京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21663号 / 医博第4469号 / 新制||医||1035(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 篠原 隆司, 教授 万代 昌紀, 教授 近藤 玄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22880号 / 医博第4674号 / 新制||医||1047(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 篠原 隆司, 教授 近藤 玄, 教授 万代 昌紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Oshima N, Yamada Y, Nagayama S, Kawada K, Hasegawa S, et al. (2014) Induction of Cancer Stem Cell Properties in Colon Cancer Cells by Defined Factors. PLoS ONE 9(7): e101735. doi:10.1371/journal.pone.0101735 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18547号 / 医博第3940号 / 新制||医||1006(附属図書館) / 31447 / 京都大学大学院医学研究科医学専攻 / (主査)教授 千葉 勉, 教授 野田 亮, 教授 武藤 学 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The repetitive sequence composes nearly half of human and mouse genome, most of which are scattered repeats of transposable elements (TEs). The non-LTR retrotransposons are the most accumulated TEs in the mammalian genome and L1s are the most active and abundant autonomous retrotransposons. L1s are highly activated during the epigenetic reprogramming of early mammalian embryos and have the highest level of expression among all retrotransposons throughout the preimplantation state. Moreover, the reprogramming of somatic cells into iPSCs is associated with an increase in L1 expression. The transcription of L1 during the early embryogenesis is necessary to regulate developmental genes and prevent heterochromatin formation to maintain cellular pluripotency state, that guarantying an appropriate future differentiation. However, the role of L1 reactivation during the somatic cell reprogramming remains unclear. Therefore, aim of this work is to study the impact of L1 transcription during the reprogramming process of the iPSCs. We used CRISPR-mediated gene activation (CRISPRa) system that fuse a deactivated Cas9 (dCas9) with transactivation domains (VPR). We confirm the ability to overexpress L1 in Human Embryonic Kidney cells (HEK293) and Human Dermal Fibroblasts (HDFs) by utilizing CRISPR activation system and this will provide a good opportunity to study the role of L1 transcripts during the reprogramming of HDFs into iPSCs. Furthermore, we established stable HDFs that able to express combinations of “Yamanaka” reprogramming factors. The model system will allow to investigate the effect of overexpressing L1 with reprogramming factors to answer the question of whether L1 can trigger or facilitate the reprogramming processes and its underlying mechanism.

Orientador: Fernanda da Cruz Landim / Resumo: As pesquisas envolvendo a biologia das células-tronco abordam um amplo espectro de fenômenos, que vão desde o nível tecidual e celular, até o seu uso em terapias celulares. Esta crescente atenção sugere que é necessário estudar conceitos básicos da biologia das células-tronco para compreender completamente os processos de diferenciação funcional. Desta forma, o instrumento da reprogramação celular por meio da manipulação gênica fornece subsídios para melhor compreender os processos de renovação e diferenciação que constituem as características fundamentais das células-tronco. A obtenção dessas células em medicina veterinária visa validar diversos modelos experimentais domésticos, como o equino, na busca de novos fármacos e terapias alternativas para reabilitação. Uma série de estudos, porém, ainda são necessários para que tais aplicações sejam viáveis, uma vez que os mecanismos fundamentais das técnicas empregadas ainda não estão totalmente elucidados. Embora a reprogramação celular por meio de vetores virais tenha sido relatada com sucesso em diversas espécies animais, outras técnicas também podem ser empregadas, como o uso de transposons, sequências de DNAs capazes de se movimentar de uma região para outra no genoma de uma célula. Não se tem conhecimento de qual o melhor tipo celular a ser utilizado, e nem tão pouco qual a metodologia de reprogramação mais eficiente. Sabe-se que o cordão umbilical possui uma reserva rica em células-tronco mesenquimais, as quais por serem mu... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Researches on the biology of stem cells cover a broad spectrum of phenomena, ranging from tissue and cellular level, to their use in cell therapy. This growing attention suggests that is necessary to study basic concepts of stem cells organization in order to fully understand the functional differentiation processes. Thus, the cell reprogramming through gene manipulation provides grants to better understand the processes of renewal and differentiation which are the essential characteristics of stem cells. Obtaining these cells in veterinary medicine aims to validate various household experimental models, such as horses, on the search for new drugs and alternative therapies for rehabilitation. However, a number of studies is still necessary for such applications to be feasible, since the fundamental mechanisms of techniques employed are not fully elucidated yet. Although cell reprogramming using viral plasmid has been reported with success in several animal species, other techniques may also be employed, such use transposons, this is, DNAs sequences capable of moving from one region to another in the cell genome. The unawereness of what the best cell type to be used, and nor what is the most efficient reprogramming methodology. It is known that the cord has rich reserves mesenchymal stem cells, which are multipotent and can improve the efficiency of obtaining the induced Pluripotent Stem Cells (iPS) compared to the use of fibroblast, inefficient to be reprogrammed. The aim of this study was to obtain iPS through viral transfection and nonviral adult fibroblasts and equine cord cells, aiming to observe which transfection and cell type is more efficient for cell reprogramming. Both cell types was infected with viral vectors and transposons containing the genes OCT-4, SOX-2, c-MYC, and KLF-4; transformed cells were evaluated for morphology, immunocytochemistry... (Complete abstract click electronic access below) / Doutor

<p>Breast cancer is the predominant cancer diagnosed among women, and the second most deadly cancer. The vast majority of cancer-related deaths is caused by the metastatic spread of cancer from the primary tumor to a distant site in the body. Therefore, new strategies which minimize breast cancer metastasis are imperative to improve patient survival. Cancer cells which acquire anchorage independence, or the ability to survive without extracellular matrix attachment, and metabolic flexibility have increased potential to metastasize. In the present studies, the ability to survive detachment and subsequent metabolic changes were determined in human Harvey-<i>ras</i> transformed MCF10A-<i>ras</i> breast cancer cells. Detachment resulted in reduced viability in a time-dependent manner with the lowest cell viability observed at forty hours. In addition, decreased cell viability was observed in both glutamine and glucose depleted detached conditions, suggesting a dependence on both nutrients for detached survival. Compared to attached cells, detached cells had reduced total pool sizes of pyruvate, lactate, α-ketoglutarate, fumarate, malate, alanine, serine, and glutamate, suggesting the metabolic stress which occurs under detached conditions. However, intracellular citrate and aspartate pools were unchanged, demonstrating a preference to maintain these pools in detached conditions. Compared to attached cells, detached cells had suppressed glutamine metabolism, as determined by decreased glutamine flux into the TCA cycle and reduced mRNA abundance of glutamine metabolizing enzymes. Further, detached glucose anaplerosis through pyruvate dehydrogenase activity was decreased, while pyruvate carboxylase (PC) expression and activity were increased. A switch in metabolism was observed away from glutamine anaplerosis to a preferential utilization of PC activity to replenish the TCA cycle, determined by reduced PC mRNA abundance in detached cells treated with a cell-permeable analog of α-ketoglutarate, the downstream metabolite of glutamine which enters the TCA cycle. These results suggest that detached cells elevate PC to increase flux of carbons into the TCA cycle when glutamine metabolism is reduced. </p> <p>Vitamin D is recognized for its role in preventing breast cancer progression, and recent studies suggest that regulation of energy metabolism may contribute to its anticancer effects. Vitamin D primarily acts on target tissue through its most active metabolite, 1α,25-dihydroxyvitamin D (1,25(OH)₂D). The present work investigated 1,25(OH)₂D’s effects on viability of detached cells through regulation of energy metabolism. Treatment of MCF10A-<i>ras</i> cells with 1,25(OH)₂D resulted in decreased viability of detached cells. While 1,25(OH)₂D treatment did not affect many of the glucose metabolism outcomes measured, including intracellular pyruvate and lactate pool sizes, glucose flux to pyruvate and lactate, and mRNA abundance of enzymes involved in glucose metabolism, 1,25(OH)₂D treatment reduced detached PC expression and glucose flux through PC. A reduction in glutamine metabolism was observed with 1,25(OH)₂D treatment, although no 1,25(OH)₂D target genes were identified. Further, PC depletion by shRNA decreased cell viability in detached conditions with no additional effect with 1,25(OH)₂D treatment. Moreover, PC overexpression resulted in increased detached cell viability and inhibited 1,25(OH)₂D’s negative effects on viability. These results suggest that 1,25(OH)₂D reduces detached cell viability through regulation of PC. Collectively this work identifies a key metabolic adaptation where detached cells increase PC expression and activity to compensate for reduced glutamine metabolism and that 1,25(OH)₂D may be utilized to reverse this effect and decrease detached cell viability. These results contribute to an increased understanding of metastatic processes and the regulation of these processes by vitamin D, which may be effective in preventing metastasis and improve breast cancer patient survival.</p>

The ability to reprogram adult somatic cells into induced pluripotent stem (iPS) cells could provide a valuable implement for in vitro disease modeling and drug discovery. More importantly, they may potentially serve as an unlimited source of cells for regenerative medicine. However, most of the iPS cells have been generated by retroviral vectors, and therefore they carry the risk of viral integration into the host genome. This problem prevents their use for clinical applications and regenerative medicine. mRNA-mediated delivery of reprogramming factors is an alternative approach for cellular reprogramming. mRNA-based reprogramming offers the advantage of being completely free of genomic integration and is therefore highly suitable for clinical translation. However, there are some limitations which must be overcome so that mRNA can be widely used for successful cellular reprogramming. In the current thesis, the attempt was to generate stable mRNA-iPS cells through overcoming those limitations. Several human donor cells were transfected with mRNA encoding reprogramming factors and the generation of two stable mRNA-iPS cell lines was shown. The resultant mRNA-iPS colonies were assessed for pluripotency markers. Their pluripotency features were evaluated by the viral-iPS cells produced by conventional retroviral vectors. It was noticed that the generation of mRNA-iPS cells was largely affected by the parental cells from which they were derived. However, characterization and evaluation of the generated mRNA-iPS cells proved their pluripotency states comparable to the viral-iPS cells. On the other hand, the aging hallmarks of the iPS cells were assessed in the second part of this thesis. The potential aging signatures of the iPS cells should be conducted before their use in clinical applications. Currently, there are controversial data regarding the ability of reprogramming to fully rejuvenate an aged somatic cell and reverse agerelated changes such as shortened telomeres, dysfunctional mitochondria and DNA damage. Moreover, mixed findings have been published regarding whether the iPS cells are fully rejuvenated or they might retain some of the aging hallmarks from the cells which they were derived. This thesis studied these controversies through the investigation of three hallmarks of aging including telomere length, mitochondrial alteration and DNA damage. Telomere elongation was indicated in the iPS cells. Furthermore, mitochondrial morphology and function were improved into more immature features in iPS cell lines than their corresponding fibroblasts. Moreover, the iPS cell lines were shown to have less amount of DNA damage compared to their parental fibroblasts. In summary, it can be concluded that generation of mRNA-iPS cells is largely affected by the primary donor cells from which they are derived. Furthermore, it seems that reprogramming enables reversion of aging signatures to a more youthful state.

Myeloid precursor cell reprogramming into a myeloid-derived suppressor cell (MDSC) contributes to high mortality rates in mouse and human sepsis. S100A9 mRNA and intracellular protein levels increase during early sepsis and remain elevated in Gr1+CD11b+ MDSCs after pro-inflammatory sepsis transitions to the later chronic anti-inflammatory and immunosuppressive phenotype. The purpose of this study was to determine whether intracellular S100A9 protein might sustain Gr1+CD11b+ MDSC repressor cell reprogramming during sepsis. We used a chronic model of sepsis in mice to show that S100A9 release from MDSCs and circulating phagocytes decreases after early sepsis and that targeting the S100a9 gene improves survival. Surprisingly, we find that intracellular S100A9 protein translocates from the cytosol to nucleus in Gr1+CD11b+ MDSCs during late sepsis and promotes expression of miR-21 and miR-181b immune repressor mediators. We further provide support of this immunosuppression pathway in human sepsis. This study may inform a new therapeutic target for improving sepsis outcome.

Somatic cell nuclear transfer (SCNT) is a useful tool for selective breeding, conservation, and production of transgenic animals. Despite the successful cloning of several species, high rates of embryo failure following SCNT prevent the wide-scale use of the technique. Embryos produced through cloning have a higher incidence of developmental arrest, decreased developmental potential, frequent implantation failures, and increased incidence of abortion. The objective of this dissertation research was to characterize the factors that lead to SCNT failures by examining epigenetic and apoptotic pathways that can negatively influence the development of cloned preimplantation embryos. Aberrant genome reprogramming is generally considered to be a key factor in the failure of SCNT embryo development. Therefore, we used bisulfite pyrosequencing technology to compare DNA methylation patterns of several genes critical for embryonic development (POU5F1, NANOG, SOX2, and KLF4) in SCNT and in vitro fertilized (IVF) blastocyst stage embryos. The methylation profiles obtained from these experiments indicate that methylation patterns of the POU5F1 gene were undermethylated compared to IVF embryos, suggesting reprogramming did occur, but that the reduced methylation was inappropriate for the blastocyst stage. Furthermore, aberrant methylation profiles were detected for SOX2 and NANOG, suggesting that problems of genome reprogramming following SCNT can be gene-specific or localized. Because high rates of apoptosis are associated with failure of preimplantation embryos, we compared the activation of the P53-mediated apoptosis pathway in individual IVF and SCNT preimplantation embryos at multiple developmental stages. This pathway is activated in response to cell stress and genomic instability, and in response to the expression of genes associated with somatic cell reprogramming. Evidence from gene expression and immunohistochemistry analyses suggests that the P53 pathway is frequently active in SCNT embryos. Also, we detected expression of several factors known to induce apoptosis more frequently and at higher levels in SCNT embryos. Collectively, the work presented here illuminates some of the molecular consequences of incomplete or inappropriate genome reprogramming in cloned embryos. The identification of these factors may lead to interventions that target the apoptosis pathway during preimplantation development and increase SCNT success rates.

Les maladies complexes comme le Cancer et la maladie d'Alzheimer sont causées par des perturbations moléculaires multiples responsables d'un comportement cellulaire pathologique.Un enjeu majeur de la médecine de précision est l'identification des perturbations moléculaires induites par les maladies complexes et les thérapies à partir de leurs conséquences sur les phénotypes cellulaire.Nous définissons un modèle des maladies complexes,appelé la reprogrammation comportementale,assimilant les perturbations moléculaires à des altérations des fonctions dynamiques locales de systèmes dynamiques discrets induisant une reprogrammation de la dynamique globale du réseau. Ce cadre de modélisation s'appuie d'une part, sur les réseaux Booléens contrôlés, qui sont des réseaux Booléens dans lesquels sont insérés des paramètres de contrôle modélisant les perturbations et, d'autre part, sur la définition de modes (Possibilité, Nécessité) permettant d'exprimer les objectifs de cette reprogrammation.A partir de ce cadre, nous démontrons que le calcul des noyaux, i.e., des ensembles minimaux d'actions permettant la reprogrammation selon un mode s'exprime comme un problème d'inférence abductive en logique propositionnelle. En nous appuyant sur les méthodes historiques de calcul d'impliquants premiers des fonctions Booléennes,nous développons deux méthodes permettant le calcul exhaustif des noyaux de la reprogrammation. Enfin, nous évaluons la pertinence du cadre de modélisation pour l'identification des perturbations responsables de la transformation d'une cellule saine en cellule cancéreuse et la découverte de cibles thérapeutiques sur un modèle du cancer du sein. Nous montrons notamment que les perturbations inférées par nos méthodes sont compatibles avec la connaissance biologique en discriminant les oncogènes des gènes suppresseurs de tumeurs et en récupérant la mutation du gène BRCA1. De plus, la méthode récupère le phénomène de létalité synthétique entre PARP1 et BRCA1, qui constitue un traitement anticancéreux optimal car il cible spécifiquement les cellules tumorales. / Complex diseases such as cancer and Alzheimer's are caused by multiple molecular perturbations responsible for pathological cellular behavior. A major challenge of precision medicine is the identification of the molecular perturbations induced by the disease and the therapies from their consequences on cell phenotypes. We define a model of complex diseases, called behavioral reprogramming, that assimilates the molecular perturbations to alterations of the dynamic local functions of discrete dynamical systems inducing a reprogramming of the global dynamics of the network. This modeling framework relies on the one hand, on Control Boolean networks, which are Boolean networks containing control parameters modeling the perturbations and, on the other hand, the definition of reprogramming modes (Possibility, Necessity) expressing the objective of the behavioral reprogramming. From this framework, we demonstrate that the computation of the cores, namely, the minimal sets of action allowing reprogramming is a problem of abductive inference in propositional logic. Using historical methods computing the prime implicants of Boolean functions, we develop two methods computing all the reprogramming cores.Finally, we evaluate the modeling framework for the identification of perturbations responsible for the transformation of a healthy cell into a cancercell and the discovery of therapeutic targets ona model of breast cancer. In particular, we showthat the perturbations inferred by our methods a recompatible with biological knowledge by discriminating oncogenes and tumor suppressor genes and by recovering the causal of the BRCA1 gene. In addition, the method recovers the synthetic lethality phenomenon between PARP1 and BRCA1 that constitutes an optimal anti-cancer treatment because it specifically targets tumor cells.