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Tracing human cancer evolution with hypermutable DNANaxerova, Kamila 04 February 2015 (has links)
Metastasis is the main cause of cancer morbidity and mortality. Despite its clinical significance, several fundamental questions about the metastatic process in humans remain unsolved. Does metastasis occur early or late in cancer progression? Do metastases emanate directly from the primary tumor or give rise to each other? How does heterogeneity in the primary tumor relate to the genetic composition of secondary lesions? Addressing these questions in representative patient populations is crucial, but has been difficult so far. Here we present a simple, scalable PCR assay that enables the tracing of tumor lineage in patient tissue specimens. Our methodology relies on somatic variation in highly mutable polyguanine (poly-G) repeats located in non-coding genomic regions. We show that poly-G mutations are present in a variety of human cancers. Using colon carcinoma as an example, we demonstrate an association between patient age at diagnosis and tumor mutational burden, suggesting that poly-G variants accumulate during normal division in colonic stem cells. We further show that poorly differentiated colon carcinomas have fewer mutations than well-differentiated tumors, possibly indicating a shorter mitotic history of the founder cell in these cancers. We collect multiple spatially separated samples from primary carcinomas and their metastases and use poly-G fingerprints to build well-supported phylogenetic trees that illuminate each patient's path of progression. Our results imply that levels of intra-tumor heterogeneity vary significantly among patients.
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Trp53 Mutation in Keratin 5 (Krt5)-Expressing Basal Cells Facilitates the Development of Basal Squamous-Like Invasive Bladder Cancer in the Chemical Carcinogenesis of Mouse Bladder / ケラチン5発現基底細胞でのTrp53遺伝子変異はマウス化学発癌モデルの基底扁平上皮様サブタイプ筋層浸潤性膀胱癌の形成を促進するMasuda, Norihiko 24 January 2022 (has links)
京都大学 / 新制・論文博士 / 博士(医学) / 乙第13466号 / 論医博第2253号 / 新制||医||1055(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 村川 泰裕, 教授 中島 貴子, 教授 藤田 恭之 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM
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Dlx Genes, Neurogenesis and Regeneration in the Adult Zebrafish BrainWeinschutz Mendes, Hellen 09 January 2020 (has links)
The Dlx homeobox genes encode homeodomain transcription factors that are involved in
multiple developmental aspects. In the brain, these genes take part in neuronal migration and
differentiation, more precisely in the migration and differentiation of GABAergic neurons.
Dysfunctions in the GABAergic system can lead to various pathological conditions, where
impaired inhibitory function is one of the main causes of several neuropathies characterized by
neuronal hyperexcitability. The Dlx genes are organized as bi-gene clusters and highly
conserved cis-regulatory elements have been previously characterized to be fundamental for
the regulation of Dlx expression in developing embryos of different vertebrates. The activity of
these regulatory elements and the Dlx genes has been well studied in developmental stages of
mice and zebrafish, but little is known about their activity in the adult brain. The extensive
neurogenesis that takes place in the adult zebrafish brain provides an ideal platform for the
visualization of mechanisms involving dlx genes during adulthood and their possible
involvement in adult neurogenesis. Here we show novel information concerning the expression
of dlx1a, dlx2a, dlx5a and dlx6a in the adult zebrafish brain and provide insight into the identity
of cells that express dlx. We also demonstrate the involvement of dlx genes in brain
regeneration and through lineage tracing, their fate determination in the adult zebrafish brain.
Analyses in the adult zebrafish has revealed that all four dlx paralogs are expressed in the
forebrain and midbrain throughout adulthood and expression is found in almost all areas
presenting continuous proliferation. Most dlx-expressing cells present GABAergic neuronal
identity in the adult forebrain where, in some areas they were identified as the Calbindin
subtype. In some areas of the midbrain, especially within the hypothalamus, many dlxexpressing
cell co-localized with a marker for neural stem cells. However, cells expressing dlx
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genes did not co-localize with markers for proliferating cells or for glia. Investigations during
brain regeneration in response to injury in the adult zebrafish brain has revealed that dlx5a
expression decreases shortly after lesion and that the dlx5a/6a bi-gene cluster, more
specifically, dlx5a, is up regulated during the peak of regeneration response proposing a
possible role for dlx during regeneration in adults. Studies of lineage tracing have shown the
progeny of dlx1a/2a-expressing cells in adults are located within small clusters in different areas
of the adult brain where they seem to become mature neurons. Our observations provide a
better understanding about the role of dlx genes during adulthood, further contributing to the
general knowledge of the molecular pathways involved in adult neurogenesis and regeneration
in the zebrafish adult brain.
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Pancreatic RECK inactivation promotes cancer formation, epithelial-mesenchymal transition, and metastasis / 膵特異的RECK発現の不活化は、膵発癌、上皮間葉転換、転移を引き起こすMasuda, Tomonori 25 March 2024 (has links)
京都大学 / 新制・課程博士 / 博士(医学) / 甲第25194号 / 医博第5080号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 藤田 恭之, 教授 小濱 和貴, 教授 川口 義弥 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM
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Analysis of cellular drivers of zebrafish heart regeneration by single-cell RNA sequencing and high-throughput lineage tracingHu, Bo 22 September 2021 (has links)
Das Herz eines Zebrafishs ist bemerkenswert, da es sich nach einer Verletzung vollständig regenerieren kann. Der Regenerationsprozess wird von Fibrose begleitet - der Bildung von überschüssigem Gewebe der extrazellulären Matrix (ECM). Anders als bei Säugetieren ist die Fibrose im Zebrafish nur transient. Viele Signalwege wurden identifiziert, die an der Herzregeneration beteiligt sind. Allerdings sind die Zelltypen, insbesondere Nicht-Kardiomyozyten, die für die Regulation des Regenerationsprozesses verantwortlich sind, weitgehend unbekannt. In dieser Arbeit haben wir systematisch alle Zelltypen des gesunden und des verletzten Zebrafischherzens mithilfe einer auf Mikrofluidik basierenden Hoch-Durchsatz- Einzelzell-RNA-Sequenzierung bestimmt. Wir fanden eine große Heterogenität von ECM-produzierenden Zellen, einschließlich einer Reihe neuer Fibroblasten, die nach einer Verletzung mit unterschiedlicher Dynamik auftreten. Wir konnten aktivierte Fibroblasten beschreiben und Fibroblasten-Subtypen mit einer pro-regenerativen Funktion identifizieren.
Darüber hinaus haben wir eine Methode entwickelt, um die Transkriptomanalyse und die Rekonstruktion von Zell-Verwandtschaften auf Einzelzellebene zu kombinieren. Unter Verwendung der CRISPR-Cas9-Technologie führten wir zufällige Mutationen in bekannte und ubiquitär transkribierte DNA-Loci während der Embryonalentwicklung von Zebrafischen ein. Diese Mutationen dienten als zellspezifische, permanente und vererbbare “Barcodes”, die zu einem späteren Zeitpunkt erfasst werden konnten. Mit maßgeschneiderten Analysealgorithmen konnten wir dann Stammbäume der sequenzierten Einzelzellen erstellen. Mit dieser neuen Methode haben wir gezeigt, dass im sich regenerierenden Zebrafischherz ECM-produzierende Zellpopulationen entweder mit dem Epi- oder mit dem Endokardium verwandt sind. Zusätzlich entdeckten wir, dass vom Endokardium abgeleitete Zelltypen vom Wnt-Signalweg abhängig sind. / The zebrafish heart has the remarkable capacity to fully regenerate after injury. The regeneration process is accompanied by fibrosis - the formation of excess extracellular matrix (ECM) tissue, at the injury site. Unlike in mammals, the fibrosis of the zebrafish heart is only transient. While many pathways involved in heart regeneration have been identified, the cell types, especially non-myocytes, responsible for the regulation of the regenerative process have largely remained elusive. Here, we systematically determined all different cell types of both the healthy and cryo-injured zebrafish heart in its regeneration process using microfluidics based high-throughput single-cell RNA sequencing. We found a considerable heterogeneity of ECM producing cells, including a number of novel fibroblast cell types which appear with different dynamics after injury. We could describe activated fibroblasts that extensively switch on gene modules for ECM production and identify fibroblast sub- types with a pro-regenerative function.
Furthermore, we developed a method that is capable of combining transcriptome analysis with lineage tracing on the single-cell level. Using CRISPR-Cas9 technology, we introduced random mutations into known and ubiquitously transcribed DNA loci during the zebrafish embryonic development. These mutations served as cell-unique, permanent, and heritable barcodes that could be captured at a later stage simultaneously with the transcriptome by high-throughput single-cell RNA sequencing. With custom tailored analysis algorithms, we were then able to build a developmental lineage tree of the sequenced single cells. Using this new method, we revealed that in the regenerating zebrafish heart, ECM contributing cell populations derive either from the epi- or the endocardium. Additionally, we discovered in a functional experiment that endocardial derived cell types are Wnt signaling dependent.
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Time to quit? : non-genetic heterogeneity in cell fate propensity after DNA damageCampbell, Callum James January 2018 (has links)
Cellular checkpoints are typically considered to both facilitate the ordered execution of the cell cycle and to act as a barrier to oncogene driven cell cycles and the transmission of unresolved genetic lesions from one phase to the next. Furthermore, these mechanisms are also believed to underpin the responses of cells, both in normal and cancerous tissues, to those therapies that either directly or indirectly generate DNA damage. In recent studies however, it has become clear these checkpoints permit the passage of significant genomic aberrations into subsequent cell cycle phases and even descendant cells, and that heterogeneous responses are apparent amongst genetically identical cells. The consequences of this checkpoint ‘negligence’ remain relatively uncharacterised despite the importance of checkpoints in current models for how genomic instability is avoided in the face of ubiquitous DNA damage. Unresolved DNA damage is presumably inherited by subsequent cell cycle phases and descendant cells yet characterisation of the consequences of this has been relatively limited to date. I therefore utilised microscopy-based lineage tracing of cells expressing genetically encoded fluorescent sensors, particularly the Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) probes (Sakaue-Sawano et al., 2008), with semi-automated image analysis to characterise the response of single cells and their descendants to DNA lesions across multiple cell cycle generations. This approach, complemented by generational tracing by flow cytometry, permitted me to characterise the timing of cell fate determination in treated and descendant cells, the non-genetic heterogeneity in checkpoint responses and overall lineage behaviour, correlations between cells (similarly to Sandler et al., 2015) and cell cycle timing dependencies in the response to DNA damaging agents. With these single cell analytical approaches I show that the consequences of DNA damage on descendant cell fate is dramatic, suggesting checkpoint mechanisms may have consequences and even cooperate across phases and generations. U2OS cell lineages traced for three generations following the induction of DNA damage in the form of strand breaks showed greatly induced cell death in the daughters and granddaughters of DNA damaged cells, termed delayed death. Furthermore, lineage behaviour was characterised as highly heterogeneous in when and whether cell death occurred. Complementary flow cytometric approaches validated the findings in U2OS cells and suggested HeLa cells may show similar behaviour. These findings indicate that checkpoint models need to incorporate multigenerational behaviour in order to better describe the response of cells to DNA damage. Understanding the processes governing cell fate determination in descendant cells will impact upon our understanding of the development of genomic instability during carcinogenesis and how DNA-damaging chemotherapeutics drive cells to ‘quit’ the cell cycle.
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Defining lineage potential and fate behaviour of progenitors during pancreas developmentSznurkowska, Magdalena Katarzyna January 2018 (has links)
No description available.
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Defining the Regional and Lineage Contribution of Early Mesp1 Cardiovascular Progenitors During Mammalian Heart DevelopmentChabab, Samira 17 May 2016 (has links)
The heart arises from two sources of mesoderm progenitors, the first (FHF) and the second heart field (SHF) progenitors. Mesp1 has been proposed to mark the most primitive multipotent cardiac progenitors (MCPs) common for both heart fields. However, it remains unclear whether at the single cell level, Mesp1 progenitors represent a common progenitor for the FHF and SHF. Using mosaic tracing and inducible clonal analysis with a multicolor reporter strategy, we investigated the contribution of Mesp1 cardiovascular progenitors in a temporally controlled manner during the early gastrulation. Our data indicated that the myocardium derives from ~250 Mesp1 expressing cardiac progenitors born during gastrulation. Temporal analysis of clonally labeled Mesp1 cells revealed the existence of temporally distinct populations of Mesp1 progenitors that are restricted to either the FHF or the SHF. FHF progenitors were unipotent, while SHF progenitors, were either uni- or bipotent. Microarray and single cell RT-PCR analysis of Mesp1 progenitors revealed the existence of molecularly distinct populations of Mesp1 progenitors, consistent with their lineage and regional contribution. Moreover biophysical analysis of clonal data revealed that, despite arising at different time points and contributing to different heart regions, the temporally distinct cardiac progenitors present very similar clonal dynamics. Altogether, these results provide insights into the number of cardiac progenitors and their mode of growth. Moreover they provide evidence that heart development arises from distinct populations of unipotent and bipotent cardiac progenitors expressing Mesp1 independently at different time points during gastrulation. Our data reveal that the regional segregation and lineage restriction of cardiac progenitors occurs very early during embryonic development. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished
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Visualization of stem cell activity in pancreatic cancer expansion by direct lineage tracing with live imaging / 細胞系譜解析とライブイメージングによる膵癌幹細胞動態の可視化Maruno, Takahisa 26 July 2021 (has links)
京都大学 / 新制・論文博士 / 博士(医学) / 乙第13427号 / 論医博第2231号 / 新制||医||1053(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 道行, 教授 渡邊 直樹, 教授 川口 義弥 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM
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Gata4-Dependent Differentiation of c-Kit+ Derived Endothelial Cells Underlies Artefactual Cardiomyocyte Regeneration in the HeartMaliken, Bryan D., B.A. 29 October 2018 (has links)
No description available.
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