@phdthesis{Dannhaeuser2021,
  author    = {Dannh{\"a}user, Sven},
  title     = {Function of the Drosophila adhesion-GPCR Latrophilin/CIRL in nociception and neuropathy},
  doi       = {10.25972/OPUS-20158},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201580},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Touch sensation is the ability to perceive mechanical cues which is required for essential behaviors. These encompass the avoidance of tissue damage, environmental perception, and social interaction but also proprioception and hearing. Therefore research on receptors that convert mechanical stimuli into electrical signals in sensory neurons remains a topical research focus. However, the underlying molecular mechanisms for mechano-metabotropic signal transduction are largely unknown, despite the vital role of mechanosensation in all corners of physiology. Being a large family with over 30 mammalian members, adhesion-type G protein-coupled receptors (aGPCRs) operate in a vast range of physiological processes. Correspondingly, diverse human diseases, such as developmental disorders, defects of the nervous system, allergies and cancer are associated with these receptor family. Several aGPCRs have recently been linked to mechanosensitive functions suggesting, that processing of mechanical stimuli may be a common feature of this receptor family - not only in classical mechanosensory structures. This project employed Drosophila melanogaster as the candidate to analyze the aGPCR Latrophilin/dCIRL function in mechanical nociception in vivo. To this end, we focused on larval sensory neurons and investigated molecular mechanisms of dCIRL activity using noxious mechanical stimuli in combination with optogenetic tools to manipulate second messenger pathways. In addition, we made use of a neuropathy model to test for an involvement of aGPCR signaling in the malfunctioning peripheral nervous system. To do so, this study investigated and characterized nocifensive behavior in dCirl null mutants (dCirlKO) and employed genetically targeted RNA-interference (RNAi) to cell-specifically manipulate nociceptive function. The results revealed that dCirl is transcribed in type II class IV peripheral sensory neurons - a cell type that is structurally similar to mammalian nociceptors and detects different nociceptive sensory modalities. Furthermore, dCirlKO larvae showed increased nocifensive behavior which can be rescued in cell specific reexpression experiments. Expression of bPAC (bacterial photoactivatable adenylate cyclase) in these nociceptive neurons enabled us to investigate an intracellular signaling cascade of dCIRL function provoked by light-induced elevation of cAMP. Here, the findings demonstrated that dCIRL operates as a down-regulator of nocifensive behavior by modulating nociceptive neurons. Given the clinical relevance of this results, dCirl function was tested in a chemically induced neuropathy model where it was shown that cell specific overexpression of dCirl rescued nocifensive behavior but not nociceptor morphology.},
  subject      = {Drosophila},
  language  = {en}
}
@phdthesis{Frenz2019,
  author    = {Frenz, Silke},
  title     = {Generierung und Evaluation von Mausmodellen f{\"u}r die auditorische Neuropathie},
  doi       = {10.25972/OPUS-17710},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-177101},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Der H{\"o}rsinn ist f{\"u}r uns Menschen von entscheidender Bedeutung, um mit der Umwelt kommunizieren zu k{\"o}nnen. H{\"o}rst{\"o}rungen werden dabei in sensorineurale Erkrankungen der neuronalen Strukturen und nicht-sensorineurale Schallleitungsschwerh{\"o}rigkeiten unterschieden. In der vorliegenden Studie sollte es darum gehen zu untersuchen, inwiefern ein neues konditionelles Mausmodell, die Brn3.1 IRES Cre Maus, und ein bestehendes Mausmodell, die pmn-Maus, sich eignen, um die Erkrankung der auditorischen Neuropathie nachzubilden. Die Brn3.1 IRES Cre Maus wurde zur Evaluation der Expression von Cre-Rekombinase unter der Aktivit{\"a}t des Brn3.1 Promotors mit gefloxten Reporter-Mauslinien verkreuzt. Die pmn-Maus ist ein anerkanntes Modell einer Motoneuronerkrankung, hatte aber in vorherigen Untersuchungen erh{\"o}hte H{\"o}rschwellen gezeigt. Alle verwendeten Mauslinien wurden mittels ABR und DPOAE frequenzspezifisch untersucht, um die Funktion der Haarzellen im Corti´schen Organ zu evaluieren. Bei der pmn-Linie wurden die audiologischen Untersuchungen w{\"o}chentlich zwischen P21 und P35 durchgef{\"u}hrt. Zus{\"a}tzlich wurde das Corti´sche Organ zu diesen Zeitpunkten morphologisch untersucht. Es zeigte sich, dass alle verwendeten Mauslinien unauff{\"a}llige H{\"o}rschwellen verglichen zur Backgroundlinie C57BL/6 hatten sowie DPOAE-Antworten zeigten. Die Verkreuzung der Brn3.1 IRES Cre Linie mit den beiden Reporterlinien zeigte eine nachweisbare Cre-Rekombinase-Expression unter der Aktivit{\"a}t des Brn3.1 Promotors nur an den postnatalen Tagen P14 und P21. Diese Expression erfolgte mosaikartig in den {\"a}ußeren Haarzellen. Mit Hilfe einer RT-PCR wurde eine Diskrepanz zwischen Genotyp und Expression des Reporterproteins im Gewebe festgestellt. Dies ließ vermuten, dass die Expression von Cre-Rekombinase durch gene silencing Prozesse unterdr{\"u}ckt wurde und Brn3.1 als Promotor nicht leistungsstark genug war, um eine Cre-Rekombinase-Expression steuern zu k{\"o}nnen. Die pmn-Linie zeigte in ABR-Untersuchungen bereits zum Zeitpunkt P21 erh{\"o}hte H{\"o}rschwellen in allen untersuchten Frequenzen im Vergleich zum Wildtyp. DPOAE-Antworten waren in der pmn-Linie nur bedingt ausl{\"o}sbar. Es zeigte sich in der morphologischen Evaluation ein Verlust von {\"a}ußeren Haarzellen {\"u}ber die gesamte L{\"a}nge des Corti´schen Organes. Durch einen TUNEL-Assay konnte das Absterben dieser Zellen durch apoptotische Vorg{\"a}nge nachgewiesen werden. Der pmn-Ph{\"a}notyp entsteht durch eine Mutation im TBCE Gen. Dieses Gen kodiert f{\"u}r ein Protein, welches einen stabilisierenden Einfluss auf die Organisation der Mikrotubuli hat. Die TBCE-Verteilung im Corti´schen Organ zeigte, dass dieses haupts{\"a}chlich in den {\"a}ußeren Haarzellen und inneren St{\"u}tzzellen exprimiert wird und damit wahrscheinlich einen bedeutenden Einfluss auf die Erhaltung der Haarzellen hat. Eine Analyse des H{\"o}rnervs zeigte einen Verlust von Mikrotubuli. Neben diesen in vivo Untersuchungen sollte außerdem eine gliazellfreie Kultur von dissoziierten auditorischen Neuronen etabliert werden. Hierf{\"u}r wurden mehrere Faktoren einer Prim{\"a}rzellkultur in Bezug auf ihren Einfluss auf die Gesamtzellzahl, den prozentualen Neuronanteil und die Axonl{\"a}nge der Neurone untersucht. Das Medium, die Beschichtung des Zellkulturgef{\"a}ßes, die Gabe von Neurotrophinen/Zytokinen und der Einsatz eines Zytostatikums wurden separat untersucht. Es zeigte sich, dass das Medium, die Beschichtung und Neurotrophin-/Zytokingabe haupts{\"a}chlich einen Einfluss auf das axonale L{\"a}ngenwachstum von Neuronen haben. Den Prozentsatz der Neurone beeinflusste nur der Einsatz des Zytostatikums Cytosin-β-D-arabinofuranosid (AraC) signifikant. Die Ergebnisse wurden auch im Zusammenhang mit der reellen Neuronanzahl in Kultur gesehen. Es ergab sich weiterhin eine Pr{\"a}ferenz f{\"u}r DMEM- {\"u}ber NB-Medium sowie f{\"u}r zus{\"a}tzliche Lamininbeschichtung, f{\"u}r den Einsatz des Zytokins LIF gegen{\"u}ber den neurotrophen Faktoren BDNF und NT-3 und die Gabe des Zytostatikums AraC ab Tag 2 nach Ausplattierung in einer Konzentration von 5-10 µM. Ein kombinatorischer Einsatz dieser Pr{\"a}ferenzen spiegelte in Summe die Ergebnisse der Versuchsreihen Neurotrophine/Zytokin und AraC wieder. Der Anteil der Neurone in der Kultur konnte im Durchschnitt auf 10-12 \% gesteigert werden. Eine Verschiebung des Glia-/Neuronenanteils zugunsten letzterer ist vermutlich nur durch den Einsatz weiterer Faktoren oder anderer Methoden m{\"o}glich. Die untersuchten Mausmodelle zeigten auf Grund der Untersuchungsergebnisse nur teilweise {\"A}hnlichkeiten mit der Erkrankung der auditorischen Neuropathie. Die Erkenntnisse zur pmn-Mauslinie {\"u}ber das frequenzspezifische H{\"o}rverm{\"o}gen und die morphologische Degeneration im Corti´schen Organ im altersabh{\"a}ngigen Verlauf k{\"o}nnen aber hilfreich sein, um neben der motorischen auch die sensorische Degeneration dieser Pathologie besser zu verstehen. Zudem konnten auch umfassende Erkenntnisse f{\"u}r die Kultur von dissoziierten auditorischen Neuronen der Maus in Bezug auf verschiedene Variablen erhalten werden.},
  subject      = {Audiologie},
  language  = {de}
}
@phdthesis{Gao2017,
  author    = {Gao, Shiqiang},
  title     = {Characterizing new photoreceptors to expand the Optogenetic toolbox},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-112941},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Optogenetics is a method to control the cell activity with light by expression of a natural or engineered photoreceptor via genetic modification technology. Optogenetics early success came with the light-gated cation channel "Channelrhodopsin-2" in neurons and expanded from neuroscience to other research fields such as cardiac research and cell signaling, also due to the enrichment by new photoreceptors. In this study, I focus on searching and characterizing new photoreceptors to expand the optogenetic tool box. In this work I characterize three newly discovered microbial rhodopsins and some engineered mutants of them. The first rhodopsin is a proton pump from the diatom Fragilariopsis cylindrus, Fragilariopsis Rhodopsin or abbreviated: FR. I cloned the full-length FR and proved it to be a light-activated proton pump with high efficacy in comparison to Bacteriorhodopsin (BR). During this study, I also developed a new method to improve the plasma membrane targeting of several microbial rhodopsins. I also obtained a FR mutant (channel-like FR or chFR) which behaves like a light-gated proton channel. FR can be used for optogenetic hyperpolarization or alkalization of a cell while the chFR could be used for depolarization or lowering of the cellular pH. The induction of FR expression under iron-limited conditions in the diatom indicated an alternative energy generation mechanism of F. cylindrus when iron-containing enzymes are scarce. I then characterized a new microbial rhodopsin with novel light-regulated Guanylyl Cyclase (GC) activity. This rhodopsin guanylyl cyclase from the fungus Blastocladiella emersonii (B.e. CyclaseOpsin or BeCyclOp) has been proven by me to be an efficient light-gated GC with high specificity and fast kinetics. BeCyclOp also has a novel structure with eight transmembrane helices, containing a long cytosolic N-terminus which participates in the tight regulation of the GC activity. In collaboration with Prof. Alexander Gottschalk (Univ. Frankfurt/M.), BeCyclOp has been tested in muscle cells and sensory neurons of Caenorhabditis elegans and proven to be a powerful optogenetic tool in a living animal. I also generated a BeCyclOp mutant with enhanced light sensitivity. Already more than ten years ago, guanylyl cyclase rhodopsins were suggested to exist in Chlamydomonas reinhardtii by analyzing genomic sequence data. But until now no functional proof existed. By further cloning and sequencing I discovered such a rhodopsin with light-regulated guanylyl cyclase activity. This functional Cyclaseopsin (COP6c) is quite different to BeCyclOp, as it was proven to be a light-inhibited GC. Cop6c is much larger than BeCyclOp with a His-Kinase and a response regulator domain between the rhodopsin and the cyclase domain. I also introduced a new strategy for generating optogenetic tools by fusing the photoactivated adenylyl cyclase bPAC to two different CNG channels. These new tools function via light-gated cAMP production and subsequent CNG channel activation. These tools combined the properties of bPAC (highly sensitive to blue light) and CNG channels (high single-channel conductance and high Ca2+ permeability), as demonstrated by expression in Xenopus oocytes. As a further benefit the fusing of bPAC to CNG channels leads to a bPAC with a more than tenfold reduced dark activity which is a valuable improvement for bPAC itself as an optogenetic tool.},
  subject      = {Photorezeptor},
  language  = {en}
}
@phdthesis{Stangl2012,
  author    = {Stangl, Christoph},
  title     = {Lichtgesteuerte Manipulation Zentraler Second Messenger in Pflanzen},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85940},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {In der vorliegenden Arbeit wurden lichtaktivierte Nucleotidcyclasen auf Basis der lichtaktivierten Adenylatcyclase bPAC (=BlaC, Ryu et al., 2010; Stierl et al., 2011), sowie der direkt lichtaktivierbare Kationenkanal Channelrhodopsin-2 (Nagel et al., 2003) eingesetzt, um lichtinduzierte und damit nicht-invasive Manipulationen der Second Messenger cAMP, cGMP und Calcium, sowie des Membranpotentials in Pflanzenzellen vorzunehmen. Nach transienter Transfektion von N. benthamiana konnte sowohl die Expression der beiden Channelrhodopsinvarianten C128A::YFP und C128T::YFP (unver{\"o}ffentlichte Daten von R. Gueta und G. Nagel, 2008, Berndt et al., 2009; Bamann et al., 2010), als auch deren Lokalisation in der Plasmamembran von Protoplasten fluoreszenzmikroskopisch gezeigt werden. Die Funktion von Channelrhodopsin als lichtaktivierbarer Kationenkanal konnte in dieser Arbeit erstmals elektrophysiologisch in Pflanzenzellen nachgewiesen werden. In Einstichmessungen im Mesophyllgewebe von N. benthamiana wurden reproduzierbar blaulichtinduzierte Depolarisationen der Plasmamembran erzielt, die in Dauer und Frequenz {\"u}ber das applizierte Lichtmuster steuerbar waren. In Patch-Clamp-Messungen an epidermalen Protoplasten von N. benthamiana, welche transient Channelrhodopsin-2-C128A und Channelrhodopsin-2-C128T exprimierten, konnten zudem blaulichtinduzierte Einw{\"a}rts-Str{\"o}me gezeigt werden. Die Expression der beiden verwendeten Channelrhodopsinvarianten schien hierbei ann{\"a}hernd unabh{\"a}ngig von der vorliegenden Konzentration des zugegebenen Retinals. Des Weiteren konnte in A. thaliana sowohl die Expression als auch die Funktion der lichtaktivierbaren Adenylatcyclase bPAC::YFP (Stierl et al., 2011), sowie einer hieraus durch gezielte Mutation (nach Ryu et al., 2010) abgeleiteten lichtaktivierbaren Guanylatcyclase (bPGC::YFP) erstmalig in h{\"o}heren Pflanzen gezeigt werden. Nach Best{\"a}tigung der Funktion dieser beiden lichtaktivierbaren Nucleotidcyclasen in transient transfizierten Protoplasten von A. thaliana wurden zwei stabil transgene Pflanzenlinien generiert. ZUSAMMENFASSUNG DER ARBEIT - 190 - Pflanzen dieser Linie exprimierten neben dem konstitutiv unter der Kontrolle des 35S-CaMV-Promotors exprimierten Calciumreporterprotein Aequorin zus{\"a}tzlich und unter der Kontrolle eines {\"o}strogeninduzierbaren Glucocorticoidpromotors (Zuo et al., 2000) die lichtaktivierten Nucleotidcyclasen bPAC::YFP bzw. bPGC::YFP. In beiden stabil transgenen Pflanzenlinien wurden Expression und Funktion der jeweiligen lichtaktivierten Nucleotidcyclase gezeigt. In Messungen an einem modifizierten Luminometer konnten weiterhin erstmals blaulichtinduzierte Calciumsignaturen in Pflanzenzellen generiert werden. Auf die Folge von Blaulichtpulsen kam es wiederholt zum Calciumeinstrom in Zellen der erstellten transgenen Pflanzenlinien. Neben der M{\"o}glichkeit, sowohl die Konzentration der cyclischen Nucleotide als auch des cytoplasmatischen Calciums {\"u}ber Licht zu manipulieren, wurde durch diese Pflanzen ein direkter Zusammenhang beider Second Messenger gezeigt. Weiterhin sind ph{\"a}notypische Auff{\"a}lligkeiten der erstellten Pflanzenlinien beobachtet worden. Es kam zur Verz{\"o}gerung des Keimungszeitpunktes bPAC::YFP-exprimierender Samen im Licht, jedoch wuchsen die Pflanzen im Anschluss an die verz{\"o}gerte Keimung normal und uneingeschr{\"a}nkt weiter. In bPAC::YFP-exprimierenden Pollen von Nicotiana SR-1 konnte zudem das Wachstum der Pollenschl{\"a}uche durch blaues Licht gestoppt werden. bPGC::YFP-exprimierende Pollen hingegen zeigten auch im blauen Licht unver{\"a}ndertes Pollenschlauchwachstum. Neben den beiden erfolgreich generierten, stabil transgenen Pflanzenlinien wurden in analogen Ans{\"a}tzen transgene A. thaliana Col-0 Aequorin Zellkulturlinien generiert, die neben dem konstitutiv aktiven Calciumreporterprotein Aequorin ebenso bPAC::YFP bzw. bPGC::YFP unter der Kontrolle des {\"o}strogeninduzierbaren Glucocorticoidpromotors (Zuo et al., 2000) exprimierten. Auch hier konnten Expression und Funktion beider Nucleotidcyclasen immunologisch und fluoreszenzmikroskopisch gezeigt werden. {\"U}ber den Einsatz einer sog. 2A-Sequenz wurde weiterhin ein funktionsf{\"a}higes Fusionskonstrukt aus dem cAMP-aktivierten Kationenkanal CNGA2 (C460W-E583M, nach Rich et al., 2001) und der lichtaktivierten Adenylatcyclase EuPACα (Iseki et al., 2002) erstellt. Die Funktion dieses Fusionskonstruktes wurde elektrophysiologisch sowie immunologisch gezeigt.},
  subject      = {Calciumion},
  language  = {de}
}
@phdthesis{Kunz2021,
  author    = {Kunz, Tobias C.},
  title     = {Expansion Microscopy (ExM) as a tool to study organelles and intracellular pathogens},
  doi       = {10.25972/OPUS-22333},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-223330},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {The resolution of fluorescence light microscopy was long believed to be limited by the diffraction limit of light of around 200-250 nm described in 1873 by Ernst Abbe. Within the last decade, several approaches, such as structured illumination microscopy (SIM), stimulated emission depletion STED and (direct) stochastic optical reconstruction microscopy (d)STORM have been established to bypass the diffraction limit. However, such super-resolution techniques enabling a resolution <100 nm require specialized and expensive setups as well as expert knowledge in order to avoid artifacts. They are therefore limited to specialized laboratories. Recently, Boyden and colleagues introduced an alternate approach, termed expansion microscopy (ExM). The latter offers the possibility to perform superresolution microscopy on conventional confocal microscopes by embedding the sample into a swellable hydrogel that is isotropically expanded. Since its introduction in 2015, expansion microscopy has developed rapidly offering protocols for 4x, 10x and 20x expansion of proteins and RNA in cells, tissues and human clinical specimens. Mitochondria are double membrane-bound organelles and crucial to the cell by performing numerous tasks, from ATP production through oxidative phosphorylation, production of many important metabolites, cell signaling to the regulation of apoptosis. The inner mitochondrial membrane is strongly folded forming so-called cristae. Besides being the location of the oxidative phosphorylation and therefore energy conversion and ATP production, cristae have been of great interest because changes in morphology have been linked to a plethora of diseases from cancer, diabetes, neurodegenerative diseases, to aging and infection. However, cristae imaging remains challenging as the distance between two individual cristae is often below 100 nm. Within this work, we demonstrate that the mitochondrial creatine kinase MtCK linked to fluorescent protein GFP (MtCK-GFP) can be used as a cristae marker. Upon fourfold expansion, we illustrate that our novel marker enables visualization of cristae morphology and localization of mitochondrial proteins relative to cristae without the need for specialized setups. Furthermore, we show the applicability of expansion microscopy for several bacterial pathogens, such as Chlamydia trachomatis, Simkania negevensis, Neisseria gonorrhoeae and Staphylococcus aureus. Due to differences in bacterial cell walls, we reveal important aspects for the digestion of pathogens for isotropic expansion. We further show that expansion of the intracellular pathogens C. trachomatis and S. negevensis, enables the differentiation between the two distinct developmental forms, catabolic active reticulate bodies (RB) and infectious elementary bodies (EB), on a conventional confocal microscope. We demonstrate the possibility to precisely locate chlamydial effector proteins, such as CPAF or Cdu1, within and outside the chlamydial inclusion. Moreover, we show that expansion microscopy enables the investigation of bacteria, herein S. aureus, within LAMP1 and LC3-II vesicles. With the introduction of the unnatural α-NH2-ω-N3-C6-ceramide, we further present the first approach for the expansion of lipids that may also be suitable for far inaccessible molecule classes like carbohydrates. The efficient accumulation and high labeling density of our functionalized α-NH2-ω-N3-C6-ceramide in both cells and bacteria enables in combination with tenfold expansion nanoscale resolution (10-20 nm) of the interaction of proteins with the plasma membrane, membrane of organelles and bacteria. Ceramide is the central molecule of the sphingolipid metabolism, an important constituent of cellular membranes and regulates many important cellular processes such as differentiation, proliferation and apoptosis. Many studies report about the importance of sphingolipids during infection of various pathogens. While the transport of ceramide to Chlamydia has been reported earlier, one of the unanswered questions remaining was if ceramide forms parts of the outer or inner bacterial membrane. Expansion of α-NH2-ω-N3-C6-ceramide enabled the visualization of ceramide in the inner and outer membrane of C. trachomatis and their distance was determined to be 27.6 ± 7.7 nm.},
  subject      = {Fluoreszenzmikroskopie},
  language  = {en}
}
@phdthesis{Ullrich2013,
  author    = {Ullrich, Sybille},
  title     = {Biochemische und biophysikalische Analyse der strukturellen Integrit{\"a}t von Channelrhodopsin 2 und dessen Mutanten},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-92006},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2013},
  abstract  = {Channelrhodopsin 2 (ChR2) aus dem Augenfleck von C. rheinhardtii geh{\"o}rt zur Gruppe der mikrobiellen Rhodopsine (Typ1-Rhodopsine). ChR2 besteht aus einem extrazellul{\"a}r gelegenen N-Terminus, 7 Transmembranhelices und einem zytosolisch gelegenen C-Terminus. Der lichtreaktive Bestandteil (Chromophor) all-trans-Retinal ist via Schiff´ Base kovalent an ein Lysinrest der siebten Transmembranhelix gebunden. Bei Applikation von Blaulicht isomerisiert all-trans- zu 13-cis-Retinal, was in einer Konformations{\"a}nderung und dem {\"O}ffnen des Kanals resultiert. Abh{\"a}ngig vom elektrochemischen Gradienten k{\"o}nnen ein- und zweiwertige Kationen in die Zelle ein- oder aus der Zelle herausstr{\"o}men. Eine retinalabh{\"a}ngige Stabilit{\"a}t konnte bereits f{\"u}r Bakteriorhodopsin (BR) best{\"a}tigt werden (Booth, Farooq et al. 1996, Turner, Chittiboyina et al. 2009, Curnow and Booth 2010), bez{\"u}glich ChR2 waren bisher nur wenige Daten verf{\"u}gbar (Hegemann, Gartner et al. 1991, Lawson, Zacks et al. 1991). Die heterologe Expression von wildtypischem und modifiziertem ChR2 in Oozyten von X. laevis erlaubte einen detaillierteren Einblick in die retinalabh{\"a}ngige Stabilit{\"a}t und pH-abh{\"a}ngige Dunkelleitf{\"a}higkeit von Guanidinium. Wildtypisches Chop2 zeigte bei Zugabe von Retinal zum Inkubationsmedium, direkt nach RNA-Injektion, Stromamplituden im µA-Bereich und deutliche Fluoreszenzintensit{\"a}ten. Ausschließlich endogen vorhandenes Retinal hatte verminderten Fluoreszenzen und Stromamplituden zur Folge, was auf ein geringes Vorhandensein von Chop2-Proteinen in der Plasmamembran hindeutete. Da die Inkubation {\"u}ber Nacht in retinalsupplementierter L{\"o}sung nur eine minimale Erh{\"o}hung des resultierenden Stromes erbrachte, deuten die in dieser Arbeit erhaltenen Ergebnisse stark auf eine verminderte Stabilit{\"a}t des Proteins bei fehlender Bindung des Kofaktors Retinal. Das Einf{\"u}gen einer aromatischen Aminos{\"a}ure (Y/F/W) an Position 159 f{\"u}hrte zu einer, von der Retinalsupplementation unabh{\"a}ngigen, in beiden Ans{\"a}tzen gleichwertigen Expressionsst{\"a}rke. Diese {\"a}usserte sich in {\"a}quivalenten Fluoreszenzintensit{\"a}ten. Die erhaltenen Stromamplituden wiesen eine starke Differenz auf: ohne Zugabe zus{\"a}tzlichen Chromophors lag die Stromst{\"a}rke bei nur wenigen Nanoampere, die bei Inkubation in einer retinalhaltigen L{\"o}sung {\"u}ber Nacht auf das Niveau von retinalsupplementierten Oozyten anstieg. Des Weiteren konnte die Zunahme der Stromamplitude innerhalb von 15 Minuten beobachtet werden, wenn die vermessenen Oozyten mit einer retinalhaltigen L{\"o}sung perfundiert wurden. Zusammengefasst weisen die Ergebnisse auf eine Stabilisierung des aromatisch substituierten Proteins hin. Bei der von Berndt et al. (2011) beschriebenen Mutante T159C konnten diese Eigenschaften nicht nachgewiesen werden. Die Modifikation der Retinalbindestelle (K257) in Verbindung mit einer aromatischen Substitution an Position 159 resultierte in deutlichen Fluoreszenzintensit{\"a}ten, unabh{\"a}ngig von der Retinalverf{\"u}gbarkeit bei, in beiden F{\"a}llen, fehlenden lichtaktivierten Str{\"o}men. Diese und die gleichwertigen Bandenst{\"a}rken des Proteinimmunoblots von aromatisch substituierten ChR2-Varianten unterst{\"u}tzen die Hypothese der retinalunabh{\"a}ngigen Stabilit{\"a}t zus{\"a}tzlich. Die Ergebnisse legen, im Falle von Chop2-WT, eine Degradation des Apoproteins nahe. Bei Einf{\"u}gen einer aromatischen AS an Position 159 ist das Apoprotein davor gesch{\"u}tzt (siehe Abb. 75). Infolge der strukturellen Similarit{\"a}t, dem Vorhandensein delokalisierter π-Elektronen und der r{\"a}umlichen Gr{\"o}ße der aromatischen AS ist eine strukturelle Ver{\"a}nderung des Apoproteins denkbar, die eine Degradation aufgrund von nunmehr unzug{\"a}nglichen Ubiquitinierungsstellen verhindert. Des Weiteren besteht die M{\"o}glichkeit, dass sich bei fehlender Bindung des Kofaktors Wassermolek{\"u}le in der N{\"a}he der Bindetasche befinden, welche von umliegenden Aminos{\"a}uren (u.a. T159, D156) unter großem Energieaufwand koordiniert werden und die strukturelle Integrit{\"a}t bis hin zur Degradation beeintr{\"a}chtigen k{\"o}nnen. Dies k{\"o}nnte durch eine Erh{\"o}hung der Hydrophobizit{\"a}t bei Einf{\"u}gen einer aromatischen Aminos{\"a}ure verhindert werden. Bei Substitutionen durch eine aromatische AS (Y/W/F) an Position 159 zeigte sich ein weiteres, bisher nicht beschriebenes, Charakteristikum. Bei Perfusion der Oozyten mit einer guanidiniumhaltigen L{\"o}sung, konnten in Abh{\"a}ngigkeit des pH-Wertes ohne die Applikation von Licht St{\"o}me im µA Bereich aufgezeichnet werden. Die Gr{\"o}ße der Stromamplitude korreliert hierbei mit dem Anstieg des pH-Wertes und der Konzentration an Guanidiniumionen der perfundierten L{\"o}sung und kann durch das Hinzuf{\"u}gen von 1mM Lanthan reversibel geblockt werden. Des Weiteren konnten die vorgenommenen Messungen die Ergebnisse der retinalabh{\"a}ngigen Degradation verifizieren, da der Einstrom von Gua+ sowohl bei retinalsupplementierter Inkubation, als auch bei ausschließlich endogen vorhandenem Retinal zu beobachten war. Des Weiteren zeigte auch die Doppelmutante T159Y/K257R trotz ihres Unverm{\"o}gens Retinal zu binden, die beschriebenen lichtunabh{\"a}ngigen Str{\"o}me. Die Ergebnisse bei Substitution durch Phenylalanin (F) stellen eine Abweichung des Musters dar. Bei Inkubation von T159F-injizierten Zellen bei ausschließlich endogen vorhandenem Retinal konnte eine stark erh{\"o}hte Guanidiniumleitf{\"a}higkeit festgestellt werden, diese kam jedoch bei retinalsupplementierter Inkubation nicht zum Tragen. Dies k{\"o}nnte ein Hinweis auf eine sterische Hinderung durch das gebundene Chromophor sein, die bei den Substitutionen durch Tyrosin und Tryptophan, m{\"o}glicherweise durch unterschiedliche chemische Eigenschaften der AS, nicht auftreten. Die hervorgerufene pH-Abh{\"a}ngigkeit kann in zwei m{\"o}glichen Ursachen begr{\"u}ndet liegen: • Vorhandensein einer (de)protonierbaren Gruppe wie Histidin, Arginin oder Lysin, die als pH-Sensor dienen k{\"o}nnte • Deprotonierung der Schiff´ Base durch Guandininium Das Vorhandensein eines pH-Sensors konnte durch die vorgenommenen Modifikationen von H114, R115, R120 und H249 nicht best{\"a}tigt werden. Bei Substitution von K257 (in Verbindung mit T159Y) zu Arginin (R) konnte weiterhin ein pH-abh{\"a}ngiger Gua+-Dunkelstrom festgestellt werden. Die Modifikation zu Alanin (A) oder Glutamin (Q) hingegen resultierte im Ausbleiben der Str{\"o}me. Der Austausch einer basischen zu einer neutralen Gruppe ohne protonierbaren Rest deutet auf die Beteiligung der Schiff´ Base bzw. der Aminos{\"a}ure an Position 257 am Mechanismus der Dunkelleitf{\"a}higkeit hin.},
  subject      = {Elektrophysiologie},
  language  = {de}
}
@phdthesis{Yang2022,
  author    = {Yang, Shang},
  title     = {Characterization and engineering of photoreceptors with improved properties for optogenetic application},
  doi       = {10.25972/OPUS-20527},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-205273},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Optogenetics became successful in neuroscience with Channelrhodopsin-2 (ChR2), a light-gated cation channel from the green alga Chlamydomonas reinhardtii, as an easy applicable tool. The success of ChR2 inspired the development of various photosensory proteins as powerful actuators for optogenetic manipulation of biological activity. However, the current optogenetic toolbox is still not perfect and further improvements are desirable. In my thesis, I engineered and characterized several different optogenetic tools with new features. (i) Although ChR2 is the most often used optogenetic actuator, its single-channel conductance and its Ca2+ permeability are relatively low. ChR2 variants with increased Ca2+ conductance were described recently but a further increase seemed possible. In addition, the H+ conductance of ChR2 may lead to cellular acidification and unintended pH-related side effects upon prolonged illumination. Through rational design, I developed several improved ChR2 variants with larger photocurrent, higher cation selectivity, and lower H+ conductance. (ii) The light-activated inward chloride pump NpHR is a widely used optogenetic tool for neural silencing. However, pronounced inactivation upon long time illumination constrains its application for long-lasting neural inhibition. I found that the deprotonation of the Schiff base underlies the inactivation of NpHR. Through systematically exploring optimized illumination schemes, I found illumination with blue light alone could profoundly increase the temporal stability of the NpHR-mediated photocurrent. A combination of green and violet light eliminates the inactivation effect, similar to blue light, but leading to a higher photocurrent and therefore better light-induced inhibition. (iii) Photoactivated adenylyl cyclases (PACs) were shown to be useful for light-manipulation of cellular cAMP levels. I developed a convenient in-vitro assay for soluble PACs that allows their reliable characterization. Comparison of different PACs revealed that bPAC from Beggiatoa is the best optogenetic tool for cAMP manipulation, due to its high efficiency and small size. However, a residual activity of bPAC in the dark is unwanted and the cytosolic localization prevents subcellular precise cAMP manipulation. I therefore introduced point mutations into bPAC to reduce its dark activity. Interestingly, I found that membrane targeting of bPAC with different linkers can remarkably alter its activity, in addition to its localization. Taken together, a set of PACs with different activity and subcellular localization were engineered for selection based on the intended usage. The membrane-bound PM-bPAC 2.0 with reduced dark activity is well-tolerated by hippocampal neurons and reliably evokes a transient photocurrent, when co-expression with a CNG channel. (iv) Bidirectional manipulation of cell activity with light of different wavelengths is of great importance in dissecting neural networks in the brain. Selection of optimal tool pairs is the first and most important step for dual-color optogenetics. Through N- and C-terminal modifications, an improved ChR variant (i.e. vf-Chrimson 2.0) was engineered and selected as the red light-controlled actuator for excitation. Detailed comparison of three two-component potassium channels, composed of bPAC and the cAMP-activated potassium channel SthK, revealed the superior properties of SthK-bP. Combining vf-Chrimson 2.0 and improved SthK-bP "SthK(TV418)-bP" could reliably induce depolarization by red light and hyperpolarization by blue light. A residual tiny crosstalk between vf-Chrimson 2.0 and SthK(TV418)-bP, when applying blue light, can be minimized to a negligible level by applying light pulses or simply lowering the blue light intensity.},
  language  = {en}
}
@phdthesis{Schubert2019,
  author    = {Schubert, Frank Klaus},
  title     = {The circadian clock network of \(Drosophila\) \(melanogaster\)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-157136},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {All living organisms need timekeeping mechanisms to track and anticipate cyclic changes in their environment. The ability to prepare for and respond to daily and seasonal changes is endowed by circadian clocks. The systemic features and molecular mechanisms that drive circadian rhythmicity are highly conserved across kingdoms. Therefore, Drosophila melanogaster with its relatively small brain (ca. 135.000 neurons) and the outstanding genetic tools that are available, is a perfect model to investigate the properties and relevance of the circadian system in a complex, but yet comprehensible organism. The last 50 years of chronobiological research in the fruit fly resulted in a deep understanding of the molecular machinery that drives circadian rhythmicity, and various histological studies revealed the neural substrate of the circadian system. However, a detailed neuroanatomical and physiological description on the single-cell level has still to be acquired. Thus, I employed a multicolor labeling approach to characterize the clock network of Drosophila melanogaster with single-cell resolution and additionally investigated the putative in- and output sites of selected neurons. To further study the functional hierarchy within the clock network and to monitor the "ticking clock" over the course of several circadian cycles, I established a method, which allows us to follow the accumulation and degradation of the core clock genes in living brain explants by the means of bioluminescence imaging of single-cells.},
  subject      = {Taufliege},
  language  = {en}
}
@phdthesis{Fei2023,
  author    = {Fei, Lin},
  title     = {Optogenetic regulation of osmolarity and water flux},
  doi       = {10.25972/OPUS-32309},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-323092},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Optogenetics is a powerful technique that utilizes light to precisely regulate physiological activities of neurons and other cell types. Specifically, light-sensitive ion channels, pumps or enzymes are expressed in cells to enable their regulation by illumination, thus allowing for precise control of biochemical signaling pathways. The first part of my study involved the construction, optimization, and characterization of two optogenetic tools, KCR1 and NCR1. Elena Govorunova et al. discovered a lightgated potassium channel, KCR1, in the protozoan Hyphochytrium catenoides. Traditional potassium ion channels are classified as either ligand-gated or voltage-gated and possess conserved pore-forming domains and K+ -selective filters. However, KCR1 is unique in that it does not contain the signature sequence of previously known K+ channels and is a channelrhodopsin. We synthesized the KCR1 plasmid according to the published sequence and expressed it in Xenopus oocytes. Due to the original KCR1 current being too small, I optimized it into KCR1 2.0 to improve its performance by fusing LR (signal peptide LucyRho, enhances expression) at the N-terminal and T (trafficking signal peptide) and E (ER export signal peptide) at the C-terminal. Additionally, I investigated the light sensitivity, action spectrum, and kinetics of KCR1 2.0 in Xenopus oocytes. The potassium permeability of KCR1 2.0, PK/Pna  24, makes KCR1 2.0 a powerful hyperpolarizing tool that can be used to inhibit neuronal firing in animals. Inspired by KCR1, we used the KCR1 sequence as a template for gene sequence alignment with the sequences in H. catenoides. We found that NCR1 and KCR1 have similar gene sequences. NCR1 was characterized by us as a light-gated sodium channel. This NCR1 was also characterized and published by Govorunova et al. very recently, with the name HcCCR. Due to the original NCR1 current being too small, I optimized it into NCR1 2.0 to improve its performance by fusing LR at the N-terminal and T and E at the C-terminal, which significantly improved the expression level and greatly increased the current amplitude of NCR1. Full-length NCR1 2.0 contains 432 amino acids. To test whether the number of amino acids changes the characteristics of NCR1 2.0, we designed NCR1 2.0 (330), NCR1 2.0 (283), and NCR1 2.0 (273) by retaining the number of amino acids at 330, 280, and 273 in NCR1 2.0, respectively. As the number of amino acids decreased, the current in NCR1 2.0 increased. I also investigated the light sensitivity, action spectrum, and kinetics of NCR1 2.0 (273) in the Xenopus Abstract 2 oocytes. We performed four point mutations at amino acid positions 133 and 116 of NCR1 2.0 and analyzed the reversal potentials of the mutants. The mutations were as follows: NCR1 2.0 (273 D116H), NCR1 2.0 (273 D116E), NCR1 2.0 (283 V133H), and NCR1 2.0 (283 D116Q). The second part of this study focuses on light-induced water transport using optogenetic tools. We explored the use of optogenetic tools to regulate water flow by changing the osmolarity in oocytes. Water flux through AQP1 is driven by the osmotic gradient that results from concentration differences of small molecules or ions. Therefore, we seek to regulate ion concentrations, using optogenetic tools to regulate the flux of water noninvasively. To achieve this, I applied the light-gated cation channels XXM 2.0 and NCR1 2.0 to regulate the concentration of Na+ , while K + channel KCR1 2.0 was used to regulate K + concentration. As Na+ flows into the Xenopus oocytes, the membrane potential of the oocytes becomes positive, and Clcan influx through the light-gated anion channel GtACR1. By combining these optogenetic tools to regulate NaCl or KCl concentrations, I can change the osmolarity inside the oocytes, thus regulating the flux of water. I co-expressed AQP1 with optogenetic tools in the oocytes to accelerate water flux. Overall, I designed three combinations (1: AQP1, XXM 2.0 and GtACR1. 2: AQP1, NCR1 2.0 and GtACR1. 3: AQP1, KCR1 2.0 and GtACR1) to regulate the flow of water in oocytes. The shrinking or swelling of the oocytes can only be achieved when AQP1, light-gated cation channels (XXM 2.0/NCR1 2.0/KCR1 2.0), and light-gated anion channels (GtACR1) are expressed together. The illumination after expression of either or both alone does not result in changes in oocyte morphology. In sum, I demonstrated a novel strategy to manipulate water movement into and out of Xenopus oocytes, non-invasively through illumination. These findings provide a new avenue to interfere with water homeostasis as a means to study related biological phenomena across cell types and organisms.},
  subject      = {Osmolarit{\"a}t},
  language  = {en}
}
@phdthesis{YuStrzelczyk2023,
  author    = {Yu-Strzelczyk, Jing},
  title     = {Generation and Characterization of novel proteins for light-activated hyperpolarization of cell membranes},
  doi       = {10.25972/OPUS-26675},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-266752},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {The light-gated cation channel Channelrhodopsin-2 was discovered and characterized in 2003. Already in 2005/2006 five independent groups demonstrated that heterologous expression of Channelrhodopsin-2 is a highly useful and simply applicable method for depolarizing and thereby activating nerve cells. The application of Channelrhodopsin-2 revolutionized neuroscience research and the method was then called optogenetics. In recent years more and more light-sensitive proteins were successfully introduced as "optogenetic tools", not only in neuroscience. Optogenetic tools for neuronal excitation are well developed with many different cation-conducting wildtype and mutated channelrhodopsins, whereas for inhibition of neurons in the beginning (2007) only hyperpolarizing ion pumps were available. The later discovered light-activated anion channels (anion channelrhodopsins) can be useful hyperpolarizers, but only at low cytoplasmic anion concentration. For this thesis, I optimized CsR, a proton-pumping rhodopsin from Coccomyxa subellipsoidea, which naturally shows a robust expression in Xenopus laevis oocytes and plant leaves. I improved the expression and therefore the photocurrent of CsR about two-fold by N-terminal modification to the improved version CsR2.0, without altering the proton pump function and the action spectrum. A light pulse hyperpolarised the mesophyll cells of CsR2.0-expressing transgenic tobacco plants (N. tabacum) by up to 20 mV from the resting membrane potential of -150 to -200 mV. The robust heterologous expression makes CsR2.0 a promising optogenetic tool for hyperpolarization in other organisms as well. A single R83H point-mutation converted CsR2.0 into a light-activated (passive) proton channel with a reversal potential close to the Nernst potential for intra-/extra-cellular H+ concentration. This light-gated proton channel is expected to become a further useful optogenetic tool, e.g. for analysis of pH-regulation in cells or the intercellular space. Ion pumps as optogenetic tools require high expression levels and high light intensity for efficient pump currents, whereas long-term illumination may cause unwanted heating effects. Although anion channelrhodopsins are effective hyperpolarizing tools in some cases, their effect on neuronal activity is dependent on the cytoplasmic chloride concentration which can vary among neurons. In nerve cells, increased conductance for potassium terminates the action potential and K+ conductance underlies the resting membrane potential in excitable cells. Therefore, several groups attempted to synthesize artificial light-gated potassium channels but 2 all of these published innovations showed serious drawbacks, ranging from poor expression over lacking reversibility to poor temporal precision. A highly potassium selective light-sensitive silencer of action potentials is needed. To achieve this, I engineered a light-activated potassium channel by the genetic fusion of a photoactivated adenylyl cyclase, bPAC, and a cAMP-gated potassium channel, SthK. Illumination activates bPAC to produce cAMP and the elevated cAMP level opens SthK. The slow diffusion and degradation of cAMP makes this construct a very light-sensitive, long-lasting inhibitor. I have successfully developed four variants with EC50 to cAMP ranging from 7 over 10, 21, to 29 μM. Together with the original fusion construct (EC50 to cAMP is 3 μm), there are five different light- (or cAMP-) sensitive potassium channels for researchersto choose, depending on their cell type and light intensity needs.},
  subject      = {Proteine},
  language  = {en}
}