京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第19907号 / 生博第354号 / 新制||生||47(附属図書館) / 32984 / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 藤田 尚志, 教授 米原 伸, 教授 朝長 啓造 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第20782号 / 生博第388号 / 新制||生||51(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 藤田 尚志, 教授 朝長 啓造, 教授 永尾 雅哉 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21022号 / 医博第4368号 / 新制||医||1028(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 朝長 啓造, 教授 萩原 正敏, 教授 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Aus dem Patientengut einer Fachklinik für Klein- und Heimtiere wurden 11 nicht vorbehandelte Katzen zwischen einem und zwölf Jahren mit mittel- bis hochgradiger Gingivo-Stomatitis ausgewählt. Diese wurden für zwölf Wochen (84 Tage) stationär aufgenommen und nach einem standardisierten Therapiekonzept behandelt: Am ersten Tag erfolgte nach dentalem Röntgen eine umfassende Zahnsanierung. An den Tagen 0, 14, 28, 42 und 84 wurde Interferon (Virbagen Omega® des Herstellers Virbac S.A.®, Carros Cedex, Frankreich) unter Sedation lokal, d.h. submukosal mit 1 ME/kg KGW injiziert. An den Tagen 56, 58, 60 und 62 erfolgte die Interferongabe systemisch. Begleittherapien wurde nach Bedarf eingesetzt, jedoch ohne die Verwendung von Glukokortikoiden und Hormonpräparaten. Verfüttert wurde ausschließlich Futter des Herstellers Royal Canin®, Köln, in den ersten 14 Tagen das Feuchtfutter Royal Canin convalescence support®, ab Tag 15 Royal Canin intestinal® Feucht- und Trockenfutter. An allen Behandlungstagen wurden zur qualitativen Virusbestimmung Tupferproben der am stärksten entzündeten Bezirke entnommen, die Maulhöhle nach einem festen System abfotografiert und die Veränderungen in Formblättern (Stärke der Faucites, Gingivitis, Buccostomatitis, Größe der Fläche und Art der Veränderung) und Grafik-charts festgehalten. Am ersten und letzten Tag wurden außerdem Biopsien zur quantitativen Bestimmung der Viruslast entnommen. Die Entwicklungen in folgenden Bereichen wurden anhand fixer Kriterien 14-tägig festgehalten: Allgemeinzustand, Schmerzen bei der Maulöffnung, Halitosis/zäher Speichel, Größe der Mandibularlymphknoten, Appetit, Schmerzen bei Futter-aufnahme oder Gähnen, Hypersalivation, Aktivität, Putztrieb und Zugänglichkeit. Die klinischen Verbesserungen waren bei allen Tieren schon nach 14 Tagen augenfällig. Der Hauptvorstellungsgrund der Besitzer, Appetitlosigkeit und Schmerzen bei der Futteraufnahme waren einer fast ungestörten Futteraufnahme gewichen, diese konnte in den folgenden Wochen kaum noch optimiert werden. Die entzündlichen Ulzerationen und Proliferationen der Maulhöhle halbierten sich innerhalb der ersten 14 Tage, nach 84 Tagen war der Heilungsprozess bei acht der elf Katzen abgeschlossen. Die persistierenden Proliferationen der restlichen Katzen waren allerdings nicht entzündlich und beeinflussten die Futteraufnahme nicht. Allgemeinzustand, Aktivität, Putztrieb und Zugänglichkeit stiegen bei zehn von elf Katzen bis zum 42. Tag etwa linear auf artspezifisches Normalniveau an und blieben hier konstant. Hypersalivation und Schwellung der Mandibularlymphknoten legte sich, so vorhanden, bei allen Tieren bis auf zwei innerhalb von 28 Tagen, bei diesen beiden war bis zum 84. Tag nur eine geringgradige Verbesserung zu beobachten. Nach der systemischen Vier-Tages-Therapie wurde ein erneutes Aufflackern der Gingivo-Stomatitis etwa auf das Niveau des 56. Tages beobachtet, allerdings ohne Folgen für die Verhaltensparameter. Eine Reduktion der Viruslast konnte trotz der eindrucksvollen Verbesserungen im klinischen Bereich in keinem Fall festgestellt werden. Die FIV/FeLV-positiven Katzen sprachen langfristig wesentlich schlechter auf das Therapiekonzept an als die übrigen Probanden. Daher gilt es bei diesen Tieren vor Interferoneinsatz kritisch zwischen den nicht unerheblichen Kosten und der zweifelhaften Prognose abzuwägen. Generell kann das Therapiekonzept Zahnsanierung – Interferon – Begleittherapie nach erfolgtem FIV/FeLV-Test bei der felinen Gingivo-Stomatitis als klinisch erfolgreich betrachtet werden.

Cleavage and polyadenylation are two necessary messenger RNA (mRNA) maturation steps for gene expression. The Cleavage and Polyadenylation Specificity Factor (CPSF) complex, which recognizes the AAUAAA polyadenylation signal and executes the cleavage reaction, is indispensable for these two processes. In this thesis, I describe my study of the regulation and functions of two non-canonical isoforms of the CPSF subunit WDR33. In addition, I provide detailed analyses on our current knowledge of CPSF subunits’ functions and their influences on a diverse collection of biological processes and conditions. In Chapter1, I provide a general introduction to cleavage and polyadenylation, WDR33, innate immune response via molecular pattern recognition, and the cGAS-STING pathway. Chapter 2 presents my original research on non-canonical WDR33 isoforms, termed WDR33v2 (V2) and WDR33v3 (V3). I determined that their mRNAs are produced by alternative polyadenylation. Both V2 and V3 proteins lack multiple WD repeats, but they can interact with and stabilize each other. This is a novel mode of protein-protein interaction, which I termed WD repeat complementation (WDRC). Unexpectedly, I found that even though V2 and V3 are isoforms of a polyadenylation factor, they are not themselves polyadenylation factors. Regulated by the NF-κB pathway, they are interestingly immune factors involved in the cGAS-STING pathway that induces immune responses against cytosolic double-stranded DNA. V2 decreases STING disulfide oligomerization and suppresses STING-mediated interferon β induction, but facilitates STING-mediated autophagy. Binding of V3 to V2 via WDRC prevents V2’s regulation of STING, suggesting that V3 is a V2 inhibitor. My findings thus further our understanding of STING-mediated immune responses. More broadly, these findings also demonstrate that isoforms produced by alternative mRNA processing can be functionally unrelated. In light of the versatility of the WDR33 gene, I performed a literature review in Chapter 3 on both the canonical and non-canonical functions of CPSF. I first summarize the general functions of CPSF subunits. Subsequently, I discuss their involvements in a variety of biological processes and conditions. This discussion reveals that different processes involve different CPSF subunits. Although CPSF is responsible for only two simple biochemical reactions, it has profound influences on cellular homeostasis. Together, my thesis studies reveal new insights into the molecular mechanism of the cGAS-STING pathway, underscore the importance of alternative mRNA processing, and provide the latest analyses of the functional significances of CPSF.

No description available.

The classical description of the innate antiviral response involves the production of type I interferon (IFN) and the subsequent expression of hundreds of interferon stimulated genes (ISGs), which cooperatively repress viral replication and spread. More recently, an IFN-independent antiviral response has also been described, in which the entry of an enveloped virus induces a subset of ISGs without requiring the production of IFN, although the details of this response remain unclear. In this work, multiple approaches were used to further characterize antiviral signaling pathways. Initially, the potential involvement in the IFN-independent response of the small GTPase Rac1, which has been implicated in both viral entry and antiviral signaling, was investigated. Here, Rac1 was shown to have a possible function in the negative regulation of ISG expression, although technical complications prevented definitive conclusions. As an alternative strategy to identify novel aspects of antiviral signaling, the mechanism of action of ICP0, a herpes simplex virus (HSV) protein involved in innate immune evasion, was investigated. Although ICP0 is generally thought to perform its actions in the nucleus, by tagging proteins for proteasome-mediated degradation via the E3 ubiquitin ligase activity of its RING finger domain, here it was shown that not only does cytoplasmic ICP0 have a RING-dependent but proteasome-independent ability to block antiviral signaling, but also that ICP0 has a previously unknown RING-independent function in the promotion of viral replication in the cytoplasm. To further investigate the cytoplasmic activities of ICP0, proteins interacting with ICP0 in the cytoplasm were identified using quantitative mass spectrometry. This revealed several intriguing binding partners for ICP0, including WDR11, a poorly-characterized cellular protein which was shown to undergo a dramatic relocation during HSV infection, although it was not required for viral replication in cultured cells. Therefore, this study has uncovered several new and unexpected insights into ICP0 behavior. / Thesis / Doctor of Philosophy (PhD)

Diarrheal disease caused by attaching and effacing pathogens, such as enteropathogenic E. coli (EPEC), is a worldwide health concern. As the second leading cause of diarrheal-related death in young children, new investigations into host defense against EPEC, as well as future therapeutics, is greatly needed. To elucidate the host immune responses to these enteric pathogens, the attaching and effacing (A/E) murine pathogen, Citrobacter rodentium, has been widely used. It is well understood that C. rodentium infection induces a robust Th1 response within the host. Yet how these pleiotropic IFNγ immune responses are initiated, propagated, and the accessory immune cell types involved remains poorly understood. In this thesis, I investigated how innate immune cell types such as natural killer cells, which are significant producers of IFNγ, mediate these Th1 directed responses. This work identified that both NK and NK-like innate lymphoid type 1 cells (ILC1s) are capable of producing IFNγ in response to C. rodentium, and NK cells rapidly increase in numbers within the colon during the early stages of infection. Depletion of these cell types causes a delayed Th1 CD4+ T cell response within the colon, resulting in increased bacterial load, and greater degree of colonic pathology at later time points. Additionally, depletion of these cells results in decreased CXCL9 chemokine expression in mice. I later determined that CXCL9 exhibited direct antimicrobial action against Citrobacter in vitro. Depletion of this chemokine in vivo, in the absence of adaptive immune responses, or its receptor CXCR3, results in increased mortality rates, elevated bacterial loads, greater degree of pathology, and deeper penetration of bacteria within the colonic crypts. These data indicate a potential direct antimicrobial role for this IFNγ-induced chemokine, independent of its known properties for the homing of T cells to the site of infection. These findings demonstrate the importance of accessory IFNγ-producing immune cells in not only mediating Th1 CD4+ T cells responses, but also other innate host defense mechanisms against A/E pathogens. / Thesis / Doctor of Philosophy (PhD)

Class II cytokine receptors are important pleiotropic regulators of the immune system that play a central role in pathogen defense, tumor surveillance and immune system homeostasis. Most of these activities are very promising for biomedical applications, which, however, have so far failed to succeed due to severe undesired side effects resulting from the pleiotropic nature of these cytokine receptors. Controlling the functional plasticity of class I/II cytokine receptor signaling by engineered cytokines has recently emerged as a promising approach to selectively reduce such side effects. In this context, systematic studies on the IFNalpha/beta receptor and other systems have identified that the binding kinetics of the ligand-receptor interaction play an important role in defining signaling specificity. This has been explained by altered equilibrium and dynamics of the signaling complex in the plasma membrane. In this work, I have investigated how the spatiotemporal organization and dynamics of signaling complexes regulate activation and signaling specificity of other members of the class II cytokine receptors. I focused on the type II IFN and IL-10 systems that supposedly form hexameric ligand-receptor signaling complexes in the plasma membrane. To this end, we developed an orthogonal multicolor anti-GFP nanobody-based labeling strategy, that allowed imaging of up to four different class II cytokine receptor subunits simultaneously. Using this labeling strategy, I investigated the spatiotemporal dynamics of IFNGR and IL-10R complex assembly by co-localization and co-tracking of single receptor subunits. Thereby, I did show that unliganded receptor subunits of IFNGR and IL-10R remain monomeric at the cell surface, whereas binding of the ligand led to fast and efficient receptor homo- and hetero-dimerization, verifying a ligand-induced receptor complex assembly model for both cytokine receptors. Moreover, I verified the hexameric ligand-receptor complex structure in cellulo. Analysis of single molecule trajectories and co-trajectories revealed a decrease in mobility and diffusion of IFNGR and IL-10R subunits upon ligand stimulation indicating receptor confinement and endocytosis. In this context, I identified an abnormal diffusion behavior of IL-10R2 that was dependent on the length of its transmembrane helix. We used partial agonists for both receptor complexes to systematically alter receptor binding stoichiometry and complex stability in the plasma membrane and correlated these with downstream signaling responses. Our analysis revealed a minor contribution of the second low affinity receptor subunit and its associated kinase to the overall signaling activity. However, the second high affinity binding subunit was indispensable to acquire full signaling potential. We managed to obtained decoupling of gene expression for both hexameric class II cytokine receptors by utilizing engineered ligands with altered receptor binding affinities. Our findings could pave the way for new biomedical approaches with engineered IFNgamma and IL-10 in the future. Furthermore, we uncovered pathogenic mechanisms behind the IFNGR2-T168N mutant and auto-IFNgamma antibodies, both of which prominently cause the Mendelian Susceptibility to Mycobacteria Disease (MSMD) syndrome, showing that both interfere with IFNGR activation by preventing recruitment of IFNGR2 into receptor complexes.