@phdthesis{Langlhofer2016,
  author    = {Langlhofer, Georg},
  title     = {{\"U}ber die Bedeutung intrazellul{\"a}rer Subdom{\"a}nen des Glycinrezeptors f{\"u}r die Kanalfunktion},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-140249},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Der zur Familie der pentameren ligandengesteuerten Ionenkan{\"a}le zugeh{\"o}rige Glycinrezeptor (GlyR) ist ein wichtiger Vermittler synaptischer Inhibition im Zentralnervensystem von S{\"a}ugetieren. GlyR-Mutationen f{\"u}hren zur neurologischen Bewegungsst{\"o}rung Hyperekplexie. Aufgrund fehlender struktureller Daten ist die intrazellul{\"a}re Loop-Struktur zwischen den Transmembransegmenten 3 und 4 (TM3-4 Loop) eine weitgehend unerforschte Dom{\"a}ne des GlyR. Innerhalb dieser Dom{\"a}ne wurden Rezeptortrunkierungen sowie Punktmutationen identifiziert. Rezeptortrunkierung geht mit Funktionslosigkeit einher, welche jedoch durch Koexpression des fehlenden Sequenzabschnitts zum Teil wiederhergestellt werden kann. Innerhalb dieser Arbeit wurde die Interaktion zwischen trunkierten, funktionslosen GlyR und sukzessiv verk{\"u}rzten Komplementationskonstrukten untersucht. Dabei wurden als Minimaldom{\"a}nen f{\"u}r die Interaktion das C-terminalen basische Motive des TM3-4 Loops, die TM4 sowie der extrazellul{\"a}re C-Terminus identifiziert. Die R{\"u}ckkreuzung transgener M{\"a}use, die das Komplementationskonstrukt iD-TM4 unter Kontrolle des GlyR-Promotors exprimierten, mit der oscillator-Maus spdot, die einen trunkierten GlyR exprimiert und 3 Wochen nach der Geburt verstirbt, hatte aufgrund fehlender Proteinexpression keinen Effekt auf die Letalit{\"a}t der Mutation. Des Weiteren wurde die Bedeutsamkeit der Integrit{\"a}t beider basischer Motive 316RFRRKRR322 und 385KKIDKISR392 im TM3-4 Loop in Kombination mit der Loop-L{\"a}nge f{\"u}r die Funktionalit{\"a}t und das Desensitisierungsverhalten des humanen GlyRα1 anhand von chim{\"a}ren Rezeptoren identifiziert. Eine bisher unbekannte Patientenmutation P366L innerhalb des TM3-4 Loops wurde mit molekularbiologischen, biochemischen und elektrophysiologischen Methoden charakterisiert. Es wurde gezeigt, dass die mutierten Rezeptorkomplexe in vitro deutlich reduzierte Glycin-induzierte Maximalstr{\"o}me sowie eine beschleunigte Schließkinetik aufweisen. P366L hat im Gegensatz zu bereits charakterisierten Hyperekplexiemutationen innerhalb des TM3-4 Loops keinen Einfluss auf die Biogenese des Rezeptors. P366 ist Teil einer m{\"o}glichen Poly-Prolin-Helix, die eine Erkennungssequenz f{\"u}r SH3-Dom{\"a}nen darstellt. Ein potenzieller Interaktionspartner des TM3-4 Loops des GlyRα1 ist Collybistin, welches eine wichtige Rolle bei der synaptischen Rezeptorintegration spielt und die Verbindung zum Zytoskelett vermittelt. An der inhibitorischen Synapse verursacht P366L durch die Reduzierung postsynaptischer Chloridstr{\"o}me, das beschleunigte Desensitisierungsverhalten des GlyRα1 sowie ein ver{\"a}ndertes Interaktionsmotiv St{\"o}rungen der glycinergen Transmission, die zur Auspr{\"a}gung ph{\"a}notypischer Symptome der Hyperekplexie f{\"u}hren.},
  subject      = {Glycinrezeptor},
  language  = {de}
}
@phdthesis{Mayer2019,
  author    = {Mayer, Rafaela},
  title     = {OxPAPC as an endogenous agonist of TRPA1 channels on nociceptors},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-175890},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Non-steroidal antiinflammatory drugs are most commonly used for inflammatory and postoperative pain. But they lack effectiveness and specificity, leading to severe side effects, like gastric ulcers, asthma and severe bleeding. Oxidized 1-palmitoyl-2-arachinidonoyl-sn-glycero-3-phosphocholine (OxPAPC) plays an important role in inflammatory pain. PAPC is a common phosphatidylcholine of membranes, which can be oxidized by reactive oxygen species. In preliminary experiments, our group found that local injection of OxPAPC in rat paws induces hyperalgesia. In this study we examined the effect of OxPAPC on transient receptor potential A1 (TRPA1), an ion channel expressed in C-fiber neurons. Furthermore, we investigated if intracellular cysteine residues of TRPA1 were necessary for agonist-channel-interactions and if a subsequent TRPA1 activation could be prevented by OxPAPC scavengers. To answer these questions, we performed calcium imaging using HEK-293 cells stably expressing hTRPA1, or transiently expressing the triple mutant channel hTRPA1-3C and na{\"i}ve DRG neurons. Cells were incubated with the ratiometric, fluorescent dye Fura-2/AM and stimulated with OxPAPC. The change of light emission after excitation with 340 and 380 nm wavelengths allowed conclusions regarding changes of intracellular calcium concentrations after TRPA1 activation. In our investigation we proved evidence that OxPAPC activates TRPA1, which caused a flow of calcium ions into the cytoplasm. The TRPA1-specific channel blocker HC-030031 eliminated this agonist-induced response. TRPA1-3C was not completely sensitive to OxPAPC. The peptide D-4F and the monoclonal antibody E06 neutralized OxPAPC-induced TRPA1 activation. In this work, the importance of OxPAPC as a key mediator of inflammatory pain and as a promising target for drug design is highlighted. Our results indicate that TRPA1 activation by OxPAPC involves cysteine-dependent mechanisms, but there are other, cysteine-independent activation mechanisms as well. Potential pharmaceuticals for the treatment of inflammatory pain are D-4F and E06, whose efficiency has recently been confirmed in the animal model by our research group.},
  subject      = {Schmerzforschung},
  language  = {en}
}
@phdthesis{Kloka2019,
  author    = {Kloka, Jan Andreas},
  title     = {Endogene Lipide als neues Behandlungstarget im TRPA1-vermittelten Entz{\"u}ndungsschmerz},
  doi       = {10.25972/OPUS-18084},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-180844},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {For nociceptive wound pain, the transient receptor potential channel(TRP) mediated calcium influx is essential. Reactive oxygen species (ROS) and their downstream oxidation products such as 4-hydroxynonenal activate the ankyrin 1 homologue TRPA1 in vivo and in vitro. The calcium imaging experiments performed in this study were carried out on stably with TRPA1 and TRPV1 transfected HEK-293 cells and spinal dorsal root ganglion neurons to further understand the mechanistic correlations of nociceptive pain development in inflammatory wound pain. E06, a monoclonal autoantibody (mAb) against oxidized phosphatidylcholine (OxPC) and D-4F, a mimetic peptide of the structural protein apolipoprotein A-I of high density lipoproteins (HDL) were previously used as a diagnostic tools and novel compounds in atherosclerosis. In this study, E06 mAb and D-4F peptide, both, reduced the TRPA1-mediated calcium influx in vitro caused by lipid peroxidation products (OxPL) such as 4-HNE and reactive oxygen species such as H2O2. In addition, we discovered that neither E06 mAb nor D-4F showed a calcium influx-relevant interaction with the Transient Receptor Potential Channel Vanillin 1 (TRPV1) activator capsaicin or the TRPV1 channel itself. Taken together, E06 mAb and D-4F peptide are two promising substances to reduce inflammatory pain and local pain relief.},
  subject      = {Entz{\"u}ndung},
  language  = {de}
}
@phdthesis{Janzen2022,
  author    = {Janzen, Dieter},
  title     = {Functional analysis of ion channels and neuronal networks in 2D and 3D \(in\) \(vitro\) cell culture models},
  doi       = {10.25972/OPUS-25170},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-251700},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {In the central nervous system, excitatory and inhibitory signal transduction processes are mediated by presynaptic release of neurotransmitters, which bind to postsynaptic receptors. Glycine receptors (GlyRs) and GABAA receptors (GABAARs) are ligand-gated ion channels that enable synaptic inhibition. One part of the present thesis elucidated the role of the GlyRα1 β8 β9 loop in receptor expression, localization, and function by means of amino acid substitutions at residue Q177. This residue is underlying a startle disease phenotype in the spontaneous mouse model shaky and affected homozygous animals are dying 4-6 weeks after birth. The residue is located in the β8 β9 loop and thus part of the signal transduction unit essential for proper ion channel function. Moreover, residue Q177 is involved in a hydrogen network important for ligand binding. We observed no difference in ion channel trafficking to the cellular membrane for GlyRα1Q177 variants. However, electrophysiological measurements demonstrated reduced glycine, taurine, and β alanine potency in comparison to the wildtype protein. Modeling revealed that some GlyRα1Q177 variants disrupt the hydrogen network around residue Q177. The largest alterations were observed for the Q177R variant, which displayed similar effects as the Q177K mutation present in shaky mice. Exchange with structurally related amino acids to the original glutamine preserved the hydrogen bond network. Our results underlined the importance of the GlyR β8 β9 loop for proper ion channel gating. GlyRs as well as GABAARs can be modulated by numerous allosteric substances. Recently, we focused on monoterpenes from plant extracts and showed positive allosteric modulation of GABAARs. Here, we focused on the effect of 11 sesquiterpenes and sesquiterpenoids (SQTs) on GABAARs. SQTs are compounds naturally occurring in plants. We tested SQTs of the volatile fractions of hop and chamomile, including their secondary metabolites generated during digestion. Using the patch-clamp technique on transfected cells and neurons, we were able to observe significant GABAAR modulation by some of the compounds analyzed. Furthermore, a possible binding mechanism of SQTs to the neurosteroid binding site of the GABAAR was revealed by modeling and docking studies. We successfully demonstrated GABAAR modulation by SQTs and their secondary metabolites. The second part of the thesis investigated three-dimensional (3D) in vitro cell culture models which are becoming more and more important in different part of natural sciences. The third dimension allows developing of complex models closer to the natural environment of cells, but also requires materials with mechanical and biological properties comparable to the native tissue of the encapsulated cells. This is especially challenging for 3D in vitro cultures of primary neurons and astrocytes as the brain is one of the softest tissues found in the body. Ultra-soft matrices that mimic the neuronal in vivo environment are difficult to handle. We have overcome these challenges using fiber scaffolds created by melt electrowriting to reinforce ultra-soft matrigel. Hence, the scaffolds enabled proper handling of the whole composites and thus structural and functional characterizations requiring movement of the composites to different experimental setups. Using these scaffold-matrigel composites, we successfully established methods necessary for the characterization of neuronal network formation. Before starting with neurons, a mouse fibroblast cell line was seeded in scaffold-matrigel composites and transfected with the GlyR. 3D cultured cells displayed high viability, could be immunocytochemically stained, and electrophysiologically analyzed. In a follow-up study, primary mouse cortical neurons in fiber-reinforced matrigel were grown for up to 21 days in vitro. Neurons displayed high viability, and quantification of neurite lengths and synapse density revealed a fully formed neuronal network already after 7 days in 3D culture. Calcium imaging and patch clamp experiments demonstrated spontaneous network activity, functional voltage-gated sodium channels as well as action potential firing. By combining ultra-soft hydrogels with fiber scaffolds, we successfully created a cell culture model suitable for future work in the context of cell-cell interactions between primary cells of the brain and tumor cells, which will help to elucidate the molecular pathology of aggressive brain tumors and possibly other disease mechanisms.},
  subject      = {Zellkultur},
  language  = {en}
}