@phdthesis{Keller2004,
  author    = {Keller, Sascha},
  title     = {Struktur- und Funktionsanalysen an BMP Ligand-Rezeptor-Komplexen},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-12467},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2004},
  abstract  = {F{\"u}r BMPs wie auch die anderen Mitglieder der TGF-beta-Superfamilie beginnt der Signalweg mit der Bindung des Liganden an zwei Typen transmembran{\"a}rer Rezeptoren. Die Ligand-Rezeptor Interaktionen sind dabei durch unterschiedliche Affinit{\"a}t und Spezifit{\"a}t gekennzeichnet und bilden wahrscheinlich die Grundlage f{\"u}r das breite Spektrum biologischer Funktionen. In dieser Arbeit wurde mittels einer Struktur- und Funktionsanalyse von BMP Ligand-Rezeptor Komplexen die molekulare Basis f{\"u}r die Affinit{\"a}t und Spezifit{\"a}t dieser Wechselwirkungen untersucht. Hierf{\"u}r wurde die Kristallstruktur des BMP-2 : BR-IAec Ligand-Rezeptor Komplexes bei einer Aufl{\"o}sung von 1,9{\AA} ermittelt. Mit der h{\"o}heren Aufl{\"o}sung war die Charakterisierung der geometrischen Parameter eines Netzwerks von zehn Wasserstoff-Br{\"u}ckenbindungen in der Interaktionsfl{\"a}che zwischen BMP-2 und BR-IAec m{\"o}glich. Deren zentrale Bedeutung f{\"u}r dieWechselwirkung konnte auch durch funktionelle Analyse best{\"a}tigt werden. So stellen die im Zentrum der Bindungsfl{\"a}che liegenden Wasserstoff-Br{\"u}ckenbindungen BMP-2 Leu51 (N) : BR-IAec Gln86 (OE1) und BMP-2 Leu51 (O) : BR-IAec Gln86 (NE1), sowie die BMP-2 Asp53 (N) : BR-IAec Cys77 (O) H-Br{\"u}cke die Hauptdeterminanten der Ligand-Rezeptor Bindung dar. Dar{\"u}ber hinaus ließ sich aus der strukturellen Analyse des "wrist"-Epitops von BMP-2 eine besondere Bedeutung der Pr{\"a}-Helix Schleife L2, sowie der im Kontakt eingeschlossenen Wassermolek{\"u}le f{\"u}r die Anpassung der Bindungsfl{\"a}che an unterschiedliche Interaktionspartner ableiten. Diese Ergebnisse bilden die Grundlage f{\"u}r ein neues Modell zur Beschreibung von Affinit{\"a}t und Spezifit{\"a}t der hochaffinen BMP-Typ I Rezeptor Interaktion. Dabei stellen die Wasserstoff-Br{\"u}ckenbindungen den Hauptanteil zur Bindungsenergie, w{\"a}hrend die hydrophobe Umgebung in der Interaktionsfl{\"a}che die Bildung von Wasserstoff-Br{\"u}ckenbindungen energetisch beg{\"u}nstigen und hydrophobe Wechselwirkungen nur geringf{\"u}gigen Einfluss auf die Affinit{\"a}t nehmen. Die vorliegenden Arbeit beschreibt zudem die Pr{\"a}paration und Kristallisation von bin{\"a}ren Ligand-Typ I Rezeptor Komplexen f{\"u}r BMP-2, BMP-6 und GDF-5, sowie die der tern{\"a}ren Komplexe von BMP-2, BR-IAec und ActR-IIec bzw. BR-IIec. Die extrazellul{\"a}ren Dom{\"a}nen der hierf{\"u}r verwendeten Rezeptoren wurden durch Expression in E.coli oder Sf-9 Insektenzellen erhalten. Ihre funktionelle Charakterisierung erfolgte durch BIAcore Interaktionsanalyse an immobilisierten Liganden, wobei in Abh{\"a}ngigkeit vom Ligand-Rezeptor Komplex unterschiedliche Affinit{\"a}ten ermittelt werden konnten. In {\"U}bereinstimmung mit den hierbei erhaltenen Daten wurden die Ligand-BMP Typ IB Rezeptor Komplexe f{\"u}r BMP-2, BMP-6 und GDF-5, sowie der GDF-5 : BR-IAec Ligand-Rezeptor Komplex pr{\"a}pariert. Des Weiteren konnte die Bildung des tern{\"a}ren BMP-2 : BR-IAec : ActR-IIec Ligand-Rezeptor Komplexes in L{\"o}sung nachgewiesen werden. F{\"u}r all diese Komplexe konnten Kristallisationsbedingungen ermittelt werden. Trotz Optimierung dieser Bedingungen reichte die Qualit{\"a}t der erhaltenen Kristalle nicht f{\"u}r eine Aufkl{\"a}rung der Struktur aus. F{\"u}r eine detailliertes Verst{\"a}ndnis der Mechanismen der Rezeptoraktivierung muss die strukturelle und funktionelle Charakterisierung von BMP Ligand-Rezeptor Komplexen fortgef{\"u}hrt werden. Die pr{\"a}sentierten Ergebnisse deuten darauf hin, dass {\"u}ber die Kenntnis der einzelnen Affinit{\"a}ten und die gezielte Modifikation der Interaktionspartner eine erfolgreiche Strukturanalyse dieser Ligand-Rezeptor Komplexe m{\"o}glich ist.},
  subject      = {Knochen-Morphogenese-Proteine},
  language  = {de}
}
@phdthesis{Kraich2008,
  author    = {Kraich, Michael},
  title     = {Strukturelle und funktionelle Untersuchungen der Interaktion zwischen Ligand und Rezeptor im Interleukin-4- und Interleukin-13-System},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-27655},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2008},
  abstract  = {Interleukin-4 (IL-4) und Interleukin-13 (IL-13) sind bedeutende Regulatorproteine des Immunsystems. Sie spielen eine entscheidende Rolle bei der Entstehung und dem Verlauf von allergischen Erkrankungen, wie z.B. Asthma. Um ihre Signale in die Zielzelle zu transduzieren, kann von beiden Zytokinen der gleiche Zelloberfl{\"a}chenrezeptor verwendet werden, wodurch sich die {\"u}berlappenden, biologischen Funktionen erkl{\"a}ren lassen. Dieser gemeinsam genutzte Rezeptor ist aus den beiden Untereinheiten IL-4Ralpha; und IL-13Ralpha1 aufgebaut. Da IL-4 und IL-13 auf Aminos{\"a}ureebene nur etwa 25\% Sequenzidentit{\"a}t besitzen und stark unterschiedliche Affinit{\"a}ten zu den beiden Rezeptorketten besitzen, stellt sich die Frage, durch welchen molekularen Erkennungsmechanismus, die Affinit{\"a}t und die Spezifit{\"a}t der Ligand-Rezeptor-Interaktion unabh{\"a}ngig voneinander reguliert werden kann. In dieser Arbeit gelang es, rekombinante Expressions- und Aufreinigungsstrategien f{\"u}r IL-13 und die extrazellul{\"a}ren Dom{\"a}nen der Rezeptorketten IL-13Ralpha1 und IL-13Ralpha2 zu entwickeln. Dadurch war es m{\"o}gliche, eine breite Mutations-/Interaktionsanalyse der IL-13Ralpha1-Kette durchzuf{\"u}hren.Es konnte gezeigt werden, dass die N-terminale FnIII-{\"a}hnliche Dom{\"a}ne von IL-13Ralpha1 sowohl an der Bindung von IL-13 als auch an der Interaktion mit IL-4 beteiligt ist. Im funktionellen Bindeepitop der IL-13Ralpha1-Kette wurden die Aminos{\"a}urereste Arg84, Phe253 und Tyr321 als Hauptbindungsdeterminanten f{\"u}r die Interaktion mit IL-13 identifiziert. Durch die Interaktionsstudien der IL-13Ralpha1-Varianten mit IL-4 wurde gezeigt, dass diese Hauptbindungsdeterminanten auch f{\"u}r die niederaffine Bindung von IL-4 von gr{\"o}ßter Bedeutung sind. Die funktionellen Bindeepitope f{\"u}r IL-4 und IL-13 auf der IL-13Ralpha1-Kette sind nahezu identisch und {\"u}berlappen in einem großen Bereich. Aufgrund der Ergebnisse aus der Mutagenesestudie war es m{\"o}glich, ein Strukturmodell der extrazellul{\"a}ren Dom{\"a}ne der IL-13Ralpha1-Kette zu erstellen. Darin wird eine neuartige Orientierung der N-terminalen FnIII-Dom{\"a}ne und deren Beteiligung an der Ligandeninteraktion dargestellt. Mit Hilfe des Strukturmodells gelang es, neue Aminos{\"a}urerest auf der Oberfl{\"a}che von IL-13 zu identifizieren, die an der Bindung zu IL-13Ralpha1 beteiligt sind, was die Relevanz des Strukturmodells weiter unterstreicht. In einem weiteren Teil dieser Arbeit wurde versucht, den molekularen Mechanismus aufzukl{\"a}ren, durch den es den superagonistischen IL-4-Varianten T13D und F82D gelingt, mit dreifach h{\"o}herer Affinit{\"a}t an die IL-4Ralpha-Kette zu binden, als wildtypischer Ligand. Durch strukturelle und funktionelle Untersuchungen wurde gezeigt, dass der Affinit{\"a}tssteigerung ein indirekter Mechanismus zugrunde liegt, bei dem eine Konformations{\"a}nderung und die Fixierung der Arg85-Seitenkette von IL-4 zur Ausbildung von zus{\"a}tzlichen Ligand-Rezeptor-Interaktionen f{\"u}hrt. Das Bindeepitop zwischen IL-4 und der IL-4Ralpha-Kette besitzt eine modulare Architektur aus drei unabh{\"a}ngig voneinander agierenden Interaktionsclustern. Bei der Interaktion von wildtypischem IL-4 mit IL-4Ralpha tragen nur zwei dieser Cluster in signifikanter Weise zur freien Bindeenergie bei. Im Falle der superagonistischen IL-4-Varianten ist jedoch auch das dritte Cluster an der Generierung von zus{\"a}tzlicher, freier Bindeenergie beteiligt, wodurch die Affinit{\"a}t zwischen Ligand und Rezeptor erh{\"o}ht wird. Damit stellt der modulare Aufbau der Interaktionsfl{\"a}che zwischen IL-4 und der IL-4Ralpha-Kette m{\"o}glicherweise einen Mechanismus dar, {\"u}ber den Proteine die Affinit{\"a}t von Wechselwirkungen {\"u}ber einen großen Bereicht variieren k{\"o}nnen, ohne dabei Spezifit{\"a}t einzub{\"u}ssen. Da IL-4 und IL-13 als interessante Zielmolek{\"u}le f{\"u}r die Therapie von allergischen und asthmatischen Erkrankungen erkannt worden sind, k{\"o}nnen die in der vorliegenden Arbeit gewonnenen Informationen {\"u}ber den Bindemechanismus und die Einblicke in den molekularen Charakter der Interaktion zwischen den beiden Zytokinen und ihren spezifischen Rezeptorketten dabei helfen, neuartige und hoch spezifische, inhibitorische Molek{\"u}le zu entwickeln.},
  subject      = {Renaturierung <Biochemie>},
  language  = {de}
}
@phdthesis{Polleichtner2006,
  author    = {Polleichtner, Johann Georg},
  title     = {Studies of structure-function relationship of components of multidrug efflux pumps and type I secretion systems},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-18671},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2006},
  abstract  = {This work deals with channel-tunnel dependent multidrug efflux pumps and type I secretion systems, more concrete with the improved classification of the adaptor protein family, the characterization of the TolC-homologue protein HI1462 of Haemophilus influenzae, and the molecular characterization of the interaction between TolC and AcrA of Escherichia coli.},
  subject      = {Gram-negative Bakterien},
  language  = {en}
}
@phdthesis{Stegmeier2006,
  author    = {Stegmeier, Johannes Friedrich},
  title     = {Study of Omp85 Family Proteins YaeT and YtfM and Multidrug Export Machineries in Escherichia coli},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-18171},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2006},
  abstract  = {In this study the Omp85 family proteins YaeT and YtfM of Escherichia coli were investigated by using biochemical and electrophysiological methods as well as bioinformatical and structural analysis. In addition, knock-out strains were constructed to further study the relevance of these proteins in vivo. The prediction that Omp85 proteins are composed of two domains, a periplasmic amino-terminal POTRA (polypeptide translocation associated) domain and a carboxy-terminal domain anchoring these proteins in the outer membrane, was confirmed by the construction of mutants. It could be shown that the carboxy-terminal part of the proteins is able to insert into the outer bacterial membrane, even if the POTRA domain is removed. Furthermore, pore-forming activity in the black-lipid bilayer was observed for both full-length proteins as well as their carboxy-terminal membrane located parts. The channels formed by both proteins in the black lipid bilayer showed variable single channel conductance states rather than a defined value for conductance. In 1M KCl, e.g. YaeT forms pores with a channel conductance of 100 to 600 pS containing a most abundant value at 400 pS. This variability is at least reasonable for YaeT due to a prerequisite flexibility of its channel for OMP insertion. YaeT was identified to form a cation selective, YtfM an anion selective channel, which is less pH dependent than YaeT. Another feature of the YaeT channel is that its selectivity and conductance is influenced by charged detergent molecules indicating an accumulation of these molecules in hydrophobic pockets inside the compact channel. YaeT revealed heat-modifiable mobility in SDS-PAGE which is characteristic for \&\#946;-barrel OMPs, whereas YtfM did not show this behaviour. This result could be explained by sequence alignment and structural comparison of YaeT and YtfM via CD and FTIR spectra displaying much higher \&\#946;-strand content for the carboxy-terminal part of YaeT compared to YtfM. Since the carboxy-terminal parts were shown to have pore forming ability and are inserted in the OM in vivo, the substitution of the essential protein YaeT by its carboxy-terminal mutant was attempted in a yaeT knock-out strain. The carboxy-terminal half of YaeT was not sufficient to compensate depletion of the full-length protein indicating an important role of the amino-terminus for cell viability. In contrary, YtfM is shown to be a non-essential protein and lack of YtfM had no effects on the composition and integrity of the OM. However, chromosomal deletion of ytfM remarkably reduced the growth rate of cells. This study provides the first detailed investigation of the structure of YaeT and describes its electrophysiological behaviour, which could be a basis for further studies of YaeT and its substrate proteins. Furthermore, YtfM was characterised and its in vivo function was investigated revealing YtfM as the second Omp85 family protein of importance in E. coli. In a second part of this study assembly and function of multidrug efflux pumps were investigated. Drug efflux pumps are tripartite export machineries in the cell envelope of Gram-negative bacteria conferring multidrug resistance and therefore causing severe problems for medical treatment of diseases. Protein structures of all three efflux pump components are solved, but the exact interaction sites are still unknown. Assembly of a hybrid exporter system composed of the Pseudomonas aeruginosa channel tunnel OprM, the E. coli adaptor protein AcrA and its associated transporter AcrB could be shown by chemical cross-linking, even though this efflux pump is not functional. Exchange of the hairpin domain of AcrA by the corresponding hairpin from the adaptor protein MexA of P. aeruginosa restored functionality tested by antibiotic sensitivity assays. This shows the importance of the MexA hairpin domain for functional interaction with the OprM channel tunnel. Interestingly, the hybrid protein was also able to assemble with TolC as outer membrane component to form a functional efflux pump indicating a higher flexibility of TolC compared to OprM concerning interaction partners. Based on these results, an interaction model of the hairpin domain and the channel tunnel on molecular level for AcrA and TolC as well as MexA and OprM, respectively, is presented. This model provides a basis for directed mutagenesis to reveal the exact contact sites of the hairpin of the adapter protein and the outer membrane component},
  subject      = {Escherichia coli},
  language  = {en}
}
@phdthesis{Barth2008,
  author    = {Barth, Enrico},
  title     = {Study of the properties of channel-forming proteins of the cell walls of different Corynebacteriae},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-36325},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2008},
  abstract  = {Die Gattung Corynebacterium geh{\"o}rt, neben Mycobacterium, Nocardia, Rhodococcus und weiteren nahverwandten Gattungen, dem unverwechselbaren, gattungs{\"u}bergreifenden Taxon Mycolata an. Viele Spezies aus dieser heterogenen Gruppe Mycols{\"a}ure-haltiger Actinomyceten sind entweder aufgrund ihrer medizinischen oder ihrer biotechnologischen Bedeutung bekannt. Beispielsweise z{\"a}hlen Mycobacterium tuberculosis, Mycobacterium leprae, Corynebacterium diphtheriae und Nocardia farcinica, welche weltweit Verursacher besonders gef{\"a}hrlicher bakterieller Infektionskrankheiten sind, zu dieser ungew{\"o}hnlichen Gruppe Gram-positiver Bakterien. Ebenso bedeutsam sind einige apathogene Mycolata-Arten, die industrielle Anwendung finden. Corynebacterium glutamicum und Corynebacterium efficiens sind leistungsf{\"a}hige Bakterien, die zum Beispiel in der Produktion des Geschmacksverst{\"a}rkers Glutamat und des Tierfuttermittelzusatzes Lysin eingesetzt werden, w{\"a}hrend verschiedene Rhodococcus Spezies Anwendung bei der Herstellung von Acryls{\"a}uren finden. Die Zellwand der Mycolata zeigt, verglichen mit der klassischer Gram-positiver Bakterien, eine außergew{\"o}hnliche Zusammensetzung und Struktur auf. Abgesehen von einem Arabinogalactan-Peptidoglycan-Komplex enth{\"a}lt die Zellwand der meisten Actinomyceten einen hohen Anteil an Mycols{\"a}uren. Diese langkettigen, verzweigten Fetts{\"a}uren formen eine, mit der {\"a}ußeren Membran Gram-negativer Bakterien vergleichbare, stark undurchl{\"a}ssige, hydrophobe {\"a}ußere H{\"u}lle, welche die Grundlage der außergew{\"o}hnlichen Medikamentenresistenz bei den Mycolata bildet. Wie die {\"a}ußere Membran Gram-negativer Bakterien enth{\"a}lt die Zellwand der Mycolata porenformende Proteine, die den Durchlass hydrophiler Substanzen gestatten. Indem sie eine Verbindung zwischen dem Zellinneren und der Umwelt, in der das Bakterium lebt, schaffen und einen kontrollierten Austausch zwischen beiden erm{\"o}glichen, tragen die Kanalproteine entscheidend zur Funktion der bakteriellen Zellh{\"u}lle bei. Das Ziel dieser Arbeit war das Wissen {\"u}ber Zellwandkan{\"a}le in Corynebakterien zu erweitern. Deshalb untersuchten wir PorA und PorH Proteine, die basierend auf fr{\"u}heren Studien Zellwandkan{\"a}len in C. glutamicum, C. efficiens und Corynebacterium callunae zugeordnet werden, um ungekl{\"a}rten Fragen nachzugehen und um Wissen {\"u}ber deren Struktur zu erlangen. Ferner inspizierten wir Zellw{\"a}nde pathogener Corynebakterien, genauer gesagt von Corynebacterium diphtheriae und Corynebacterium jeikeium, um herauszufinden, ob diese Spezies wie ihre harmlosen Verwandten Kanalproteine besitzen. In dieser Arbeit wiesen wir mit C. diphtheriae und C. jeikeium in zwei weiteren Corynebacterium-Arten offene, mit Wasser gef{\"u}llte Zellwandkan{\"a}le nach. Des Weiteren stellten wir fest, dass sich die Zellwandkan{\"a}le von C. glutamicum, C. efficiens und C. diphtheriae aus zwei Proteinen zusammensetzen, einem zugeh{\"o}rig zu der Gruppe der PorH Proteine und einem weiteren aus der Gruppe der PorA Proteine. Diese heteromere Struktur von Zellwandkan{\"a}len bei Corynebakterien stellt ein Novum f{\"u}r Zellwandkan{\"a}le bei den Mycolata dar. Indessen besteht der Zellwandkanal von C. jeikeium aus nur einem Protein, CjPorA, angeordnet zu einem Oligomer. Obgleich das Molekulargewicht dieses Proteins (4 kDa) mit dem von PorH und PorA Proteinen vergleichbar ist (5-7 kDa), weißt seine Prim{\"a}rsequenz keine eindeutige Homologie zu diesen auf. Dennoch deutet vieles auf eine Verwandtschaft zwischen CjPorA und PorH/PorA Proteinen hin, da das Gen jk0268, welches f{\"u}r CjPorA kodiert, sich in einer Region des C. jeikeium Chromosoms befindet, die der Genomregion entspricht in welcher die porH/porA Gene der anderen Corynebakterien lokalisiert sind. Dies l{\"a}sst vermuten, dass jk0268 (welches f{\"u}r den homomeren Zellwandkanal in C. jeikeium kodiert) und die porH/porA Gene von C. glutamicum, C. efficiens und C. diphtheriae (die einen heteromeren Zellwandkanal kodieren) wahrscheinlich Nachkommen eines gemeinsamen Vorl{\"a}ufergens sind. Phylogenetische Analysen der Gattung Corynebacterium unterst{\"u}tzen diese Annahme. Desweitern legen sie nahe, dass die hier untersuchten Zellwandkan{\"a}le innerhalb dieser Gattung wahrscheinlich weit verbreitet sind. Ein umfassendes Wissen {\"u}ber Zellwandkan{\"a}le, denen beim Transport gel{\"o}ster Stoffe {\"u}ber die {\"a}ußere Membran in Corynebakterien und anderen Mitgliedern der Mycolata eine entscheidende Rolle zukommt, k{\"o}nnte von großem wirtschaftlichem und medizinischem Nutzen sein.},
  subject      = {Zellwand},
  language  = {en}
}
@phdthesis{DeğirmencineePoelloth2023,
  author    = {Değirmenci [n{\´e}e P{\"o}lloth], Laura},
  title     = {Sugar perception and sugar receptor function in the honeybee (\(Apis\) \(mellifera\))},
  doi       = {10.25972/OPUS-32187},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-321873},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {In the eusocial insect honeybee (Apis mellifera), many sterile worker bees live together with a reproductive queen in a colony. All tasks of the colony are performed by the workers, undergoing age-dependent division of labor. Beginning as hive bees, they take on tasks inside the hive such as cleaning or the producing of larval food, later developing into foragers. With that, the perception of sweetness plays a crucial role for all honeybees whether they are sitting on the honey stores in the hive or foraging for food. Their ability to sense sweetness is undoubtedly necessary to develop and evaluate food sources. Many of the behavioral decisions in honeybees are based on sugar perception, either on an individual level for ingestion, or for social behavior such as the impulse to collect or process nectar. In this context, honeybees show a complex spectrum of abilities to perceive sweetness on many levels. They are able to perceive at least seven types of sugars and decide to collect them for the colony. Further, they seem to distinguish between these sugars or at least show clear preferences when collecting them. Additionally, the perception of sugar is not rigid in honeybees. For instance, their responsiveness towards sugar changes during the transition from in-hive bees (e.g. nurses) to foraging and is linked to the division of labor. Other direct or immediate factors changing responsiveness to sugars are stress, starvation or underlying factors, such as genotype. Interestingly, the complexity in their sugar perception is in stark contrast to the fact that honeybees seem to have only three predicted sugar receptors. In this work, we were able to characterize the three known sugar receptors (AmGr1, AmGr2 and AmGr3) of the honeybee fully and comprehensively in oocytes (Manuscript II, Chapter 3 and Manuscript III, Chapter 4). We could show that AmGr1 is a broad sugar receptor reacting to sucrose, glucose, maltose, melezitose and trehalose (which is the honeybees' main blood sugar), but not fructose. AmGr2 acts as its co-receptor altering AmGr1's specificity, AmGr3 is a specific fructose receptor and we proved the heterodimerization of all receptors. With my studies, I was able to reproduce and compare the ligand specificity of the sugar receptors in vivo by generating receptor mutants with CRISPR/Cas9. With this thesis, I was able to define AmGr1 and AmGr3 as the honeybees' basis receptors already capable to detect all sugars of its known taste spectrum. In the expression analysis of my doctoral thesis (Manuscript I, Chapter 2) I demonstrated that both basis receptors are expressed in the antennae and the brain of nurse bees and foragers. This thesis assumes that AmGr3 (like the Drosophila homologue) functions as a sensor for fructose, which might be the satiety signal, while AmGr1 can sense trehalose as the main blood sugar in the brain. Both receptors show a reduced expression in the brain of foragers when compared with nurse bees. These results may reflect the higher concentrated diet of nurse bees in the hive. The higher number of receptors in the brain may allow nurse bees to perceive hunger earlier and to consume the food their sitting on. Forager bees have to be more persistent to hunger, when they are foraging, and food is not so accessible. The findings of reduced expression of the fructose receptor AmGr3 in the antennae of nurse bees are congruent with my other result that nurse bees are also less responsive to fructose at the antennae when compared to foragers (Manuscript I, Chapter 2). This is possible, since nurse bees sit more likely on ripe honey which contains not only higher levels of sugars but also monosaccharides (such as fructose), while foragers have to evaluate less-concentrated nectar. My investigations of the expression of AmGr1 in the antennae of honeybees found no differences between nurse bees and foragers, although foragers are more responsive to the respective sugar sucrose (Manuscript I, Chapter 2). Considering my finding that AmGr2 is the co-receptor of AmGr1, it can be assumed that AmGr1 and the mediated sucrose taste might not be directly controlled by its expression, but indirectly by its co-receptor. My thesis therefore clearly shows that sugar perception is associated with division of labor in honeybees and appears to be directly or indirectly regulated via expression. The comparison with a characterization study using other bee breeds and thus an alternative protein sequence of AmGr1 shows that co-expression of different AmGr1 versions with AmGr2 alters the sugar response differently. Therefore, this thesis provides first important indications that alternative splicing could also represent an important regulatory mechanism for sugar perception in honeybees. Further, I found out that the bitter compound quinine lowers the reward quality in learning experiments for honeybees (Manuscript IV, Chapter 5). So far, no bitter receptor has been found in the genome of honeybees and this thesis strongly assumes that bitter substances such as quinine inhibit sugar receptors in honeybees. With this finding, my work includes other molecules as possible regulatory mechanism in the honeybee sugar perception as well. We showed that the inhibitory effect is lower for fructose compared to sucrose. Considering that sugar signals might be processed as differently attractive in honeybees, this thesis concludes that the sugar receptor inhibition via quinine in honeybees might depend on the receptor (or its co-receptor), is concentration-dependent and based on the salience or attractiveness and concentration of the sugar present. With my thesis, I was able to expand the knowledge on honeybee's sugar perception and formulate a complex, comprehensive overview. Thereby, I demonstrated the multidimensional mechanism that regulates the sugar receptors and thus the sugar perception of honeybees. With this work, I defined AmGr1 and AmGr3 as the basis of sugar perception and enlarged these components to the co-receptor AmGr2 and the possible splice variants of AmGr1. I further demonstrated how those sugar receptor components function, interact and that they are clearly involved in the division of labor in honeybees. In summary, my thesis describes the mechanisms that enable honeybees to perceive sugar in a complex way, even though they inhere a limited number of sugar receptors. My data strongly suggest that honeybees overall might not only differentiate sugars and their diet by their general sweetness (as expected with only one main sugar receptor). The found sugar receptor mechanisms and their interplay further suggest that honeybees might be able to discriminate directly between monosaccharides and disaccharides or sugar molecules and with that their diet (honey and nectar).},
  subject      = {Biene},
  language  = {en}
}
@phdthesis{Thum2006,
  author    = {Thum, Andreas Stephan},
  title     = {Sugar reward learning in Drosophila : neuronal circuits in Drosophila associative olfactory learning},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-17930},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2006},
  abstract  = {Genetic intervention in the fly Drosophila melanogaster has provided strong evidence that the mushroom bodies of the insect brain act as the seat of memory traces for aversive and appetitive olfactory learning (reviewed in Heisenberg, 2003). In flies, electroshock is mainly used as negative reinforcer. Unfortunately this fact complicates a comparative consideration with other inscets as most studies use sugar as positive reinforcer. For example, several lines of evidence from honeybee and moth have suggested another site, the antennal lobe, to house neuronal plasticity underlying appetitive olfactory memory (reviewed in Menzel, 2001; Daly et al., 2004). Because of this I focused my work mainly on appetitive olfactory learning. In the first part of my thesis, I used a novel genetic tool, the TARGET system (McGuire et al., 2003), which allows the temporally controlled expression of a given effector gene in a defined set of cells. Comparing effector genes which either block neurotransmission or ablate cells showed important differences, revealing that selection of the appropriate effector gene is critical for evaluating the function of neural circuits. In the second part, a new engram of olfactory memory in the Drosophila projection neurons is described by restoring Rutabaga adenlylate cyclase (rut-AC) activity specifically in these cells. Expression of wild-type rutabaga in the projection neurons fully rescued the defect in sugar reward memory, but not in aversive electric shock memory. No difference was found in the stability of the appetitive memories rescued either in projection neurons or Kenyon cells. In the third part of the thesis I tried to understand how the reinforcing signals for sugar reward are internally represented. In the bee Hammer (1993) described a single octopaminergic neuron - called VUMmx1 - that mediates the sugar stimulus in associative olfactory reward learning. Analysis of single VUM neurons in the fly (Selcho, 2006) identified a neuron with a similar morphology as the VUMmx1 neuron. As there is a mutant in Drosophila lacking the last enzymatic step in octopamine synthesis (Monastirioti et al., 1996), Tyramine beta Hydroxylase, I was able to show that local Tyramine beta Hydroxylase expression successfully rescued sugar reward learning. This allows to conclude that about 250 cells including the VUM cluster are sufficient for mediating the sugar reinforcement signal in the fly. The description of a VUMmx1 similar neuron and the involvement of the VUM cluster in mediating the octopaminergic sugar stimulus are the first steps in establishing a neuronal map for US processing in Drosophila. Based on this work several experiments are contrivable to reach this ultimate goal in the fly. Taken together, the described similiarities between Drosophila and honeybee regarding the memory organisation in MBs and PNs and the proposed internal representation of the sugar reward suggest an evolutionarily conserved mechanism for appetitive olfactory learning in insects.},
  subject      = {Taufliege},
  language  = {en}
}
@phdthesis{Nayak2007,
  author    = {Nayak, Arnab},
  title     = {Sumoylation Modulates NFATc1-mediated Lymphokine Gene Expression},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-24722},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2007},
  abstract  = {Die Aktivit{\"a}t von Transkriptionsfaktoren kann durch die Modifikation mit SUMO positiv oder negativ beeinflusst werden, indem Protein-Protein-Interaktionen als auch die subzellul{\"a}re bzw. subnukle{\"a}re Lokalisation ver{\"a}ndert werden. In T-Zellen spielt die Familie der NFAT (Nuclear Factor of Activated T cells)-Transkriptionsfaktoren eine wichtige Rolle bei der Zytokingenregulation. NFATc1 wird durch die Verwendung zwei verschiedener Promotoren (P1 \& P2) bzw. Polyadenylierungsstellen (pA1 \& pA2) und alternativen Spleißens in sechs Isoformen exprimiert. Sie werden als NFATc1/alphaA, betaA, alphaB, betaB, alphaC und betaC bezeichnet, wobei alpha und beta sich auf die beiden unterschiedlichen 1. Exons und A, B, C sich auf die differentiell gespleißten und unterschiedlich langen C-Termini beziehen. Die NFATc1/A-Isoformen umfassen einen relativ kurzen C-Terminus, w{\"a}hrend die langen Isoformen B und C extra-C-terminale Peptide von 128 bzw. 246 Aminos{\"a}uren aufweisen. Um die spezifischen, biologischen Effekte der NFATc1-Isoformen zu untersuchen, wurde ein sog. ‚Yeast two Hybrid screen' mit einer humanen Milz-cDNA-Bibliothek und dem NFATc1/C-spezifischen C-Terminus durchgef{\"u}hrt. Am Ende wurden Ubc9 und PIAS1, Proteine, die an der Sumoylierung beteiligt sind, am h{\"a}ufigsten dedektiert. Anschließend konnte gezeigt werden, dass NFATc1 tats{\"a}chlich sumoyliert wird. Das Ausmaß an Sumoylierung ist Isoformen abh{\"a}ngig. W{\"a}hrend NFATc1/A, das eine einzige Sumoylierungsstelle besitzt, nur eine geringe Sumoylierung aufweist, f{\"u}hren die beiden zus{\"a}tzlichen Stellen in NFATc1/C zu einer effizienten Modifikation mit SUMO. Diese C-terminale Modifikation dirigiert NFATc1/C in SUMO-1-K{\"o}rperchen, die mit PML-nbs kolokalisieren. Dar{\"u}ber hinaus rekrutiert sumoyliertes NFATc1/C die transkriptionellen Korepressoren HDAC (sowohl Klasse I wie Klasse II HDACs), was zu einer signifikanten Verringerung der Histonazetylierung am IL-2-Promotor, eines wichtigen NFATc1-Zielgens, f{\"u}hrt. Konsequenterweise wurde eine Verminderung der IL-2-Produktion beobachtet, w{\"a}hrend NFATc1/C, das wegen Mutation der entscheidenden Lysine nicht mehr sumoyliert werden kann, ein dramatisch erh{\"o}htes Transaktivierungspotential am IL-2-Promotor aufwies. Das unterst{\"u}tzt unsere Daten, die mit einem IL-2-Promotor getriebenen Reporterassay gewonnen wurden und zeigen, dass das Transaktivierungspotential von NFATc1/C durch Sumoylierung herabgesetzt wird. Demzufolge {\"u}bt Sumoylierung einen negativen Effekt auf die transkriptionelle NFATc1-Aktivit{\"a}t aus. Immunfluoreszenzversuche zeigten, dass die Modifikation mit SUMO außerdem zur Relokalisation von NFATc1/C in transkriptionell inaktive, heterochromatische Regionen f{\"u}hrt, was durch die F{\"a}rbung von trimethyliertem Histon mit anti-H3K9 m3 nachgewiesen wurde. Interessanterweise war in Abwesenheit von Sumoylierung NFATc1 teilweise mit transkriptionellen Hotspots im Kern lokalisiert. Das mag zu dem h{\"o}heren Transkriptionspotential des nicht-sumoylierten NFATc1 beitragen. Es ist wichtig zu erw{\"a}hnen, dass die transkriptionelle Aktivit{\"a}t auf andere NFATc1-Zielgene durch die Sumoylierung von NFATc1 positiv verst{\"a}rkt war. Dies deutet auf einen nicht-universalen Effekt der Sumoylierung auf die NFATc1/C-Funktion hin. Demzufolge dirigiert Sumoylierung NFATc1 in Kernk{\"o}rperchen, wo es mit transkriptionellen Korepressoren interagiert und selbst ans Heterochromatin relokalisiert, was zu einer Repression der NFATc1/C vermittelten Transkription f{\"u}hrt. Als sehr wichtig erscheint, dass der Effekt der NFATc1/C-Sumoylierung Promotor spezifisch ist. Zusammengenommen ver{\"a}ndet die Modifikation mit SUMO die NFATc1-Funktion von einem Transaktivator zu einem DNA-Bindungsstellen spezifischen Repressor. Daher wird hier ein neuer regulatorischer Mechanismus aufgezeigt, der die Isoform spezifische NFAT-Funktion kontrolliert.},
  language  = {en}
}
@phdthesis{Eiring2021,
  author    = {Eiring, Patrick},
  title     = {Super-resolution microscopy of plasma membrane receptors},
  doi       = {10.25972/OPUS-25004},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-250048},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Plasma membrane receptors are the most crucial and most commonly studied components of cells, since they not only ensure communication between the extracellular space and cells, but are also responsible for the regulation of cell cycle and cell division. The composition of the surface receptors, the so-called "Receptome", differs and is characteristic for certain cell types. Due to their significance, receptors have been important target structures for diagnostic and therapy in cancer medicine and often show aberrant expression patterns in various cancers compared to healthy cells. However, these aberrations can also be exploited and targeted by different medical approaches, as in the case of personalized immunotherapy. In addition, advances in modern fluorescence microscopy by so-called single molecule techniques allow for unprecedented sensitive visualization and quantification of molecules with an attainable spatial resolution of 10-20 nm, allowing for the detection of both stoichiometric and expression density differences. In this work, the single molecule sensitive method dSTORM was applied to quantify the receptor composition of various cell lines as well as in primary samples obtained from patients with hematologic malignancies. The focus of this work lies on artefact free quantification, stoichiometric analyses of oligomerization states and co localization analyses of membrane receptors. Basic requirements for the quantification of receptors are dyes with good photoswitching properties and labels that specifically mark the target structure without generating background through non-specific binding. To ensure this, antibodies with a predefined DOL (degree of labeling) were used, which are also standard in flow cytometry. First background reduction protocols were established on cell lines prior analyses in primary patient samples. Quantitative analyses showed clear expression differences between the cell lines and the patient cells, but also between individual patients. An important component of this work is the ability to detect the oligomerization states of receptors, which enables a more accurate quantification of membrane receptor densities compared to standard flow cytometry. It also provides information about the activation of a certain receptor, for example of FLT3, a tyrosine kinase, dimerizing upon activation. For this purpose, different well-known monomers and dimers were compared to distinguish the typical localization statistics of single bound antibodies from two or more antibodies that are in proximity. Further experiments as well as co localization analyses proved that antibodies can bind to closely adjacent epitopes despite their size. These analytical methods were subsequently applied for quantification and visualization of receptors in two clinically relevant examples. Firstly, various therapeutically relevant receptors such as CD38, BCMA and SLAMF7 for multiple myeloma, a malignant disease of plasma cells, were analyzed and quantified on patient cells. Furthermore, the influence of TP53 and KRAS mutations on receptor expression levels was investigated using the multiple myeloma cell lines OPM2 and AMO1, showing clear differences in certain receptor quantities. Secondly, FLT3 which is a therapeutic target receptor for acute myeloid leukemia, was quantified and stoichiometrically analyzed on both cell lines and patient cells. In addition, cells that have developed resistance against midostaurin were compared with cells that still respond to this type I tyrosine-kinase-inhibitor for their FLT3 receptor expression and oligomerization state.},
  subject      = {Fluoreszenzmikroskopie},
  language  = {en}
}
@phdthesis{Goetz2020,
  author    = {G{\"o}tz, Ralph},
  title     = {Super-resolution microscopy of plasma membrane receptors and intracellular pathogens},
  doi       = {10.25972/OPUS-20716},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-207165},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Humans tend to believe in what they can see with their own eyes. Hence, visualization methods like microscopy have always been extremely popular since their invention in the 17th century. With the advent of super-resolution microscopy, the diffraction limit of ~200 - 250 nm could be overcome to enable more detailed insights into biological samples. Especially the single molecule localization microscopy method dSTORM offers the possibility of quantitative bioimaging. Hereby, the repetitive photoswitching of organic dyes in the presence of thiols is exploited to enable a lateral resolution of 20 nm. Another, recently introduced super-resolution method is expansion microscopy (ExM) which physically expands the sample to increase the resolution by the expansion factor from four to even twenty. To enable this, the sample is embedded into a hydrogel, homogenized using an unspecific proteinase and expanded in distilled water. Within this thesis, both methods were used to shed light on plasma membrane receptor distributions and different bacterial and fungal pathogens. In the first part of this thesis dSTORM was used to elucidate the "Receptome", the entirety of all membrane receptors, of the cell line Jurkat T-cells and primary T-cells. Within this project we could successfully visualize and quantify the distribution of the plasma membrane receptors CD2, CD3, CD4, CD5, CD7, CD11a, CD20, CD28, CD45, CD69 and CD105 with receptor densities ranging from 0.8 cluster/µm² in case of CD20 and 81.4 cluster/µm² for the highly abundant CD45 in activated primary T-cells at the basal membrane. Hereby, we could also demonstrate a homogeneous distribution of most receptors, while only few were clustered. In the case of CD3-clusters were detected in Jurkat T-cells and in primary activated T-cells, but not in na{\"i}ve ones, demonstrating the activation of this receptor. This was followed by the application of dSTORM to three different clinical projects involving the receptors CD38, BCMA and CD20 which are immunotherapeutic targets by monoclonal antibodies and CAR T-cells. In the first two projects dSTORM was applied to determine the receptor upregulation upon exposure of various drugs to MM1.S cells or primary multiple myeloma patient cells. This increase in membrane receptor expression can subsequently enhance the efficacy of therapies directed against these receptors. Within the CD20-project, the superior sensitivity of dSTORM compared to flow cytometry could be demonstrated. Hereby, a substantially higher fraction of CD20-positive patient cells was detected by dSTORM than by flow cytometry. In addition, we could show that by dSTORM CD20-positive evaluated cells were eradicated by immunotherapeutic CAR T-cell treatment. These studies were followed by whole cell super-resolution imaging using both LLS-3D dSTORM and 10x ExM to exclude any artifacts caused by interactions with the glass surface. In 10x ExM signal amplification via biotinylated primary antibodies and streptavidin ATTO 643 was essential to detect even single antibodies directed against the heterodimer CD11a with standard confocal microscopes. Albeit probably not quantitative due to the process of gelation, digestion and expansion during the ExM protocol, even some putative dimers of the receptor CD2 could be visualized using 10x ExM-SIM, similar to dSTORM experiments. Within the second part of this thesis, expansion microscopy was established in bacterial and fungal pathogens. ExM enabled not only an isotropic fourfold expansion of Chlamydia trachomatis, but also allowed the discrimination between the two developmental forms by the chlamydial size after expansion into reticulate and elementary bodies. Hereafter, a new α-NH2-ω-N3-C6-ceramide was introduced enabling an efficient fixation and for the first time the use of lipids in both, 4x and 10x ExM, termed sphingolipid ExM. This compound was used to investigate the ceramide uptake and incorporation into the cell membrane of Chlamydia trachomatis and Simkania negevensis. For Chlamydia trachomatis the combined resolution power of 10x ExM and SIM even allowed the visualization of both bacterial membranes within a distance of ~30 nm. Finally, ExM was applied to the three different fungi Ustilago maydis, Fusarium oxysporum and Aspergillus fumigatus after enzymatic removal of the fungal cell wall. In case of Ustilago maydis sporidia this digestion could be applied to both, living cells resulting in protoplasts and to fixed cells, preserving the fungal morphology. This new protocol could be demonstrated for immunostainings and fluorescent proteins of the three different fungi.},
  subject      = {Mikroskopie},
  language  = {en}
}