Cancer, despite significant advances in diagnosis and treatment, is the second most common cause of death in economically advanced countries. The main reason for the failure of anticancer therapy is the development of chemoresistance, which can be either internal or acquired, and is primarily mediated by the activation of various key regulators (eg MDR, PI3K/Akt, etc.). Genetic and epigenetic mechanisms are involved in activating these pathwa- ys. Significant epigenetic mechanisms that can participate in chemoresistance include regula- tion of gene expression by microRNA (miRNA) and long noncoding RNA (lncRNA). Dere- gulated expression of these non-coding RNAs has been observed in many diseases and their involvement in the initiation and progression of malignant tumors has been demonstrated. In this study, we investigated the expression of long non-coding RNA MIAT in hypoxia (1% O2) in chemosensitive and chemoresistant neuroblastoma cell lines (NBL), as hypoxia is a significant negative prognostic factor of many tumors and is involved in chemoresistance. Relative expression of MIAT was influenced by the number of cultured cells, where expression was increased by culturing more cells. MIAT expression was also significantly increased after 6 hours of NBL culture UKF-NB-4 in hypoxic conditions, and...

L’hypoxie tumorale est l’une des principales causes de l’agressivité des glioblastomes (GB). Plusieurs études attestent de l’implication de l’isoforme HIF-1α (hypoxia inducible factor-1α) dans la progression de ces tumeurs et dans leur résistance à la radiothérapie (RT). Plus récemment, il a été établi que l’isoforme HIF-2α régule la capacité tumorigénique des cellules souches de GB (CSG). Cependant, le rôle de ce facteur dans la croissance des cellules de GB différenciées et leur réponse à la RT est moins documenté. Dans ce contexte, l’objectif de ce travail a été de renforcer ces connaissances à l’échelle préclinique en utilisant deux approches d’ARN interférence (ARNi) pour moduler l’expression de HIF-2 : cibler directement HIF-2α ou cibler HAF (hypoxia associated factor), un facteur impliqué dans le switch de HIF-1α vers HIF-2α. Les résultats obtenus sur un modèle orthotopique de cellules humaines de GB (U251-MG) différenciées montrent que l’invalidation de HAF conduit à un fort ralentissement de la croissance de ces tumeurs mais indépendamment de HIF-1α ou HIF-2α. L’effet de l’invalidation de HIF-2α serait, quant à lui, dépendant de l’environnement tumoral. En effet, la diminution d’expression de HIF-2α dans les cellules U251 ne modifie pas la croissance tumorale dans un modèle de greffe sous-cutanée, alors que celle-ci favorise la croissance tumorale lorsque les cellules de GB sont implantées en intracérébral. Par comparaison aux tumeurs contrôles, ces tumeurs sont plus invasives et mieux perfusées. In vitro, l’inhibition de l’expression de HIF-2α n’a aucun effet sur la survie des cellules U251 alors qu’elle diminue la mort apoptotique de ces cellules exposées aux rayons X.L’ensemble des données présentées dans cette étude suggère que HAF et HIF-2α pourraient réguler la capacité tumorigénique des cellules de GB différenciées, tout comme observé pour les CSG. En outre, ces résultats soulignent la nécessité de prendre en compte le microenvironnement cellulaire afin de mieux comprendre le comportement de la tumeur dans son environnement hypoxique. / Hypoxia is one of the main causes of glioblastoma (GB) aggressiveness. Various studies attest on the involvement of the HIF-1α isoform (hypoxia inducible factor-1α) in the progression of these tumors and in their resistance to radiation therapy (RT). More recently, it was established that the HIF-2α isoform regulates the tumorigenic capacity of GB stem cells (GSC). However, the role of this factor in the growth of differentiated GB cells and their response to RT is less documented. In this context, the goal of this work was to strengthen this knowledge at the preclinical level by using two RNA interference (RNAi) strategies to modulate the expression of HIF-2: one directly targets HIF-2α, the other one targets HAF (hypoxia associated factor), a factor involved in the switch of HIF-1α to HIF-2α. Our results obtained on an orthotopic model of differentiated human GB (U251-MG) cells showed that the invalidation of HAF leads to a strong slowdown in tumor growth but independently of HIF-1α or HIF-2α. On the other hand, the effect of HIF-2α silencing seems dependent on the tumor environment. Indeed, the extinction ofHIF-2α expression in U251 cells does not modify tumor growth in a subcutaneous model, whereas it promotes tumor growth when GB cells are intracerebrally grafted. Compared to control tumors, these tumors are more invasive and highly perfused. In vitro, the inhibition of HIF-2α expression has no effect on GB cell survival whereas decreasing the X-rays induced apoptotic death.Collectively, these data suggest that HAF and HIF-2α could regulate the tumorigenic capacity of differentiated GB cells, like it does in CSGs. In addition, these results highlight the need to take into account the cellular microenvironment to better understand the behavior of the tumor in its hypoxic environment.

Understanding species-specific physiological tolerances to environmental extremes is key in determining the factors that contribute to regulating species distribution. This understanding will aid in determining which species will manage to thrive in a changing global climate. According to the IPCC (2013) it is expected that, in the coming years, many different types of abiotic factors will change as a result of global climate change. The intertidal habitat is a model habitat for studying environmental extremes as it is located at the interface between the marine and terrestrial environments, making it one of the most stressful marine habitats. It is characterized by a 24 hr light: dark cycle and a 12.4 tidal ebb and flow that exposes animals inhabiting this habitat to a wide array of aerial-associated stressors such as changes in temperature, aerial exposure, low oxygen or hypoxic conditions and desiccation stress. Sessile organisms such as marine mussels of the genus Mytilus, are an ideal study species for studying physiological tolerance at the environmental extremes of the intertidal habitat. In particular, M. trossulus and M. galloprovincialis are an excellent study system for examining physiological tolerance at environmental extremes due to the recent change in biogeographic range of both species. M. galloprovincialis, a native of the Mediterranean, has been taking over the coast of California and has been displacing the heat sensitive native M. trossulus. The effects of salinity stress and heat stress on the physiologies of these species have been investigated by Braby and Somero (2006a and 2006b), Tomanek and Zuzow (2010) and Tomanek et al. (2012). The results of these studies indicate that the invasive M. galloprovincialis is more heat tolerant but is sensitive to hyposalinity while the reverse is true for the native M. trossulus.. The next logical environmental stress to study is low tide or aerial-emersion as both species can be found both tidally and subtidally. According to Grieshaber et al. (1994) and Müller et al. (2012) Mytilus edulis mussels have mechanisms for mitigating aerial-emersion hypoxia; however, very few studies have been performed using the study system of M. trossulus and M. galloprovincialis in regard to aerial-emersion hypoxia. This study aimed to observe the responses of both M. trossulus and M. galloprovincialis to aerial-emersion hypoxic stress or low tide. The study also looked to see if the recent habitat history (tidal or subtidal) can play a role in the response of the mussels to hypoxia. The results of the experiment indicate that the invasive M. galloprovincialis may be less sensitive to hypoxic stress when compared to the native M. trossulus. This difference in sensitivity may be due to the difference in mechanisms of energy metabolism proteins and proteostasis proteins used to mitigate the effects of hypoxic stress. Moreover, tidal acclimation appears to better prepare the mussels for subsequent aerial exposure in both species, possibly based upon the principles of stress-hardening outlined by Kültz (2005).

Indiana University-Purdue University Indianapolis (IUPUI) / The human genome generates a vast number of functionally and structurally diverse noncoding transcripts, incorporated into complex networks which modulate the activity of classic pathways. Long noncoding RNAs (lncRNA) have been shown to exhibit diverse regulatory roles in various physiological and pathological processes. Hypoxia, a key feature of the tumor microenvironment, triggers adaptive responses in cancer cells that involve hundreds of genes. While the coding component of hypoxia signaling has been extensively studied, much less information is available regarding its noncoding arm. My doctoral work identified and functionally characterized a novel hypoxia-inducible lncRNAs encoded from the miR193b-host gene (MIR193BHG) locus, on chromosome 16. In the pursuit of understanding how MIR193BHG responds to hypoxia, we discovered a more complex transcriptional control of MIR193BHG by hypoxia. Ectopic expression of MIR193BHG in breast cancer cell lines in vitro and in xenografts significantly represses cell invasion, as well as the metastasis to lung and liver. Conversely, inhibition of MIR193BHG promotes cancer cell invasiveness and metastasis. RNAseq followed by pathway analysis revealed that MIR193BHG is a negative modulator of cholesterol biosynthesis pathway. MIR193BHG exerts a highly coordinated effect on the expression of cholesterol biosynthetic genes which leads to a measurable impact on the total cellular cholesterol content. The role of MIR193BHG in cholesterol metabolism also provided a mechanistic explanation for the sex maturation associated SNPs located in vicinity of this gene locus. Our work also provided preliminary insights into the functional mechanism of MIR193BHG by showing that its modulation of genes in cholesterol synthesis is predominantly at transcriptional level. Overall, my dissertation project identified a non-canonical hypoxia-inducible lncRNA, MIR193BHG, which modulates breast cancer invasion and metastasis via finetuning of cholesterol synthesis.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19369号 / 医博第4046号 / 新制||医||1012(附属図書館) / 32383 / 新制||医||1012 / 京都大学大学院医学研究科医学専攻 / (主査)教授 宮本 享, 教授 柳田 素子, 教授 松原 和夫 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

http://pubs.rsna.org/doi/full/10.1148/radiol.2015150416 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19575号 / 医博第4082号 / 新制||医||1013(附属図書館) / 32611 / 京都大学大学院医学研究科医学専攻 / (主査)教授 平岡 眞寛, 教授 YOUSSEFIAN Shohab, 教授 増永 慎一郎 / 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20974号 / 医博第4320号 / 新制||医||1026(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 増永 慎一郎, 教授 高田 穣, 教授 武田 俊一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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The purpose of this study was to investigate the effects of Pb and hypoxia co-exposure on Pb toxicity and Ca homeostasis in early life stage (ELS) zebrafish (Danio rerio). Previous evidence indicates that exposure of ELS zebrafish to hypoxia (~20% air saturation) reduces Ca uptake, likely through down-regulation of the apical epithelial Ca channel (ECaC). Considering that Pb and Ca are known antagonists and compete for uptake pathways, it was hypothesized that co-exposure of Pb with hypoxia would decrease Pb toxicity by reducing Pb uptake (likely mediated through a reduced number of ECaCs). However, it was shown that at 96 hpf, whole body accumulation of both Pb and Ca was lower at 40% air saturation compared to 100% and 20% air saturation. This result closely aligned with the 96h LC50 results which showed the highest mortality of zebrafish at 40% compared to the other air saturation levels. This suggests that toxicity is likely the result of exacerbated hypocalcemia at 40% air saturation due to both Pb competition for Ca binding to Ca uptake channels/transporters, such as ECaC, and potentially reduced expression of such channels/transporters in response to this level of hypoxia. Overall, it appears that ELS zebrafish respond differentially to the 40% and 20% hypoxia levels when co-exposed with Pb. Further investigation is needed to illustrate the physiological and molecular mechanisms underlying this response.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24190号 / 医博第4884号 / 新制||医||1060(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 伊藤 能永, 教授 安達 泰治, 教授 椛島 健治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM