microarranjo de Tecido (TMA) contendo diferentes lesões prostáticas. Foi realizada Imuno-histoquímica com os anticorpos c-MYC, NKX3.1 e E-caderina pelo método da peroxidase e DAB. A análise estatística foi realizada pelo teste de Qui-quadrado ou teste de Fischer para determinar associação entre as variáveis. A proteína c-MYC apresentou maior expressão no CaP e na PIA quando comparada com a próstata normal e com HPB (p= 0,0068). Observou-se maior número de amostras com ausência da expressão do NKX3.1 no CaP (94.75% ) e PIA (100%) quando comparando com a próstata normal e HPB (p=0,0022). Em relação a E-caderina houve maior número de amostras positivas na HPB e próstata normal quando comparado CaP e a PIA, no entanto sem diferença estatística. O processo de gênese tumoral na próstata canina esta associado com o aumento de expressão proteica do c-MYC e a perda da expressão proteica do NKX3.1
Abstract: The dog is the only species besides humans that develop spontaneous and naturally prostatic carcinoma (PCa) with high frequency. The canine model is primarily used for studies of molecular mechanisms of PCa and provides a natural animal model for studies of potential therapies. PCa in men presents CMYC oncogene mutation and reduced E-cadherin and NKX3.1 protein expression. In this context our objective was to evaluate NKX3.1, C-MYC and E-cadherin expression in benign prostatic hyperplasia (BPH), prostatic inflammatory atrophy (PIA) and PCa in dogs and verify the role of these proteins in the progression to prostate cancer. A tissue microarray (TMA) slide was constructed and immunohistochemistry with antibodies C-MYC, NKX3.1 and E-cadherin was performed with peroxidase method and DAB. Chi-square or Fisher exact test was used to determine the association between the categorical variables. The evaluation of the C-MYC oncogene protein showed higher cytoplasmic positivity in canine PCa and PIA compared to BPH (p= 0.0068). We observed a decrease of NKX3.1 expression in 94.75% of PCa, 100% of PIA in comparison to BPH (p=0.0022). E-cadherin expression was higher in BPH and PIA in comparison to PCa, with no statistical difference. The carcinogenesis process of canine prostatic lesion may be related to gain of CMYC and loss of NKX3.1
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Ersözlü, Sara [Verfasser]. "Effects of pre- and postnatal deletion of the transcription factor Nkx2-1 on the expression of NGF, trkA, trkB and p75NTR in mice and a clinical update on NKX2-1 haploinsufficiency in humans / Sara Ersözlü." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2021. http://d-nb.info/1234985268/34.
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Choquet, Caroline. "Lineage analysis of ventricular trabeculations to decipher the role of Nkx2-5 in conduction system development." Thesis, Aix-Marseille, 2018. http://www.theses.fr/2018AIXM0217.
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La coordination des battements cardiaques est assurée par la propagation rapide de l’activité électrique dans le système de conduction ventriculaire (SCV). Etudier la formation du SCV est crucial pour comprendre l’origine des troubles de conduction de l’adulte. Au cours de l’embryogénèse le SCV est issu des trabécules, des projections myocardiques à la surface interne des ventricules. Les trabécules subissent une compaction avant la naissance qui est nécessaire à la maturation du myocarde. Des défauts au cours des étapes embryonnaires seraient en cause dans l’apparition d’une cardiomyopathie rare nommée Non-Compaction du Ventricule Gauche (LVNC). LVNC et troubles de conduction observés chez des patients et des souris mutantes sont associés au gène NKX2-5, qui code pour un facteur de transcription clé pour le développement du cœur.Mon premier objectif de thèse consiste à étudier le rôle de Nkx2-5 dans l’origine et l’évolution pathologique de la LVNC. Mon second objectif consiste à définir le rôle de Nkx2-5 au cours du développement trabéculaire et de la formation du SCV afin de comprendre l’origine de l’hypoplasie du SCV chez les souris Nkx2-5 hétérozygotes.Des systèmes génétiques complexes ont été utilisés pour induire la délétion de Nkx2-5 dans les trabécules à plusieurs étapes du développement et suivre le destin des trabécules afin d’établir la fenêtre de ségrégation du lignage conducteur. L’ensemble de mes résultats ont permis d’identifier les étapes clés du développement du SCV et un rôle majeur de Nkx2-5 afin de mieux appréhender les troubles de conduction. Enfin mes résultats ont mis en évidence une nouvelle cible potentielle pour des perspectives thérapeutiquesThe rapid propagation of electrical activity through the ventricular conduction system (VCS) controls the spatiotemporal contraction of the ventricles. A better understanding of VCS development is crucial to comprehend the etiology of conduction disturbances observed in adults. During embryogenesis, the VCS originates from ventricular trabeculae that are myocardial protrusions in the lumen of the ventricles. Before birth, trabeculae undergo a compaction step required for maturation of the myocardial wall. Impairment of these developmental steps can lead to the apparition of a rare cardiomyopathy referred as Left Ventricular Non-Compaction (LVNC). LVNC and conduction defects have been observed in patients and mutant mice carrying mutations in NKX2-5, encoding a key transcriptional regulator of heart development.The first objective of my thesis is to decipher the involvement of Nkx2-5 in the origin and pathological evolution of the LVNC. The second objective is to decipher the temporal requirement of Nkx2-5 during trabecular morphogenesis and VCS development and to understand the origin of the VCS hypoplasia observed in Nkx2-5 heterozygous mice. Complex genetic technics were used to induce the deletion of Nkx2-5 in ventricular trabeculae at different developmental time points and to trace the fate of trabeculae and establish the temporal window of the conductive lineage segregation during development.Altogether, my results identify key steps in the VCS development, demonstrate a crucial role of Nkx2-5 and contribute to improve understanding of conduction defects. Interestingly, my results potentially identify new target cells for therapeutic intervention
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Välimäki, M. (Mika). "Discovery of cardioprotective isoxazole-amide compounds targeting the synergy of transcription factors GATA4 and NKX2-5." Doctoral thesis, M. Välimäki, 2018. http://urn.fi/urn:isbn:9789529412525.
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Abstract Acute myocardial infarction is a life-threatening condition that occurs as a result of reduced blood flow in the cardiac muscle, eventually leading to tissue damage. In infarcted areas, cardiomyocytes have insufficient ability to proliferate and replace the injured cells, which is associated with a deficient pumping capacity. A strictly regulated combinatorial interplay of transcription factors, e.g., GATA4, NKX2-5, TBX5, and MEF2C, orchestrates cardiac type gene expression during the cardiomyocyte differentiation and maturation processes. The aim of the present study was to (i) characterize the protein-protein interaction of the cardiac transcription factors GATA4-NKX2-5, (ii) evaluate the chemical agents that modify the synergy of GATA4-NKX2-5 in vitro, (iii) examine the capacity of the lead compound to promote myocardial repair in vivo after myocardial infarction and other cardiac injuries and (iv) study the structural features of the compound important for metabolism and cytotoxicity. Integration of the experimental mutagenic data with computational modeling suggests that the structural architecture of the GATA4-NKX2-5 interaction resembles the protein structure of the conserved DNA binding domain of nuclear receptors. Fragment-based screening, reporter gene-based optimization and pharmacophore searching were utilized to identify the most potent lead compound targeting the GATA4-NKX2-5 interaction: N-[4-(diethylamino)phenyl]-5-methyl-3-phenylisoxazole-4-carboxamide. This compound presented anti-hypertrophic effects in vitro and cardioprotective effects in vivo. In addition, structural analysis of the lead compound revealed the signature molecular features for metabolism and cytotoxicity. Current drug treatments are able to delay, but not prevent the progress of the heart failure; therefore, modulators of protein-protein interactions of key transcription factors may represent a novel class of pharmaceuticals for cardiac remodeling and repairTiivistelmä Sydäninfarkti on henkeä uhkaava verenkierron häiriö, joka syntyy veren virtauksen äkillisen vähentymisen seurauksena sydänlihaksessa aiheuttaen kudosvaurion. Vaurioituneen sydänlihaskudoksen kyky uusiutua tai korvata kuolleet sydänlihassolut uusilla on puutteellinen, ja tämän seurauksena sydämen pumppauskyky heikkenee. Transkriptiotekijöiden GATA4, NKX2-5, TBX5 ja MEF2C muodostamat ja koordinoimat proteiinikompleksit säätelevät sydänsolujen geenien ilmenemistä solujen elinkaaren aikana. Väitöskirjatyön tavoitteena oli (i) karakterisoida geeninsäätelytekijöiden GATA4-NKX2-5 molekyylirakenteet ja niiden keskinäinen vuorovaikutus, (ii) seuloa kemiallisia yhdisteitä, jotka muokkaavat GATA4-NKX2-5 proteiinikompleksin aikaansaamaa geeniaktivaatiota, (iii) tutkia johtoyhdisteen vaikutuksia in vivo sydäninfarktia ja painekuormitusta kuvaavissa eläinmalleissa, ja (iv) tutkia johtoyhdisteen molekyylirakenteen yhteyttä yhdisteen metaboliaan ja sytotoksisuuteen. Väitöskirjatyö osoittaa molekyylimallinuksen ja kokeellisten tulosten perusteella, että geeninsäätelytekijöiden GATA4-NKX2-5 proteiinikompleksin orientaatio matkii tumareseptoriperheen DNA domeenin tertiäärirakennetta. Molekyylifragmenttien, lusiferaasi-reportterikokeen ja farmakoforimallin avulla seulottiin ja optimoitiin sitoutumisvoimakkuudeltaan lupaavin GATA4-NKX2-5 proteiinikompleksin toimintaan vaikuttava johtoyhdiste: N-[4-(dietyyliamino)fenyyli]-5-metyyli-3-fenyyli-isoksatsoli-4-karboksamidi. Johtoyhdisteellä havaittiin solu- ja eläinmalleissa hypertrofiaa estäviä vaikutuksia in vitro ja sydäntä suojaavia vaikutuksia in vivo. Väitöskirjatyö osoitti lisäksi aktiivisten molekyylien rakenneominaisuuksia, jotka keskeisesti vaikuttavat yhdisteiden metaboliaan ja sytotoksisuuteen. Nykyinen lääkehoito hidastaa, mutta ei pysäytä sydänlihasvaurioon liittyvän kroonisen sydämen vajaatoiminnan etenemistä. Lääkevaikutuksen kohdentaminen sydämen keskeisten transkriptiotekijöiden yhteisvaikutukseen avaa uuden mahdollisen tutkimuslinjan sydänlihasvaurion estossa ja korjauksessa
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Jullian, Estelle. "Myogenic fate choice in the cardiopharyngeal mesoderm." Thesis, Aix-Marseille, 2019. http://www.theses.fr/2019AIXM0363.
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Le mésoderme cardiopharyngé (CPM) est localisé au niveau crânial de l’embryon de souris, et contribue aux muscles de la tête et du cou, dérivés des arcs pharyngés, et aux cellules progénitrices du second champ cardiaque qui donne naissance au muscle cardiaque. L’étude du CPM permet de comprendre les malformations congénitales cardiaques et crâniofaciales, comme celles observées chez les patients atteints du syndrome de microdélétion 22q11.2. Chez la souris, une analyse de clonale rétrospective a établi qu’il existe une relation clonale entre certaines parties du cœur, dérivant du second champ cardiaque et certains muscles branchiomériques. Bien que, chez le protochordé Ciona, une cellule progénitrice du CPM a été identifiée, capable de contribuer au cœur et aux muscles squelettiques pharyngés, les cellules progénitrices communes entre le cœur et les muscles de la tête n’ont pas été localisées dans l’embryon de souris. L’objectif de ma thèse consiste à étudier le destin du cœur contre celui des muscles de la tête dans le CPM. Le premier chapitre des résultats adresse la localisation spatiotemporelle des potentielles cellules progénitrices bipotentes du cœur et des muscles de la tête dans le CPM murin et comment elles sont régulées. Les résultats démontrent que bien que les composants conservés soient présents, leur régulation diffère entre la souris et Ciona. Le second et le troisième chapitres de résultats présentent une analyse de l’hétérogénéité à l’intérieur du CPM et entre les arcs pharyngés. Des domains ont été déterminés dans les arcs, et le destin cellulaire reste à explorerCardiopharyngeal mesoderm is localized at the cranial level of the mouse embryo, and contributes to head and neck muscles, derived from pharyngeal arches, and cardiac muscle. Study cardiopharyngeal mesoderm allows to understand some congenital abnormalities, which have cardiac and craniofacial defects, like DiGeorge syndrome. In mouse, retrospective clonal analysis allows to determinate a relationship between second heart field and specific branchiomeric muscles. Each pharyngeal arch gives rise to a specific branchiomeric muscles group which is linked to a part of the heart. Indeed, it has been showed in Chordates, a progenitor cell which is able to contribute to the heart and head muscles. My thesis objective is to investigate heart versus head muscles fate in cardiopharyngeal mesoderm. I wanted to understand the mechanism underlying heart and head muscles specification. The first part of the thesis will undercover the localization and the timeline of the potential bipotent myogenic progenitor cells present in cardiopharyngeal mesoderm and how they are regulated. The results showed that the conserved components are present but the regulation between each component seemed to be different in the mouse compared to Ciona. The second part and the three part of the thesis will undercover the heterogeneity intra- and inter-pharyngeal arches. Domains through the core of the arches could be observed and the fate of each domain needs to be explored
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Gotoh, Shimpei. "Generation of Alveolar Epithelial Spheroids via Isolated Progenitor Cells from Human Pluripotent Stem Cells." Kyoto University, 2015. http://hdl.handle.net/2433/195967.
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Final publication is available at http://dx.doi.org/10.1016/j.stemcr.2014.07.005. Shimpei Gotoh, Isao Ito, Tadao Nagasaki, Yuki Yamamoto, Satoshi Konishi, Yohei Korogi, Hisako Matsumoto, Shigeo Muro, Toyohiro Hirai, Michinori Funato, Shin-Ichi Mae, Taro Toyoda, Aiko Sato-Otsubo, Seishi Ogawa, Kenji Osafune, Michiaki Mishima, Generation of Alveolar Epithelial Spheroids via Isolated Progenitor Cells from Human Pluripotent Stem Cells, Stem Cell Reports, Volume 3, Issue 3, 9 September 2014, Pages 394-403, ISSN 2213-6711.Kyoto University (京都大学)
0048
新制・課程博士
博士(医学)
甲第18681号
医博第3953号
新制||医||1007(附属図書館)
31614
京都大学大学院医学研究科医学専攻
(主査)教授 妻木 範行, 教授 江藤 浩之, 教授 瀬原 淳子
学位規則第4条第1項該当
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Altekoester, Ann-Kristin [Verfasser], Thorsten [Gutachter] Hoppe, and Niels H. [Gutachter] Gehring. "Decoding the transcriptional landscape of Nkx2-5 in heart development and disease / Ann-Kristin Altekoester ; Gutachter: Thorsten Hoppe, Niels H. Gehring." Köln : Universitäts- und Stadtbibliothek Köln, 2019. http://d-nb.info/1182533337/34.
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Liu, Jillian Mei-ling. "Developmental Mechanisms of Central Hypoventilation." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1523759415675165.
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Velupandian, Uma Maheshwari. "The diagnosis of Patent Foramen Ovale, its importance in migraine, and an insight into its genetic basis." Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/the-diagnosis-of-patent-foramen-ovale-its-importance-in-migraine-andan-insight-into-its-genetic-basis(d13d4a0b-b1f3-437a-899a-960015f9b33f).html.
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Background: Patent Foramen Ovale (PFO), a remnant of the foetal circulation, is emerging as a new cause of disease. It has been found to be associated with cryptogenic stroke in young adults, peripheral arterial embolism and neurological decompression sickness in divers. The detection of PFO remains a diagnostic challenge; transoesophageal echocardiogram being currently considered the ‘gold standard’. The development of a non-invasive technique is crucial for the identification of a venous-to-arterial shunt (v-aCS) which may permit paradoxical embolism. Little is known about the genetic basis of PFO and our limited knowledge is based on animal studies and gene mutations detected in patients with other cardiac septal defects. Methods: Study 1: PFO Detection and Evaluation: This study was designed to evaluate transcranial Doppler (TCD), transthoracic echocardiogram (TTE) and transoesophageal echocardiogram (TOE) with administration of contrast via arm and femoral veins. We then developed a standardized protocol for PFO detection and quantification using TCD. Study 2: PFO and Migraine: The PFO detection protocol developed from the first study formed the diagnostic technique to detect v-aCS in an adequately powered matched case control study to explore the association between PFO and migraine. Study 3: The Genetic basis of PFO: This study was designed to explore the genetic basis of a PFO using a candidate gene approach. Results: Study 1 - PFO Detection Study: When compared with TOE with femoral vein contrast injection as the ‘gold standard’, TCD with arm vein contrast was 100% sensitive and 97.4% specific for detecting a PFO. We defined a PFO positive (+ve) study on TCD as > 15 microbubbles entering the cerebral circulation, on TCD following arm vein injection and >16 microbubbles with a femoral contrast injection. A ‘major’ PFO+ve v-aCS was defined as >35 microbubbles with arm vein injection or >90 microbubbles with femoral vein injection. We then developed a new diagnostic pathway for PFO detection in clinical practice. Study 2 - PFO Migraine study: A significant difference in prevalence of v-aCS between migraine with aura M+A) and their matched controls was demonstrated with adjusted OR=3.72 (1.48-9.38) p=0.005 for a PFO+ve v-aCS, and a highly significant difference between M+A and controls for a ‘major’ PFO+ve v-aCS with adjusted OR = 6.38 (1.89 – 21.48) p = 0.003. There was significant association with APC resistance and migraine on thrombophilia screen. Study 3 - The PFO Genetics Study: This study detected mutations of GATA4 and NKX2-5 in both PFO+ve cases and PFO-ve controls. Two novel non synonymous mutations of GATA4, c.461T>A and c.994G>A were found only in PFO positive individuals and may be associated with a PFO. All the PFO+ve cases with a GATA4 gene mutation had a major PFO+ve v-aCSConclusion:TCD detects PFO with a sensitivity of 100% and specificity of 92.3% and is the most reliable non-invasive technique for PFO detection. When arm vein injections are used both cough and valsalva provocation is essential. There was a highly significant association between PFO+ve v- aCS and M+A, especially with a ‘major’ PFO+ve v-aCS. GATA 4 mutations though infrequent were found PFO+ve cases and all had major v-aCS.
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Humphrey, Peter Saah. "Signal transduction mechanisms for stem cell differentation into cardiomyocytes." Thesis, University of Hertfordshire, 2009. http://hdl.handle.net/2299/3760.
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Cardiovascular diseases are among the leading causes of death worldwide and particularly in the developed World. The search for new therapeutic approaches for improving the functions of the damaged heart is therefore a critical endeavour. Myocardial infarction, which can lead to heart failure, is associated with irreversible loss of functional cardiomyocytes. The loss of cardiomyocytes poses a major difficulty for treating the damaged heart since terminally differentiated cardiomyocytes have very limited regeneration potential. Currently, the only effective treatment for severe heart failure is heart transplantation but this option is limited by the acute shortage of donor hearts. The high incidence of heart diseases and the scarcity donor hearts underline the urgent need to find alternative therapeutic approaches for treating cardiovascular diseases. Pluripotent embryonic stem (ES) cells can differentiate into functional cardiomyocytes. Therefore the engraftment of ES cell-derived functional cardiomyocytes or cardiac progenitor cells into the damaged heart to regenerate healthy myocardial tissues may be used to treat damaged hearts. Stem cell-based therapy therefore holds a great potential as a very attractive alternative to heart transplant for treating heart failure and other cardiovascular diseases. A major obstacle to the realisation of stem cell-based therapy is the lack of donor cells and this in turn is due to the fact that, currently, the molecular mechanisms or the regulatory signal transduction mechanisms that are responsible for mediating ES cell differentiation into cardiomyocytes are not well understood. Overcoming this huge scientific challenge is absolutely necessary before the use of stem cell-derived cardiomyocytes to treat the damaged heart can become a reality. Therefore the aim of this thesis was to investigate the signal transduction pathways that are involved in the differentiation of stem cells into cardiomyocytes. The first objective was the establishment and use of cardiomyocyte differentiation models using H9c2 cells and P19 stem cells to accomplish the specific objectives of the thesis. The specific objectives of the thesis were, the investigation of the roles of (i) nitric oxide (ii) protein kinase C (PKC), (iii) p38 mitogen-activated protein kinase (p38 MAPK) (vi) phosphoinositide 3-kinase (PI3K) and (vi) nuclear factor-kappa B (NF-kB) signalling pathways in the differentiation of stem cells to cardiomyocytes and, more importantly, to identify where possible any points of convergence and potential cross-talk between pathways that may be critical for differentiation to occur. P19 cells were routinely cultured in alpha minimal essential medium (α-MEM) supplemented with 100 units/ml penicillin /100 μg/ml streptomycin and 10% foetal bovine serum (FBS). P19 cell differentiation was initiated by culturing the cells in microbiological plates in medium containing 0.8 % DMSO to form embryoid bodies (EB). This was followed by transfer of EBs to cell culture grade dishes after four days. H9c2 cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% FBS. Differentiation was initiated by incubating the cells in medium containing 1% FBS. In both models, when drugs were employed, they were added to cells for one hour prior to initiating differentiation. Cell monolayers were monitored daily over a period of 12 or 14 days. H9c2 cells were monitored for morphological changes and P19 cells were monitored for beating cardiomyocytes. Lysates were generated in parallel for western blot analysis of changes in cardiac myosin heavy chain (MHC), ventricular myosin chain light chain 1(MLC-1v) or troponin I (cTnI) using specific monoclonal antibodies. H9c2 cells cultured in 1% serum underwent differentiation as shown by the timedependent formation of myotubes, accompanied by a parallel increase in expression of both MHC and MLC-1v. These changes were however not apparent until 4 to 6 days after growth arrest and increased with time, reaching a peak at day 12 to 14. P19 stem cells cultured in DMSO containing medium differentiated as shown by the timedependent appearance of beating cardiomyocytes and this was accompanied by the expression of cTnI. The differentiation of both P19 stem cells and H9c2 into cardiomyocytes was blocked by the PI3K inhibitor LY294002, PKC inhibitor BIM-I and the p38 MAPK inhibitor SB2035800. However when LY294002, BIM-I or SB2035800 were added after the initiation of DMSO-induced P19 stem cell differentiation, each inhibitor failed to block the cell differentiation into beating cardiomyocytes. The NF-kB activation inhibitor, CAPE, blocked H9c2 cell differentiation into cardiomyocytes. Fast nitric oxide releasing donors (SIN-1 and NOC-5) markedly delayed the onset of differentiation of H9c2 cells into cardiomyocytes while slow nitric oxide releasing donors (SNAP and NOC-18) were less effective in delaying the onset of differentiation or long term differentiation of H9c2 cells into cardiomyocytes. Akt (protein kinase B) is the key downstream target of PI3K. Our cross-talk data also showed that PKC inhibition and p38 MAPK inhibition respectively enhanced and reduced the activation of Akt, as determined by the phosphorylation of Akt at serine residue 473. In conclusion, PKC, PI3K, p38 MAPK and NF-kB are relevant for the differentiation of stem cells into cardiomyocytes. Our data also show that the PKC, PI3K and p38 MAPK signalling pathways are activated as very early events during the differentiation of stem cells into cardiomyocytes. Our data also suggest that PKC may negatively regulate Akt activation while p38 MAPK inhibition inhibits Akt activation. Our fast NO releasing donor data suggest that nitric oxide may negatively regulate H9c2 cell differentiation.
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Papizan, James. "Structure-function analysis of the essential islet regulatory factor Nkx2.2." Thesis, 2013. https://doi.org/10.7916/D8V12BX4.
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The specification and differentiation of the pancreatic beta cell lineage requires guidance by spatiotemporally regulated signaling cues and a highly orchestrated set of transcription factors. Defining the factors and their regulatory functions that are required for proper beta cell development will enhance our ability to recapitulate these developmental events in vitro to generate beta cells from alternate cell sources. The homeodomain transcription factor Nkx2.2 is essential for pancreatic endocrine cell development; Nkx2.2-/- mice lack all beta cells and have reductions in alpha and pancreatic polypeptide (PP) cells. In place of these cell populations, the Nkx2.2-/- null islet is replete with ghrelin-producing epsilon cells. An Nkx2.2-repressor fusion protein derivative (Pdx1:Nkx2.2-EnR) expressed in the Nkx2.2-/- background can fully rescue the alpha cell population, but can only specify a few immature beta cells, suggesting that Nkx2.2 must contain both repressor and activator functions to properly guide beta cell development. Accordingly, Nkx2.2 has been shown to be an activator of several beta-cell targets. It has also been demonstrated that the corepressor Grg3 is expressed in the endocrine population and can physically interact with Nkx2.2, which points toward a mechanism by which Nkx2.2 confers transcriptional repression; however, the genes targeted by Nkx2.2/Grg3 are unknown. Additionally, how Nkx2.2 can both repress and activate genes in the same cellular context, and differentially regulate the same gene in different cellular contexts, is not understood. In this dissertation, I sought to determine the regulatory role of Nkx2.2 in the developing pancreas and its dependence on Grg interactions, and to elucidate whether post-translational modifications play a role in modulating Nkx2.2 regulatory activities. By analyzing mice carrying knock-in mutations in the Nkx2.2 Grg-interaction domain (Nkx2.2TNmut/TNmut), I show that the interaction between Nkx2.2 and Grg protein is required at two developmental stages of beta cell development: 1) Grg-mediated Nkx2.2 repression is necessary for correct beta-cell specification, and 2) the recruitment of Grg by Nkx2.2 is required to repress Arx in the beta cells to prevent beta-to-alpha cell reprogramming. Additionally, by analyzing the Nkx2.2TNmut/TNmut and Nkx2.2TNmut/TNmut;Ins:Cre;Arxfl/fl mice, I have identified several additional genes that may be regulated by Grg-mediated Nkx2.2 repression. Finally, I also present data to suggest that Nkx2.2 protein is phosphorylated, and that the phosphorylation state determines whether Nkx2.2 functions as an activator or a repressor in a promoter-specific context. These studies have begun to elucidate the complex regulatory roles that Nkx2.2 plays in specifying and maintaining the beta-cell lineage. Future analyses will help us to better understand the spatiotemporal regulatory activities that are required to make and maintain functional beta cells.
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Churchill, Angela Josephine. "Spatiotemporal and Mechanistic Analysis of Nkx2.2 Function in the Pancreatic Islet." Thesis, 2016. https://doi.org/10.7916/D84M94N2.
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Pancreatic beta cell specification is a complex process, requiring proper function of numerous transcription factors. Nkx2.2 is a transcription factor that is crucial for beta cell formation, and is expressed early and throughout pancreatic development. Nkx2.2-/- mice display complete loss of the beta cell lineage and defects in the specification of other endocrine cell types, demonstrating the importance of Nkx2.2 in establishing proper endocrine cell ratios. Recent studies have also demonstrated a role for Nkx2.2 within the mature beta cell to maintain identity and function.
This thesis work investigated the timing of pancreatic beta cell specification and the mechanism of this process. In these studies, Nkx2.2 was ablated specifically within the Ngn3-expressing endocrine progenitor population in vivo. These mice displayed defects similar to Nkx2.2-/- mice. Surprisingly, the disruption of endocrine cell specification did not require loss of expression of multiple essential transcription factors known to function downstream of Nkx2.2, including Ngn3, Rfx6, and NeuroD1. While these factors are all necessary for beta cell specification, their preserved expression did not rescue beta cell formation. ChIP-Seq analyses also revealed co-occupancy of Nkx2.2, Rfx6, and NeuroD1 near endocrine-related genes, suggesting Nkx2.2 may cooperate with its downstream targets to regulate beta cell fate. These results have revealed a unique requirement for Nkx2.2 during a critical window of beta cell development.
In addition, the role of a conserved domain of Nkx2.2, the specific domain (SD), was assessed using Nkx2.2SDmutant mice. Transcriptional profiling of Nkx2.2SDmutant endocrine progenitors revealed a critical role for the SD domain in regulating the transcription of endocrine fate genes early in the process of endocrine differentiation. In addition, beta cell-specific deletion of the Nkx2.2 SD domain resulted in hyperglycemia, glucose intolerance and dysregulation of beta cell functional genes. This suggests the SD domain is important for mediating Nkx2.2 function within the beta cell to maintain glucose homeostasis.
Together, these results have elucidated a critical developmental window for beta cell specification and demonstrated an essential role for Nkx2.2 and specifically its SD domain in this process. Furthermore, these studies suggest that beta cell transcription factors may also regulate endocrine fate in a combinatorial manner, and exert changes within the endocrine progenitor lineage. These findings have provided us with a better understanding of in vivo pancreatic development, and will improve current research efforts to differentiate beta cells in vitro from hPSCs.
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Abarinov, Elena. "Progressive restriction of CNS cell-fate potential by the transiently expressed transcription factor Nkx2.2." Thesis, 2019. https://doi.org/10.7916/d8-shpq-2d83.
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The progressive loss of developmental potential is a hallmark of all differentiating cells in multicellular organisms. At the chromatin level, this restriction in cell-fate plasticity is established through the silencing of active and poised lineage-specific genes that are incompatible with the terminal fate of the maturing cell type. The effective and stable inhibition of gene expression relies on the coordinated action of transcriptional repressors. These repressors are often transiently expressed only at the time of cell-fate specification and direct lineage decisions by suppressing alternative developmental programs. However, compared to the numerous studies examining the mechanisms by which cell-type specific transcriptional activators program cellular identity, little is currently known regarding how transient repressors execute permanent silencing of gene regulatory networks. To address this question, I have examined the mechanisms through which the transiently expressed transcription factor (TF) Nkx2.2 represses the acquisition of motor neuron (MN) identity in V3 neuronal progenitors. While it is well-established that Nkx2.2 functions as a transcriptional repressor through its interactions with the Groucho (Grg) family of co-repressors, how these interactions manifest in gene silencing has remained unknown. Moreover, the effects of Nkx2.2 occupancy on chromatin modifications have not been determined. In this dissertation, I demonstrate that surprisingly, Nkx2.2 decommissions enhancers of the MN developmental program not through the recruitment of additional co-repressor proteins but rather through the eviction of co-activator complexes. While this displacement is dependent upon an intact Grg-interacting domain, Nkx2.2 binding does not increase Grg enrichment. In addition, extensive profiling of Nkx2.2 genome-wide binding events in neural precursors unexpectedly revealed that Nkx2.2 occupies not only enhancers of MN progenitor genes acutely repressed by Nkx2.2 but also enhancers of genes expressed exclusively in postmitotic MNs, long after Nkx2.2 expression has been down- regulated. In vivo lineage tracing experiments and in vitro genomic analyses demonstrated that Nkx2.2 also functions in a repressive capacity at these poised regulatory regions. Here, Nkx2.2 binding prevents the activation of postmitotic genetic networks through a preferential enlistment of histone deacetylase complex 2 (HDAC2) proteins. However, this binding is not accompanied by the deposition of repressive chromatin modifications, and removal of Nkx2.2 in differentiating V3 neurons leads to the ectopic expression of the postmitotic MN TFs Isl1 and Hb9. Collectively, these studies indicate that transiently expressed repressors may establish gene suppression by counteracting the activities of transcriptional activators, rather than by directly establishing repressive chromatin signatures. As transcriptional reprogramming of differentiated cell linages often fails to adequately silence the expression programs of the starting population, these results may help to inform new methodologies for instructing cell conversions.
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Kordowich, Simon. "Funktionelle Charakterisierung der Transkriptionsfaktoren Nkx2.2 und Arx in der Entwicklung der endokrinen Zellen im murinen Pankreas." Doctoral thesis, 2011. http://hdl.handle.net/11858/00-1735-0000-0006-AE27-C.
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Bing-Sheng-Lu and 呂秉昇. "Functional analysis of NKX2.5 gene in Nasopharyngeal Carcinoma." Thesis, 2018. http://ndltd.ncl.edu.tw/handle/g588p4.
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碩士國立臺灣大學
病理學研究所
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Nasopharyngeal carcinoma (NPC) is one of common head and neck cancers in Southeast Asia, particularly Southern China, Hong Kong and Taiwan, believed to have a multifactorial etiology. Environmental factors, consumption of Cantonese salted food and infections of Epstein-Barr virus (EBV) are most documented. The pathogenesis of NPC, like that of most solid tumors, remains elusive. The aim of this study was to identify the significant genes that may be altered during NPC progression. Previously, using cDNA microarray analysis, and we found that the transcription factor NKX2.5. NKX2.5 showed a significant different expression and highly expressed in NPC cell lines than normal nasal mucosal epithelial cells. In order to investigate that whether NKX2.5 gene could affect NPC progression, we observed at first the gene expression in different NPC cells lines using qRT-PCR analysis and found that expression of NKX2.5 in NPC cell lines, especially in NPC-TW01 cell line was highly upregulated. We also demonstrate that the expression level of NKX2.5 protein was increased in NPC-TW01 NPC cell line and NPC biopsy specimens by immunohistochemical staining and western bloting. In order to analysis of the functional role of NKX2.5 in NPC-TW01 cell line, the expression of NKX2.5 was significantly reduced that compared with the control in NPC-TW01 after knockdown of NKX2.5 by NKX2.5 shRNA-lentiviral infection. This in turn resulted in a significant reduction of cell proliferation, invasion, and migration activity of NPC cells. However, in NOD/SCID mice bearing NKX2.5 transfected NPC xenograft, the tumor growth was significantly decreased. Our findings strongly suggest that NKX2.5 may play a role to promote the formation of nasopharyngeal carcinoma of nasopharynx and as a gene in NPC pathogenesis to affect NPC migration, proliferation and invasion. These novel findings of functional role of NKX2.5 may have a potential implication in molecular targeted therapy for NPC.
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Santos, Fernando Augusto Gonçalves dos. "Validation of hiPSC-NKX2.5-GFP line for cardiac differentiation." Master's thesis, 2021. http://hdl.handle.net/10348/10586.
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Dissertation for the purpose of obtaining a Master's degree in Biotechnology for Health SciencesThe induced pluripotent stem cell (iPSC) technology has created great expectations to advance the development of therapies for cardiovascular diseases, the leading cause of death worldwide. Accordingly, human iPSCs (hiPSCs) efficiently differentiate to cardiomyocytes (hiPSC-CM) which has been already applied in pre-clinical studies, among others, to improve heart regeneration after myocardial infarction. Nevertheless, it is suggested that therapeutic efficiency of hiPSC-CM may be related to the stage of their development. Thus, the main aim of this study was to validate the hiPSC-NKX2.5-GFP line, that enables expression of a cardiacspecific transcription factor, NKX2.5 which is also an established marker of cardiac progenitor cells, and a reporter gene, green fluorescent protein (GFP) from the same promoter, allowing the identification and facilitating the isolation of early cardiomyocytes in the first days of hiPSCs differentiation. Using polymerase chain reaction (PCR) we confirmed the insertion of the GFP-encoding sequence into the locus of the NKX2.5 gene. hiPSCs were subjected to cardiac differentiation. Flow cytometric analysis revealed the presence of cardiac troponin T- and GFP-positive cells among differentiating hiPSCs. The sorted population of GFP-positive cells also confirmed the insertion of the GFP-encoding sequence identified through reverse transcription PCR. The results proved the possibility of the detection of cardiac progenitor cells based on GFP fluorescence dependent on NKX2.5 activation. A quantitative measurement of the differentiation efficiency confirmed that the method used for the genetic construction of tested cell line does not interfere with its cardiac differentiation potential. Therefore, it could be concluded that the hiPSC-NKX2.5-GFP line can be used for identification and isolation of early cardiomyocytes being a valuable tool for studies of therapeutic approaches to treat cardiovascular diseases.
A tecnologia de células estaminais pluripotentes induzidas (iPSCs) criou grandes expectativas no que diz respeito ao avanço do desenvolvimento de terapias para as doenças cardiovasculares, consideradas como a principal causa de morte em todo o mundo. Assim, as iPSCs humanas (hiPSCs) diferenciam-se de forma eficiente em cardiomiócitos (hiPSC-CM), o que já foi aplicado em estudos pré-clínicos, entre outros, para melhorar a regeneração cardíaca após a ocorrência de um enfarte do miocárdio. No entanto, sugere-se que a eficácia terapêutica dos hiPSC-CM pode estar relacionada com o seu estágio de desenvolvimento. Assim, o objetivo principal deste estudo foi validar a linha hiPSC-NKX2.5-GFP, que permite a expressão de um fator de transcrição específico para o coração, NKX2.5, que também é um marcador estabelecido de células progenitoras cardíacas e um gene repórter, a proteína fluorescente verde (GFP) de um mesmo promotor, permitindo a identificação e facilitando o isolamento de cardiomiócitos precoces nos primeiros dias de diferenciação das hiPSCs. Usando a reação em cadeia da polimerase (PCR), confirmamos a inserção da sequência codificadora de GFP no locus do gene NKX2.5. As hiPSCs foram submetidas a um protocolo de diferenciação cardíaca. A análise de citometria de fluxo revelou a presença de células positivas para a troponina T e GFP entre hiPSCs em diferenciação. A população classificada de células positivas para GFP também confirmou a inserção da sequência de codificação de GFP, identificada por PCR de transcrição reversa. Os resultados comprovaram a possibilidade de detecção de células progenitoras cardíacas com base na fluorescência GFP. Uma medição quantitativa da eficiência de diferenciação confirmou que o método utilizado para a construção genética da linha celular testada não interfere no seu potencial de diferenciação cardíaca. Portanto, pode-se concluir que a linha hiPSC-NKX2.5-GFP pode ser utilizada para identificação e isolamento de cardiomiócitos precoces, sendo uma ferramenta valiosa para estudos de abordagens terapêuticas no tratamento das doenças cardiovasculares.