@phdthesis{Kunz2017,
  author    = {Kunz, Meik},
  title     = {Systembiologische Analysen von Interaktionen: Zytokinine (Pflanzenpathogene), 3D-Zellkulturen (Krebstherapie) und Drugtargets},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-134911},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Der Einsatz von computergest{\"u}tzten Analysen hat sich zu einem festen Bestandteil der biowissenschaftlichen Forschung etabliert. Im Rahmen dieser vorliegenden Arbeit wurden systembiologische Untersuchungen auf verschiedene biologische Themengebiete und Organismen angewendet. In diesem Zusammenhang liefert die Arbeit einen innovativen und interdisziplin{\"a}ren methodischen Ansatz. Die grundlegende Frage lautet: Wie verstehe und beschreibe ich Signalwege und wie kann ich sie beeinflussen? Der Ansatz verkn{\"u}pft verschiedene biologische Datens{\"a}tze und Datenebenen miteinander, beginnend vom Genom und Interaktionskontext {\"u}ber semiquantitative Simulationen hin zu neuen Interventionen und Experimenten, welche therapeutisch und biotechnologisch genutzt werden k{\"o}nnen. Die Analysen k{\"o}nnen auf diese Weise - zu einem besseren Verst{\"a}ndnis experimenteller Daten und biologischer Fragestellungen beitragen und erm{\"o}glichen ein systematisches Verst{\"a}ndnis der zugrunde liegenden Signalwege und Netzwerkeffekte (z.B. in Pflanzen). - Dar{\"u}ber hinaus erm{\"o}glichen sie die Identifizierung wichtiger funktioneller Hubproteine und die Entwicklung neuer therapeutischer Strategien f{\"u}r weitere experimentelle Testungen (z.B. Tumormodelle), - stellen zudem einen hilfreichen Schritt auf dem Weg zur personalisierten Medizin (z.B. lncRNAs und Tumormodelle) und Medikamentenentwicklung (z.B. Datenbank DrumPID) dar. (i) Als Grundlage wurde hierzu eine integrierte systembiologische Methode entwickelt, welche experimentelle Daten (z.B. Transkriptomdaten) hinsichtlich ihrer biologischen Funktionen untersucht und die Identifizierung relevanter funktioneller Cluster und Hubproteine erm{\"o}glicht. In einem ersten Teil wurden Analysen zum pflanzlichen Immunsystem durchgef{\"u}hrt. Mithilfe der entwickelten Methode wurden Genexpressionsdatens{\"a}tze von A. thaliana, die mit dem Pathogen Pst DC3000 infiziert wurden, untersucht, um den Einfluss verschiedener Virulenzfaktoren auf das Interaktom der Wirtspflanze zu untersuchen und neue Modulatoren einer CK-vermittelten Immunabwehr zu finden. In diesem Zusammenhang konnte gezeigt werden, dass die von Pst DC3000 sekretierten Abwehrstoffe wichtige pflanzliche Hormonsignalwege f{\"u}r die Immunabwehr in A. thaliana beeinflussen. Die Ergebnisse zeigen zudem, dass sich der Einfluss auf das Netzwerkverhalten der Effektorproteine und COR-Phytotoxine von dem der PAMPs unterscheidet, sich jedoch auch eine Regulierung gemeinsamer Signalwege und eine {\"U}berlappung der beiden Phasen der Immunantwort (PTI und ETI) in A. thaliana finden lassen. Die komplexe Immunantwort auf eine Infektion spiegelt sich zudem in einer h{\"o}heren Anzahl an funktionellen Clustern und Hubproteinen in Pst DC3000 gegen{\"u}ber den beiden untersuchten Mutanten wider, wobei sich f{\"u}r Pst DC3000 insbesondere ein stark vernetztes immunrelevantes Cluster um den JA-Signalweg zeigt. Weiterhin wurden anhand der entwickelten Methode wichtige Hubproteine f{\"u}r die Immunabwehr identifiziert. Als bedeutende Vertreter sind AHK2 und AAR14 zu nennen, welche Teil des Zweikomponentensystems der Signal{\"u}bertragung von CK sind und hierbei wichtige Modulatoren f{\"u}r eine CK-vermittelte Immunabwehr darstellen. (ii) Im zweiten Teil der Arbeit schließen sich Untersuchungen an einem in vitro-Experiment einer 2D- und 3D-Zellkultur einer HSP90-Behandlung in einem Lungentumormodell an. In diesem Zusammenhang wurden mithilfe der entwickelten Methode Unterschiede zwischen den beiden Zellkultursystemen gefunden, die das unterschiedliche Behandlungsansprechen erkl{\"a}ren, und f{\"u}r die beiden KRAS-mutierten Zelllinien A549 und H441 des 3D-Testsystems neue prognostische und therapeutische Kandidaten identifiziert. Hierbei haben die durchgef{\"u}hrten Analysen zwei funktionelle Cluster von Protein-Interaktionen um p53 und die STAT-Familie gefunden, welche eine Verbindung zu HSP90 haben und die entsprechenden Behandlungsunterschiede nach einer HSP90-Inhibierung zwischen den beiden Zellkultursystemen erkl{\"a}ren k{\"o}nnen. Unter Ber{\"u}cksichtigung des zelllinien-spezifischen Mutationshintergrunds wurde eine prognostische Markersignatur und daraus abgeleitet HIF1A f{\"u}r die H441-Zelllinie und AMPK f{\"u}r die A549-Zelllinie als neue therapeutische Targets gefunden, wobei die anschließend durchgef{\"u}hrten in silico-Simulationen einen potentiellen therapeutischen Effekt aufzeigen konnten. Weiterhin wurden wichtige experimentelle Readout-Parameter in ein in silico-Lungentumormodell integriert, wobei unter Einbeziehung des Mutationshintergrunds f{\"u}r die verwendeten Zelllinien die HSP90-Behandlung des 3D-Testsystems computergest{\"u}tzt abgebildet werden konnte. Im weiteren Verlauf wurden im in silico-Lungentumormodell Resistenzmechanismen nach einer Gefitinib-Behandlung mit bekanntem Mutationsstatus f{\"u}r die Zelllinien HCC827 und A549 untersucht und daraus folgend neue Therapieans{\"a}tze abgeleitet, die von potentieller klinischer Bedeutung sein k{\"o}nnen. Die durchgef{\"u}hrten in silico-Simulationen f{\"u}r HCC827 konnten hierbei zeigen, dass eine EGFR- und c-MET-Koaktivierung zu einer Gefitinib-Resistenz f{\"u}hren kann, wohingegen bei den A549 eine Komutation von KRAS und IGF-1R zu einem geringen Behandlungsansprechen beitr{\"a}gt. Die Simulationen lassen zudem erkennen, dass eine direkte Inhibierung der an der Resistenzentwicklung beteiligten Rezeptoren c-MET und IGF-1R in beiden F{\"a}llen nicht die bestm{\"o}gliche Therapiestrategie darstellt. In beiden Zelllinien konnte gezeigt werden, dass eine kombinierte Inhibierung von PI3K und MEK den bestm{\"o}glichen therapeutischen Effekt liefert, was demnach einen vielversprechenden Therapieansatz bei Gefitinib-resistenten Lungentumorpatienten darstellt. In einem weiteren Schritt wurde das therapeutische Potential der miRNA-21 im in silico-Modell f{\"u}r die HCC827-Zelllinie untersucht. Die durchgef{\"u}hrten Simulationen zeigen, dass eine miRNA-21-{\"U}berexpression zu einer Resistenzentwickung nach Gefitinib-Behandlung beitragen kann, wobei eine Inhibierung der miRNA-21 diesen Effekt umkehren kann. Die Ergebnisse lassen zudem erkennen, dass eine PTEN-Aktivierung als potentieller Marker einer erfolgreichen therapeutischen Inhibierung der miRNA-21 fungieren kann, wohingegen eine reduzierte miRNA-21-Expression als m{\"o}glicher Marker f{\"u}r eine erfolgreiche Gefitinib-Behandlung dienen kann. (iii) Im dritten Teil der Arbeit wurden systematisch RNA- und Protein-Interaktionen untersucht. Hierzu wurden integrierte systembiologische Analysen an neu identifizierten und funktionell bislang unbekannten lncRNAs durchgef{\"u}hrt. Die Analysen f{\"u}r die infolge einer Herzhypertrophie hochregulierte lncRNA Chast haben umfassend gezeigt, dass diese Proteine und Transkriptionsfaktoren regulieren und binden kann, welche die Signal{\"u}bertragung und Genexpression regulieren, aber auch eine Verbindung zum kardiovaskul{\"a}ren System und stressinduzierter Herzhypertrophie besitzt. Anhand der Ergebnisse l{\"a}sst sich schlussfolgern, dass Chast direkt und indirekt (a) Proteine binden und die Translation beeinflussen kann, zudem eine Chromatin-modifizierende Funktion besitzt und so die Transkription, z.B. f{\"u}r herz- und stress-assoziierte Gene, reguliert, und/oder (b) in einem negativen Feedbackloop seine eigene Transkription reguliert. Obwohl lncRNAs meist eine geringe Konservierung aufweisen, konnten die durchgef{\"u}hrten Analysen f{\"u}r Chast eine Sequenz-Struktur-Konservierung in S{\"a}ugetieren aufzeigen. Weiterhin haben die Untersuchungen an zwei hypoxie-induzierten lncRNAs in Endothelzellen gezeigt, dass die lncRNA MIR503HG eine hohe Sequenz-Struktur-Konservierung in S{\"a}ugetieren besitzt, wohingegen die LINC00323-003 eine geringe Konservierung aufzeigt. Dies untermauert die Tatsache, dass lncRNAs h{\"a}ufig eine geringe Konservierung aufweisen, was Untersuchungen in Modellorganismen hinsichtlich einer therapeutischen Nutzung schwierig machen. Da sich zahlreiche Untersuchungen auf Interaktionen und Signalwege konzentriert haben, wurde abschließend eine Datenbank entwickelt, welche Analysen von Protein-Interaktionen und Signalwegen nachhaltig voranbringt. Die entwickelte DrumPID-Datenbank stellt insbesondere die Interaktion zwischen einem Medikament und seinem Target in den Fokus und erm{\"o}glicht Analysen einzelner Interaktionen und beteiligter Signalwege, bietet zus{\"a}tzlich aber auch verschiedene Links zu anderen Datenbanken f{\"u}r individuelle weiterf{\"u}hrende Analysen. DrumPID erm{\"o}glicht ein geeignetes Medikament u. a. f{\"u}r ein vorgegebenes Zielprotein zu finden und dessen Wirkmechanismus und Interaktionskontext zu untersuchen, was zu einem besseren experimentellen Verst{\"a}ndnis beitragen kann. Zudem erlaubt DrumPID eine potentielle chemische Leitstruktur f{\"u}r ein Zielprotein zu entwickeln, was z.B. spezifisch ein parasitisches Protein inhibiert, ohne dabei einen toxischen Effekt im Menschen zu haben. Zahlreiche weitere Pharmakabeispiele belegen, dass DrumPID f{\"u}r den t{\"a}glichen wissenschaftlichen Gebrauch auf dem Gebiet der Analyse von Protein-Pharmaka-Interaktionen und der Medikamentenentwicklung geeignet ist. Die beschriebenen Ergebnisse der Promotionsarbeit wurden in f{\"u}nf Originalarbeiten, zwei {\"U}bersichtsartikeln und einem Buchteil, u. a. in Science Translational Medicine, ver{\"o}ffentlicht, sechs dieser Publikationen erfolgten im Rahmen von Erstautorschaften.},
  subject      = {Systembiologie},
  language  = {de}
}
@phdthesis{Kohlert2001,
  author    = {Kohlert, Claudia},
  title     = {Systemische Verf{\"u}gbarkeit und Pharmakokinetik von Thymol nach oraler Applikation einer thymianhaltigen Zubereitung im Menschen},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-1621},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2001},
  abstract  = {{\"A}therische {\"O}le bzw. {\"A}therisch-{\"O}l Komponenten sind etabliert in der Therapie der chronischen und akuten Bronchitis. Eine klinische Studie, die mit Thymianextrakt durchgef{\"u}hrt wurde, ließ auf die klinische Wirksamkeit bei akuter Bronchitis schließen. Zahlreiche pharmakodynamische Effekte konnten in vitro f{\"u}r Thymianextrakt bzw. das {\"a}therische Thymian{\"o}l gezeigt werden, jedoch wurde bis jetzt die systemische Verf{\"u}gbarkeit der betreffenden Verbindungen am Zielorgan noch nicht untersucht. Diesbez{\"u}gliche Untersuchungen sind notwendig, um die Verkn{\"u}pfung zwischen den in vitro beobachteten Effekten und der klinischen Wirksamkeit herstellen zu k{\"o}nnen. Eine pharmakokinetische Studie wurde nach Einnahme einer Einzeldosis BronchipretTP (Filmtabletten mit 60 mg Primelwurzelextrakt und 160 mg Thymianextrakt) durchgef{\"u}hrt, um festzustellen, ob Thymol, das den Hauptbestandteil des {\"a}therischen Thymian{\"o}ls darstellt, zur Wirksamkeit dieser Zubereitung beitragen kann. Dazu wurde eine Extraktionsmethode f{\"u}r die Bestimmung von Thymol nach enzymatischer Spaltung des Phase-II-Konjugates Thymolsulfat im Plasma sowie von Thymolsulfat und Thymolglucuronid im Urin entwickelt. Es wurde eine neuartige, automatisierte headspace Festphasenmikroextraktionsmethode (HS-SPME) entwickelt, die Extraktion, Anreicherung und Probenaufgabe in einem einzigen Schritt erm{\"o}glichte. Die Quantifizierung von Thymol im unteren ng.mL-1 Bereich wurde durch die Verwendung von Gaschromatographie in Kombination mit Flammenionisationsdetektion durchgef{\"u}hrt. Die Automatisierung der Methode war notwendig um sie nach internationalen Richtlinien zur Validierung bioanalytischer Methoden validieren zu k{\"o}nnen. Dies ist das erste Mal, dass eine Bioverf{\"u}gbarkeitsstudie bzw. eine pharmakokinetische Studie mit Hilfe der HS-SPME durchgef{\"u}hrt wurde. Thymol selbst war nicht systemisch verf{\"u}gbar. Es war kein freies Thymol oberhalb von 1,4 ng/mL im Plasma detektierbar. Der systemisch verf{\"u}gbare Phase-II-Metabolit wurde per LC-MS/MS als Thymolsulfat identifiziert. Thymolsulfat wurde nur im Plasma detektiert, wohingegen sowohl Thymolsulfat als auch Thymolglucuronid im Urin nachgewiesen wurden. Im Urin wurde kein freies Thyxmol oberhalb von 2,1 ng/mL detektiert. Da Thymol nur in Form von Thymolsulfat systemisch verf{\"u}gbar war, wurde die pharmakokinetische Auswertung an Hand der sich nach enzymatischer Spaltung von Thymolsulfat ergebenden Daten vorgenommen. Die pharmakokinetische Studie wurde nach Verabreichung einer Einzeldosis BronchipretTP (1,08 mg Thymol) mit 12 Probanden durchgef{\"u}hrt. Die Resorption von Thymol war fr{\"u}hzeitig. Bereits nach 20 min konnte Thymol im hydrolisierten Plasma nachgewiesen werden. Maximale Plasmakonzentrationen (cmax) von 93,1±24,5 ng/mL (MW±SD) wurden nach 1,97±0,77 h (tmax) (MW±SD) erreicht. Die Plasmakonzentrationen zeigten einen biphasischen Verlauf und die terminale Eliminationphase setzte nach ca. 10 h ein. Die Eliminationshalbwertszeit errechnete sich zu 10,2 h. Dies betont die Wichtigkeit entsprechend langer Meßzeitr{\"a}ume in Verbindung mit einer hoch sensitiven Analytik, um die Elimination vollst{\"a}ndig erfassen zu k{\"o}nnen. Bezogen auf die verabreichte Thymoldosis (1,08 mg) wurden 16,2±4,5 Prozent (MW±SD) mit unver{\"a}ndertem Thymolgrundger{\"u}st renal eliminiert. Die renale Clearance von 271±156 mL/h (MW±SD) indiziert eine hohe Plasmaeiweißbindung und/oder tubul{\"a}re Reabsorption. Die Daten deuten darauf hin, dass die klinische Wirksamkeit nach Applikation einer thymianextrakthaltigen Zubereitung auf Thymolsulfat oder m{\"o}gliche Phase-I-Metabolite zur{\"u}ckzuf{\"u}hren sein k{\"o}nnte. Die pharmakodynamischen Effekte dieser Verbindungen wurden bislang noch nicht untersucht. Andererseits k{\"o}nnte die Aktivit{\"a}t von Sulfatasen im Lungengewebe die pulmonale Elimination von Thymol erkl{\"a}ren. Freies Thymol k{\"o}nnte somit am Respirationstrakt wirksam sein, obwohl es im Plasma nicht detektiert wurde.},
  subject      = {Mensch},
  language  = {de}
}
@phdthesis{Boyanova2012,
  author    = {Boyanova, Desislava Veselinova},
  title     = {Systems biological analysis of the platelet proteome and applications of functional module search in proteome networks},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72165},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {Recent development of proteomic approaches and generation of large-scale proteomic datasets calls for new methods for biological interpretation of the obtained results. Systems biological approaches such as integrated network analysis and functional module search have become an essential part of proteomic investigation. Proteomics is especially applied in anucleate cells such as platelets. The underlying molecular mechanisms of platelet activation and their pharmacological modulation are of immense importance for clinical research. Advances in platelet proteomics have provided a large amount of proteomic data, which has not yet been comprehensively investigated in a systems biological perspective. To this end, I assembled platelet specific data from proteomic and transcriptomic studies by detailed manual curation and worked on the generation of a comprehensive human platelet repository for systems biological analysis of platelets in the functional context of integrated networks (PlateletWeb) (http:/PlateletWeb.bioapps.biozentrum.uni-wuerzburg.de). I also added platelet-specific experimentally validated phosphorylation data and generated kinase predictions for 80\% of the newly identified platelet phosphosites. The combination of drug, disease and pathway information with phosphorylation and interaction data makes this database the first integrative platelet platform available for platelet research. PlateletWeb contains more than 5000 platelet proteins, which can also be analyzed and visualized in a network context, allowing identification of all major signaling modules involved in platelet activation and inhibition. Using the wealth of integrated data I performed a series of platelet-specific analyses regarding the platelet proteome, pathways, drug targets and novel platelet phosphorylation events involved in crucial signaling events. I analyzed the statistical enrichment of known pathways for platelet proteins and identified endocytosis as a highly represented pathway in platelets. Further results revealed that highly connected platelet proteins are more often targeted by drugs. Using integrated network analysis offered by PlateletWeb, I analyzed the crucial activation signaling pathway of adenosine diphosphate (ADP), visualizing how the signal flow from receptors to effectors is maintained. My work on integrin inside-out signaling was also based on the integrated network approach and examined new platelet-specific phosphorylation sites and their regulation using kinase predictions. I generated hypothesis on integrin signaling, by investigating the regulation of Ser269 phosphorylation site on the docking protein 1 (DOK1). This phosphorylation site may influence the inhibiting effect of DOK1 on integrin a2bb3. Extending the integrated network approach to further cell lines, I used the assembled human interactome information for the analysis of functional modules in cellular networks. The investigation was performed with a previously developed module detection algorithm, which finds maximum-scoring subgraphs in transcriptomic datasets by using assigned values to the network nodes. We extended the algorithm to qualitative proteomic datasets and enhanced the module search by adding functional information to the network edges to concentrate the solution onto modules with high functional similarity. I performed a series of analyses to validate its performance in small-sized (virus-infected gastric cells) and medium-sized networks (human lymphocytes). In both cases the algorithm extracted characteristic modules of sample proteins with high functional similarity. The functional module search is especially useful in site-specific phosphoproteomic datasets, where kinase regulation of the detected sites is often sparse or lacking. Therefore, I used the module detection algorithm in quantitative phosphoproteomic datasets. In a platelet phosphorylation dataset, I presented a pipeline for network analysis of detected phosphorylation sites. In a second approach, the functional module detecting algorithm was used on a phosphoproteome network of human embryonic stem cells, in which nodes represented the maximally changing phosphorylation sites in the experiment. Additional kinases from the human phosphoproteome in PlateletWeb were included to the network to investigate the regulation of the signal flow. Results indicated important phosphorylation sites and their upstream kinases and explained changes observed in embryonic stem cells during differentiation. This work presents novel approaches for integrated network analysis in cells and introduces for the first time a systematic biological investigation of the human platelet proteome based on the platelet-specific knowledge base PlateletWeb. The extended methods for optimized functional module detection offer an invaluable tool for exploring proteomic datasets and covering gaps in complex large-scale data analysis. By combining exact module detection approaches with functional information data between interacting proteins, characteristic functional modules with high functional resemblance can be extracted from complex datasets, thereby focusing on important changes in the observed networks.},
  subject      = {Netzwerkanalyse},
  language  = {en}
}
@phdthesis{Schrama2004,
  author    = {Schrama, David},
  title     = {T-Zell-priming außerhalb sekund{\"a}rer lymphatischer Gewebe},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-15060},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2004},
  abstract  = {T-Zellimmunantworten werden normalerweise durch folgenden Weg initiiert: unreife dendritische Zellen nehmen Antigen in der Peripherie auf, wandern in die sekund{\"a}ren lymphatischen Organe, wobei sie auf ihrem Weg sowohl reifen als auch das Antigen prozessieren. In den sekund{\"a}ren lymphatischen Organen angekommen, pr{\"a}sentieren sie als reife dendritische Zellen den T-Zellen die Antigene in Form von Peptiden zusammen mit kostimulierenden Molek{\"u}len. Dadurch rufen sie eine spezifische T-Zellantwort hervor. In der vorliegenden Arbeit wurde untersucht, ob nicht Situationen herbeigef{\"u}hrt werden k{\"o}nnen, die ein T-Zell priming außerhalb der sekund{\"a}ren lymphatischen Organe erlauben. Dazu wurden ein murines Modell, bei dem das Zytokin Lymphotoxin-alpha spezifisch am Tumor angereichert wurde, und ein humanes Modell, bei dem reife, antigenbeladene DC intradermal appliziert wurden, untersucht. Im murinen Modell zeigte sich, dass die gerichtete Anreicherung von Lymphotoxin-alpha am Tumor zu dessen Zerst{\"o}rung f{\"u}hrte, welche durch T-Zellen vermittelt wurde, und mit der Induktion eines terti{\"a}ren lymphatischen Gewebes am Tumor assoziiert war. Dieses terti{\"a}re lymphatische Gewebe war durch die Kompartimentalisierung von T- und B-Zellen und der Pr{\"a}senz von high endothelial venules charakterisiert und besaß zudem mit dendritischen Zellen und na{\"i}ven T-Zellen alle Voraussetzungen f{\"u}r ein in loco priming. Dementsprechend konnte in der Folge der gerichteten Lymphotoxin-alpa Therapie im Tumor ein Anstieg am T-Zellinfiltrat, welches sich oligoklonal zusammensetzte, beobachtet werden. In vitro Experimente verdeutlichte die Tumorspezifit{\"a}t der Therapie-induzierten T-Zellantwort, da die T-Zellen auf ein Tumorantigen mit der Aussch{\"u}ttung von Interferon gamma reagierten und die Tumorzellen lysierten. Im humanen Modell wurden Hautbiopsien von Melanompatienten untersucht, denen im Rahmen einer klinischen Studie autologe, in vitro generierte und antigenbeladene DC intradermal appliziert wurden. Die Patienten erlaubten die Entnahme von Hautbiopsien aus den Injektionsstellen f{\"u}r wissenschaftliche Untersuchungen. Eine Induktion bzw. Verst{\"a}rkung einer spezifischen T-Zellantwort durch die Vakzinierung mit antigenbeladenen dendritischen Zellen konnte bereits in zahlreichen Arbeiten und auch in dem in dieser Arbeit untersuchten Patientenkollektiv gezeigt werden. Bei der Analyse der Injektionsstellen zeigt sich, dass ein großer Teil der injizierten dendritischen Zellen in der Vakzinierungsstelle verharren und dass diese unabh{\"a}ngig von einer Beladung mit Antigen zu einer Induktion von high endothelial venules Charakteristika f{\"u}hrte. Waren die dendritischen Zellen mit Antigen beladen, so f{\"u}hrte dies zu einem st{\"a}rkeren T-Zellinfiltrat in den Injektionsstellen, wobei sowohl na{\"i}ve als auch central memory T-Zellen nachgewiesen wurde. Diese Zellen wurden vermutlich durch die {\"U}berexpression der DC CK1 und SDF1 Chemokinen in den Injektionsstellen, die chemotaktisch auf T-Zellen wirken, angezogen. Das Infiltrat in den Injektionsstellen war oligoklonal und wies tumorspezifische T-Zellen auf. Nachdem diese T-Zellklone im Blut der Patienten vor der Vakzinierung nicht nachweisbar waren, m{\"u}ssen sie zumindest in den Injektionsstellen expandiert sein. Interessanterweise konnte einer dieser Klone in Metastasen nachgewiesen werden, die nach der Vakzinierung dem Patienten entfernt wurden. In beiden Modellen wurde also durch die Manipulation des Mikromilieus, d.h. Lymphotoxin-alpa Anreicherung am Tumor bzw. Injektion von reifen dendritischen Zellen in die Haut, Strukturen wie z.B. high endothelial venules induziert, die ein in loco priming erm{\"o}glichen sollten. Dementsprechend riefen diese Ver{\"a}nderungen ein Tumorantigen-spezifisches Infiltrat hervor. Diese Ergebnisse deuten darauf hin, dass T-Zell priming auch außerhalb sekund{\"a}rer lymphatischer Organe erfolgen kann. Prinzipiell scheint also nur der Kontakt von reifen, antigenbeladenen dendritischen Zellen mit den entsprechenden antigenspezifischen, na{\"i}ven T-Zellen entscheiden zu sein. Die M{\"o}glichkeit des in vitro primings bekr{\"a}ftigt diese These. In vivo erfolgt dieses Aufeinandertreffen normalerweise in den sekund{\"a}ren lymphatischen Organen, doch konnte in der vorliegenden Arbeit gezeigt werden, dass Ver{\"a}nderungen des Mikromilieus diesen Kontakt auch in anderen Geweben erm{\"o}glicht.},
  subject      = {T-Lymphozyt},
  language  = {de}
}
@phdthesis{Bruehlmann2017,
  author    = {Br{\"u}hlmann, David},
  title     = {Tailoring Recombinant Protein Quality by Rational Media Design},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-147345},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Nowadays, more than half of the biotherapeutics are produced in mammalian cell lines as a result of correct protein folding and assembly as well as their faculty to bring about a variety of post-translational modifications. The widespread progression of biosimilars has moved the focus in mammalian cell-culture process development. Thereby, the modulation of quality attributes of recombinant therapeutic proteins has increasingly gained importance from early process development stages. Protein quality directly shapes the clinical efficacy and safety in vivo, and therefore, the control of the complex post-translational modifications, such as glycosylation (e.g. high mannose, fucosylation, galactosylation and sialylation), charge variants, aggregates and low-molecular-weight species formation, is pivotal for efficient receptor binding and for triggering the desired immune responses in patients. In the frame of biosimilar development, product quality modulation methods using the potential of the host cell line are particularly sought after to match the quality profile of the targeted reference medicinal product (RMP) as closely as possible. The environment the cell is dwelling in directly influences its metabolism and the resulting quality profile of the expressed protein. Thereby the cell culture medium plays a central role in upstream manufacturing. In this work, concentration adjustment of selected media components and supplementation with a variety of compounds was performed to alter various metabolic pathways, enzyme activities and in some cases the gene expression levels of Chinese Hamster Ovary (CHO) cells in culture. The supplementation of cell culture medium with the trisaccharide raffinose in fed-batch cultures entailed an increase of the abundance of high mannose glycans in two different CHO cell lines. Raffinose especially favored mannose 5 glycans. At the same time, it impaired cell culture performance, induced changes on the intracellular nucleotide levels and even varied the expression levels of glycosylation-related genes. Supplementation with a number of galactosyltransferase inhibiting compounds, in particular fluorinated galactose analogs (alpha- and beta-2F-peracetyl-galactose), consistently decreased the production of galactosylated monoclonal antibodies (mAb). By means of targeted addition during the culture rather than at the beginning, the inhibition was further increased, while limiting detrimental effects on both growth and productivity. High-throughput screening in 96-deepwell plates showed that spermine and L-ornithine also reduced the level of galactosylation. On the other hand, exploratory screening of a variety of potentially disulfide-bridge-reducing agents highlighted that the inherent low-molecular-species level of the proprietary platform cell culture process was likely due to favored reduction. This hypothesis was reinforced by the observation that supplementation of cysteine and N-acetylcysteine promoted fragmentation. Additionally, fragmentation decreased with higher protein expression. At that point, aiming to improve the efficiency in process development, a rational experimental design method was developed to identify and to define the optimal concentration range of quality modulating compounds by calling on a combination of high throughput fed-batch testing and multivariate data analysis. Seventeen medium supplements were tested in five parallel 96-deepwell plate experiments. The selection process of promising modulators for the follow-up experiment in shake tubes consisted in a three-step procedure, including principal component analysis, quantitative evaluation of their performance with respect to the specifications for biosimilarity and selection following a hierarchical order of decisions using a decision tree. The method resulted in a substantial improvement of the targeted glycosylation profile in only two experimental rounds. Subsequent development stages, namely validation and transfer to industrial-scale facilities require tight control of product quality. Accordingly, further mechanistic understanding of the underlying processes was acquired by non-targeted metabolomic profiling of a CHO cell line expressing a mAb cultured in four distinct process formats. Univariate analysis of intra- and extracellular metabolite and temporal glycosylation profiles provided insights in various pathways. The numerous of parameters were the main driver to carry out principal component analysis, and then, using the methodology of partial-least-square (PLS) projection on latent structures, a multivariate model was built to correlate the extracellular data with the distinct glycosylation profiles. The PLS observation model proved to be reliable and showed its great benefit for glycan pattern control in routine manufacturing, especially at large scale. Rather than relying on post-production interpretation of glycosylation results, glycosylation can be predicted in real-time based on the extracellular metabolite levels in the bioreactor. Finally, for the bioactivity assessment of the glycan differences between the biosimilar and the reference medicinal product (RMP), the health agencies may ask for in the drug registration process, extended ranges of glycan variants need to be generated so that the in vitro assays pick up the changes. The developed glycosylation modulator library enabled the generation of extreme glycosylation variants, including high mannose, afucosylated, galactosylated as well as sialic acid species of both a mAb and an antibody fusion molecule with three N-glycosylation sites. Moreover, to create increased variety, enzymatic glycoengineering was explored for galactosylation and sialylation. The glyco variants induced significant responses in the respective in vitro biological activity assays. The data of this work highlight the immense potential of cell culture medium optimization to adjust product quality. Medium and feed supplementation of a variety of compounds resulted in reproducible and important changes of the product quality profile of both mAbs and a fusion antibody. In addition to the intermediate modulation ranges that largely met the requirements for new-biological-entity and biosimilar development, medium supplementation even enabled quick and straightforward generation of extreme glycan variants suitable for biological activity testing.},
  subject      = {Zellkultur},
  language  = {en}
}
@article{DracopoliFeltquateSametal.1990,
  author    = {Dracopoli, Nicholas C. and Feltquate, David M. and Sam, Brigitta and Schartl, Manfred},
  title     = {Taql and Mspl RFLPs are detected by the human 2,3-biphosphoglycerate mutase (BPGM) cDNA},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-61763},
  year      = {1990},
  abstract  = {No abstract available},
  subject      = {Physiologische Chemie},
  language  = {en}
}
@article{HeisigWeberEnglbergeretal.2012,
  author    = {Heisig, Julia and Weber, David and Englberger, Eva and Winkler, Anja and Kneitz, Susanne and Sung, Wing-Kin and Wolf, Elmar and Eilers, Martin and Wei, Chia-Lin and Gessler, Manfred},
  title     = {Target Gene Analysis by Microarrays and Chromatin Immunoprecipitation Identifies HEY Proteins as Highly Redundant bHLH Repressors},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-75341},
  year      = {2012},
  abstract  = {HEY bHLH transcription factors have been shown to regulate multiple key steps in cardiovascular development. They can be induced by activated NOTCH receptors, but other upstream stimuli mediated by TGFß and BMP receptors may elicit a similar response. While the basic and helix-loop-helix domains exhibit strong similarity, large parts of the proteins are still unique and may serve divergent functions. The striking overlap of cardiac defects in HEY2 and combined HEY1/HEYL knockout mice suggested that all three HEY genes fulfill overlapping function in target cells. We therefore sought to identify target genes for HEY proteins by microarray expression and ChIPseq analyses in HEK293 cells, cardiomyocytes, and murine hearts. HEY proteins were found to modulate expression of their target gene to a rather limited extent, but with striking functional interchangeability between HEY factors. Chromatin immunoprecipitation revealed a much greater number of potential binding sites that again largely overlap between HEY factors. Binding sites are clustered in the proximal promoter region especially of transcriptional regulators or developmental control genes. Multiple lines of evidence suggest that HEY proteins primarily act as direct transcriptional repressors, while gene activation seems to be due to secondary or indirect effects. Mutagenesis of putative DNA binding residues supports the notion of direct DNA binding. While class B E-box sequences (CACGYG) clearly represent preferred target sequences, there must be additional and more loosely defined modes of DNA binding since many of the target promoters that are efficiently bound by HEY proteins do not contain an Ebox motif. These data clearly establish the three HEY bHLH factors as highly redundant transcriptional repressors in vitro and in vivo, which explains the combinatorial action observed in different tissues with overlapping expression.},
  subject      = {Biologie},
  language  = {en}
}
@article{EiringMcLaughlinMatikondaetal.2021,
  author    = {Eiring, Patrick and McLaughlin, Ryan and Matikonda, Siddharth S. and Han, Zhongying and Grabenhorst, Lennart and Helmerich, Dominic A. and Meub, Mara and Beliu, Gerti and Luciano, Michael and Bandi, Venu and Zijlstra, Niels and Shi, Zhen-Dan and Tarasov, Sergey G. and Swenson, Rolf and Tinnefeld, Philip and Glembockyte, Viktorija and Cordes, Thorben and Sauer, Markus and Schnermann, Martin J.},
  title     = {Targetable conformationally restricted cyanines enable photon-count-limited applications},
  series = {Angewandte Chemie Internationale Edition},
  volume    = {60},
  journal   = {Angewandte Chemie Internationale Edition},
  number    = {51},
  doi       = {10.1002/anie.202109749},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-256559},
  pages     = {26685-26693},
  year      = {2021},
  abstract  = {Cyanine dyes are exceptionally useful probes for a range of fluorescence-based applications, but their photon output can be limited by trans-to-cis photoisomerization. We recently demonstrated that appending a ring system to the pentamethine cyanine ring system improves the quantum yield and extends the fluorescence lifetime. Here, we report an optimized synthesis of persulfonated variants that enable efficient labeling of nucleic acids and proteins. We demonstrate that a bifunctional sulfonated tertiary amide significantly improves the optical properties of the resulting bioconjugates. These new conformationally restricted cyanines are compared to the parent cyanine derivatives in a range of contexts. These include their use in the plasmonic hotspot of a DNA-nanoantenna, in single-molecule F{\"o}rster-resonance energy transfer (FRET) applications, far-red fluorescence-lifetime imaging microscopy (FLIM), and single-molecule localization microscopy (SMLM). These efforts define contexts in which eliminating cyanine isomerization provides meaningful benefits to imaging performance.},
  language  = {en}
}
@phdthesis{Adhikari2024,
  author    = {Adhikari, Bikash},
  title     = {Targeted degradation of Myc-interacting oncoproteins},
  doi       = {10.25972/OPUS-31732},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-317326},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {The hallmark oncoprotein Myc is a major driver of tumorigenesis in various human cancer entities. However, Myc's structural features make it challenging to develop small molecules against it. A promising strategy to indirectly inhibit the function of Myc is by targeting its interactors. Many Myc-interacting proteins have reported scaffolding functions which are difficult to target using conventional occupancy- driven inhibitors. Thus, in this thesis, the proteolysis targeting chimera (PROTAC) approach was used to target two oncoproteins interacting with Myc which promote the oncogenicity of Myc, Aurora-A and WDR5. PROTACs are bifunctional small molecules that bind to the target protein with one ligand and recruit a cellular E3- ligase with the other ligand to induce target degradation via the ubiquitin- proteasome system. So far, the most widely used E3-ligases for PROTAC development are Cereblon (CRBN) and von Hippel-Lindau tumor suppressor (VHL). Furthermore, there are cases of incompatibility between some E3-ligases and proteins to bring about degradation. Hence there is a need to explore new E3- ligases and a demand for a tool to predict degradative E3-ligases for the target protein in the PROTAC field. In the first part, a highly specific mitotic kinase Aurora-A degrader, JB170, was developed. This compound utilized Aurora-A inhibitor alisertib as the target ligand and thalidomide as the E3-ligase CRBN harness. The specificity of JB170 and the ternary complex formation was supported by the interactions between Aurora-A and CRBN. The PROTAC-mediated degradation of Aurora-A induced a distinct S- phase defect rather than mitotic arrest, shown by its catalytic inhibition. The finding demonstrates that Aurora-A has a non-catalytic role in the S-phase. Furthermore, the degradation of Aurora-A led to apoptosis in various cancer cell lines. In the second part, two different series of WDR5 PROTACs based on two protein- protein inhibitors of WDR5 were evaluated. The most efficient degraders from both series recruited VHL as a E3-ligase and showed partial degradation of WDR5. In addition, the degradation efficiency of the PROTACs was significantly affected by the linker nature and length, highlighting the importance of linker length and composition in PROTAC design. The degraders showed modest proliferation defects at best in cancer cell lines. However, overexpression of VHL increased the degradation efficiency and the antiproliferative effect of the PROTACs. In the last part, a rapamycin-based assay was developed to predict the degradative E3-ligase for a target. The assay was validated using the WDR5/VHL and Aurora- A/CRBN pairs. The result that WDR5 is degraded by VHL but not CRBN and Aurora-A is degraded by CRBN, matches observations made with PROTACs. This technique will be used in the future to find effective tissue-specific and essential E3-ligases for targeted degradation of oncoproteins using PROTACs. Collectively, the work presented here provides a strategy to improve PROTAC development and a starting point for developing Aurora-A and WDR5 PROTACs for cancer therapy.},
  subject      = {Degradation},
  language  = {en}
}
@article{KuehnischHerbstAl‐Wakeel‐Marquardetal.2019,
  author    = {K{\"u}hnisch, Jirko and Herbst, Christopher and Al-Wakeel-Marquard, Nadya and Dartsch, Josephine and Holtgrewe, Manuel and Baban, Anwar and Mearini, Giulia and Hardt, Juliane and Kolokotronis, Konstantinos and Gerull, Brenda and Carrier, Lucie and Beule, Dieter and Schubert, Stephan and Messroghli, Daniel and Degener, Franziska and Berger, Felix and Klaassen, Sabine},
  title     = {Targeted panel sequencing in pediatric primary cardiomyopathy supports a critical role of TNNI3},
  series = {Clinical Genetics},
  volume    = {96},
  journal   = {Clinical Genetics},
  number    = {6},
  doi       = {10.1111/cge.13645},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-213958},
  pages     = {549 -- 559},
  year      = {2019},
  abstract  = {The underlying genetic mechanisms and early pathological events of children with primary cardiomyopathy (CMP) are insufficiently characterized. In this study, we aimed to characterize the mutational spectrum of primary CMP in a large cohort of patients ≤18 years referred to a tertiary center. Eighty unrelated index patients with pediatric primary CMP underwent genetic testing with a panel-based next-generation sequencing approach of 89 genes. At least one pathogenic or probably pathogenic variant was identified in 30/80 (38\%) index patients. In all CMP subgroups, patients carried most frequently variants of interest in sarcomere genes suggesting them as a major contributor in pediatric primary CMP. In MYH7, MYBPC3, and TNNI3, we identified 18 pathogenic/probably pathogenic variants (MYH7 n = 7, MYBPC3 n = 6, TNNI3 n = 5, including one homozygous (TNNI3 c.24+2T>A) truncating variant. Protein and transcript level analysis on heart biopsies from individuals with homozygous mutation of TNNI3 revealed that the TNNI3 protein is absent and associated with upregulation of the fetal isoform TNNI1. The present study further supports the clinical importance of sarcomeric mutation—not only in adult—but also in pediatric primary CMP. TNNI3 is the third most important disease gene in this cohort and complete loss of TNNI3 leads to severe pediatric CMP.},
  language  = {en}
}
@article{FrankKesnerLibertietal.2023,
  author    = {Frank, Erik T. and Kesner, Lucie and Liberti, Joanito and Helleu, Quentin and LeBoeuf, Adria C. and Dascalu, Andrei and Sponsler, Douglas B. and Azuma, Fumika and Economo, Evan P. and Waridel, Patrice and Engel, Philipp and Schmitt, Thomas and Keller, Laurent},
  title     = {Targeted treatment of injured nestmates with antimicrobial compounds in an ant society},
  series = {Nature Communications},
  volume    = {14},
  journal   = {Nature Communications},
  doi       = {10.1038/s41467-023-43885-w},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-358081},
  year      = {2023},
  abstract  = {Infected wounds pose a major mortality risk in animals. Injuries are common in the ant Megaponera analis, which raids pugnacious prey. Here we show that M. analis can determine when wounds are infected and treat them accordingly. By applying a variety of antimicrobial compounds and proteins secreted from the metapleural gland to infected wounds, workers reduce the mortality of infected individuals by 90\%. Chemical analyses showed that wound infection is associated with specific changes in the cuticular hydrocarbon profile, thereby likely allowing nestmates to diagnose the infection state of injured individuals and apply the appropriate antimicrobial treatment. This study demonstrates that M. analis ant societies use antimicrobial compounds produced in the metapleural glands to treat infected wounds and reduce nestmate mortality.},
  language  = {en}
}
@article{PauliPaulProppertetal.2021,
  author    = {Pauli, Martin and Paul, Mila M. and Proppert, Sven and Mrestani, Achmed and Sharifi, Marzieh and Repp, Felix and K{\"u}rzinger, Lydia and Kollmannsberger, Philip and Sauer, Markus and Heckmann, Manfred and Sir{\´e}n, Anna-Leena},
  title     = {Targeted volumetric single-molecule localization microscopy of defined presynaptic structures in brain sections},
  series = {Communications Biology},
  volume    = {4},
  journal   = {Communications Biology},
  doi       = {10.1038/s42003-021-01939-z},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259830},
  pages     = {407},
  year      = {2021},
  abstract  = {Revealing the molecular organization of anatomically precisely defined brain regions is necessary for refined understanding of synaptic plasticity. Although three-dimensional (3D) single-molecule localization microscopy can provide the required resolution, imaging more than a few micrometers deep into tissue remains challenging. To quantify presynaptic active zones (AZ) of entire, large, conditional detonator hippocampal mossy fiber (MF) boutons with diameters as large as 10 mu m, we developed a method for targeted volumetric direct stochastic optical reconstruction microscopy (dSTORM). An optimized protocol for fast repeated axial scanning and efficient sequential labeling of the AZ scaffold Bassoon and membrane bound GFP with Alexa Fluor 647 enabled 3D-dSTORM imaging of 25 mu m thick mouse brain sections and assignment of AZs to specific neuronal substructures. Quantitative data analysis revealed large differences in Bassoon cluster size and density for distinct hippocampal regions with largest clusters in MF boutons. Pauli et al. develop targeted volumetric dSTORM in order to image large hippocampal mossy fiber boutons (MFBs) in brain slices. They can identify synaptic targets of individual MFBs and measured size and density of Bassoon clusters within individual untruncated MFBs at nanoscopic resolution.},
  language  = {en}
}
@article{OttoSchmidtKastneretal.2019,
  author    = {Otto, C. and Schmidt, S. and Kastner, C. and Denk, S. and Kettler, J. and M{\"u}ller, N. and Germer, C.T. and Wolf, E. and Gallant, P. and Wiegering, A.},
  title     = {Targeting bromodomain-containing protein 4 (BRD4) inhibits MYC expression in colorectal cancer cells},
  series = {Neoplasia},
  volume    = {21},
  journal   = {Neoplasia},
  number    = {11},
  doi       = {10.1016/j.neo.2019.10.003},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-202451},
  pages     = {1110-1120},
  year      = {2019},
  abstract  = {The transcriptional regulator BRD4 has been shown to be important for the expression of several oncogenes including MYC. Inhibiting of BRD4 has broad antiproliferative activity in different cancer cell types. The small molecule JQ1 blocks the interaction of BRD4 with acetylated histones leading to transcriptional modulation. Depleting BRD4 via engineered bifunctional small molecules named PROTACs (proteolysis targeting chimeras) represents the next-generation approach to JQ1-mediated BRD4 inhibition. PROTACs trigger BRD4 for proteasomale degradation by recruiting E3 ligases. The aim of this study was therefore to validate the importance of BRD4 as a relevant target in colorectal cancer (CRC) cells and to compare the efficacy of BRD4 inhibition with BRD4 degradation on downregulating MYC expression. JQ1 induced a downregulation of both MYC mRNA and MYC protein associated with an antiproliferative phenotype in CRC cells. dBET1 and MZ1 induced degradation of BRD4 followed by a reduction in MYC expression and CRC cell proliferation. In SW480 cells, where dBET1 failed, we found significantly lower levels of the E3 ligase cereblon, which is essential for dBET1-induced BRD4 degradation. To gain mechanistic insight into the unresponsiveness to dBET1, we generated dBET1-resistant LS174t cells and found a strong downregulation of cereblon protein. These findings suggest that inhibition of BRD4 by JQ1 and degradation of BRD4 by dBET1 and MZ1 are powerful tools for reducing MYC expression and CRC cell proliferation. In addition, downregulation of cereblon may be an important mechanism for developing dBET1 resistance, which can be evaded by incubating dBET1-resistant cells with JQ1 or MZ1.},
  language  = {en}
}
@phdthesis{Gotthard2023,
  author    = {Gotthard, Hannes},
  title     = {Targeting Colorectal Cancer Stem Cells with Hemibodies},
  doi       = {10.25972/OPUS-30309},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-303090},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {The cancer stem cell hypothesis is a cancer development model which elicited great interest in the last decades stating that cancer heterogeneity arises from a stem cell through asymmetrical division. The Cancer Stem Cell subset is described as the only population to be tumorigenic and having the potential to renew. Conventional therapy often fails to eradicate CSC resulting in tumor relapse. Consequently, it is of great inter-est to eliminate this subset of cells to provide the best patient outcome. In the last years several approaches to target CSC were developed, one of them being immunotherapeu-tic targeting with antibodies. Since markers associated with CSC are also expressed on normal stem cells or healthy adjacent tissue in colorectal cancer, dual targeting strate-gies are preferred over targeting only a single antigen. Subsequently, the idea of dual targeting two CSC markers in parallel by a newly developed split T cell-engaging anti-body format termed as Hemibodies emerged. In a preliminary single cell RNA sequenc-ing analysis of colorectal cancer cells CD133, CD24, CD166 and CEA were identified as suitable targets for the combinatorial targeting strategy. Therefore, this study focused on trispecific and trivalent Hemibodies comprising a split binding moiety against CD3 and a binding moiety against either CD133, CD24, CD166 or CEA to overcome the occurrence of resistance and to efficiently eradicate all tumor cells including the CSC compartment. The study showed that the Hemibody combinations CD133xCD24, CD133xCD166 and CD133xCEA are able to eliminate double positive CHO cells with high efficacy while having a high specificity indicated by no killing of single antigen positive cells. A thera-peutic window ranging between one to two log levels could be achieved for all combina-tions mentioned above. The combinations CD133xCD24 and CD133xCD166 further-more proved its efficacy and specificity on established colorectal cancer cell lines. Be-sides the evaluation of specificity and efficacy the already introduced 1st generation of Hemibodies could be improved into a 2nd generation Hemibody format with increased half-life, stability and production yield. In future experiments the applicability of above-mentioned Hemibodies will be proven on patient-derived micro tumors to also include variables like tumor microenvironment and infiltration.},
  subject      = {Monoklonaler bispezifischer Antik{\"o}rper},
  language  = {en}
}
@article{KhayenkoMaric2019,
  author    = {Khayenko, Vladimir and Maric, Hans Michael},
  title     = {Targeting GABA\(_A\)R-associated proteins: new modulators, labels and concepts},
  series = {Frontiers in Molecular Neuroscience},
  volume    = {12},
  journal   = {Frontiers in Molecular Neuroscience},
  number    = {162},
  doi       = {10.3389/fnmol.2019.00162},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201876},
  year      = {2019},
  abstract  = {γ-aminobutyric acid type A receptors (GABA\(_A\)Rs) are the major mediators of synaptic inhibition in the brain. Aberrant GABA\(_A\)R activity or regulation is observed in various neurodevelopmental disorders, neurodegenerative diseases and mental illnesses, including epilepsy, Alzheimer's and schizophrenia. Benzodiazepines, anesthetics and other pharmaceutics targeting these receptors find broad clinical use, but their inherent lack of receptor subtype specificity causes unavoidable side effects, raising a need for new or adjuvant medications. In this review article, we introduce a new strategy to modulate GABAeric signaling: targeting the intracellular protein interactors of GABA\(_A\)Rs. Of special interest are scaffolding, anchoring and supporting proteins that display high GABA\(_A\)R subtype specificity. Recent efforts to target gephyrin, the major intracellular integrator of GABAergic signaling, confirm that GABA\(_A\)R-associated proteins can be successfully targeted through diverse molecules, including recombinant proteins, intrabodies, peptide-based probes and small molecules. Small-molecule artemisinins and peptides derived from endogenous interactors, that specifically target the universal receptor binding site of gephyrin, acutely affect synaptic GABA\(_A\)R numbers and clustering, modifying neuronal transmission. Interference with GABA\(_A\)R trafficking provides another way to modulate inhibitory signaling. Peptides blocking the binding site of GABA\(_A\)R to AP2 increase the surface concentration of GABA\(_A\)R clusters and enhance GABAergic signaling. Engineering of gephyrin binding peptides delivered superior means to interrogate neuronal structure and function. Fluorescent peptides, designed from gephyrin binders, enable live neuronal staining and visualization of gephyrin in the post synaptic sites with submicron resolution. We anticipate that in the future, novel fluorescent probes, with improved size and binding efficiency, may find wide application in super resolution microscopy studies, enlightening the nanoscale architecture of the inhibitory synapse. Broader studies on GABA\(_A\)R accessory proteins and the identification of the exact molecular binding interfaces and affinities will advance the development of novel GABA\(_A\)R modulators and following in vivo studies will reveal their clinical potential as adjuvant or stand-alone drugs.},
  language  = {en}
}
@phdthesis{Jung2016,
  author    = {Jung, Lisa Anna},
  title     = {Targeting MYC Function as a Strategy for Tumor Therapy},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-146993},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {A large fraction of human tumors exhibits aberrant expression of the oncoprotein MYC. As a transcription factor regulating various cellular processes, MYC is also crucially involved in normal development. Direct targeting of MYC has been a major challenge for molecular cancer drug discovery. The proof of principle that its inhibition is nevertheless feasible came from in vivo studies using a dominant-negative allele of MYC termed OmoMYC. Systemic expression of OmoMYC triggered long-term tumor regression with mild and fully reversible side effects on normal tissues. In this study, OmoMYC's mode of action was investigated combining methods of structural biology and functional genomics to elucidate how it is able to preferentially affect oncogenic functions of MYC. The crystal structure of the OmoMYC homodimer, both in the free and the E-box-bound state, was determined, which revealed that OmoMYC forms a stable homodimer, and as such, recognizes DNA via the same base-specific DNA contacts as the MYC/MAX heterodimer. OmoMYC binds DNA with an equally high affinity as MYC/MAX complexes. RNA-sequencing showed that OmoMYC blunts both MYC-dependent transcriptional activation and repression. Genome-wide DNA-binding studies using chromatin immunoprecipitation followed by high-throughput sequencing revealed that OmoMYC competes with MYC/MAX complexes on chromatin, thereby reducing their occupancy at consensus DNA binding sites. The most prominent decrease in MYC binding was seen at low-affinity promoters, which were invaded by MYC at oncogenic levels. Strikingly, gene set enrichment analyses using OmoMYC-regulated genes enabled the identification of tumor subgroups with high MYC levels in multiple tumor entities. Together with a targeted shRNA screen, this identified novel targets for the eradication of MYC-driven tumors, such as ATAD3A, BOP1, and ADRM1. In summary, the findings suggest that OmoMYC specifically inhibits tumor cell growth by attenuating the expression of rate-limiting proteins in cellular processes that respond to elevated levels of MYC protein using a DNA-competitive mechanism. This opens up novel strategies to target oncogenic MYC functions for tumor therapy.},
  subject      = {Myc},
  language  = {en}
}
@article{SchmidtDenkWiegering2020,
  author    = {Schmidt, Stefanie and Denk, Sarah and Wiegering, Armin},
  title     = {Targeting protein synthesis in colorectal cancer},
  series = {Cancers},
  volume    = {12},
  journal   = {Cancers},
  number    = {5},
  issn      = {2072-6694},
  doi       = {10.3390/cancers12051298},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-206014},
  year      = {2020},
  abstract  = {Under physiological conditions, protein synthesis controls cell growth and survival and is strictly regulated. Deregulation of protein synthesis is a frequent event in cancer. The majority of mutations found in colorectal cancer (CRC), including alterations in the WNT pathway as well as activation of RAS/MAPK and PI3K/AKT and, subsequently, mTOR signaling, lead to deregulation of the translational machinery. Besides mutations in upstream signaling pathways, deregulation of global protein synthesis occurs through additional mechanisms including altered expression or activity of initiation and elongation factors (e.g., eIF4F, eIF2α/eIF2B, eEF2) as well as upregulation of components involved in ribosome biogenesis and factors that control the adaptation of translation in response to stress (e.g., GCN2). Therefore, influencing mechanisms that control mRNA translation may open a therapeutic window for CRC. Over the last decade, several potential therapeutic strategies targeting these alterations have been investigated and have shown promising results in cell lines, intestinal organoids, and mouse models. Despite these encouraging in vitro results, patients have not clinically benefited from those advances so far. In this review, we outline the mechanisms that lead to deregulated mRNA translation in CRC and highlight recent progress that has been made in developing therapeutic strategies that target these mechanisms for tumor therapy.},
  language  = {en}
}
@phdthesis{Reintjes2004,
  author    = {Reintjes, Norbert},
  title     = {Taxonomy, faunistics and life-history traits of Dytiscidae and Noteridae (Coleoptera) in a West African savannah},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-10287},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2004},
  abstract  = {The studies inventoried the species of the families Dytiscidae and Noteridae (Coleoptera) in Como{\´e} National Park in northern Ivory Coast, West Africa and investigated the ecological role of temporary and permanent water bodies for the aestivation of these aquatic beetles. The ecological studies focused on the question how the beetles cope with the temporary loss of their aquatic habitats during dry season. The climate in the study area is characterised by a pronounced dry season from about November to March/April, in which the temporary ponds and creeks in the savannah entirely desiccate. The only available water bodies during dry season in Como{\´e} National Park are the Como{\´e} River, pools in some of its tributaries, and a few of the large savannah ponds. The taxonomic and faunistic analysis revealed a high species richness in the study area and yielded a total of twelve species of Noteridae in four genera and 95 species of Dytiscidae in 22 genera. Thirty of these species had not yet been reported from the Ivory Coast. A description of a new species in the genus Laccophilus is given, named L. comoensis in honour of the National Park. Strong incidences exist that the material includes more species yet unknown to science. Concerning the mode of aestivation, observations in pilot studies led to the working hypothesis that the beetles pass the dry season as adults in aquatic habitats. Consequently, presence of adults in aquatic habitats throughout the dry season and cyclic migration of adults between temporary and permanent water bodies was expected. Regular sampling of water bodies throughout the dry season and beginning rainy season yielded 33,705 individuals in 72 species and 26 genera. In all the sample periods Noteridae and / or Dytiscidae were recorded. The number of species per period was between 36 and 58. It is concluded that in Como{\´e} National Park a) at least parts of the populations of the recorded species pass the dry season as adults and b) aquatic habitats serve as a refuge for aestivation of these adult beetles. In a rocky area in the riverbed of the permanent Como{\´e} River four sets of studies were performed during dry and beginning rainy season. According to the working hypothesis beetles should be searching for adequate aquatic habitats as long as temporary savannah waters are becoming inhospitable and are falling dry. Seven rock pools in the riverbed of the Como{\´e} River were artificially filled and thus offered for colonization at the peak of the dry season (end of January). After five days the rock pools were quantitatively sampled by completely emptying them. All the rock pools were colonized by Dytiscidae and / or Noteridae and with a total of 1,507 individuals in 26 species abundance and diversity were high. Habitats for aestivation are needed most, when the majority of the savannah waters are fallen dry. Little precipitation on February 18th 1999 had filled rock pools in the riverbed of the Como{\´e} River but no pools in the savannah, where the rain was immediately absorbed by the very dry soil. An inventory of beetles was performed in 21 naturally filled rock pools five to 20 days after this precipitation. The sampling yielded 8,456 individuals in 41 species. Except the smallest, all rock pools contained beetles. The result showed that Dytiscidae and Noteridae utilise the rock pools as aquatic habitat during dry season. Beetles adapted to a highly seasonal environment like the aquatic system in the study area should be good colonizers. Sampling of four, respectively five rock pools at two occasions within 24 hours after the start of precipitation examined the potential of colonizers at that period (March). Prior to these precipitations the pools had been completely dry. Dytiscidae were already present in all rock pools and a total of 434 Dytiscidae in 14 species was found. The working hypothesis of cyclic migration suggests that the beetles should leave the rock pools at the onset of the rainy season when precipitation had filled temporary water bodies in the savannah. After several precipitation events an inventory of 13 rock pools of the Como{\´e} River in May controlled for adult beetles. Only four species with 126 individuals were still found, of which Yolina chopardi contributed 81.7\%. This species seems to differ from the other recorded species in the use of habitats, since it was never recorded in the savannah. In general, however, diversity and abundance of Dytiscidae and Noteridae in the rock pools, as expected, was low after the onset of the rainy season. During the entire study of the rock pools in the riverbed of the Como{\´e} River 10,523 individuals in 44 species and 18 genera were collected. Thus, more than half of the species recorded in Como{\´e} National Park were found in the rock pools. The results suggest that the Como{\´e} River and the rock pools in the riverbed serve as aquatic retreat for adult Dytiscidae and Noteridae during dry season when temporary water bodies in the savannah are desiccated. The suggested cyclic migration between water bodies predicts that newly formed savannah waters are recolonized by the beetles at the onset of the rainy season. This colonization should be a) by adults and b) airborne. Two artificial ponds in the open savannah were offered only for aerial colonization at the beginning of the rainy season. The ponds were controlled for adult Noteridae and Dytiscidae daily during one continuous phase of eleven and a second one of 16 days (end of March to end of April). On every sampling date Noteridae or Dytiscidae were recorded. In the entire study 2,744 individuals in 44 species and 16 genera were collected. After precipitation, abundance and species richness increased. Thirty-five of the encountered species had been recorded in rock pools of the Como{\´e} River before. The principal species in the artificial savannah ponds had been principal species in samplings of the rock pools as well. The results support the hypothesis of cyclic migration: most species of Dytiscidae and Noteridae of the Como{\´e} National Park fly from desiccating savannah waters to permanent water bodies or water bodies holding water for extended times during dry season. They pass the dry season in these waters and fly back into the savannah after precipitation at the onset of the rainy season. Exceptions from this general rule are discussed.},
  subject      = {Schwimmk{\"a}fer},
  language  = {en}
}
@article{AnkenbrandWeberBeckeretal.2016,
  author    = {Ankenbrand, Markus J. and Weber, Lorenz and Becker, Dirk and F{\"o}rster, Frank and Bemm, Felix},
  title     = {TBro: visualization and management of de novo transcriptomes},
  series = {Database},
  volume    = {2016},
  journal   = {Database},
  doi       = {10.1093/database/baw146},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-147954},
  pages     = {baw146},
  year      = {2016},
  abstract  = {RNA sequencing (RNA-seq) has become a powerful tool to understand molecular mechanisms and/or developmental programs. It provides a fast, reliable and cost-effective method to access sets of expressed elements in a qualitative and quantitative manner. Especially for non-model organisms and in absence of a reference genome, RNA-seq data is used to reconstruct and quantify transcriptomes at the same time. Even SNPs, InDels, and alternative splicing events are predicted directly from the data without having a reference genome at hand. A key challenge, especially for non-computational personnal, is the management of the resulting datasets, consisting of different data types and formats. Here, we present TBro, a flexible de novo transcriptome browser, tackling this challenge. TBro aggregates sequences, their annotation, expression levels as well as differential testing results. It provides an easy-to-use interface to mine the aggregated data and generate publication-ready visualizations. Additionally, it supports users with an intuitive cart system, that helps collecting and analysing biological meaningful sets of transcripts. TBro's modular architecture allows easy extension of its functionalities in the future. Especially, the integration of new data types such as proteomic quantifications or array-based gene expression data is straightforward. Thus, TBro is a fully featured yet flexible transcriptome browser that supports approaching complex biological questions and enhances collaboration of numerous researchers.},
  language  = {en}
}
@article{ReisSchwebsDietzetal.2018,
  author    = {Reis, Helena and Schwebs, Marie and Dietz, Sabrina and Janzen, Christian J. and Butter, Falk},
  title     = {TelAP1 links telomere complexes with developmental expression site silencing in African trypanosomes},
  series = {Nucleic Acids Research},
  volume    = {46},
  journal   = {Nucleic Acids Research},
  number    = {6},
  doi       = {10.1093/nar/gky028},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-225180},
  pages     = {2820-2833},
  year      = {2018},
  abstract  = {During its life cycle, Trypanosoma brucei shuttles between a mammalian host and the tsetse fly vector. In the mammalian host, immune evasion of T. brucei bloodstream form (BSF) cells relies on antigenic variation, which includes monoallelic expression and periodic switching of variant surface glycoprotein (VSG) genes. The active VSG is transcribed from only 1 of the 15 subtelomeric expression sites (ESs). During differentiation from BSF to the insect-resident procyclic form (PCF), the active ES is transcriptionally silenced. We used mass spectrometry-based interactomics to determine the composition of telomere protein complexes in T. brucei BSF and PCF stages to learn more about the structure and functions of telomeres in trypanosomes. Our data suggest a different telomere complex composition in the two forms of the parasite. One of the novel telomere-associated proteins, TelAP1, forms a complex with telomeric proteins TbTRF, TbRAP1 and TbTIF2 and influences ES silencing kinetics during developmental differentiation.},
  language  = {en}
}