@phdthesis{Dwertmann2012,
  author    = {Dwertmann, Anne},
  title     = {Impact of the Tumor Suppressor Arf on Miz1 and Sumoylation of Myc and Miz1},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-71876},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {Upon oncogenic stress, the tumor suppressor Arf can induce irreversible cell cycle arrest or apoptosis, depending on the oncogenic insult. In this study, it could be shown that Arf interacts with Myc and the Myc-associated zinc-finger protein Miz1 to facilitate repression of genes involved in cell adhesion. Formation of a DNA-binding Arf/Myc/Miz1 complex disrupts interaction of Miz1 with its coactivator nucleophosmin and induces local heterochromatinisation, causing cells to lose attachment and undergo anoikis. The assembly of the complex relies on Myc, which might explain why high Myc levels trigger apoptosis and not cell cycle arrest in the Arf response. This mechanism could play an important role in eliminating cells harboring an oncogenic mutation. Arf furthermore induces sumoylation of Miz1 at a specific lysine by repressing the desumoylating enzyme Senp3. A sumoylation-deficient mutant of Miz1 however does not show phenotypic differences under the chosen experimental conditions. Myc can also be modified by Sumo by multisumoylation at many different lysines, which is unaffected by Arf. The exact mechanism and effect of this modification however stays unsolved.},
  subject      = {Apoptosis},
  language  = {en}
}
@phdthesis{Koetschan2012,
  author    = {Koetschan, Christian},
  title     = {The Eukaryotic ITS2 Database - A workbench for modelling RNA sequence-structure evolution},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73128},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {In den vergangenen Jahren etablierte sich der Marker „internal transcribed spacer 2" (ITS2) zu einem h{\"a}ufig genutzten Werkzeug in der molekularen Phylogenetik der Eukaryoten. Seine schnell evolvierende Sequenz eignet sich bestens f{\"u}r den Einsatz in niedrigeren phylogenetischen Ebenen. Die ITS2 faltet jedoch auch in eine sehr konservierte Sekund{\"a}rstruktur. Diese erm{\"o}glicht die Unterscheidung weit entfernter Arten. Eine Kombination aus beiden in einer Sequenzstrukturanalyse verbessert die Aufl{\"o}sung des Markers und erm{\"o}glicht die Rekonstruktion von robusteren B{\"a}umen auf h{\"o}herer taxonomischer Breite. Jedoch war die Durchf{\"u}hrung solch einer Analyse, die die Nutzung unterschiedlichster Programme und Datenbanken vorraussetzte, f{\"u}r den klassischen Biologen nicht einfach durchf{\"u}hrbar. Um diese H{\"u}rde zu umgehen, habe ich den „ITS2 Workbench" entwickelt, eine im Internet nutzbare Arbeitsplattform zur automatisierten sequenzstrukturbasierten phylogenetischen Analyse basierend auf der ITS2 (http://its2.bioapps.biozentrum.uni-wuerzburg.de). Die Entwicklung begann mit der L{\"a}ngenoptimierung unterschiedlicher „Hidden Markov Model" (HMM)-Topologien, die erfolgreich auf ein Modell zur Sequenzstrukturvorhersage der ITS2 angewandt wurden. Hierbei wird durch die Analyse von Sequenzbestandteilen in Kombination mit der L{\"a}ngenverteilung verschiedener Helixregionen die Struktur vorhergesagt. Anschließend konnte ich HMMs auch bei der Sequenzstrukturgenerierung einsetzen um die ITS2 innerhalb einer gegebenen Sequenz zu lokalisieren. Dieses neu implementierte Verfahren verdoppelte die Anzahl vorhergesagter Strukturen und verk{\"u}rzte die Laufzeit auf wenige Tage. Zusammen mit weiteren Optimierungen des Homologiemodellierungsprozesses kann ich nun ersch{\"o}pfend Sekund{\"a}rstrukturen in mehreren Interationen vorhersagen. Diese Optimierungen liefern derzeit 380.000 annotierte Sequenzen einschließlich 288.000 Strukturvorhersagen. Um diese Strukturen f{\"u}r die Berechnung von Alignments und phylogenetischen B{\"a}umen zu verwenden hab ich das R-Paket „treeforge" entwickelt. Es erm{\"o}glicht die Generierung von Sequenzstrukturalignments auf bis zu vier unterschiedlich kodierten Alphabeten. Damit k{\"o}nnen erstmals auch strukturelle Basenpaarungen in die Alignmentberechnung mit einbezogen werden, die eine Sch{\"a}tzung neuer Scorematrizen vorraussetzten. Das R-Paket erm{\"o}glicht zus{\"a}tzlich die Rekonstruktion von „Maximum Parsimony", „Maximum Likelihood" und „Neighbour Joining" B{\"a}umen auf allen vier Alphabeten mittels weniger Zeilen Programmcode. Das Paket wurde eingesetzt, um die noch umstrittene Phylogenie der „chlorophyceae" zu rekonstruieren und k{\"o}nnte in zuk{\"u}nftigen Versionen des ITS2 workbench verwendet werden. Die ITS2 Plattform basiert auf einer modernen und sehr umfangreichen Web 2.0 Oberfl{\"a}che und beinhaltet neuste AJAX und Web-Service Technologien. Sie umfasst die HMM basierte Sequenzannotation, Strukturvorhersage durch Energieminimierung bzw. Homologiemodellierung, Alignmentberechnung und Baumrekonstruktion basierend auf einem flexiblen Datenpool, der {\"A}nderungen am Datensatz automatisch aktualisiert. Zus{\"a}tzlich wird eine Detektion von Sequenzmotiven erm{\"o}glicht, die zur Kontrolle von Annotation und Strukturvorhersage dienen kann. Eine BLAST basierte Suche auf Sequenz- und Strukturebene bietet zus{\"a}tzlich eine Vereinfachung des Taxonsamplings. Alle Funktionen sowie die Nutzung der ITS2 Webseite sind in einer kurzen Videoanleitung dargestellt. Die Plattform l{\"a}sst jedoch nur eine bestimmte Gr{\"o}ße von Datens{\"a}tzen zu. Dies liegt vor allem an der erheblichen Rechenleistung, die bei diesen Berechnungen ben{\"o}tigt wird. Um die Funktion dieses Verfahrens auch auf großen Datenmengen zu demonstrieren, wurde eine voll automatisierte Rekonstruktion des Gr{\"u}nalgenbaumes (Chlorophyta) durchgef{\"u}hrt. Diese erfolgreiche, auf dem ITS2 Marker basierende Studie spricht f{\"u}r die Sequenz-Strukturanalyse auf weiteren Daten in der Phylogenetik. Hier bietet der ITS2 Workbench den idealen Ausgangspunkt.},
  subject      = {Ribosomale RNA},
  language  = {en}
}
@phdthesis{Koethe2012,
  author    = {K{\"o}the, Susanne},
  title     = {Masernvirus-Infektion von Dendritischen Zellen und Virus-Transmission an T-Zellen},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73108},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {Dendritische Zellen (DCs) sind Antigen-pr{\"a}sentierende Zellen, die Pathogene erkennen und nach erfolgreicher Reifung spezifische adaptive Immunit{\"a}t induzieren. Die Infektion unreifer DCs durch Masernviren (MV) erfolgt CD150-abh{\"a}ngig und DC-SIGN-unterst{\"u}tzt. Infizierte DCs vermitteln wahrscheinlich den MV-Transport vom Respirationstrakt in sekund{\"a}re lym-phatische Gewebe, wo die MV-spezifische Immunit{\"a}t und die generalisierte Immunsuppressi-on initiiert werden sowie die MV-Transmission an T-Zellen stattfinden kann, die wesentlich f{\"u}r die Dissemination des Virus ist. Die MV-Infektion von iDCs initiierte deren Ausreifung begleitet von der moderaten Hochre-gulierung der CD150-Oberfl{\"a}chenexpression. Die Akkumulation viraler Proteine als auch die Freisetzung viraler Partikel waren in DCs im Vergleich zu Virus-produzierenden B-Zelllinie B95a beeintr{\"a}chtigt. Diese Arbeit verglich die subzellul{\"a}re Verteilung der viralen Proteine in DCs und B95a-Zellen. In DC wiesen Matrix (M)-Proteine eine prominente Assoziation mit den Komponenten des Ribonukleoprotein (RNP)-Komplexes auf. Die ausgepr{\"a}gte Relokali-sierung des Tetraspanins CD81 zu Phospho (P)-Protein-Kompartimenten und die Inhibition der r{\"a}umliche Interaktion der untersuchten Tetraspanine waren spezifisch f{\"u}r B95a-Zellen. Weder in B95a-Zellen noch f{\"u}r DC konnte f{\"u}r MV ein virus-containing compartment (VCC) detektiert werden, das f{\"u}r HIV-1 zuvor beschrieben wurde. Um den zellul{\"a}ren Transport des M-Proteins in infizierten, lebenden DCs untersuchen zu k{\"o}nnen, wurde das Protein carboxyterminal mit dem Tetracystein (TC)-Tag fusioniert. Das M-TC Fusionsprotein zeigte alle untersuchten biologischen Eigenschaften des Wildtyp-Proteins bez{\"u}glich seiner subzellul{\"a}ren Verteilung, der Assoziation mit DRMs sowie der Generierung und Freisetzung von virus-like particles (VPLs). Innerhalb des Viruskontextes interferierte der TC-Tag allerdings stark mit der Virusreplikation bzw. Freisetzung. Durch die Verminderung der Partikelproduktion in DCs wird eine spezielle MV-Transmissionsstruktur f{\"u}r die effiziente {\"U}bertragung an T-Zellen ben{\"o}tigt. Die MV-Transmission an autologe T-Zellen basierte vorwiegend auf Infektion von DCs (cis-Infektion) und weniger auf DC-SIGN-gebundenen Virus (trans-Infektion). Die Interaktion zwischen dem MV-Glykoprotein H mit seinem Rezeptor CD150 war wichtig f{\"u}r die Transmission. Die Transmission von MV erfolgte haupts{\"a}chlich durch die Bildung von Kontaktfl{\"a}chen, entspre-chend den beschriebenen virologischen Synapsen, wo virale Proteine akkumulierten und CD150 aktinabh{\"a}ngig rekrutiert wurde, und seltener {\"u}ber aktinreiche Filopodien. Die HIV-VS Markerproteine ICAM-1, aktiviertes LFA-1, CD81, DC-SIGN und der phosphorylierte Ezrin / Radixin / Moesin (ERM)-Proteinkomplex polarisierten zur MV-VS. Moesin und der Substanz P Rezeptor (SPR), die Prozesse des MV-Eintritts oder der Aufnahme unterst{\"u}tzen, akkumulierten ebenfalls in den Transmissionsstrukturen. Zusammengefasst zeigte diese Arbeit, dass die gebildete Plattform f{\"u}r MV-Transmission (MV-VS) wichtige Gemeinsamkeiten mit der HIV-VS teilt. In der MV-VS akkumulierten Proteine, die Aktindynamiken regulieren, die die Konjugatstabilit{\"a}t verst{\"a}rken und die die Membranfusion unterst{\"u}tzen, die einen effizienten Eintritt des MV in T-Zellen erm{\"o}glichen.},
  subject      = {Masernvirus},
  language  = {de}
}
@phdthesis{Simon2012,
  author    = {Simon, Nina Monica},
  title     = {Molecular interactions of the malaria parasite Plasmodium falciparum during the sexual reproduction in the mosquito midgut},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72403},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {The sexual phase of Plasmodium falciparum begins with the differentiation of intraerythrocytic sexual stages, termed gametocytes, in the human host. Mature gametocytes circulate in the peripheral blood and are taken up by the mosquito during the blood meal. These stages are essential for the spread of the malaria disease and form gametes in the mosquito midgut within minutes. A highly conserved family of six secreted proteins has been identified in Plasmodium falciparum. They comprise multiple adhesive domains and are termed PfCCp1 through PfCCp5, and PfFNPA. It was revealed in this work that PfCCp multi-domain adhesion proteins form protein complexes in gametocytes and on the surface of newly emerged macrogametes by adhesion domain-mediated binding. Co-Immunoprecipitation assays with activated gametocyte lysates show interactions between PfCCp proteins and indicate surface association via Pfs230 and Pfs25. Pfs230 is connected with the plasma membrane of the parasite by its interaction partner Pfs48/45. This protein is linked to the plasma membrane by a GPI anchor and presumably retains the multi-protein complex on the surface of newly emerged macrogametes in the mosquito midgut. A WD40 domain containing protein was identified to be part of this protein complex. It might serve as platform for the assembly of the multi protein complex or mediate the interplay among proteins, as suggested from known functions of the WD40 domain repeats. During egress from the host erythrocyte, the emerging gametes become vulnerable to factors of the human complement, which is taken up with the blood meal. In this thesis it was found that the complement system is active for about one hour post feeding. Macrogametes defend against complement-mediated lysis by co-opting the human complement regulators Factor H and FHL-1 from the blood-meal. These serum proteins bind via its SCR domains 5-7 to the surface of macrogametes. Once bound, they trigger complement inactivation of the alternative pathway, which prevents induction of complement lysis on the surface of the malaria parasite. Antibodies against Factor H are able to impair the sexual development in vitro and are able to block transmission to the mosquito. Interaction studies on endogenous proteins and immobilized recombinant proteins revealed the PfGAP50 protein as binding partner of Factor H and FHL-1. This protein was hitherto described as a glideosome-associated protein in invasive parasite stages, but has not yet been characterized in gametes. First localization studies indicate a relocation of PfGAP50 from the inner membrane complex to the surface of macrogametes. Malaria still persists as one of the deadliest infectious diseases worldwide. Investigations on the essential transmissive stages, gametocytes and gametes of Plasmodium falciparum, stood in the background of research for a long time. This work deciphered details on protein interactions on the surface of the malaria parasite and provides first information about coactions between the parasite and the human complement in the mosquito midgut.},
  subject      = {Plasmodium falciparum},
  language  = {en}
}
@phdthesis{Froelich2012,
  author    = {Fr{\"o}lich, Nadine},
  title     = {Analyse der µ-Opiatrezeptoraktivierung und Signaltransduktion in lebenden Zellen mittels FRET-Mikroskopie},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-71009},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {Der Fluoreszenz-Resonanz-Energie-Transfer ist ein Ph{\"a}nomen, welches erstmals 1948 von Theodor F{\"o}rster beschrieben wurde. Mit der Entwicklung von Fluoreszenzproteinen konnten in Kombination mit Mikroskopietechniken Einblicke in zellbiologische Vorg{\"a}nge gewonnen werden, die durch biochemische oder physiologische Experimente nicht m{\"o}glich sind. Dabei spielt die hohe zeitliche und r{\"a}umliche Aufl{\"o}sung eine wichtige Rolle. Auf dem Forschungsgebiet der GPCR, welche die gr{\"o}ßte Gruppe von Membranproteinen bei den S{\"a}ugetieren darstellen, wurden insbesondere Erkenntnisse {\"u}ber Konformations{\"a}nderungen der Rezeptoren, die Kinetik der Rezeptoraktivierung und die Interaktion mit intrazellul{\"a}ren Signalproteinen gewonnen. Der µ-Opioidrezeptor geh{\"o}rt zur Familie der GPCR und stellt aufgrund seiner analgetischen Wirkungen eine wichtige pharmakologische Zielstruktur dar. Das Ziel dieser Arbeit war sowohl den Rezeptor als auch seine Signalwege mittels FRET-Mikroskopie zu untersuchen. Zun{\"a}chst sollte ein intramolekularer FRET-Sensor des µ-Opioidrezeptors entwickelt werden, dazu wurden basierend auf den Kenntnissen {\"u}ber die Terti{\"a}rstruktur und dem Aufbau bereits bekannter GPCR-Sensoren verschiedene Rezeptorkonstrukte kloniert. Bei den Konstrukten wurden entweder zwei Fluoreszenzproteine oder ein Fluoreszenzprotein und ein Fluorophor-bindendes Tetracysteinmotiv kombiniert. Auch die Positionen der eingef{\"u}gten Sequenzen wurden in den intrazellul{\"a}ren Dom{\"a}nen variiert, da der Rezeptor auf die Modifikationen mit beeintr{\"a}chtigter Membranlokalisation reagierte. Durch die Optimierung wurden Rezeptoren konstruiert, die an der Zellmembran lokalisiert waren. Jedoch zeigte keines der Rezeptorkonstrukte Funktionalit{\"a}t im Hinblick auf die Rezeptoraktivierung. Im zweiten Teil wurden die pharmakologischen Effekte der Metabolite von Morphin am humanen µ-Opioidrezeptor systematisch analysiert. Dazu wurde die F{\"a}higkeit der Metabolite, Gi-Proteine zu aktivieren und β-Arrestin2 zu rekrutieren, mittels FRET-basierter Messungen an lebenden Zellen untersucht. Außerdem wurde die Affinit{\"a}t der Metabolite zum humanen µ Opioidrezeptor anhand der Verdr{\"a}ngung eines radioaktiven Liganden analysiert. Meine Experimente identifizierten eine Gruppe mit stark agonistischen und eine mit schwach agonistischen Eigenschaften. Die starken Partialagonisten aktivieren den Rezeptor bereits bei nanomolaren Konzentrationen, w{\"a}hrend die schwachen Metabolite den Rezeptor erst bei Konzentrationen im mikromolaren Bereich aktivieren. Die Metabolite Normorphin, Morphin-6-Glucuronid und 6-Acetylmorphin zeigen geringere Potenz als Morphin bei der Gi-Aktivierung aber {\"u}berraschenderweise h{\"o}here Potenz und Effizienz f{\"u}r die β-Arrestin-Rekrutierung. Dies deutet auf eine bevorzugte Aktivierung von β-Arrestin2 hin. Die aus diesen Studien gewonnenen Ergebnisse liefern Hinweise darauf, welche Metabolite bei der Signalverarbeitung am µ Opioidrezeptor in vivo beteiligt sind.},
  subject      = {Opiatrezeptor},
  language  = {de}
}
@phdthesis{AlcantarinoMenescal2012,
  author    = {Alcantarino Menescal, Luciana},
  title     = {In vivo characterization of genetic factors involved in Xmrk driven melanoma formation in Medaka (Oryzias latipes): a closer look at braf, Stat5 and c-myc},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-70762},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {Melanoma arises from the malignant transformation of melanocytes and is one of the most aggressive forms of human cancer. In fish of the genus Xiphophorus, melanoma development, although very rarely, happens spontaneously in nature and can be induced by interspecific crossing. The oncogenic receptor tyrosine kinase, Xmrk, is responsible for melanoma formation in these fishes. Since Xiphophorus are live-bearing fishes and therefore not compatible with embryonic manipulation and transgenesis, the Xmrk melanoma model was brought to the medaka (Oryzias latipes) system. Xmrk expression under the control of the pigment cell specific mitf promoter leads to melanoma formation with 100\% penetrance in medaka. Xmrk is an orthologue of the human epidermal growth factor receptor (EGFR) and activates several downstream signaling pathways. Examples of these pathways are the direct phosphorylation of BRAF and Stat5, as well as the enhanced transcription of C-myc. BRAF is a serine-threonine kinase which is found mutated at high frequencies in malignant melanomas. Stat5 is a transcription factor known to be constitutively activated in fish melanoma. C-myc is a transcription factor that is thought to regulate the expression of approximately 15\% of all human genes and is involved in cancer progression of a large number of different tumors. To gain new in vivo information on candidate factors known to be involved in melanoma progression, I identified and analysed BRAF, Stat5 and C-myc in the laboratory fish model system medaka. BRAF protein motifs are highly conserved among vertebrates and the results of this work indicate that its function in the MAPK signaling is maintained in medaka. Transgenic medaka lines carrying a constitutive active version of BRAF (V614E) showed more pigmented skin when compared to wild type. Also, some transiently expressing BRAF V614E fishes showed a disrupted eye phenotype. In addition, I was able to identify two Stat5 copies in medaka, named Stat5ab/a and Stat5ab/b. Sequence analysis revealed a higher similarity between both Stat5 sequences when compared to either human Stat5a or Stat5b. This suggests that the two Stat5 copies in medaka arose by an independent duplication processes. I cloned these two Stat5 present in medaka, produced constitutive active and dominant negative gene versions and successfully established transgenic lines carrying each version under the control of the MITF promoter. These lines will help to elucidate questions that are still remaining in Stat5 biology and its function in melanoma progression, like the role of Stat5 phosphorylation on tumor invasiveness. In a third project during my PhD work, I analysed medaka C-myc function and indentified two copies of this gene in medaka, named c-myc17 and c-myc20, according to the chromosome where they are located. I produced conditional transgenic medaka lines carrying the c-myc17 gene coupled to the hormone binding domain of the estrogen receptor to enable specific transgene activation at a given time point. Comparable to human C-myc, medaka C-myc17 is able to induce proliferation and apoptosis in vivo after induction. Besides that, C-myc17 long-term activation led to liver hyperplasia. In summary, the medaka models generated in this work will be important to bring new in vivo information on genes involved in cancer development. Also, the generated transgenic lines can be easily crossed to the melanoma developing Xmrk medaka lines, thereby opening up the possibility to investigate their function in melanoma progression. Besides that, the generated medaka fishes make it possible to follow the whole development of melanocytes, since the embryos are transparent and can be used for high throughput chemical screens.},
  subject      = {Japank{\"a}rpfling},
  language  = {en}
}
@phdthesis{Hupp2012,
  author    = {Hupp, Sabrina},
  title     = {Modulation of Actin Dynamics by the Cholesterol-Dependent Cytolysin Pneumolysin - a novel mechanism beyond pore formation},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-70889},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {Streptococcus pneumoniae is one of the major causes of bacterial meningitis, which mainly affects young infants in the developing countries of Africa, Asia (esp. India) and South America, and which has case fatality rates up to 50\% in those regions. Bacterial meningitis comprises an infection of the meninges and the sub-meningeal cortex tissue of the brain, whereat the presence of pneumolysin (PLY), a major virulence factor of the pneumococcus, is prerequisite for the development of a severe outcome of the infection and associated tissue damage (e. g. apoptosis, brain edema, and ischemia). Pneumolysin belongs to the family of pore forming, cholesterol-dependent cytolysins (CDCs), bacterial protein toxins, which basically use membrane-cholesterol as receptor and oligomerize to big aggregates, which induce cell lysis and cell death by disturbance of membrane integrity. Multiple recent studies, including this work, have revealed a new picture of pneumolysin, whose cell-related properties go far beyond membrane binding, pore formation and the induction of cell death and inflammatory responses. For a long time, it has been known that bacteria harm the tissues of their hosts in order to promote their own survival and proliferation. Many bacterial toxins aim to rather hijack cells than to kill them, by interacting with cellular components, such as the cytoskeleton or other endogenous proteins. This study was able to uncover a novel capacity of pneumolysin to interact with components of the actin machinery and to promote rapid, actin-dependent cell shape changes in primary astrocytes. The toxin was applied in disease-relevant concentrations, which were verified to be sub-lytic. These amounts of toxin induced a rapid actin cortex collapse in horizontal direction towards the cell core, whereat membrane integrity was preserved, indicating an actin severing function of pneumolysin, and being consistent with cell shrinkage, displacement, and blebbing observed in live cell imaging experiments. In contrast to neuroblastoma cells, in which pneumolysin led to cytoskeleton remodeling and simultaneously to activation of Rac1 and RhoA, in primary astrocytes the cell shape changes were seen to be primarily independent of small GTPases. The level of activated Rac1 and RhoA did not increase at the early time points after toxin application, when the initial shape changes have been observed, but at later time points when the actin-dependent displacement of cells was slower and less severe, probably presenting the cell's attempt to re-establish proper cytoskeleton function. A GUV (giant unilamellar vesicle) approach provided insight into the effects of pneumolysin in a biomimetic system, an environment, which is strictly biochemical, but still comprises cellular components, limited to the factors of interest (actin, Arp2/3, ATP, and Mg2+ on one side, and PLY on the other side). This approach was able to show that the wildtype-toxin, but not the Δ6 mutant (mutated in the unfolding domain, and thus non-porous), had the capacity to exhibit its functions through a membrane bilayer, meaning it was able to aggregate actin, which was located on the other side of the membrane, either via direct interaction with actin or in an Arp2/3 activating manner. Taking a closer look at these two factors with the help of several different imaging and biochemical approaches, this work unveiled the capacity of pneumolysin to bind and interact both with actin and Arp2 of the Arp2/3 complex. Pneumolysin was capable to slightly stabilize actin in an actin-pyrene polymerization assay. The same experimental setup was applied to show that the toxin had the capacity to lead to actin polymerization through activation of the Arp2/3 complex. This effect was additionally confirmed with the help of fluorescent microscopy of rhodamine (TRITC)-tagged actin. Strongest Arp2/3 activation, and actin nucleation/polymerization is achieved by the VCA domain of the WASP family proteins. However, addition of PLY to the Arp2/3-VCA system led to an enhanced actin nucleation, suggesting a synergistic activation function of pneumolysin. Hence, two different effects of pneumolysin on the actin cytoskeleton were observed. On the one hand an actin severing property, and on the other hand an actin stabilization property, both of which do not necessarily exclude each other. Actin remodeling is a common feature of bacterial virulence strategies. This is the first time, however, that these properties were assigned to a toxin of the CDC family. Cytoskeletal dysfunction in astrocytes leads to dysfunction and unregulated movement of these cells, which, in context of bacterial meningitis, can favor bacterial penetration and spreading in the brain tissue, and thus comprises an additional role of pneumolysin as a virulence factor of Streptococcus pneumonia in the context of brain infection.},
  subject      = {Hirnhautentz{\"u}ndung},
  language  = {en}
}
@phdthesis{Foertsch2012,
  author    = {F{\"o}rtsch, Christina},
  title     = {Pneumolysin: the state of pore-formation in context to cell trafficking and inflammatory responses of astrocytes},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-70892},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {Pneumolysin, a protein toxin, represents one of the major virulence factors of Streptococcus pneumoniae. This pathogen causes bacterial meningitis with especially high disease rates in young children, elderly people and immunosuppressed patients. The protein toxin belongs to the family of cholesterol-dependent cytolysins, which require membrane cholesterol in order to bind and to be activated. Upon activation, monomers assemble in a circle and undergo conformational change. This conformational change leads to the formation of a pore, which eventually leads to cell lysis. This knowledge was obtained by studies that used a higher concentration compared to the concentration of pneumolysin found in the cerebrospinal fluid of meningitis patients. Thus, a much lower concentration of pneumolysin was used in this work in order to investigate effects of this toxin on primary mouse astrocytes. Previously, a small GTPase activation, possibly leading to cytoskeletal changes, was found in a human neuroblastoma cell line. This led to the hypothesis that pneumolysin can lead to similar cytoskeletal changes in primary cells. The aim of this work was to investigate and characterise the effects of pneumolysin on primary mouse astrocytes in terms of a possible pore formation, cellular trafficking and immunological responses. Firstly, the importance of pore-formation on cytoskeletal changes was to be investigated. In order to tackle this question, wild-type pneumolysin and two mutant variants were used. One variant was generated by exchanging one amino acid in the cholesterol recognising region, the second variant was generated by deleting two amino acids in a protein domain that is essential for oligomerisation. These variants should be incapable of forming a pore and were compared to the wild-type in terms of lytic capacities, membrane binding, membrane depolarisation, pore-formation in artificial membranes (planar lipid bilayer) and effects on the cytoskeleton. These investigations resulted in the finding that the pore-formation is required for inducing cell lysis, membrane depolarisation and cytoskeletal changes in astrocytes. The variants were not able to form a pore in planar lipid bilayer and did not cause cell lysis and membrane depolarisation. However, they bound to the cell membrane to the same extent as the wild-type toxin. Thus, the pore-formation, but not the membrane binding was the cause for these changes. Secondly, the effect of pneumolysin on cellular trafficking was investigated. Here, the variants showed no effect, but the wild-type led to an increase in overall endocytotic events and was itself internalised into the cell. In order to characterise a possible mechanism for internalisation, a GFP-tagged version of pneumolysin was used. Several fluorescence-labelled markers for different endocytotic pathways were used in a co-staining approach with pneumolysin. Furthermore, inhibitors for two key-players in classical endocytotic pathways, dynamin and myosin II, were used in order to investigate classical endocytotic pathways and their possible involvement in toxin internalisation. The second finding of this work is that pneumolysin is taken up into the cell via dynamin- and caveolin-independent pinocytosis, which could transfer the toxin to caveosomes. From there, the fate of the toxin remains unknown. Additionally, pneumolysin leads to an overall increase in endocytotic events. This observation led to the third aim of this work. If the toxin increases the overall rate of endocytosis, the question arises whether toxin internalisation favours bacterial tissue penetration of the host or whether it serves as a defence mechanism of the cell in order to degrade the protein. Thus, several proinflammatory cytokines were investigated, as previous studies describe an effect of pneumolysin on cytokine production. Surprisingly, only interleukin 6-production was increased after toxin-treatment and no effect of endocytotic inhibitors on the interleukin 6-production was observed. The conclusion from this finding is that pneumolysin leads to an increase of interleukin 6, which would not depend on the endocytotic uptake of pneumolysin. The production of interleukin 6 would enhance the production of acute phase proteins, T-cell activation, growth and differentiation. On the one hand, this activation could serve pathogen clearance from infected tissue. On the other hand, the production of interleukin 6 could promote a further penetration of pathogen into host tissue. This question should be further investigated.},
  subject      = {Streptococcus pneumoniae},
  language  = {en}
}
@phdthesis{Tyagi2012,
  author    = {Tyagi, Anu},
  title     = {Role of SWI/SNF in regulating pre-mRNA processing in Drosophila melanogaster},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72253},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {ATP dependent chromatin remodeling complexes are multifactorial complexes that utilize the energy of ATP to rearrange the chromatin structure. The changes in chromatin structure lead to either increased or decreased DNA accessibility. SWI/SNF is one of such complex. The SWI/SNF complex is involved in both transcription activation and transcription repression. The ATPase subunit of SWI/SNF is called SWI2/SNF2 in yeast and Brahma, Brm, in Drosophila melanogaster. In mammals there are two paralogs of the ATPase subunit, Brm and Brg1. Recent studies have shown that the human Brm is involved in the regulation of alternative splicing. The aim of this study was to investigate the role of Brm in pre-mRNA processing. The model systems used were Chironomus tentans, well suited for in situ studies and D. melanogaster, known for its full genome information. Immunofluorescent staining of the polytene chromosome indicated that Brm protein of C. tentans, ctBrm, is associated with several gene loci including the Balbiani ring (BR) puffs. Mapping the distribution of ctBrm along the BR genes by both immuno-electron microscopy and chromatin immunoprecipitation showed that ctBrm is widely distributed along the BR genes. The results also show that a fraction of ctBrm is associated with the nascent BR pre-mRNP. Biochemical fractionation experiments confirmed the association of Brm with the RNP fractions, not only in C. tentans but also in D. melanogaster and in HeLa cells. Microarray hybridization experiments performed on S2 cells depleted of either dBrm or other SWI/SNF subunits show that Brm affects alternative splicing and 3´ end formation. These results indicated that BRM affects pre-mRNA processing as a component of SWI/SNF complexes. 1},
  subject      = {Taufliege},
  language  = {en}
}
@phdthesis{Leikam2012,
  author    = {Leikam, Claudia},
  title     = {Oncogene-induced senescence in melanocytes},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-79316},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {Melanoma is the most aggressive skin cancer with very limited treatment options. Upon appearance of metastases chemotherapeutics are used to either kill or slow down the growth of cancer cells by inducing apoptosis or senescence, respectively. With melanomas originating from melanocytes, it is vital to elucidate the mechanisms that distinguish senescence induction from proliferation and tumourigenicity. Xmrk (Xiphophorus melanoma receptor kinase), the fish orthologue of the human epidermal growth factor receptor (EGFR), causes highly aggressive melanoma in fish. Using an inducible variant, HERmrk, I showed that high receptor levels result in melanocyte senescence, whereas low and medium expression allows for cell proliferation and tumourigenicity. Mechanistically, HERmrk leads to increased reactive oxygen species (ROS) levels, which trigger a DNA damage response. Consequently, multinucleated, senescent cells develop by both endomitosis and fusion. Furthermore, oncogenic N-RAS (N--RAS61K) induces a similar multinucleated phenotype in melanocytes. In addition, I found that both overexpression of C-MYC and the knockdown of miz­-1 (Myc­-interacting zinc finger protein 1) diminished HERmrk-induced senescence entry. C-MYC prevent ROS induction, DNA damage and senescence, while acting synergistically with HERmrk in conveying tumourigenic features to melanocytes. Further analyses identified cystathionase (CTH) as a novel target gene of Myc and Miz-­1 crucial for senescence prevention. CTH encodes an enzyme involved in the synthesis of cysteine from methionine, thereby allowing for increased ROS detoxification. Even though senescence was thought to be irreversible and hence tumour protective, I demonstrated that prolonged expression of the melanoma oncogene N­-RAS61K in pigment cells overcomes initial OIS by triggering the emergence of tumour-initiating, mononucleated stem-like cells from multinucleated senescent cells. This progeny is dedifferentiated, highly proliferative, anoikis­-resistant and induces fast­-growing, metastatic tumours upon transplantation into nude mice. Our data demonstrate that induction of OIS is not only a cellular failsafe mechanism, but also carries the potential to provide a source for highly aggressive, tumour­-initiating cells.},
  subject      = {Melanom},
  language  = {en}
}