Die Rolle von Connexinen und Gap Junction-vermittelter Kommunikation in pluripotenten Stammzellen sowie der frühen Embryonalentwicklung sind bis heute nicht vollständig aufgeklärt. Mutationen in humanen Connexinen verursachen eine Vielzahl von Krankheiten. Connexin-defiziente iPS Zellen stellen eine gute Basis für die Erforschung der Rolle von Connexinen während der Embryonalentwicklung und bei der Krankheitsentstehung dar. Das Ziel der vorliegenden Arbeit war es, das CRISPR/Cas9-System in pluripotenten Stammzellen erfolgreich anzuwenden und ein Protokoll zur Erstellung verschiedener Cx43-Defektmutanten zu entwerfen. Nach der Etablierung der CRSIPR/Cas9-Methode in HEK293T-Zellen konnte in der vorliegenden Arbeit darüber hinaus erfolgreich eine Cx43-Defizienz in FSiPS-Zellen erzeugt werden. Weiterhin wurden mehrere Cx43-Mutanten geschaffen und initial auf Pluripotenzmarker und ihr Differenzierungspotential untersucht. Diese Arbeit bildet die Basis für weitere Untersuchungen des Cx43 in iPS-Zellklonen und davon abgeleiteten Zelltypen sowie artifiziellen 3D-Gewebekulturen. Darüber hinaus bildet sie die Grundlage für die Bildung weiterer Connexin-Defektmutanten sowie von iPS-Zellen mit krankheitsrelevanten Mutationen. / The roles of connexins and gap junction-mediated communication in pluripotent stem cells and early embryonic development have not been fully elucidated to date. Mutations in human connexins cause a variety of diseases. Connexin-deficient iPS cells provide a good basis for studying the role of connexins during embryonic development and in disease development. The aim of the present work was to successfully apply the CRISPR/Cas9 system in pluripotent stem cells and to design a protocol to generate different Cx43 defective mutants. Furthermore, after establishing the CRSIPR/Cas9 method in HEK293T cells, a Cx43 deficiency in FSiPS cells was successfully generated. Furthermore, several Cx43 mutants were created and initially screened for pluripotency markers and their differentiation potential. This work forms the basis for further studies of Cx43 in iPS cell clones and derived cell types as well as artificial 3D tissue cultures. Furthermore, it forms the basis for the generation of further connexin defect mutants as well as iPS cells with disease-relevant mutations.

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Reverse genetics is a discipline that involves the use of genetic manipulation and modification to study an organism's altered phenotype. In this study, infectious recombinant viruses were rescued from altered cDNA clones encoding the antigenome of human parainfluenza virus type 3 and the resulting phenotypes were examined. In one clone, the gene for the enhanced green fluorescent protein was inserted into the virus antigenome to be expressed during viral replication, resulting in infected cells emitting green fluorescence. Viral titers, mRNA replication, and genomic replication for the virus expressing the enhanced green fluorescent protein were reduced when compared to the human parainfluenza virus type 3 wild-type strain. In addition, the sensitivity of the virus expressing the enhanced green fluorescent protein to antiviral compounds is increased when compared to the wild-type strain, which may lead to the identification of false positive antiviral compounds. An assay that measures the enhanced green fluorescent protein as a direct indicator of virus replication can be shortened to 3 days in duration and is a more robust assay compared to assays that measure cellular viability. In other clones, mutations were introduced into the phosphoprotein gene to eliminate the expression of the D domain of the PD protein in order to understand its function. The titers of two recombinant knockout viruses that are deficient in the expression of the D domain are reduced when compared to the wild-type strain in both MA-104 and A549 cells. In MA 104 cells, viral mRNA transcription and genomic replication of the two knockout viruses are reduced when compared to the wild-type strain. In A549 cells, cellular expression and secretion of antiviral cytokines infected with the two knockout viruses are either reduced or remain unchanged when compared to the wild-type strain. These results suggest that the D domain may play a role in viral RNA synthesis and not in counteracting the host cell's antiviral response. The results of these studies shed light on the influence an additional gene has on viral replication and possible functions of the D domain.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18177号 / 医博第3897号 / 新制||医||1004(附属図書館) / 31035 / 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 中畑 龍俊, 教授 斎藤 通紀 / 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(薬科学) / 甲第20059号 / 薬科博第66号 / 新制||薬科||8(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 竹島 浩, 教授 中山 和久, 教授 根岸 学 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

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Osteoarthritis is the most common form of arthritis. Genetic models have been developed to determine if and how a targeted gene may influence cartilage degenerative changes. The itga1-null mouse model has an inhibited integrin α1B1 through a ubiquitous integrin α1 subunit knockout, which leads to fibrosis in articular cartilage through excessive signalling from transforming growth factor beta (TGFB). Depleting this TGFB signalling is proposed to have a protective effect on cartilage. This project is part of a foregoing study where a cartilage-specific knockout of TGFB receptor type II (TBRII) was used to deplete TGFB signalling in articular cartilage of the itga1-null mice to reduce the severity of cartilage degradation. This project continues the analysis of the genetic model into bone architecture at the knee. Mouse hindlimbs were scanned at a 13μm resolution using micro-computed tomography and segmented into 3D datasets containing calcified tissues and bone of the knee and surroundings. Quantification methods for trabecular bone parameters (bone volume fraction, trabecular separation, and trabecular thickness) and ectopic calcification of soft tissues were developed. Loss of trabecular bone around the involved joint is a hallmark of post-traumatic osteoarthritis. However, the results from this study showed no significant changes in trabecular bone of itga1-null mouse knees despite observing severe osteoarthritic changes in the adjacent cartilage. There were no significant effects in peri-articular trabecular bone when the TBRII knockout in cartilage was activated, but there were significant increases in ectopic calcifications of the menisci and collateral ligaments. These ectopic calcifications were also seen in tamoxifen control mice, suggesting that tamoxifen, along with TBRII depletion in cartilage, had a role in increased abnormal calcifications. Although integrin α1B1 inhibition appears to have an important role in cartilage degeneration, it does not appear to influence the bony changes that normally accompany post-traumatic arthritis. / Thesis / Master of Applied Science (MASc) / Osteoarthritis is a common joint disorder, associated mainly with cartilage degradation. Some genes have been identified that cause or prevent osteoarthritis. A previous study used two of these genes in a genetic mouse model to explore how osteoarthritis may develop. Removing the integrin α1 subunit from mice caused osteoarthritic changes in the cartilage of the mouse knee. When the transforming growth factor beta gene was removed from the cartilage, these changes were less severe. This project continued the study by exploring changes in bone around the mouse knee. We quantified bone changes around the mouse knee using high-resolution micro-computed tomography scans. Contrary to common findings in post-traumatic osteoarthritis, we found that there were no significant changes in the bone around the knees even where severe cartilage changes had been identified. However, there were significant increases in calcifications of soft tissues including the meniscus and ligaments around the knee.