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Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths worldwide with a five-year survival rate of only 9%. The mevalonate (MVA) pathway, which results in synthesis of cholesterol, has been demonstrated to be overexpressed in numerous cancers, leading to its involvement in various aspects of the disease, including proliferation, invasion, and metastasis. In recent years, the MVA pathway has been implicated in PDAC as well, however the underlying mechanisms behind its upregulation in this context, as well as its role in tumor progression, remain largely unknown. An initial analysis of The Cancer Genome Atlas (TCGA) datasets using the Gene Expression Profiling Interactive Analysis (GEPIA) tool revealed one mevalonic gene in particular, SQLE—encoding squalene epoxidase (SQLE)—to be significantly overexpressed in PDAC compared to other genes within the pathway. We find that patients with higher SQLE expression profiles have significantly reduced five-year survival rates that can be further correlated to tumor stage and grade, and we demonstrate that knockdown of SQLE, a branchpoint enzyme of the cholesterol pathway, reduces tumor proliferation in both two-dimensional and three-dimensional assays. We further characterize the SQLE promoter landscape by demonstrating an interesting dynamic of gene activation between the sterol regulatory element binding protein-2 (SREBP2), BRG1-specific SWI/SNF chromatin remodelers, and mutant p53. These results highlight SQLE as a therapeutic target in PDAC and provide a better understanding of its regulation in the context of this devastating disease.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13586号 / 論医博第2306号 / 新制||医||1070(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 川口 義弥, 教授 波多野 悦朗, 教授 齋藤 潤 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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No description available.

Major depressive disorder (MDD) is one of the most common psychiatric illnesses worldwide, with pharmacotherapy as a first-line option for the management of this illness. The National Center for Health Statistics found that the use of antidepressants has increased by more than 4 fold in the last 20 years. While SSRI’s act centrally to treat MDD, their peripheral effects are often overlooked. Interestingly, components of the serotonergic system including the serotonin transporter (SERT), serotonin receptors, and enzymes important for serotonin synthesis (tryptophan hydroxylase 1 and 2; Tph1 and Tph2) are affected by SSRI treatment both centrally and peripherally. This disruption of serotonin signaling in the pancreas is of particular interest as there is a considerable link between the serotonin and hedgehog signaling pathways, both of which are important for pancreatic beta cell function. I hypothesize that pancreatic beta cell exposure to the SSRI fluoxetine in vitro will lead to altered hedgehog signaling ultimately resulting in a disruption in insulin secretion. / Thesis / Master of Science in Medical Sciences (MSMS)

Bakgrund: Pankreascancer är en aggressiv cancersjukdom och uppskattas vara den tolfte dödligaste cancerformen. Sjukdomen drabbar bukspottskörtelns förmåga att producera exokrina och endokrina enzymer. Cancersjukdomen upptäcks vanligtvis i ett sent skede vilket innebär att behandlingsalternativen blir begränsade. Den teoretiska referensramen som appliceras i litteraturöversikten är livskvalitet hos personer med pankreascancer och deras upplevelser av sjukdomen. Syfte:  Beskriva personers upplevelser av pankreascancer.  Metod: Kvalitativ metod med induktiv ansats tillämpades för att genomföra litteraturöversikten. Artikelsökningarna gjordes i databaserna CINAHL och MEDLINE. Elva kvalitativa artiklar utgjorde resultatet och i analysprocessen användes en modell enligt Friberg.  Resultat: Resultatet belyser personers upplevelser av pankreascancer och presenteras i två teman: Fysisk påverkan och Psykosociala förändringar samt sex subteman: Att känna fatigue, Nutritionsbesvär, Att ha smärta, Känslomässigt lidande, En annan kroppsbild och Det sociala samspelet. Livskvaliteten för personer med pankreascancer påverkades av fysiska och psykiska symtom samt psykosociala påfrestningar. Slutsatser: Pankreascancer är en komplex och allvarlig sjukdom vars symtom ställer höga krav på sjukvården. Personers upplevelser bör vara utgångspunkten för att kunna förbättra deras livskvalitet i den mån det är möjligt.    Nyckelord: Livskvalitet, omvårdnad, pankreascancer och upplevelse. / Background: Pancreatic cancer is an aggressive form of cancer and is estimated to be the twelfth deadliest form of cancer. The disease affects the pancreas and its ability to produce exocrine and endocrine enzymes. Typically pancreatic cancer is detected at a late stage resulting in limited treatment options. The theoretical framework applied in this literature review was quality of life among individuals with pancreatic cancer and their experiences of the disease. Purpose: Describe peoples' experiences of pancreatic cancer.  Method: Qualitative method with an inductive approach was used to conduct the literature review. Article searches were done in the databases CINAHL and MEDLINE. Eleven qualitative articles formed the result which were analyzed according to a model by Friberg. Results: The results highlight peoples´ experiences of pancreatic cancer and consist of two themes: Physical impact and Psychosocial changes and six subthemes: To sense fatigue, Nutritional problems, Experiencing pain, Emotional distress, Body image changes and Social interactions. The quality of life of individuals with pancreatic cancer was affected by physical and phycological symptoms and as well as psychosocial distresses. Conclusions: Pancreatic cancer is a complex and severe disease with symptoms that place high demands on healthcare. Individuals' experiences should be the starting point for improving their quality of life to the extent possible.   Keywords: Experiences, nursing care, pancreas cancer and quality of life.

Diabetes mellitus (DM) ist eine Stoffwechselerkrankung, die durch eine chronische Hyperglykämie (erhöhter Blutzuckerspiegel) gekennzeichnet ist. Derzeit leiden mehr als 540 Millionen Menschen, die überwiegend in Ländern mit niedrigem und mittlerem Einkommen leben, an Diabetes, und es wird prognostiziert, dass die Inzidenzrate bis 2045 auf über 783 Millionen ansteigen wird, was auf eine große Krise im öffentlichen Gesundheitswesen hindeutet. Typ-1-Diabetes (T1D), eine chronische Autoimmunerkrankung, entsteht durch die von T-Zellen vermittelte Zerstörung der insulinproduzierenden β-Zellen in den Pankreasinseln, wodurch der Blutzuckerspiegel der Patienten nur unzureichend kontrolliert wird. T1D ist eine der häufigsten endokrinen und metabolischen Störungen im Kindesalter, und die Behandlungsmöglichkeiten umfassen die Verabreichung verschiedener Formen von exogenem Insulin durch unterschiedliche Modalitäten. Inseltransplantationen oder Transplantationen der gesamten Bauchspeicheldrüse sind nach wie vor die Standardtherapien, aber die Organknappheit stellt eine erhebliche Einschränkung dar. Als Alternative bieten sich humane pluripotente Stammzellen (hPSCs) für eine Diabetes- Zellersatztherapie an, aber die erzeugten inselähnlichen Cluster (SC-Inseln) sind funktionell noch nicht ausgereift. Zahlreiche Bemühungen, den Differenzierungsprozess zu verbessern, haben erhebliche Fortschritte gezeigt, aber die Herausforderungen in Bezug auf Effizienz, Funktionalität und Reifung bleiben bestehen. Ich stellte die Hypothese auf, dass (a) die Heterogenität von hPSC-abgeleiteten Pankreasvorläuferzellen (PPs) und (b) ihre Expansion sowie (c) die Förderung der Gefäßentwicklung in den SC-Inseln die β-Zellreifung und die geringe Umwandlungseffizienz von hPSCs in β-Zellen verbessern könnten. Um der Heterogenität der PP entgegenzuwirken, habe ich zunächst das Verfahren zur Differenzierung von hPSCs zu β-Zellen überarbeitet und dabei einen sehr hohen Anteil von 80-90% PDX1+/SOX9+/NKX6.1+ Zellen im PP-Stadium erreicht. Außerdem habe ich ein GMP-konformes, kostengünstiges und effizientes Expansionsverfahren verfeinert und vollständig etabliert, das eine konsistente exponentielle Expansion von PP-Zellen um das 2000-fache über 40 Tage ermöglicht. Bemerkenswerterweise kann dieses Verfahren die Anreicherung von expandierten PPs (ePPs) auf bis zu 80-90% PDX1+/SOX9+/NKX6.1+ Zellen vermitteln. Wichtig ist, dass ePPs mit ähnlicher Effizienz wie nicht-expandierte PP- Zellen in SC-Inseln mit funktionellen β-Zellen differenziert werden können. Ich stellte dann die Hypothese auf, dass die Einbeziehung sowohl von Endothelzellen (ECs) als auch von Perizyten (PCs) während der Differenzierung von PP-Zellen in einem Prozess, den ich als Tri-Kultur bezeichnete, die In-vivo-Entwicklung besser nachahmen und zu einer effizienten Differenzierung und Reifung von SC-Inseln führen würde. Um diese Hypothese der Vaskularisierung von SC-Inseln zu untersuchen, habe ich erfolgreich Verfahren zur gleichzeitigen Erzeugung von hochreinen (>95%) ECs und PCs etabliert, indem ich veröffentlichte Verfahren anpasste und modifizierte. So konnte ich sowohl PP-Zellen als auch vaskuläre Zellen aus derselben hPSC-Linie erzeugen. Im nächsten Schritt ermittelte ich das geeignete Zellverhältnis bei der gemeinsamen Ansammlung von PP-Zellen mit ECs und PCs sowie die geeignete Medienzusammensetzung, die das Überleben und die gemeinsame Entwicklung aller drei Zelltypen während S5-S7 ermöglichte. In den daraus resultierenden 'vaskularisierten' SC-Inseln (vSC-Inseln) waren die vaskulären Zellen gleichmäßig über die SC-Insel verteilt, was tatsächlich auf die Möglichkeit der Bildung von Mikrovaskulatur hindeutet. Die starke Expression endokriner Marker wurde beibehalten und die funktionelle Kompetenz der β-Zellen in den vSC-Inseln schien verbessert, was auf die potenziellen Vorteile der Vaskularisierung bei der Förderung der Inselfunktionalität hinweist. Die Entwicklung dieser vSC-Inseln ist ein wichtiger Schritt zur korrekten Modellierung der Inselentwicklung und eröffnet spannende Möglichkeiten zur Förderung der endokrinen Zellreifung in vitro sowie ihrer Integration nach der transplantation.:Summary ii Zusammenfassung iv Acknowledgements x List of Figures xii List of tables xiv List of abbreviations xv 1 INTRODUCTION 1 1.1 Classification and pathogenesis of diabetes 1 1.1.1 Type 1 Diabetes 2 1.1.2 Complications and challenges arising from diabetes 8 1.1.3 Approaches to address diabetes 9 1.1.4 Available therapeutic options and their constraints 12 1.2 Gastrulation 14 1.2.1 Patterning and germ layer specification in mammalian embryo 15 1.2.2 Patterning of the developing endoderm and gut tube formation 17 1.2.3 Emergence of alternative (hepatic and gut) lineages instead of pancreatic fate determination 19 1.2.4 Initiation of Pancreatic Fate and Morphogenesis 20 1.2.5 Primary transition 21 1.2.6 Secondary transition 23 1.2.7 Tertiary transition 27 1.2.8 Maturation of murine α and β cells during development 27 1.2.9 Differences in the developmental processes between humans and mice 28 1.2.10 Role of vasculature in islet development 30 1.3 Stem cells and their types: 33 1.3.1 Pluripotent stem cells: a promising source for β-cell replacement therapy 33 1.3.2 A long journey of hPSC towards generating β-cells 35 1.3.3 Significant differentiation protocols since last decade 37 1.3.4 Implementation of Stem cell therapies for T1D 41 1.3.5 Present status; clinical trials for the treatment of T1D, past and present 43 2 Material and methods 45 2.1 Cell culture 45 2.1.1 Coating of a matrix for the cell culture 45 2.1.2 Passaging of hESC cells 45 2.1.3 Freezing of and thawing of hPSCs 45 2.1.4 Freezing and thawing of PPs, ECs and PCs: 46 2.2 Differentiation of hPSCs to SC-islets using the Gavalas lab protocol 47 2.2.1 Seeding of hPSCs for PP differentiation 47 2.3 Expansion of PPs 49 2.4 Differentiation of hESCs into vascular cells 50 2.4.1 Differentiation of hESCs into ECS using ETV2 inducible line 50 2.4.2 Simultaneous differentiation of hESCs to ECs and PCs 50 2.5 RNA extraction, cDNA synthesis and RT-qPCR 51 2.5.1 RNA extraction 51 2.5.2 cDNA synthesis 52 2.5.3 RT-qPCR 52 2.6 Immunofluorescence analyses of monolayers cells, cryosections, and whole mounts 52 2.7 Flow cytometry (FC) of fixed (nuclear and surface markers) and live cells 54 2.8 Functionality assay for SC-islets 56 2.8.1 Static glucose-stimulated insulin secretion (GSIS) assay 56 2.8.2 C-peptide ELISA kit detection 56 2.9 Live Ca2+ imaging 57 2.10 Automated confocal imaging and analysis 57 2.11 Statistical analysis and graphs 57 3 Generation of SC-islets from hPSCs 58 3.1 hPSCs retained their pluripotency before the start of differentiation 58 3.2 Optimal seeding density is key to the successful differentiation 59 3.3 Efficient generation of definitive endoderm (DE) cells (S1) 60 3.4 Successful differentiation of DE cells into primitive gut tube (PGT) and posterior foregut (PF) 61 3.5 Generation of PDX1+/SOX9+/NKX6.1+ enriched PP population (S4) 64 3.6 Stagewise transcriptomic analysis of hPSCs to PP differentiation 66 3.7 PP cells differentiated into pancreatic endocrine progenitor (PEP) (S5) cells 68 3.8 Generation of SC-islets containing functional β cells 69 3.9 Inclusion of N21 in S7 increases INS expression, numbers of β-cells and β-cell responsiveness 71 4 Expansion of PPs under GMP-compatible condition 74 4.1 C6 expansion selects PDX1+/SOX9+/NKX6-1+ cells population across lines 74 4.2 GMP-compliant C6 shows similar growth kinetics in all three lines while maintaining chromosomal stability 77 4.3 The transcriptome of ePPs revealed their stabilization just after five passages 78 4.4 C6 promoted strong upregulation of GP2, a pancreatic progenitor marker, during expansion 79 4.5 Impact of expansion on progenitor transcription factors and ductal program activation 80 4.6 Expansion with C6 represses the endocrine program and alternative lineages 82 4.7 C6 ePPs differentiate into SC-islets containing functional β-cells 83 5 Generation of SC-islets containing endothelial cells and pericytes 85 5.1 The ETV2 inducible expression hPSC line 85 5.2 Efficient differentiation of hPSCs to ECs using the ETV2 inducible expression line 86 5.3 Co-culturing PPs with ECs till S7 89 5.4 Simultaneous generation of PCs and ECs from hPSCs 90 5.5 ECs and PCs can be expanded for three passages with similar efficiency in four different matrices 92 6 Discussion 101 7 Future outlook 110 8 Supplementary information 112 9 Bibliography 121 10 Anlage 1 144 11 Anlage 2 145

Cell therapy is a novel potential approach to treat many diseases including diabetes. Embryonic stem cells have been examined in various diabetic and non-diabetic heart studies. However, the role of pancreas transcription factor 1 alpha (ptfla) over expressing embryonic stem (ES) cells has not been defined. We hypothesize that transplanted over expressing ptfla-ES cells in streptozotocin (STZ) induced diabetic mice will attenuate cardiac hypertrophy, fibrosis, and improve cardiac function. In this investigation we divided C57/bl6 mice into three groups: Control, STZ, and STZ + ptflaES cells. Diabetes was induced with STZ (lO0mg/kg, body weight), with two separate injections on day 1 (D1) and D2. Following STZ injections, mice were transplanted with 1.2 million ptfla-ES cells in three days. Control group received normal saline. After injections, animals were examined for glucose levels, cardiac hypertrophy, fibrosis, and heart function. Our data shows that glucose levels were significantly increased following STZ injections, suggesting diabetes, and this increase was reversed with transplanted ptfl a-ES cell. Our H&E qualitative data suggest that there was increase in cardiac hypertrophy in STZ-induced diabetic animals compared with control. Moreover, Massan's trichrome staining shows increased amount of cardiac fibrosis in STZ-induced diabetic animals compared with control. This data suggests that animals have developed diabetic cardiomyopathy. Interestingly, the increased cardiac hypertrophy and fibrosis was attenuated in the animals transplanted with ptfl a-ES cells. Furthermore, cardiac function examined by echocardiography was reduced in the STZ treated animals which was reversed following ptfla-ES cell treatment. In conclusion, our data suggests that

Ptf1a/p48 is a pancreas specific bHLH transcription factor that is required at early stages of embryonic development for pancreas formation and, during adulthood, for the proper exocrine pancreatic function. P48 also exerts an antiproliferative effect, which may exert a tumor suppressor activity. In this study, based on a yeast two-hybrid approach, we have identified new p48 partners that modulate the activity of p48. Among the newly identified putative interactors we found p/CAF, which is a coactivator that potentiates its transcriptional activity, and ICAT, an inhibitor of the β-catenin/TCF signaling pathway. ICAT binds to p48 and is coexpressed with it in the pancreas during development and postnatally. Using different cellular models, ICAT overexpression in acinar tumor cells resulted in changes of the pancreatic specific gene expression pattern. Furthermore, high levels of ICAT inhibited the interaction between p48 and p/CAF. While this hetero-oligomeric complex is required for the acinar gene expression, ICAT itself is shown to be present in a reconstituted PTF1 complex in vivo. Importantly, altered ICAT expression is demonstrated in several histological types of pancreatic tumors, possibly contributing to their differentiation phenotype and neoplastic properties. / Ptf1a/p48 es un factor de transcripción bHLH específico del páncreas necesario durante los estadios tempranos del desarrollo embrionario para la formación del mismo, y para el correcto funcionamiento del páncreas exocrino en el adulto. P48 desempeña también una función antiproliferativa, la cual puede resultar en una actividad de supresión tumoral. En el presente estudio, basado en una estrategia de cribado de doble-híbrido en levadura, han sido identificadas nuevas proteínas que interaccionan y que modulan la actividad específica de p48. Entre las posibles proteínas que interaccionan y han sido identificadas de novo se encuentra p/CAF, un co-activador que potencia la actividad transcripcional de p48, y ICAT, un inhibidor de la vía de señalización de la β-catenina. Se ha demostrado que ICAT se une a p48 y ambos son co-expresados en el páncreas durante el desarrollo y en el adulto. Utilizando diferentes modelos celulares, la sobreexpresión de ICAT en células tumorales acinares resultó en un cambio en el patrón de expresión de genes específicos del páncreas. Al mismo tiempo, se observó que niveles elevados de ICAT inhiben la interacción entre p48 y su co-activador p/CAF. Mientras que este complejo hetero-oligomérico es necesario para la expresión de los genes acinares, se demostró que ICAT está presente en un complejo PTF1 reconstituido in vivo. Finalmente, se observaron alteraciones en la expresión de ICAT en varios tipos histológicos de tumores pancreáticos, que posiblemente contribuyen a su fenotipo de diferenciación y propiedades neoplásicas.