No description available.

Recognition of non-self, pathogen-associated molecular patterns is a central component of host immune response to pathogens like viruses. Intracellular detection of viral nucleic acids leads to the production of type I interferons (IFN-I) and subsequent establishment of an antiviral state in infected and neighboring cells. Viruses have evolved multiple mechanisms to counteract IFN-I responses in infected cells, however, viral nucleic acids released from dying cells can stimulate IFN-I production in surrounding or distal uninfected cells. This thesis examines the mechanisms by which cells recognize extracellular viral nucleic acids and the subsequent downstream antiviral signaling. Class A scavenger receptors (SR-As) internalize extracellular viral double-stranded RNA (dsRNA) to mediate IFN-I responses, but little is known about extracellular viral DNA. We observed that extracellular DNA is recognized and internalized by SR-As in a manner like extracellular dsRNA. Furthermore, we established that SR-A1 is sufficient in mediating extracellular dsRNA-induced cellular responses and other nucleic acid receptors like SR-J1 and DEC-205 are dispensable. Finally, a direct interaction of RNA and DNA species was demonstrated with the coiled-coil collagenous domain of SR-A1, but not the scavenger receptor cysteine rich domain of SR-A6.We elaborated the role of SR-A1 by identifying the cellular processes activated through SR-A1 following uptake of extracellular dsRNA. Cytosolic sensors are essential in mediating an antiviral response to the endocytosed dsRNA, but the mechanism of endoplasmic release and cytoplasmic entry of dsRNA remains an enigma. We demonstrated that the lack of a dsRNA-channel, SIDT2, impaired the ability of the cells to mediate an antiviral response to extracellular dsRNA. Understanding host responses to extracellular viral nucleic acids will enable the development of novel vaccines and antiviral therapeutics against RNA and DNA viruses that efficiently counteract these responses in infected cells. / Thesis / Doctor of Philosophy (PhD) / Viral infections remain a threat to global health as new diseases continue to emerge. To develop effective vaccines and antivirals to combat viruses and alleviate human disease require a deeper understanding of virus-host interactions. Host cells identify virus-associated molecules to detect viruses and eliminate them whereas, viruses employ tactics to prevent the activation of the immune system. However, virus-induced cell lysis releases viral molecules that can stimulate immune responses in neighbouring uninfected cells. This thesis examines the mechanism by which cells respond to extracellular viral nucleic acids. We showed that a protein present at the cell surface called ‘class A scavenger receptor 1’ is sufficient to internalize extracellular viral nucleic acids, leading to immune responses. The response is impaired when a channel protein, SIDT2, is absent in the cells. Further work is necessary to understand how this knowledge can be harnessed to develop vaccines and antiviral therapeutics.

<p>Skeletal muscle is dependent upon its microvasculature to deliver oxygen and substrates to support the metabolic demands of muscle contraction. Skeletal muscle capillary density is determined by a variety of factors including muscle fiber metabolic phenotype and mitochondrial volume as well as prior exercise training status. Additionally, muscle microvascular density and function can diminish with age, contributing to several age-related muscle dysfunctions. Skeletal muscle fibers regulate their surrounding microvasculature through the release of angiogenic and angiostatic signaling factors. A robust increase in angiogenic signaling from skeletal muscle facilitates increases in muscle capillarization following endurance exercise. Extracellular vesicles (EV) are membrane bound signaling factors secreted by every cell type. Skeletal muscle-derived EVs (SkM-EVs) may help facilitate numerous signaling functions of skeletal muscle including between skeletal muscle and its microvasculature.</p> <p>The primary aim of my dissertation research was to determine the signaling roles that SkM-EVs in regulating endothelial cell homeostasis and angiogenesis in states of aging and health. Chapter 1 provides an overview of the relevant literature. Chapter 2 represents an investigation into how age-related cellular senescence impacts the angiogenic potential of skeletal muscle progenitor cells. We found that stress-induced senescence increases release of small EVs and has pro-senescent and angiostatic effects on culture endothelial cells. In Chapter 3 we compared the release, contents, and angiogenic potential of SkM-EVs collected from primarily oxidative or primarily glycolytic skeletal muscle tissue in mice. We found that oxidative muscle tissue secretes more EVs than glycolytic muscle tissue, and the miR contents of EVs differ greatly between the two phenotypes. Additionally, EVs from oxidative tissue enhanced endothelial cell migration and tube formation compared to glycolytic tissue EVs, in a potentially nitric oxide mediated fashion. In Chapter 4, we tested how PGC-1α overexpression effected myotube EV release and angiogenic potential. We found that PGC-1α overexpression did not impact myotube EV release, but increased the angiogenic signaling potential of SkM-EVs. Chapter 5 is a brief summary of the results and limitations of the projects presented in Chapters 2-4, with a short discussion of potential future research directions.</p>

No description available.

No description available.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13533号 / 論医博第2273号 / 新制||医||1065(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 斎藤 通紀, 教授 遊佐 宏介 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

The CXC chemokine receptor 4 (CXCR4) and the atypical chemokine receptor 3 (ACKR3) are seven transmembrane receptors that are involved in numerous pathologies, including several types of cancers. Both receptors bind the same chemokine, CXCL12, leading to significantly different outcomes. While CXCR4 activation generally leads to canonical GPCR signaling, involving Gi proteins and β‐arrestins, ACKR3, which is predominantly found in intracellular vesicles, has been shown to signal via β‐arrestin‐dependent signaling pathways. Understanding the dynamics and kinetics of their activation in response to their ligands is of importance to understand how signaling proceeds via these two receptors. In this thesis, different Förster resonance energy transfer (FRET)‐based approaches have been combined to individually investigate the early events of their signaling cascades. In order to investigate receptor activation, intramolecular FRET sensors for CXCR4 and ACKR3 were developed by using the pair of fluorophores cyan fluorescence protein and fluorescence arsenical hairpin binder. The sensors, which exhibited similar functional properties to their wild‐type counterparts, allowed to monitor their ligand-induced conformational changes and represent the first RET‐based receptor sensors in the field of chemokine receptors. Additional FRET‐based settings were also established to investigate the coupling of receptors with G proteins, rearrangements within dimers, as well as G protein activation. On one hand, CXCR4 showed a complex activation mechanism in response to CXCL12 that involved rearrangements in the transmembrane domain of the receptor followed by rearrangements between the receptor and the G protein as well as rearrangements between CXCR4 protomers, suggesting a role of homodimers in the activation course of this receptor. This was followed by a prolonged activation of Gi proteins, but not Gq activation, via the axis CXCL12/CXCR4. In contrast, the structural rearrangements at each step of the signaling cascade in response to macrophage migration inhibitory factor (MIF) were dynamically and kinetically different and no Gi protein activation via this axis was detected. These findings suggest distinct mechanisms of action of CXCL12 and MIF on CXCR4 and provide evidence for a new type of sequential signaling events of a GPCR. Importantly, evidence in this work revealed that CXCR4 exhibits some degree of constitutive activity, a potentially important feature for drug development. On the other hand, by cotransfecting the ACKR3 sensor with K44A dynamin, it was possible to increase its presence in the plasma membrane and measure the ligand‐induced activation of this receptor. Different kinetics of ACKR3 activation were observed in response to CXCL12 and three other agonists by means of using the receptor sensor developed in this thesis, showing that it is a valuable tool to study the activation of this atypical receptor and pharmacologically characterize ligands. No CXCL12‐induced G protein activation via ACKR3 was observed even when the receptor was re-localized to the plasma membrane by means of using the mutant dynamin. Altogether, this thesis work provides the temporal resolution of signaling patterns of two chemokine receptors for the first time as well as valuable tools that can be applied to characterize their activation in response to pharmacologically relevant ligands. / Der CXC Chemokin‐Rezeptor 4 (CXCR4) und der atypische Chemokin‐Rezeptor 3 (ACKR3) sind heptatransmembranäre Rezeptoren, die in zahlreichen Krankheitsbildern eine Rolle spielen, wie in einigen Krebsarten. Beide Rezeptoren werden zwar von dem gleichen Chemokin CXCL12 aktiviert, allerdings mit unterschiedlichen Signalweiterleitungsmustern. Die Aktivierung von CXCR4 führt zu kanonischer GPCR Signaltransduktion über Gi‐Proteine und β‐Arrestine. Die Signalweiterleitung des Rezeptors ACKR3 hingegen, welcher hauptsächlich in intrazellulären Vesikeln vorliegt, erfolgt über ß‐Arrestinabhängige Signalwege. Es ist von großer Wichtigkeit die Dynamik und Kinetik dieser beiden Rezeptoren hinsichtlich der Aktivierung durch ihre Liganden und der Signalweiterleitung zu verstehen. In dieser Arbeit wurden verschiedene Förster‐Resonanzenergietransfer (FRET) Anwendungen kombiniert, um die frühen Phasen der Signal‐Kaskade von CXCR4 und ACKR3 zu untersuchen. Zur genaueren Aufklärung der Rezeptoraktivierung wurden intramolekulare FRET‐Sensoren entwickelt, hierzu wurden die Fluorophore Cyan‐fluoreszierendes Protein und engl. fluorescence arsenical hairpin binder verwendet. Die generierten Sensoren zeigten ähnliche funktionelle Eigenschaften wie die unveränderten Rezeptoren. Liganden‐induzierte Änderungen der Rezeptorkonformation können mittels dieser Sensoren beobachtet werden und stellen die ersten RET‐basierten Sensoren auf dem Forschungsgebiet der Chemokin‐Rezeptoren dar. Weitere FRET‐basierte Methoden wurden zur Untersuchung von Interaktionen zwischen Rezeptor und G‐Protein, Neuanordnung von Dimeren, sowie der G‐Protein Aktivierung eingesetzt und für beide Chemokin‐Rezeptoren etabliert. CXCR4 zeigte einen komplexen Aktivierungsmechanismus nach Stimulation durch CXCL12, bei welchem zunächst eine Neuordnung der Rezeptor‐Transmembrandomäne gefolgt von Neuordnungen zwischen Rezeptor und G‐Protein und zuletzt eine Neuordnung zwischen CXCR4 Protomeren erfolgte. Dies impliziert, dass im Aktivierungsprozess des Rezeptors Homodimere eine Rolle spielen. Zudem wurde eine verlängerte Gi ‐Protein Aktivierung gegenüber der Gq‐Protein Aktivierung bei CXCL12 stimuliertem CXCR4 beobachtet. Hingegen zeigte eine Stimulierung mit dem Macrophage Migration Inhibitory Factor (MIF) bei jedem Schritt der frühen Singal‐Kaskade veränderte Dynamiken und Kinetiken im Vergleich zu CXCL12. Darüber hinaus konnte keine Gi ‐Protein Aktivierung festgestellt werden. Dieser Befund zeigt individuelle Mechanismen für MIF und CXCL12 am CXCR4‐Rezeptor und liefert Belege für eine neuer Art von sequenziellen Signalweiterleitungen an GPCRs. Eine wichtige Beobachtung dieser Arbeit für eine potentielle Medikamentenentwicklung ist das CXCR4 ligandenunabhängige Aktivität zeigt. Um die Aktivierung des ACKR3 Sensors messen zu können wurde durch eine Co‐Transfektion mit K44A Dynamin eine höhere Membranständigkeit erreicht. CXCL12 und drei weiteren Agonisten zeigten am hier entwickelten ACKR3‐Sensor unterscheidbare Kinetiken. Mit diesem wertvollen Werkzeug können Liganden an diesem atypischen Rezeptor pharmakologisch charakterisiert werden. Es konnte keine CXCL12‐induzierte G‐Protein Aktivierung gemessen werden, trotz der stärkeren Präsenz an der Plasmamembran mit Hilfe der Dynamin‐Mutante. In Summe liefert diese Arbeit zum ersten Mal eine zeitliche Auflösung von Signalweiterleitungsmustern von zwei Chemokin‐Rezeptoren sowie wertvolle Werkzeuge zur Charakterisierung der frühen Phase der Signal‐Kaskade durch andere pharmakologisch relevanten Liganden.

No description available.

No description available.

INTRODUCTION: Following damaging stimuli, skeletal muscle exhibits coordinated interplay between intra- and extra-cellular processes resulting in satellite cell (SC) recruitment. SCs are known to play a central role in muscle plasticity post-injury by differentiating into myoblasts (MBL) and fusing with damaged tissue to donate myonuclei. Yet, their role within skeletal muscle remodeling through paracrine signaling remains to be fully elucidated. Thus, the purpose of this project was two-fold: 1) develop an in vitro model of MBL intercellular communication following myotube damage and 2) to determine if MBL proximity alone is adequate for improving tissue repair and reducing cellular stress during recovery. METHODS: C2C12 myotubes were exposed to 1 hour of electrical pulse stimulation (EPS) with 15Hz pulse for 5s and 5Hz pulse for 5s, separated by a 5s break. Myotubes were then introduced to non-electrically stimulated (NS) MBL adhered to a porous cell insert to allow paracrine signaling and samples were collected at varying timepoints post-EPS. RESULTS: EPS induced Z line sarcomeric disorganization and creatine kinase release into the cell culture media, which was mitigated in MBL+ groups (p<0.05). A significant main effect of MBL exposure was observed in EPS myotubes where MBL+ myotubes had greater Hsp70 gene expression, calpain 3 protein and gene expression, and t-ACC, p-ACCSer79, t-ULK, p-ULKSer555 protein expression than MBL- myotubes when recovering from EPS (p<0.05). A main effect of time was observed where B-dystroglycan and p-mTORSer2448 protein expression decreased in the EPS myotubes, and myotube diameter only decreased in the MBL+ condition (p<0.05). CONCLUSION: MBL signaling to damaged myotubes is evident and may increase catabolic processes through upregulating contraction-mediated protease activity and autophagy, as well as increase ATP generation through oxidative phosphorylation during regeneration. / Thesis / Master of Science (MSc) / When muscle damage occurs, whether through rigorous exercise or physical trauma, the muscle relies on a specific group of stem cells to help repair itself. These stem cells, termed satellite cells, can migrate to specific sites of muscle damage, differentiate into myoblasts, and donate nuclei and genetic material to the injured muscle. This increase in nuclear content helps the muscle synthesize more protein to rebuild and regenerate and promotes muscle growth. However, when the satellite cell becomes dysfunctional, as seen in aging muscle and certain genetic conditions, the muscle struggles to repair itself in response to damage and cannot grow in response to exercise. Satellite cell biology has clearly defined the role of nuclear donation in muscle function, however very little is known about how this stem cell ‘talks’ to the muscle through signaling molecules. As such, this thesis elucidates the effect of myoblast signaling on electrically stimulated damaged immature muscle fibers, otherwise known as myotubes, by preventing myoblast-myotube physical interactions in cell culture experimentation. Interestingly, the data presented here demonstrate that myoblast exposure to damaged myotubes may increase muscle protein breakdown as myotube diameters are reduced in size acutely post-damage, likely resulting from the increase in protease and autophagy protein expression markers. Additionally, myoblast exposure to damaged myotubes may increase mitochondrial fatty acid oxidation to generate energy, which is the fuel of choice during muscle regeneration.