Morphine is a clinically very important drug from the opioid group that is used for treatment of severe pain because of its strong analgetic effect. Opioid receptors mediating the morphine effect interact with the Gi/o class of trimeric G-proteins. Opioid receptors also occur in heart tissue and morphine can thus potentially exercise its effect on the function of this organ. The major aim of this project was to pursue consequences of long-term treatment with morphine on expression and distribution of selected heterotrimeric G-protein subunits in the rat heart. Potential cardioprotective effects of this drug have also been studied. Laboratory rats of the Wistar strain were treated with morphine (1 mg/kg/day or 10 mg/kg/day) for 10 or 28 days. The control group was treated with saline solution. Prolonged treatment with morphine did not cause any effects on Gs, Gi, Gz, Gq/11, G subunits, but the expression of Go rather decreased. The results of subsequent experiments showed that prolonged administration of high doses of morphine may reduce the area affected by infarction and reduced the frequency of ventricle arrhythmias depending on dose and duration of morphine administration. Key words: morphine, myocardium, opioid receptor, G-protein subunits, infarction.

The ability of organisms to sense – and properly respond to – their environment is crucial to their survival. Higher organisms communicate with conspecifics to ensure the survival of the species. Nematodes, such as the roundworm Caenorhabditis elegans, are ubiquitous across all biomes, and rely on chemical communication to convey information with one another. The small molecules they utilize in this communication are called ascarosides. These modular pheromones are employed by all taxa, ranging from Caenorhabditis to Ascaris. The ascaroside, ascr#8, is release by hermaphroditic C. elegans to attract potential mates. Previous work has shown that a class of male specific neurons are required for sensation of this pheromone. Here, we show that these neurons initiate a neural circuit modulated by the FMRFamide-like neuropeptide, flp-3. This neuropeptide is sensed by a set of G protein-coupled receptors (GPCRs), NPR-10 and FRPR-16. Together, these components determine the behavioral valence of males to ascr#8. Within the male-specific sensory neurons, the CEM, we show that another group of GPCRs sense the ascr#8. Two of these receptors, DMSR-12 and SRW-97, are expressed in the cilia, suggesting their involvement in direct sensation of the cue. As a targeted approach to identifying and confirming receptors for ascr#8, we have developed a bioactive photoaffinity probe. We have also confirmed that the ability of ascr#8 to attract males is conserved across the genus. Together, these studies coalesce to deepen our understanding of sex-specific chemosensation and neuronal processing. These results can be used to better understand the defects that are seen in neurodegenerative diseases – many of which exhibit sex-specific defects in neuronal processing.

In order to function, proteins must be folded into their native shape. While this can sometimes occur spontaneously, the process can be hindered by thermodynamic barriers, trapped intermediates, and aggregation prone hydrophobic interactions. Molecular chaperones are proteins that help client proteins or substrates overcome these barriers so that they can be folded properly. One such chaperone is the chaperonin CCT, a large MDa protein made up of 16 paralogous subunits that form a double ring structure. CCT encapsulates its substrates in a central cavity, where they are sequestered and folded, using ATP binding and hydrolysis to drive conformational changes in the CCT-substrate complex. CCT mediates the folding of many substrates involved in a variety of cellular process, including the cytoskeletal proteins actin and tubulin, and the G protein subunit Gabg, which signals downstream of GPCRs in a variety of cellular processes. We showed that CCT is responsible for folding the b-propeller containing proteins, mLST8 and Raptor, which are subunits of the mTOR complexes. The mTOR complexes (mTORC1 and mTORC2) are master regulators of cell growth and survival by controlling processes such as protein synthesis, energy metabolism, cell survival pathways and autophagy. CCT folds mLST8 and Raptor and help them assemble into the mTOR complexes. As a result, CCT is required for functional mTOR signaling. Furthermore, we solved a 4.0 Ǻ resolution structure of mLST8 bound to CCT. Surprisingly, mLST8 is found in the center of the folding cavity, in between the rings, despite previous evidence suggesting that substrates bind only in the apical domains. Given its role in folding and assembling the mTOR complexes, G proteins, and many other proteins involved in cell survival pathways, CCT has been implicated in cancer. CCT upregulation often correlates with a worse prognosis, likely because uncontrolled growth requires increased chaperone capacity. The peptide CT20P has been shown to have cytotoxic effects in cancer cells, likely through its binding to CCT. We characterized CT20P, showing that it binds to CCT and inhibits its substrate-folding functions in cells. We specifically showed that a GFP-CT20P fusion protein inhibited the assembly of two important signaling complexes Gbg and mTORC1. These results show that CT20P is a useful inhibitor for the study of CCT function.

Die Kommunikation von Zellen untereinander und mit ihrer Umgebung stellt die Grundlage für die Existenz lebender uni- sowie multizellulärer Organismen dar. Dabei müssen Zellen zwischen diversen chemischen und physikalischen Stimuli differenzieren. Ermöglicht wird dies durch eine individuelle Ausstattung mit membranständigen und zytosolischen Rezeptoren. Eine der größten und diversesten Superfamilien membranständiger Rezeptoren bildet die Gruppe der G-Protein-gekoppelten Rezeptoren (GPCR). GPCR weisen eine charakteristische, sehr ähnliche räumliche Grundstruktur auf. Sie besitzen eine Kerndomäne aus sieben transmembranären, im Wesentlichen hydrophoben α-Helices, die durch drei intrazelluläre sowie drei extrazelluläre Schlaufen miteinander verbunden sind. Eine häufig auftretende vierte Schlaufe wird durch die Palmitoylierung eines intrazellulär lokalisierten C-Terminus an einem Cysteinrest und die hohe Affinität des Palmitats zur Lipiddoppelschicht gebildet. Der N-Terminus des Rezeptors befindet sich extrazellulär, während der C-Terminus in den Intrazellularraum reicht. Im Vergleich zu anderen Rhodopsin-ähnlichen GPCR zeichnen sich die Glykoproteinhormonrezeptoren (GPHR) durch einen großen extrazellulären N-Terminus aus, der das aktivierende Hormon bindet. Die Funktion des N-Terminus beschränkt sich jedoch nicht auf die Ligandenbindung. Er ist auch an der direkten Aktivierung der Transmembrandomäne (TMD) beteiligt. Die Signalvermittlung erfolgt über einen intramolekularen Agonist. Durch Deletions- und Mutagenesestudien sowie identifizierte pathogene Mutationen ist dessen Existenz in GPHR nachgewiesen. Stimulationsversuche mit von der Hinge-Region innerhalb des extrazellulären N-Terminus abgeleiteten Peptiden, die alle drei GPHR aktivieren, führten in Vorarbeiten zur Spezifizierung der hochkonservierten agonistischen Sequenz FNPCEDIMGY (p10-Region). Diese ist am Übergang des N-Terminus zur ersten Transmembranhelix lokalisiert und zwischen den paralogen und orthologen GPHR hochkonserviert. Mit dieser Arbeit wird die Identität des intramolekularen Agonists bestätigt. Es wird gezeigt, dass die Einnahme des aktiven Zustandes aller GPHR die Integrität der p10-Region und ihre spezifischen intramolekularen Interaktionen mit weiteren Rezeptordeterminanten erfordert. Folglich können die GPHR, wie auch Rhodopsin, Protease-aktivierte Rezeptoren (PARs) und Adhäsions-GPCR, einer Gruppe der G-Protein-gekoppelten Rezeptoren zugeordnet werden, deren Ligand kovalent an den Rezeptor gebunden vorliegt oder selbst Teil des Rezeptorproteins ist. Auf die GPHR trifft dabei letzteres zu.:Abbildungsverzeichnis Tabellenverzeichnis Abkürzungsverzeichnis 1 Einführung 1.1 Struktur und Signaltransduktion G-Protein-gekoppelter Rezeptoren 1.2 Die Glykoproteinhormonrezeptoren 1.2.1 Topologie und Struktur der GPHR 1.2.2 Die Physiologie der GPHR und ihrer Liganden 1.2.3 GPHR-assoziierte Pathogenese 1.2.3.1 Mutationen des LH-Rezeptors 1.2.3.2 Mutationen im TSH-Rezeptor 1.2.3.3 Morbus Basedow und Hashimoto-Thyreoditis 1.2.4 Die Signalweiterleitung der Glykoproteinhormonrezeptoren 1.2.5 Modelle des Aktivierungsmechanismus der Glykoproteinhormonrezeptoren 1.3 Aufgabenstellung 2 Material und Methoden 2.1 Materialien 2.2 Zelllinien und Mikroorganismen 2.3 Medien, Puffer und Lösungen 2.3.1 Medien 2.3.2 Medien und Puffer für den cAMP-Akkumulationsassay 2.3.3 Puffer und Lösungen für die Expressionsanalysen 2.3.4 Sonstige Lösungen 2.4 Software 2.5 Kits 2.6 Vektoren 2.7 Methoden 2.7.1 Allgemeine Kulturbedingungen von Bakterien und Zelllinien 2.7.2 Molekularbiologische Standardmethoden 2.7.3 Transiente Transfektion von HEK293-T Zellen 2.7.4 Expressionsanalyse mittels Enzyme-linked Immunosorbent Assay (ELISA) 2.7.4.1 Analyse der Gesamtexpression mittels Sandwich-ELISA 2.7.4.2 Analyse der Oberflächenexpression mithilfe des Oberflächen-ELISA 2.7.5 cAMP-Akkumulationsassay 2.7.6 Luciferase-Reportergenassay 3 Ergebnisse 3.1 Herstellung der Rezeptormutanten und deren funktionelle Charakterisierung im humanen LHR 3.2 Die Erkenntnisse aus der hLHR-Mutagenesestudie sind nicht vollständig auf den hTSHR übertragbar 3.3 Mutationen innerhalb der p10-Region haben keinen Einfluss auf die Integrität der hTSHR-Transmembrandomäne 3.4 Die Integrität des Rezeptor-N-Terminus ist grundlegend relevant für die Einnahme einer aktivierbaren Konformation der GPHR 3.5 Die Deletion des N-Terminus wirkt sich nicht auf die Stabilität und Funktionalität der GPHR-Transmembrandomäne aus 3.6 Trypsin führt nicht zur vollen Aktivierung des humanen TSH-Rezeptors 3.7 Mutationen innerhalb der p10-Region können die maximale Rezeptoraktivierung induzieren 3.8 Erstellung eines Homologiemodells anhand der funktionellen Eigenschaften der generierten Mutanten 4 Diskussion 4.1 Die Aktivierung der GPHR stellt hohe Anforderungen an die strukturellen und physikochemischen Eigenschaften des intramolekularen Agonists 4.2 Das Zusammenspiel verschiedener Rezeptorregionen reguliert die Signalaktivität der GPHR 4.3 Die Integrität des N-Terminus der GPHR ist hochrelevant für die Positionierung des intramolekularen Agonists 4.4 Die Rezeptoraktivierung durch den niedermolekularen, synthetischen Agonist E2 erfolgt unabhängig von der p10-Region 4.5 Die Aktivierungsmechanismen des hLHR und hTSHR weisen Übereinstimmungen und Unterschiede auf 4.6 Ausblick Zusammenfassung der Arbeit Literaturverzeichnis Anlagen Erklärung über die eigenständige Abfassung der Arbeit Publikationen Danksagung

Heterotrimeric Gi proteins play a role in lipopolysaccharide (LPS) and Staphylococcus aureus (SA) activated signaling leading to inflammatory mediator production. We hypothesized that genetic deletion of Gi proteins would alter cytokine and chemokine production induced by LPS and SA. LPS- and heat killed SA-induced cytokine and chemokine production in splenocytes from wild type (WT), Gαi2 (-/-) or Gαi1/3 (-/-) mice were investigated. LPS- or SA-induced production of TNFα, IL-6, IFNγ, IL-12, IL-17, GM-CSF, MIP-1α, MCP-1, MIG and IP-10 were significantly increased (1.2 to 33 fold, p < 0.05) in splenocytes harvested from Gαi2(-/-) mice compared with WT mice. The effect of Gαi protein depletion was remarkably isoform specific. In splenocytes from Gαi1/3 (-/-) mice relative to WT mice, SA-induced IL-6, IFNγ, GM-CSF, and IP-10 levels were decreased (59% to 86%, p < 0.05), whereas other LPS- or SA-stimulated cytokines and chemokines were not different relative to WT mice. LPS- and SA-induced production of KC were unchanged in both groups of the genetic deficient mice. Splenocytes from both Gαi2 (-/-) and Gαi1/3 (-/-) mice did not exhibit changes in TLR2 and TLR4 expression. Also analysis of splenic cellular composition by flow cytometry demonstrated an increase in splenic macrophages and reduced CD4 T cells in both Gαi2 (-/-) and Gαi1/3 (-/-) mice relative to WT mice. The disparate response of splenocytes from the Gαi2 (-/-) relative to Gαi1/3 (-/-) mice therefore cannot be attributed to major differences in spleen cellular composition. These data demonstrate that Gi2 and Gi1/3 proteins are both involved and differentially regulate splenocyte inflammatory cytokine and chemokine production in a highly Gi isoform specific manner in response to LPS and Gram-positive microbial stimuli.

Screening compounds with image-based analysis is an important part in the processof drug discovery. It is an efficient way to screen compounds as it gives moreinformation than for example HTS. High-content screening as it is also called, hasreally progressed in recent years, as the field of data science evolves, and with it sodoes the efficiency of how images can be processed into information. Anotherimportant part of the drug discovery field is the family of receptors GPCRs, a largefamily of over 800 different receptors in humans. The reason GPCRs are importantin drug discovery is because of the large number of drugs targeting them. In thisexperiment we wanted to use image-based analysis to challenge drugs orcompounds that were said to be specific and see if they actually are that specific, orif we can see indications of the drug also working somewhere else. While the drugswe tested did not appear to cause any morphological perturbations large enough todistinguish them from the control, some drugs appear to cluster differently. Thismight suggest that they affect multiple targets, but it needs to be followed up upon inorder to draw any substantial conclusions.

The current epidemic of obesity has been linked to an increase in fat intake associated with the Western diet. Nutrient-induced stimulation of enteroendocrine cells in the small intestine leads to the release of hormones that contribute to satiety and the control of food intake. In particular, ingested fat, specifically in the form of free fatty acids, is potent activator of enteroendocrine cells in the proximal small intestine. However, the underlying signaling cascade that free fatty acids initiate in these enteroendocrine cells, which leads to secretion of satiety hormones, is not known. In general, my research is focused on identifying nutrient-responsive pathways in enteroendocrine cells involved with the release of satiety signals and using this information to begin to develop novel drug delivery strategies to reduce food intake. In general, my results revealed that activation of the fatty acid receptor GPR120 was ecessary for the linoleic acid-induced intracellular calcium rise, a necessary precursor for hormone release. Using patch clamp recording, I discovered that linoleic acid activated enteroendocrine cells by inducing membrane depolarization, a process requiring the calcium-activated, monovalent cation permeable channel TRPM5, which is activated downstream of GPR120. To validate the unexpected finding that TRPM5 was involved in fattyacid signaling, I performed experiments using bitter compounds, whose transduction pathway is known to involve TRPM5. Enteroendocrine cells express the bitter taste receptors and release cholecystokinin in response to bitter stimuli, suggesting the probable role of gut in initiation of protective behavior against ingestion of potentially harmful substances. Armed with the data on the specifics of the fatty acid transduction, I performed experiments using nanoparticles to determine their utility for delivering pharmaceuticals specifically to the enteroendocrine cells. I fabricated and characterized PLGA nanoparticles and performed intracellular uptake studies in order to optimally delivery payloads inside cells. Finally, I validated their use by using cell-based assays to determine the effects of internalized PLGA nanoparticles on ion channels and signaling pathways involved in CCK release. Taken together, this dissertation research has identified the signaling pathways (pharmacological targets) involved in fatty acid-mediated satiety hormone release and validated the potential therapeutic use of nanoparticle-mediated drug delivery for the eventual control of food intake.

Indiana University-Purdue University Indianapolis (IUPUI) / Metabolic survival mechanisms that defend body weight and conserve energy are currently at odds with modernized society which has a food supply that is ubiquitous, calorie dense, and highly palatable. Chronic overnutrition leads to a metabolic syndrome of obesity, insulin resistance, inflammation, and cardiovascular diseases that is increasingly prevalent and threatens health on a global scale. The brain is both a victim and culprit of metabolic diseases, and prolonged metabolic dysfunction can exacerbate the pathological mechanisms underlying both metabolic and neurodegenerative diseases. Since neuroendocrine pathways comprise an essential feedback mechanism that detects circulating hormones and nutrients in order to regulate satiety, energy expenditure, and glucose homeostasis, our research goals were to characterize molecular mechanisms within neuroendocrine pathways that could be leveraged for treating obesity, diabetes, and Alzheimer’s disease. First, we identified the expression of a G protein-coupled receptor, Gpr17, in POMC neurons and discovered that it protects aged mice from high-fat diet (HFD)-induced metabolic derangements. We examined the electrophysiological properties of POMC neurons and found Gpr17 deficiency led to increased spontaneous action potentials. Moreover, Pomc-Cre-driven Gpr17 knockout (PGKO) mice, especially female knockouts, had increased POMC-derived alpha-melanocyte stimulating hormone and beta-endorphin despite a comparable level of prohormone POMC in their hypothalamic extracts. Second, we generated a highly insulin resistant mouse model with human GLUT4 promoter-driven insulin receptor knockout (GIRKO) in muscle, adipose, and GLUT4-expressing neuronal subpopulations. This genetic approach recapitulates the primary defect preceding type 2 diabetes (T2D) and revealed additional factors/mechanisms that drive the ultimate progression of overt diabetes. Third, we used 5xFAD mice as a model of Alzheimer’s disease and showed that they were more susceptible to HFD-induced metabolic dysregulation and expression of AD pathological markers in the hippocampus. Our results helped elucidate the molecular and cellular mechanisms responsible for increased AD pathology in high-fat diet-fed 5xFAD mice and suggest that metabolic dysfunctions are a therapeutic target to ameliorate AD pathology. In conclusion, metabolic diseases are pervasive and require nuanced approaches that target the neuroendocrine system in order to restore metabolic homeostasis and protect the brain from neurodegenerative processes that are associated with obesity and diabetes.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第20691号 / 工博第4388号 / 新制||工||1682(附属図書館) / 京都大学大学院工学研究科都市環境工学専攻 / (主査)教授 田中 宏明, 教授 高野 裕久, 教授 米田 稔 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第13146号 / 論農博第2852号 / 新制||農||1056(附属図書館) / 学位論文||H30||N5093(農学部図書室) / (主査)教授 植田 和光, 教授 加納 健司, 教授 三芳 秀人 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM