<strong>Background:</strong> Malignant transformation of cells is typically characterised by aerobic glycolysis, resulting in supressed mitochondrial function, a state that helps resistance to apoptosis. This characteristic has been widely accepted as a hallmark of cancer and has been shown to be of critical importance in tumour development. The bioenergetic differences between normal and malignant cells are being exploited to identify potential cancer specific therapeutics. Improved in-vitro models are required to aid the identification and assessment of candidate drugs. In this project, we investigated the bioenergetic phenotypes of a panel of adult and paediatric cancer cell lines and evaluated the potential of 3D models as a platform for testing drugs that target cancer metabolism. We also investigated a novel method to assess mitochondrial function that enables the quantification of the level of oxygenation within the cell. <strong>Results:</strong> The results presented in this thesis show that not all cancers display this aerobic glycolytic phenotype. We found that while some cell lines displayed the Warburg phenotype others displayed high levels of oxidative metabolism. These bioenergetic profiles need to be considered when deciding which anti-cancer drugs to use in a chemotherapeutic regime. If a bioenergetic pattern can be identified it may one day form the basis of a screening strategy for tumours. Dichloroacetate (DCA) is a small molecule PDK inhibitor that was investigated in this study. It was found to be relatively non-toxic to cells cultured in 2D but had improved toxicity when the cells were cultured in a 3D environment. Lastly, we evaluated a new oxygen sensing nanoprobe, Mito-Xpress Intra, and the results demonstrate its potential as a non-invasive means of measuring oxygen concentrations within the cell in real time as well as highlighting some striking differences between applied ambient and measured intracellular oxygen concentrations. <strong>Conclusion:</strong> The findings suggest that not all cancers display the characteristic glycolytic phenotype. They also highlight the importance of controlling oxygen and glucose levels when evaluating metabolism and when drug testing.

This thesis in intellectual history examines the work of art historian Ernst Gombrich (1909-2001), one-time Director of London's Warburg Institute, on that institute's founder, Aby Warburg (1866-1929). The memory of War burg, as evoked in Gombrich's scholarship, is investigated as a focal point for contemporary concerns on the part of Gombrich and his peers, and as an influence on Warburg's reception in 20th century scholarship. The thesis gives a close account of Gombrich's particular intellectual achievements, in order better to understand his status as a figure of great popular and academic significance in mid-to-Iate 20th century art history and art theory. Gombrich was an emigre who left his native Austria for the United Kingdom in the 1930s and this thesis also considers the impact on intellectual history of the mid-20th century emigration from Central Europe, which was driven by ethnonationalist and above all Nazi persecution. Specifically, the thesis examines the significance for Gombrich's work of his Austrian background, in terms of both the German-language humanist culture of Bi/dung and Gombrich's sense, as a person of Jewish background, of Jewish identity. Using a methodology informed by the anthropology of emotions and the discipline of memory studies, Warburg is approached specifically as a lieu de memoire on Pierre Nora's model. The argument is that Gombrich invested his own concerns in his scholarly representations of the older art historian. The means by which this investment was made, and the negotiation of this investment amongst Gombrich's colleagues at the Warburg Institute, are traced through archival research. The impact of Gombrich's investment in Warburg on the older art historian's subsequent, posthumous reception in academia is examined, and the potential for alternative visions of Warburg marginalised by Gombrich's representation is also considered.

The Warburg effect describes the survival advantage of cancer cells in that they can proliferate under low oxygen/hypoxic conditions via a less efficient pathway known as glycolysis. It has not yet been documented at which point, in an oxygen gradient, phenotypic changes occur. Measuring the intracellular redox potential (IRP) and its impact on cellular dynamics would provide greater insight into how disruption of redox homeostasis caused by changes in oxygen concentration leads to aberrant cell signalling and diseases such as cancer. Current techniques in measuring IRP include redox-sensitive fluorescent proteins such as roGFP which is glutathione-specific. Measuring the concentration of one redox couple is, however, not an accurate representation of IRP as it does not necessarily inform about the state of other redox couples. Furthermore, fluorescent biosensors can suffer from photobleaching and may interact with other oxidants. The IRP was measured, in this work, using our newly developed novel-class of surface enhanced Raman scattering nanoparticles which can quantitatively measure the redox potential of cells in vitro. A 'homemade' device was created to keep the cells under fixed pO2 whilst obtaining measurements. The IRP was correlated with the transcriptomic and downstream metabolic profiles of MCF7 breast cancer cells, under perturbed pO2, using 1H NMR spectroscopy (NMR), mass spectrometry (MS) and RNA-sequencing. Discriminatory metabolites were all associated with energy and glucose metabolism. Discriminatory microRNAs were all affiliated with the hallmarks of cancer; the regulation of some is controlled by transcription factors containing redox-sensitive motifs in their DNA binding domains. Multivariate analysis techniques were used to analyse the different data streams in a holistic way that allows the correlation of redox potential, metabolism and transcription.

Whilst progress has been made in reducing mortality in some cancers, mortality rates remain high in many cancers and there is a need to develop novel therapeutic strategies. In this thesis, various pharmacological strategies designed to enhance the activity of existing therapeutic drugs were evaluated. Cancer cells are dependent upon aerobic glycolysis (the Warburg effect) and glutamine uptake. Using clinically approved tyrosine kinase inhibitors and Bortezomib, significant enhancement of chemosensitivity was observed when used in combination with inhibitors of lactate dehydrogenase (Gossypol) and pyruvate kinase dehydrogenase (Dichloroacetate). In contrast, depletion of glutamine from media had to be extensive in order to induce cell death and cell death only occurred after prolonged exposure to glutamine-deprived conditions. This suggests that glutamine depletion strategies alone are unlikely to be successful but may be useful in combination with other agents targeting glutamine addiction in cancer cells. Finally, Temozolomide (TMZ) is an important drug in the treatment of glioblastomas but its activity is reduced by resistance mechanisms including O6 methyl guanine methyltransferase (MGMT) and mismatch repair (MMR). This thesis has identified analogues of TMZ (EA02-45, EA02-59, EA02-64 and EA02-65) that are MGMT and MMR independent in terms of inducing cell kill in vitro. These compounds are promising leads for future development. In conclusion, this thesis has demonstrated that interfering with the metabolic phenotype of cancer can enhance the activity of existing drugs and identified novel analogues of TMZ that circumvent drug resistance mechanisms that hamper the efficacy of TMZ.

The doctoral thesis focuses on the Warburg's concept of thought space [Denkraum] and the related concept of artistic creation as an interface between the affections and abstract thinking [Zwischenraum], representing the key concepts of his late work (especially between the years 1920- 1929), culminating in the pictorial atlas Mnemosyne. In this context, the thesis focuses on the motive of ascent as well, and concludes that the pathos formula of ascent plays a crucial historical role in the constitution of modern Denkraum and forms one of the fundamental constitutive parameters of the thought-space, whose own metaphorical perspective is the contemporary aviation. For an adequate reflection of the historical development of this Denkraum, Warburg turns to the physical models of thought and artistic creation, including an attempt to use Einstein's theories of relativity. The key role of the motive of ascent in the constitution of Denkraum subsequently shapes this thought space within the confines of Renaissance humanism.

Glioblastome sind die aggressivsten und häufigsten Hirntumore beim Menschen und entziehen sich weiterhin einem kurativen Therapieansatz. Wie die meisten malignen Tumore zeigen Glioblastome den sogenannten Warburg Effekt, eine gesteigerte aerobe Glykolyse. Ein Schlüsselenzym der Glykolyse ist die 6-Phosphofrukto-1-kinase (PFK-1), deren stärkster allosterischer Aktivator Fruktose-2,6-bisphosphat (F2,6BP) ist. Die zelluläre Konzentration von F2,6BP wird von dem bifunktionalen Enzym 6-Phosphofrukto-2-kinase/Fruktose-2,6-bisphosphatase (PFK-2) reguliert. Im Menschen existieren vier PFK-2-Isoenzyme (PFKFB1-4), die gewebespezifisch exprimiert werden. Die PFKFB3 hat das höchste Kinase/Bisphosphatase- Aktivitätsverhältnis von 710:1 innerhalb der PFK-2-Familie und wird in Tumorzelllinien und verschiedenen malignen Tumoren überexprimiert. In humanem Hirngewebe wurden sechs alternative Spleißvarianten der PFKFB3-mRNA (UBI2K1–6) beschrieben, welche sich in der C-terminalen Region unterscheiden. Neuere Untersuchungen im Verlauf dieser Arbeit ergaben, dass es auch Spleißvarianten gibt, die in der N-terminalen Region variieren, sodass insgesamt 10 Spleißvarianten der PFKFB3 bekannt sind. Die spezifischen Funktionen im Zellstoffwechsel wurden bisher nur für die Spleißvariante UBI2K5 untersucht, die der anderen Spleißvarianten sind weitestgehend unbekannt. Das Ziel der vorliegenden Arbeit war, die Bedeutung der PFKFB3 Spleißvariante UBI2K4 in Glioblastomen für den Stoffwechsel und das Wachstum dieser Tumore am Modell der humanen U87-Glioblastomzelllinie aufzuklären und die These, dass die UBI2K4 eine proliferationshemmende Funktion hat, zu überprüfen. Im ersten Teil dieser Arbeit wurde mittels stabiler Transfektion von HEK-293-Zelllen mit einem pTER-Vektor und gleichzeitiger transienter Transfektion bei U87- und HEK-293-Zellen mit synthetischer siRNA ein Knockdown der UBI2K4 mRNA und des Proteins erzielt. Es stellte sich heraus, dass der UBI2K4 Knockdown in beiden Zelllinien zu einer reduzierten Viabilität und Zellproliferation führte. Die Verdopplungszeiten waren prolongiert und auch das dreidimensionale Wachstum in Soft-Agar-Kulturen war reduziert. Bei HEK-293-Zellen wurde der UBI2K4 Knockdown durch einen signifikanten Anstieg der UBI2K5 mRNA Expression kompensiert. Die UBI2K6 Expression blieb unverändert. Bei U87-Zellen, deren native UBI2K4 mRNA Expression sehr gering ist, wurde eine UBI2K5 mRNA Reduktion bei gleichzeitiger Hemmung der UBI2K4 Expression festgestellt, während die UBI2K6 mRNA leicht zunahm. Weiterhin konnte im Western Blot gezeigt werden, dass in HEK-293-Zellen neben der Spleißvariante UBI2K4, auch als Variante 4 bezeichnet, auch die neu entdeckte Variante 5 exprimiert wird. In U87-Zellen konnte nur die Expression der Variante 4 nachgewiesen werden. In einem Antikörper-Mikroarray wurde gezeigt, dass die UBI2K4 die Expression insbesondere von Proteinen mit immunmodulatorischen Eigenschaften, apoptose-induzierenden Proteinen und Proteinen der Zellkommunikation und des Metabolittransports beeinflusst. Im zweiten Teil dieser Arbeit wurde das native Protein der UBI2K4 in HEK-293- und U87-Zellen überexprimiert. Die Zellen mit einer gesteigerten UBI2K4 Proteinkonzentration konnten schneller proliferieren und zeigten eine gesteigerte Zellviabilität. DIese Ergebnisse korrelieren mit den bereits beschriebenen Ergebnissen des UBI2K4 Knockdowns im ersten Teil dieser Arbeit. Die vermutete inverse Korrelation der UBI2K4 Konzentration und der Proliferationsrate konnte hier nicht bestätigt werden. Vielmehr scheint die UBI2K4 so wie die Spleißvariante UBI2K5 der PFKFB3 ebenfalls in höheren Konzentrationen proliferationsfördernd zu wirken. Dies scheint überaus wahrscheinlich, da die im Antikörper-Mikroarray beeinflussten Proteine auch darauf hindeuten.:1 EINFÜHRUNG UND AUFGABENSTELLUNG 8 1.1 GLIOBLASTOME 8 1.2 DIE GLYKOLYTISCHE AKTIVITÄT IN TUMORZELLEN – DER WARBURG-EFFEKT 12 1.2.1 REGULATION DER GLYKOLYSE IN TUMORZELLEN 16 1.2.2 DIE 6-PHOSPHOFRUKTO-2-KINASE/FRUKTOSE-2,6-BISPHOSPHATASE ISOENZYME 18 1.3 AUFGABENSTELLUNG 26 2 MATERIAL UND METHODEN 27 2.1 LABORAUSSTATTUNG 27 2.2 CHEMIKALIEN UND REAGENZIEN 27 2.3 VERBRAUCHSMATERIALIEN 29 2.4 STANDARDS 29 2.5 ENZYME 29 2.6 ANTIKÖRPER 29 2.7 REAGENZIENSYSTEME 30 2.8 BAKTERIENSTÄMME 30 2.9 ZELLLINIEN 31 2.10 PLASMIDE 31 2.11 OLIGONUKLEOTIDE 31 2.12 RIBONUKLEINSÄUREN 34 2.13 ZELLBIOLOGISCHE METHODEN 34 2.13.1 KULTIVIERUNG UND PASSAGIEREN VON ZELLEN 34 2.13.2 BESTIMMUNG DER ZELLZAHL 35 2.13.3 KONSERVIERUNG VON ZELLEN 35 2.13.4 TRANSFEKTION UND SELEKTION 35 2.13.5 PROLIFERATIONSTESTS 38 2.14 MOLEKULARBIOLOGISCHE ARBEITEN 40 2.14.1 ARBEITEN MIT BAKTERIENKULTUREN 40 2.14.2 PRÄPARATION VON NUKLEINSÄUREN 42 2.14.3 KONZENTRATIONSBESTIMMUNG VON NUKLEINSÄUREN 43 2.14.4 AGAROSEGEL-ELEKTROPHORESE 43 2.14.5 POLYMERASE-KETTENREAKTION (PCR) 44 2.14.6 REVERSE TRANSKRIPTION 45 2.14.7 QUANTITATIVE REAL-TIME PCR 45 2.14.8 DNA-SEQUENZIERUNG 47 2.14.9 GEN-SILENCING DURCH SIRNA 48 2.14.10 ÜBEREXPRESSION DER SPLEIßVARIANTE UBI2K4 51 2.14.11 MYCOPLASMENTEST 52 2.15 PROTEINCHEMISCHE METHODEN 53 2.15.1 ZELLLYSE UND BESTIMMUNG DER PROTEINKONZENTRATION 53 2.15.2 SDS-PAGE 53 2.15.3 WESTERNBLOT-ANALYSE 54 2.16 ANTIKÖRPER MIKROARRAY 55 2.17 STATISTISCHE ANALYSEN 55 2.18 SOFTWARE 56 3 ERGEBNISSE 57 3.1 AUSWAHL DER ZELLLINIE 57 3.2 KNOCKDOWN DER UBI2K4 DURCH STABILE UND TRANSIENTE TRANSFEKTION 59 3.2.1 KLONIERUNG DER PTER-EXPRESSIONSPLASMIDE FÜR DIE STABILE TRANSFEKTION 59 3.2.2 STABILE TRANSFEKTION MITTELS PTER-PLASMIDEN 62 3.3 NACHWEIS DES KNOCKDOWN DER UBI2K4 AUF DIE MRNA EXPRESSION 64 3.3.1 KNOCKDOWN DER UBI2K4 IN STABIL TRANSFIZIERTEN HEK-293-ZELLEN 64 3.3.2 KNOCKDOWN DER UBI2K4 IN STABIL TRANSFIZIERTEN HEK-293-ZELLEN MIT ZUSÄTZLICHER TRANSIENTER TRANSFEKTION MITTELS SIRNA 65 3.3.3 KNOCKDOWN DER UBI2K4 IN TRANSIENT TRANSFIZIERTEN U87-ZELLEN 67 3.4 EXPERIMENTE MIT ZELLEN BEI KNOCKDOWN DER UBI2K4 70 3.4.1 EINFLUSS DES KNOCKDOWN DER UBI2K4 AUF DAS WACHSTUM VON HEK-293-ZELLEN 70 3.4.2 EINFLUSS DES KNOCKDOWN DER UBI2K4 AUF DIE KOLONIEBILDUNG VON HEK-293-ZELLEN IN SOFT-AGAR-KULTUREN 71 3.5 KOLORIMETRISCHE TESTS BEI KNOCKDOWN DER UBI2K4 73 3.5.1 EINFLUSS DES UBI2K4 KNOCKDOWN AUF DIE VIABILITÄT UND DIE PROLIFERATION VON HEK-293-ZELLEN 73 3.5.2 EINFLUSS DES UBI2K4 KNOCKDOWN AUF DIE VIABILITÄT UND DIE PROLIFERATION VON U87-ZELLEN 74 3.6 ERGEBNISSE DES ANTIKÖRPER MIKROARRAY 75 3.7 ÜBEREXPRESSION DER UBI2K4 DURCH STABILE / TRANSIENTE TRANSFEKTION 78 3.7.1 KLONIERUNG DER PCDNA3.1-EXPRESSIONSPLASMIDE 78 3.8 NACHWEIS DER ÜBEREXPRESSION DER UBI2K4 AUF DIE MRNA EXPRESSION 79 3.9 KOLORIMETRISCHE TESTS BEI ÜBEREXPRESSION DER UBI2K4 81 3.9.1 EINFLUSS DER UBI2K4 ÜBEREXPRESSION AUF DIE VIABILITÄT UND DIE PROLIFERATION VON HEK-293-ZELLEN 81 3.9.2 EINFLUSS DER UBI2K4 ÜBEREXPRESSION AUF DIE VIABILITÄT UND DIE PROLIFERATION VON U87-ZELLEN 82 4 DISKUSSION 85 4.1 AUSWAHL DER ZELLLINIEN 85 4.2 GENERIERUNG STABILER ZELLLINIEN MIT UBI2K4-, UBI2K5- UND UBI2K6-KNOCKDOWN 87 4.2.1 KNOCKDOWN DER PFKFB3 SPLEIßVARIANTEN DURCH STABILE TRANSFEKTION IN HEK-293- UND U87-ZELLEN 89 4.3 KNOCKDOWN DER UBI2K4 IN HEK-293- UND U87-ZELLEN MITTELS TRANSIENTER TRANSFEKTION 90 4.4 ÜBEREXPRESSION DER UBI2K4 IN HEK-293 UND U87-ZELLEN 95 4.5 MIKROARRAY 97 4.5.1 PROTEINE MIT EINER SIGNIFIKANTEN KONZENTRATIONSERHÖHUNG 98 4.5.2 PROTEINE MIT EINER SIGNIFIKANTEN KONZENTRATIONSERNIEDRIGUNG 100 5 ZUSAMMENFASSUNG DER ARBEIT 104 6 LITERATURVERZEICHNIS 107 7 ANLAGEN 129 7.1 ABBILDUNGSVERZEICHNIS 129 7.2 TABELLENVERZEICHNIS 131 8 ERKLÄRUNG ÜBER DIE EIGENSTÄNDIGE ABFASSUNG DER ARBEIT 132 9 LEBENSLAUF 133 10 DANKSAGUNG 134

Objective: Challenging differential diagnosis Background: B cell lymphoma constitutes 80-85% of cases of Non Hodgkin's lymphoma in the Untied States. Metabolic complications may arise from the disease itself or through its end organ involvement. Case Report: We describe a case of a diffuse large B cell lymphoma diagnosed by abdominal computed tomography after it initially presented as hypoglycemia not correctable by dextrose infusion that instead resulted in increased anion gap metabolic acidosis with elevated lactate levels. Conclusions: The case illustrates how lymphomas can present unusually with hypoglycemia and lactic acidosis, the latter being an ominous sign that can occur without liver involvement. In this regard, the case demonstrates the metabolic sequelae of lymphoma that should raise suspicion for an underlying process. This has implications for diagnosis, treatment, and patient survival. Attention should be paid especially in the primary care setting in order to minimize delays in diagnosis.

Objective: Challenging differential diagnosis Background: B cell lymphoma constitutes 80-85% of cases of Non Hodgkin's lymphoma in the Untied States. Metabolic complications may arise from the disease itself or through its end organ involvement. Case Report: We describe a case of a diffuse large B cell lymphoma diagnosed by abdominal computed tomography after it initially presented as hypoglycemia not correctable by dextrose infusion that instead resulted in increased anion gap metabolic acidosis with elevated lactate levels. Conclusions: The case illustrates how lymphomas can present unusually with hypoglycemia and lactic acidosis, the latter being an ominous sign that can occur without liver involvement. In this regard, the case demonstrates the metabolic sequelae of lymphoma that should raise suspicion for an underlying process. This has implications for diagnosis, treatment, and patient survival. Attention should be paid especially in the primary care setting in order to minimize delays in diagnosis.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19591号 / 医博第4098号 / 新制||医||1014(附属図書館) / 32627 / 京都大学大学院医学研究科医学専攻 / (主査)教授 野田 亮, 教授 武田 俊一, 教授 小川 修 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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