Pediatric medulloblastoma (MB) is the most common solid malignant brain neoplasm, with group 3 (G3) MB representing the most aggressive subgroup. Despite MYC amplification representing an independent poor prognostic factor in G3 MB, efforts to target the MYC pathway have met with limited therapeutic success. As such, alternative mediators of G3 MB continue to be sought. The RNA binding protein and neural stem cell determinant Musashi-1 (MSI1) has been implicated in a number of adult stem cells in various organs (e.g., brain, gut, ovaries/testes) with mounting evidence that MSI1 is an essential regulator of cancer stem cells (e.g., brain, gut, lung). Early studies in MB have shown MSI1 to be essential for tumour maintenance, however the direct interactions and specific mechanisms conferring tumours with high MSI1 expression (i.e., G3 MB) are yet to be determined. Here, I show MSI1 is an essential moderator of G3 MB in both a MYC amplified and p53 mutated (MP) mouse model of G3 MB and patient-derived xenograft (PDX) models. MSI1 inhibition resulted in an abrogation of tumour initiation in both models, translating to a significantly prolonged survival. To determine how MSI1 regulates the post-transcriptional landscape of human G3 MB, an unbiased multiplatform approach was undertaken, using enhanced cross-linking and immunoprecipitation (eCLIP), and differential analyses post-MSI1 inhibition at the transcriptome-, proteome-, and translatome-wide scale, revealing MSI1's key role in moderating G3 MB-associated cancer driving genes. In summary, employing innovational multi-platform integrative approach to stem cell cancer biology, I show the neural RNA binding protein MSI1, an essential master stem cell regulator, is hijacked from its normal neural developmental function to orchestrate the aberrant translational landscape of G3 MB. / Thesis / Doctor of Philosophy (PhD) / Brain tumours are the leading cause of childhood cancer death with medulloblastoma (MB) representing the most frequent malignant childhood brain tumour. Analysis of the data retrieved from multiple genetic studies of MB, we have determined that there are 4 genetic subgroups of MB: Wnt, Shh, Group 3 (G3) and Group 4 (G4). The subgroup with the worse prognosis is Group 3, and unique to this subgroup is the overproduction of the MYC gene products (i.e., MYC amplification). In fact, MYC amplification alone is associated with a poor prognosis in these children. As such many researchers and clinicians have been working together to find a way to target MYC. Although many pre-clinical experimental studies have cured MYC-amplified G3 MB using gene-targeting therapy, these results unfortunately have not translated into early clinical trials. Therefore, alternative targets that mediate the aggressiveness of MYC-amplified G3 MB is being sought. As cancer stem cells (CSC) have been implicated in tumour development and maintenance, a gene worthy of investigation in a neurodevelopmental tumour such as MB, is Musashi-1 (MSI1). MSI1 protein has been identified in high levels in many human cancers, been observed to play a crucial role in promoting normal stem cell features, and is also implicated in driving cancer. The protein that the MSI1 gene produces binds to genes and modifies them to either stabilize or destabilize their path to becoming a protein. By manipulating MSI1 in both NSC and MB CSC, I will observe how these cells either display greater or less cancer associated features. Further, with a new technology allowing researchers to identify MSI1 binding sites, we aim to determine how MSI1 modifies cancer causing and normal neural stem cell genes. Moreover, I will be studying both the gene-, pre-protein- and protein-level changes after experimentally manipulating MSI1 gene levels to tease out its’ main cancer associated function. Altogether, we found a core list of genes that MSI1 modulates with functional significance giving us clues for a therapeutic targeting strategy for G3 MB.

Musashi-2 (MSI2), a member of the Musashi family of RNA-binding proteins, is thought to play a critical role in the maintenance of stem cell populations and in the formation of aggressive tumours. Multiple studies indicate that MSI2 plays an important role in the maintenance of hematopoietic stem cell (HSC) populations and recent studies in humans identify MSI2 as an independent prognostic factor for overall survival in patients with Acute Myeloid Leukemia (AML). Importantly, though correlative studies implicate MSI2 as a contributor to aggressive disease in human AML, no study to date has attempted to analyze the functional role of MSI2 in primary human AML samples. Furthermore, though MSI2 is critical for the maintenance of HSCs, the mechanisms through which MSI2 functions are unknown. The work presented in this thesis elucidates the biochemical mechanisms through which MSI2 functions and examines the functional role of MSI2 in human AML. Using a lentiviral-mediated shRNA knockdown of MSI2, I demonstrate that MSI2 is critical for the maintenance of human AML. A loss of MSI2 greatly impairs the ability of AML samples to maintain disease in a xenotransplantation assay. MSI2 is an RNA binding protein that is thought to repress the translation of target mRNAs in the cytoplasm and prevent the maturation of microRNAs (miRNAs) in the nucleus. The targets of MSI2 are believed to be potent regulators of stem-ness and dysregulation of these targets could very well contribute to neoplastic transformation. Cross-linking immunoprecipitation followed by next generation sequencing (CLIP-Seq), revealed the RNA binding properties of MSI2 and the RNA targets bound by MSI2. To identify novel MSI2 protein interactors, the MSI2 locus was endogenously tagged with the promiscuous biotin ligase BirA* and subjected to BioID analysis. When compared to appropriate controls, we were able to robustly identify proteins that associate with MSI2. The analysis of one of these protein binding partners, Insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) reveals a critical role in the normal function of HSCs. / Thesis / Doctor of Philosophy (PhD) / The hematopoietic system is responsible for the production of billions of mature cells everyday. These mature cells are “differentiated”, meaning that they have gone through a process that has allowed them to become specialized to perform a very specific role. Throughout the process of differentiation, most functional cells lose their ability to proliferate. The continued production of these functional cells comes from a pool of rare, quiescent, hematopoietic stem cells (HSC). These cells maintain the production of mature cells throughout the lifetime of an organism. The Musashi-2 (MSI2) protein has been identified as a protein that is critical for the normal function of HSCs. By altering the levels of the MSI2, it is possible to greatly impair or enhance the activity of HSCs. Moreover, correlative studies implicate MSI2 as a contributor to aggressive Acute Myeloid Leukemia (AML), a disease that occurs when HSCs become dysregulated. Despite its important roles in normal and abnormal hematopoiesis, very little is known about how MSI2 functions and whether it actually has a functional role in AML. We set forth to identify mechanisms through which the MSI2 protein functions and to prove that MSI2 contributes to the maintenance of human AML. We reveal that the MSI2 protein plays a critical role for the maintenance of human AML and identify novel pathways through which the protein functions. Importantly, MSI2 is known to interact with mRNA in order to alter post-transcriptional gene expression. We thoroughly characterize the RNA-binding characteristics of MSI2 and identify a plethora of MSI2 RNA targets. In an unbiased manner, we also identify a list of MSI2-protein interactors. We identify one MSI2 protein-binding partner, Insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) that is preferentially expressed in the most immature fraction of HSCs and is critical for the proper function of HSCs.

Musashi (1935-1939), Yoshikawa Eiji\'s novel serialized by the newspa-per Asahi Shinbun and considered by John Scott Miller (2009) as a Bild-ungsroman, it narrates from Miyamoto Musashis return from the battle of Sekigahara (1600) to his victory against Sasaki Kojir in Ganry is-land (1612), when he becomes of the best swordsmen at that age. The early seventeenth century Japan that underwent a major transition is the stage for the formation of Musashi in light of the Japanese world and his own feelings and choices. All these changes are intertwined in the decision of self-exile after the seclusion amid the range of erudition, as previously to this watershed, the protagonist himself felt misunder-stood and excluded by his family members and the other members of society. In order to develop this study, we will make a clipping of Mu-sashis path to understand the essence of his isolation, and his choice to preserve his individuality so that it makes it allowed him to exercise his creativity. Thus, based on the panorama on the Bildgunsroman (Ro-mance of Formation) made by Wilma Maas (2000), we will be able to follow the stimuli and aspirations that guides him toward his formation, until reaching a totally unique development. In order to understand Mu-sashi\'s motivation to adopt exile as a way of life, in addition to attesting his gains, we base on the studies of Kat Shichi (2012) and Edward Said (2003). / Musashi (1935-1939), Yoshikawa Eiji\'s novel serialized by the newspa-per Asahi Shinbun and considered by John Scott Miller (2009) as a Bild-ungsroman, it narrates from Miyamoto Musashis return from the battle of Sekigahara (1600) to his victory against Sasaki Kojir in Ganry is-land (1612), when he becomes of the best swordsmen at that age. The early seventeenth century Japan that underwent a major transition is the stage for the formation of Musashi in light of the Japanese world and his own feelings and choices. All these changes are intertwined in the decision of self-exile after the seclusion amid the range of erudition, as previously to this watershed, the protagonist himself felt misunder-stood and excluded by his family members and the other members of society. In order to develop this study, we will make a clipping of Mu-sashis path to understand the essence of his isolation, and his choice to preserve his individuality so that it makes it allowed him to exercise his creativity. Thus, based on the panorama on the Bildgunsroman (Ro-mance of Formation) made by Wilma Maas (2000), we will be able to follow the stimuli and aspirations that guides him toward his formation, until reaching a totally unique development. In order to understand Mu-sashi\'s motivation to adopt exile as a way of life, in addition to attesting his gains, we base on the studies of Kat Shichi (2012) and Edward Said (2003).

Musashi (1935-1939), Yoshikawa Eiji\'s novel serialized by the newspa-per Asahi Shinbun and considered by John Scott Miller (2009) as a Bild-ungsroman, it narrates from Miyamoto Musashis return from the battle of Sekigahara (1600) to his victory against Sasaki Kojir in Ganry is-land (1612), when he becomes of the best swordsmen at that age. The early seventeenth century Japan that underwent a major transition is the stage for the formation of Musashi in light of the Japanese world and his own feelings and choices. All these changes are intertwined in the decision of self-exile after the seclusion amid the range of erudition, as previously to this watershed, the protagonist himself felt misunder-stood and excluded by his family members and the other members of society. In order to develop this study, we will make a clipping of Mu-sashis path to understand the essence of his isolation, and his choice to preserve his individuality so that it makes it allowed him to exercise his creativity. Thus, based on the panorama on the Bildgunsroman (Ro-mance of Formation) made by Wilma Maas (2000), we will be able to follow the stimuli and aspirations that guides him toward his formation, until reaching a totally unique development. In order to understand Mu-sashi\'s motivation to adopt exile as a way of life, in addition to attesting his gains, we base on the studies of Kat Shichi (2012) and Edward Said (2003). / Musashi (1935-1939), Yoshikawa Eiji\'s novel serialized by the newspa-per Asahi Shinbun and considered by John Scott Miller (2009) as a Bild-ungsroman, it narrates from Miyamoto Musashis return from the battle of Sekigahara (1600) to his victory against Sasaki Kojir in Ganry is-land (1612), when he becomes of the best swordsmen at that age. The early seventeenth century Japan that underwent a major transition is the stage for the formation of Musashi in light of the Japanese world and his own feelings and choices. All these changes are intertwined in the decision of self-exile after the seclusion amid the range of erudition, as previously to this watershed, the protagonist himself felt misunder-stood and excluded by his family members and the other members of society. In order to develop this study, we will make a clipping of Mu-sashis path to understand the essence of his isolation, and his choice to preserve his individuality so that it makes it allowed him to exercise his creativity. Thus, based on the panorama on the Bildgunsroman (Ro-mance of Formation) made by Wilma Maas (2000), we will be able to follow the stimuli and aspirations that guides him toward his formation, until reaching a totally unique development. In order to understand Mu-sashi\'s motivation to adopt exile as a way of life, in addition to attesting his gains, we base on the studies of Kat Shichi (2012) and Edward Said (2003).

Die Neubildung von Neuronen persistiert lebenslang in der Subgranularzellschicht des Hippocampus und der Subventrikularzone des Großhirns und wird als adulte Neuroge-nese bezeichnet. Es wird vermutet, dass diese beim erwachsenen Menschen einen rele-vanten Einfluss auf degenerative Veränderungen, verschiedene neurologische Krank-heitsbilder und auf die (Dys-)Funktion des Gedächtnisses hat. Im Tiermodell wurde eine Verringerung der Neurogenese nach chronischer Morphingabe nachgewiesen. Vorarbeiten zeigten einen Zusammenhang zwischen chronischem Heroinmissbrauch und reaktiver Astrogliose, Mikrogliose und einer vermehrten Expression des polysialylated neural cell adhesion molecule im humanen Hippocampus. Daraus leitet sich die Hypothese ab, dass chronischer Heroinmissbrauch, als Modell für eine Abhängigkeitserkrankung, einen Einfluss auf die adulte humane Neurogenese hat. Es wurden in Formalin fixierte Gewebeproben aus dem Hippocampus von Verstorbenen mit einer letalen Heroinintoxikation und mit bekanntem Heroinmissbrauch (n = 20) un-tersucht und mit einer nach Alter und Geschlecht angepassten Kontrollgruppe (n = 28) verglichen. Hierbei wurden spezifische Neurogenesemarker mittels immunhistochemi-scher Methoden angewendet und ausgewertet. Es bestand eine generell sehr geringe zelluläre Proliferationsrate und eine signifikante Reduktion Musashi-1 positiver neuro-naler Vorläuferzellen bei gleichzeitig unveränderter Anzahl Nestin positiver reifender und Calretinin positiver migrierender postmitotischer Neurone. Zudem wurde ein ver-ändertes Calretinin-Expressionsmuster als Hinweis auf eventuelle funktionelle neuronale Defizite bei Drogenabhängigen festgestellt. Der potentielle Einfluss von chronischem Heroinmissbrauch auf die adulte humane Neurogenese wird erstmals gezeigt. Die Ergebnisse weisen auf eine negative Beeinflus-sung im Stadium neuronaler Vorläuferzellen und der Zellfunktion migrierender Neurone in der Fallgruppe im Vergleich zu einer gesunden Kontrollgruppe hin. Diese Hemmung der Neurogenese könnte eine Erklärungsmöglichkeit für kognitive Defizite und Funktionsstörungen des Gedächtnisses infolge chronischen Drogenkonsums bieten und zugleich eine Bedeutung bei der Entstehung von Abhängigkeitserkrankungen haben. Insofern könnte sich hier ein Ansatzpunkt für zukünftige Therapiestrategien derartiger Erkrankungen oder ihrer Folgen bieten.

Die Neubildung von Neuronen persistiert lebenslang in der Subgranularzellschicht des Hippocampus und der Subventrikularzone des Großhirns und wird als adulte Neuroge-nese bezeichnet. Es wird vermutet, dass diese beim erwachsenen Menschen einen rele-vanten Einfluss auf degenerative Veränderungen, verschiedene neurologische Krank-heitsbilder und auf die (Dys-)Funktion des Gedächtnisses hat. Im Tiermodell wurde eine Verringerung der Neurogenese nach chronischer Morphingabe nachgewiesen. Vorarbeiten zeigten einen Zusammenhang zwischen chronischem Heroinmissbrauch und reaktiver Astrogliose, Mikrogliose und einer vermehrten Expression des polysialylated neural cell adhesion molecule im humanen Hippocampus. Daraus leitet sich die Hypothese ab, dass chronischer Heroinmissbrauch, als Modell für eine Abhängigkeitserkrankung, einen Einfluss auf die adulte humane Neurogenese hat. Es wurden in Formalin fixierte Gewebeproben aus dem Hippocampus von Verstorbenen mit einer letalen Heroinintoxikation und mit bekanntem Heroinmissbrauch (n = 20) un-tersucht und mit einer nach Alter und Geschlecht angepassten Kontrollgruppe (n = 28) verglichen. Hierbei wurden spezifische Neurogenesemarker mittels immunhistochemi-scher Methoden angewendet und ausgewertet. Es bestand eine generell sehr geringe zelluläre Proliferationsrate und eine signifikante Reduktion Musashi-1 positiver neuro-naler Vorläuferzellen bei gleichzeitig unveränderter Anzahl Nestin positiver reifender und Calretinin positiver migrierender postmitotischer Neurone. Zudem wurde ein ver-ändertes Calretinin-Expressionsmuster als Hinweis auf eventuelle funktionelle neuronale Defizite bei Drogenabhängigen festgestellt. Der potentielle Einfluss von chronischem Heroinmissbrauch auf die adulte humane Neurogenese wird erstmals gezeigt. Die Ergebnisse weisen auf eine negative Beeinflus-sung im Stadium neuronaler Vorläuferzellen und der Zellfunktion migrierender Neurone in der Fallgruppe im Vergleich zu einer gesunden Kontrollgruppe hin. Diese Hemmung der Neurogenese könnte eine Erklärungsmöglichkeit für kognitive Defizite und Funktionsstörungen des Gedächtnisses infolge chronischen Drogenkonsums bieten und zugleich eine Bedeutung bei der Entstehung von Abhängigkeitserkrankungen haben. Insofern könnte sich hier ein Ansatzpunkt für zukünftige Therapiestrategien derartiger Erkrankungen oder ihrer Folgen bieten.:I. Inhaltsverzeichnis 1 II. Bibliografische Zusammenfassung 2 III. Abkürzungsverzeichnis 3 1. Einführung 4 1.1. Drogenabhängigkeit und Epidemiologie 4 1.2. Heroin 6 1.3. Hippocampus 9 1.4. Adulte Neurogenese 11 1.5. Aufgabenstellung und Ziel der Arbeit 14 2. Materialen und Methoden 18 2.1. Fall- und Kontrollgruppe 18 2.2. Toxikologisch-chemische Untersuchungen 20 2.3. Immunhistochemie 21 2.4. Immunfluoreszenz und konfokale Mikroskopie 25 2.5. Quantifizierung, Datenanalyse und Statistik 26 3. Ergebnisse 28 3.1. Deskriptive Datenanalyse 28 3.2. Musashi-1 30 3.3. Nestin 31 3.4. Calretinin 32 3.5. Ki-67 34 3.6. Doublecortin 35 3.7. Doppelimmunfluoreszenz 36 4. Diskussion 37 4.1. Neurogenese – Proliferation (Ki-67) 38 4.2. Neurogenese – Differenzierung (MSI-1, Nestin) 39 4.3. Neurogenese – Reifung (Calretinin) 42 4.4. Methodische Grenzen und Fehlerbetrachtung 43 4.5. Fazit und Ausblick 46 5. Zusammenfassung der Arbeit 48 6. Literaturverzeichnis 52 7. Anlagen 1-8 65 IV. Selbständigkeitserklärung 73 V. Curriculum vitae 74 VI. Publikationen 75 VII. Danksagung 76

京都大学 / 0048 / 新制・課程博士 / 博士(エネルギー科学) / 甲第20729号 / エネ博第357号 / 新制||エネ||70(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 片平 正人, 教授 森井 孝, 教授 木下 正弘 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM

Miyamoto Musashi (1584-1645) wrote the Gorinsho ("Book of Five Rings") at the end of his life. The text is divided into five sections, “Earth,” “Water,” “Fire,” “Wind,” and “Space;” the first three introduce and explain both military strategy and warfare of his school, Niten-ichi-ryū. “Wind” is a critique of the tendencies Musashi noticed in other sword schools, and “Space” describes the concept of warfare and how to embody its “true way” (though this scroll is evidently incomplete). There are many English translations, yet I make the claim that one more is necessary. Since its first translation in 1974 to its most recent in 2009, the Gorinsho’s meaning has been ill represented in English and no extant translation is suitable for scholarly reference. These translations suffer primarily from three flaws: fundamental translation errors, the effacing of cultural references, and an apparent lack of knowledge concerning the Gorinsho’s textual history. The source text for these problematic English translations is invariably the Hosokawa family manuscript, a wholly unsuitable manuscript for translation. In recent years, the Harima Musashi Kenkyūkai, a Japan-based research group, has done much worthwhile scholarship on the Gorinsho and has compiled a new annotated edition of the text based on a thorough examination of all extant manuscripts. My translation is based on their authoritative edition and it benefits greatly from their research. This thesis endeavors to make clear the case that a new scholarly Gorinsho translation is necessary and provide a preliminary, annotated translation to fulfill that need.

Acute myeloid leukemia (AML) is an aggressive malignancy of the hematopoietic system, characterized by the accumulation of abnormally differentiated blast cells that is driven by leukemic stem cells (LSCs). In murine AML, Musashi-2 (MSI2), an RNA-binding protein and positive regulator of stemness, has been implicated in the propagation of disease. While its enhanced expression correlated with poor disease outcome for human AML patients, no study has yet examined its actual functional role in human leukemia. In normal human hematopoietic stem cells (HSCs), we have recently reported the inhibitory effects of MSI2 on the pro-differentiative aryl hydrocarbon receptor (AHR) signaling pathway as a mechanism for promoting self-renewal in HSCs. We hypothesized that elevated MSI2 is critical for maintenance of human AML and promotes unrestrained self-renewal of LSCs in part through constitutive repression of AHR signaling. Our work aimed to unravel the relationship between MSI2 and AHR in the human leukemic context and to determine if activation of AHR signaling can promote differentiation. Results confirmed that MSI2 is preferentially expressed in primary patient LSCs and is negatively correlated with the expression of AHR gene targets. Upon lentiviral knockdown of MSI2 in-vitro and in-vivo, leukemic growth was compromised and increased AHR signaling was observed. Circumventing the inhibitory role of MSI2 in AML, activation of AHR with a potent agonist impaired leukemic progenitor activity and proliferation. In-vivo studies employing reconstitution of immunodeficient mice with primary AML samples showed impairment of AML engraftment for a significant proportion of tested samples upon treatment with an AHR agonist. Overall, our findings from this project indicated that MSI2 is required for human AML propagation and that a decrease in MSI2 inhibitory effects on AHR signaling or direct activation of the AHR signaling pathway via a potent agonist can promote AML cell differentiation and loss. / Thesis / Master of Science (MSc) / The human blood system is sustained by a population of blood stem cells that are tightly regulated in their production of stem and differentiated cells. The Musashi-2 (MSI2) protein is a key regulator of blood stem cell identity through its inhibition of the aryl hydrocarbon receptor (AHR) signaling pathway. When there is dysregulation of blood cell homeostasis, blood malignancies such as acute myeloid leukemia (AML) may arise. In this work, the relationship between MSI2 and the AHR signaling pathway was explored within a myeloid leukemic context. It was shown that MSI2 imposes inhibitory effects on AHR to promote disease progression and that its reduction could help alleviate disease burden. Additionally, it was found that activation of the AHR signaling pathway could overcome the MSI2 differentiation block to create a therapeutic effect. Overall, the results of this project shed light on novel therapeutic strategies and targets for the treatment of AML.

What do we mean by “seeing”? Although we may see the same object in front of us, we each consciously or unconsciously select what we wish to see, eliminating information we find unnecessary. An artist or poet can see in even a tiny flower, which others barely notice, a wealth of colors or countless words. How then do our own eyes and those of others differ? This thesis aims to explore how the act of seeing shapes one’s life and influences it through a consideration of the works of Kobayashi Hideo 小林秀雄 (1902-1983), a literary critic in modern Japan. In 1949 Kobayashi published a long essay entitled “Watakushi no jinseikan” 私の人生観(My View of Life), originally given as a speech in 1948 when he was forty-six years old. In this work Kobayashi analyzes the word kan 観 (vision) with reference to more than forty historical figures from both the West and the East. The thesis selects for discussion two of these in particular, namely Miyamoto Musashi 宮本武蔵(1584-1645), a Japanese warrior of the early Edo era, and Henri Bergson (1859-1941), a major French philosopher of the twentieth century upon whom Kobayashi places special significance. While the primary focus is on interpreting this speech of Kobayashi’s, the thesis also discusses his earlier and later works in order to show the various transitions his philosophy went through over the course of his long career. The strong belief to which Kobayashi held on throughout his life as a literary critic is that the only way to see the essence of any object is to reject all rational and analytical interpretation and instead to unite one’s self with the objects: this was the ultimate approach that Kobayashi adopted in order to understand the word kan. This thesis finally addresses the question of whether this vision enabled Kobayashi to achieve his potential as a critic and as an individual.