@article{ZhangSiPahl2012, author = {Zhang, Shaowu and Si, Aung and Pahl, Mario}, title = {Visually guided decision making in foraging honeybees}, series = {Frontiers in Neuroscience}, volume = {6}, journal = {Frontiers in Neuroscience}, number = {88}, doi = {10.3389/fnins.2012.00088}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-124228}, year = {2012}, abstract = {Honeybees can easily be trained to perform different types of discrimination tasks under controlled laboratory conditions. This review describes a range of experiments carried out with free-flying forager honeybees under such conditions. The research done over the past 30 or so years suggests that cognitive abilities (learning and perception) in insects are more intricate and flexible than was originally imagined. It has become apparent that honeybees are capable of a variety of visually guided tasks, involving decision making under challenging situations: this includes simultaneously making use of different sensory modalities, such as vision and olfaction, and learning to use abstract concepts such as "sameness" and "difference." Many studies have shown that decision making in foraging honeybees is highly flexible. The trained animals learn how to solve a task, and do so with a high accuracy, but when they are presented with a new variation of the task, they apply the learnt rules from the earlier setup to the new situation, and solve the new task as well. Honeybees therefore not only feature a rich behavioral repertoire to choose from, but also make decisions most apt to the current situation. The experiments in this review give an insight into the environmental cues and cognitive resources that are probably highly significant for a forager bee that must continually make decisions regarding patches of resources to be exploited.}, language = {en} } @phdthesis{Nguyen2012, author = {Nguyen, Hoang Duong}, title = {Vaccinia virus mediated expression of human erythropoietin in colonized human tumor xenografts results in faster tumor regression and increased red blood cell biogenesis in mice}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85383}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Cancer-related anemia is prevalent in cancer patients. Anemia negatively affects normal mental and physical function capacity with common symptoms s like fatigue, headache, or depression. Human erythropoietin (hEPO), a glycoprotein hormone regulating red blood cell formation, is approved for the treatment of cancer-related anemia. It has shown benefits in correcting anemia, and subsequently improving health-related quality of life and/or enhancing radio-, and chemotherapy. Several recent clinical trials have suggested that recombinant hEPO (rhEPO) may promote tumor growth that raises the questions concerning the safety of using rhEPO for cancer treatment. However in others, such effects were not indicated. As of today, the direct functional effect of rhEPO in tumor models remains controversial and needs to be further analyzed. Based on the GLV-1h68 backbone, the hEPO-expressing recombinant VACV strains (EPO-VACVs) GLV-1h210, GLV-1h211, GLV-1h212 and GLV-1h213 were generated by replacing the lacZ expression cassette at the J2R locus with hEPO under the control of different vaccinia promoters p7.5, pSE, pSEL, pSL, respectively. Also, GLV-1h209 was generated, which is similar to GLV-1h210 but expresses a mutated non-functinal EPO (R103A). The EPO-VACV strains were characterized for their oncolytic efficacy in lung (A549) cancer cells in culture and tumor xenografts. Concomitantly, the effects of locally expressed hEPO in tumors on virus replication, host immune infiltration, tumor vascularization and tumor growth were also evaluated. As expected, EPO-VACVs enhanced red blood cell (RBC) formation in xenograft model. The number of RBCs and hemoglobin (Hb) levels were significantly increased in EPO-VACVs-treated mice compared to GLV-1h68-treated or untreated control mice. However, the mean size of RBC or Hb content per RBC remained normal. Furthermore, over-expression of hEPO did not significantly affect numbers of lymphocytes, monocytes, leucocytes or platelets in the peripheral blood stream. The expression of hEPO in colonized tumors of mice treated with EPO-VACVs was demonstrated by immunohistological staining. Interestingly, there were 9 - 10 hEPO isoforms detected either in tumors, cells, or supernatant, while 3-4 basic isoforms were missing in blood serum, where only six hEPO isoforms were found. Tumor-bearing mice after treatment with EPO-VACVs showed enhanced tumor regression compared to GLV-1h68. The virus titers in tumors in EPO-VACVs-treated mice were 3-4 fold higher compared to GLV-1h68-treated mice. Nevertheless, no significant difference in virus titers among EPO-VACVs was found. The blood vessels in tumors were significantly enlarged while the blood vessel density remained unchanged compared to the GLV-1h68 treated mice, indicating that hEPO did not affect endothelial cell proliferation in this model. Meanwhile, rhEPO (Epoetin alfa) alone or in combination with GLV-1h68 did not show any signs of enhanced tumor growth when compared to untreated controls and GLV-1h68 groups, while doses used were clinical relevant (500 U/kg). These findings suggested that hEPO did not promote angiogenesis or tumor growth in the A549 tumor xenograft model. Human EPO has been reported to function as an immune modulator. In this study, however, we did not find any involvement of hEPO in immune cytokine and chemokine expression or innate immune cell infiltration (leucocytes, B cells, macrophages and dendritic cells) into infected tumors. The degree of immune infiltration and cytokine expression was directly correlated to the number of virus particles. Increased virus replication, led to more recruited immune cells and secreted cytokines/chemokines. It was proposed that tumor regression was at least partially mediated through activation of innate immune mechanisms. In conclusion, the novel EPO-VACVs were shown to significantly increase the number of RBCs, Hb levels, and virus replication in tumors as well as to enhance tumor regression in the A549 tumor xenograft model. Moreover, locally expressed hEPO did not promote tumor angiogenesis, tumor growth, and immune infiltration but was shown to causing enlarged tumoral microvessels which facilitated virus spreading. It is conceivable that in a possible clinical application, anemic cancer patients could benefit from the EPO-VACVs, where they could serve as "wellness pills" to decrease anemic symptoms, while simultaneously destroying tumors.}, subject = {Erythropoietin}, language = {en} } @phdthesis{Goehler2012, author = {G{\"o}hler, Antonia}, title = {Untersuchung Karbohydrat-bindender Proteine mit hoher zeitlicher und r{\"a}umlicher Aufl{\"o}sung}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-76665}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Das menschliche Genom verschl{\"u}sselt 30000 bis 40000 Proteine, von denen ein Großteil kovalent gebundene Karbohydrat-Gruppen an Asparagin-, Serin-, Threonin- oder Hydroxylysin-Resten tr{\"a}gt. Diese sogenannten Glykoproteine sind allgegenw{\"a}rtige Bestandteile der extrazellul{\"a}ren Matrix von Zelloberfl{\"a}chen. Sie steuern Zell-Zell- und Zell-Matrix-Kommunikationen, k{\"o}nnen bei der roteinfaltung helfen bzw. die Proteinstabilit{\"a}t erh{\"o}hen oder Immunantworten regulieren. Die Ausl{\"o}sung von biologischen Prozesse erfordert aber {\"U}bersetzer der zuckerbasierten Informationen. Solche Effektoren sind die Lektine, unter ihnen auch die Galektine. Galektine binden spezifisch β-Galaktosen, weisen strukturelle {\"U}bereinstimmungen in der Aminos{\"a}uresequenz ihrer Zuckererkennungsdom{\"a}nen (CRDs) auf und zeigen ein „jelly-roll"-Faltungsmuster, bestehend aus einem β-Sandwich mit zwei antiparallelen Faltbl{\"a}ttern. Strukturell werden die CRDs in drei verschiedenen, topologischen Formen pr{\"a}sentiert. Proto-Typen existieren als nicht-kovalent verkn{\"u}pfte Dimere der CRDs, Chimera-Typen besitzen neben der CRD eine Nicht-Lektin-Dom{\"a}ne und bei den Tandem-Repeat-Typen sind zwei verschiedene CRDs {\"u}ber ein kurzes Linker-Peptid kovalent verbunden. Galektine werden sowohl in normalem wie auch pathogenem Gewebe exprimiert und das zunehmende Wissen {\"u}ber die Beteiligung an verschiedenen Krankheiten und Tumorwachstum liefert die Motivation, strukturelle Aspekte und die Vernetzung von Lektinen detailliert, insbesondere im Hinblick auf ihre intrafamili{\"a}ren Unterschiede, zu untersuchen. Durch die Kombination verschiedener Spektroskopie-Techniken mit hoher zeitlicher und r{\"a}umlicher Aufl{\"o}sung, basierend auf der Verwendung von Fluorophoren (intrinsisch und extrinsisch), werden in dieser Arbeit die Eigenschaften von Galektinen n{\"a}her untersucht. Mit Fluoreszenz-Korrelations-Spektroskopie (FCS) und Anisotropie-Messungen wird gezeigt, dass eine Liganden-Bindung bei Proto-Typ-Galektinen mit einer Verringerung des hydrodynamischen Radius einhergeht. Bei Tandem-Repeat- und Chimera-Typen bleibt der Radius konstant. Daf{\"u}r skaliert die Diffusionskonstante von Tandem-Repeat-Typen anormal mit der molaren Masse. Die Anisotropie-Messungen werden parallel zu den FCS-Messungen durchgef{\"u}hrt, um einen Einfluss des Fluoreszenzmarkers auszuschließen. Mit Hilfe dieser Technik wird außerdem gezeigt, dass unterschiedliche Dissoziationskonstanten und Kinetiken f{\"u}r den Bindungsprozess innerhalb der Proto-Typ-Gruppe m{\"o}glichweise auf unterschiedliche Konformationsdynamiken zur{\"u}ckgehen. Der Vergleich von hGal-1 und cG-1B verdeutlicht, dass strukturelle {\"A}hnlichkeiten zwar ein identisches Bindungsverhalten hervorrufen k{\"o}nnen, der Oxidationsprozess der Proteine aber unterschiedlich ablaufen kann. Beide Methoden k{\"o}nnen so als sehr sensitive Techniken zur Untersuchung von Strukturmerkmalen bei Galektinen etabliert werden, wobei die {\"U}bertragbarkeit auf andere Glykoproteine gew{\"a}hrleistet ist. Weiterhin gilt Quervernetzung als eine der wichtigsten Eigenschaften von Galektinen, da durch die Vernetzung von Glykoproteinen auf der Zelloberfl{\"a}che Signalwege aktiviert und Immunantworten reguliert werden. Um die r{\"a}umliche organisation und Quervernetzung von hGal-1 auf den Oberfl{\"a}chen von Neuroblastomzellen nachzuweisen, eignet sich das hochaufl{\"o}sende Mikroskopieverfahren dSTORM sehr gut. Durch Verwendung des photoschaltbaren Fluorophors Alexa647 als spezifischem Marker f{\"u}r hGal-1, einem Standard-Weitfeld-Aufbau und verschiedenen Analyseverfahren, kann eine Clusterformation von hGal-1 auf der Zelloberfl{\"a}che best{\"a}tigt werden. hGal-1 bildet Cluster mit einem mittleren Durchmesser von 81±7 nm aus. Der Durchmesser ist unabh{\"a}ngig von der Konzentration, w{\"a}hrend die Anzahl der Cluster davon abh{\"a}ngt. F{\"u}r die Clusterausbildung ist ein Startpunkt, also eine minimale Dichte der Galektin-Molek{\"u}le, notwendig. Durch Blockierung der CRDs mit Laktose wird die Clusterbildung unterdr{\"u}ckt und die Spezifit{\"a}t der CRDs gegen{\"u}ber β-Galaktosen erneut herausgestellt. Anders als dimeres hGal-1 binden Monomere deutlich schlechter an die Membranrezeptoren. Es werden keine Cluster ausgebildet, eine Quervernetzung von Membranrezeptoren ist nicht m{\"o}glich. Außerdem kann es durch die Monomere zu einer vollst{\"a}ndigen Markierung und damit Abkugellung der Zellen kommen. M{\"o}glicherweise wird der Zelltod induziert. Hochaufl{\"o}sende Mikroskopieverfahren sind durch den Markierungsprozess limitiert. Die bioorthogonale Click-Chemie er{\"o}ffnet jedoch neue M{\"o}glichkeiten zur Markierung und Visualisierung von Biomolek{\"u}len, ohne die Notwenigkeit genetischer Manipulationen. Es werden modifizierte Zuckermolek{\"u}le in die Zellmembranen eingebaut, {\"u}ber eine 1,3-polare Cycloaddition mit einem Alkin markiert und ihre Verteilung mit Hilfe von dSTORM untersucht. Es wird nachgewiesen, dass die Zuckermolek{\"u}le in Clustern auftreten und Click-Chemie trotz dem Katalysator Kupfer an lebenden Zellen durchf{\"u}hrbar ist. Die Bewegung der Gesamtcluster wird mittels Mean Square Displacement aufgeschl{\"u}sselt und eine Diffusionskonstante f{\"u}r Cluster im Bereich von 40 - 250 nm bestimmt. Zusammenfassend stellt die Kombination verschiedener Spektroskopie-Techniken ein gutes Werkzeug zur Untersuchung von Karbohydrat-bindendenden Proteinen mit hoher r{\"a}umlicher und zeitlicher Aufl{\"o}sung dar und erm{\"o}glicht einen neuen Einblick in die Biologie der Galektine.}, subject = {Fluoreszenz}, language = {de} } @phdthesis{Oberlaender2012, author = {Oberl{\"a}nder, Uwe}, title = {Untersuchung der immunstimulatorischen Effekte von Neuromelanin (NM) auf dendritische Zellen und deren Bedeutung in der Pathogenese von Morbus Parkinson}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73684}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Hintergrund: Das Absterben Neuromelanin (NM)-haltiger Zellen in der substantia nigra (SN), und die daraus resultierende Erniedrigung des Dopaminspiegels im striatum, ist ein pathologisches Hauptmerkmal der Parkinsonschen Krankheit. Ein neuerlicher Nachweis von Anti-Melanin-Antik{\"o}rpern gibt Anlass zur Vermutung, dass NM ein Autoantigen sein k{\"o}nnte. In dieser Arbeit wurde gezeigt, dass NM tats{\"a}chlich von dendritischen Zellen (DZ), die in vivo hauptverantwortlich f{\"u}r die Ausl{\"o}sung von T- und B-Zellantworten sind, erkannt wird. Die Erkennung von NM durch DZ ist eine unabdingbare Voraussetzung f{\"u}r die Einleitung einer adaptiven Immunantwort. Methoden: Murine dendritische Zellen (mDZ) wurden aus Knochenmarkszellen generiert und mit NM aus humaner SN oder synthetischem Dopaminmelanin (DAM) behandelt, nachdem beide Melanine endotoxinfrei getestet wurden. Die Phagozytose von NM wurde mittels konfokaler Mikroskopie dokumentiert. Die Expression von MHC II und CD86 wurde mittels Durchflusszytometrie (FACS) analysiert. Zytokinkonzentrationen von TNF- und dem Interleukin IL-6 wurden mit ELISA-Assays bestimmt. Abschließend wurde die Funktion der durch NM aktivierten DZ mit einer allogenen mixed lymphocyte reaction (MLR) {\"u}berpr{\"u}ft. Ergebnisse: NM wurde von den mDZ effektiv phagozytiert, woraufhin die mDZ einen reifen Phenotyp (CD86high/MHC IIhigh) zeigten. Zus{\"a}tzlich sekretierten durch NM aktivierte mDZ die Zytokine IL-6 and TNF-. Schließlich ließen die mDZ T-Zellen in einer MLR proliferieren, und beweisen so ihre Funktionalit{\"a}t und die F{\"a}higkeit eine prim{\"a}re T-Zellantwort auszul{\"o}sen. Im Gegenteil dazu konnte DAM, dem die Protein- und Lipidkomponenten von NM fehlen und nur das Melaninr{\"u}ckrat mit NM gemeinsam hat, nur einen kleinen Effekt bei den mDZ hervorrufen. Diskussion: NM wird von DZ in vitro erkannt und bewirkt deren Reifung. Sollte der Vorgang auch in vivo stattfinden, besteht die M{\"o}glichkeit, dass SN-Antigene dem adaptiven Immunsystem pr{\"a}sentiert werden, was in einzelnen F{\"a}llen zur Einleitung einer adaptiven Immunantwort f{\"u}hren k{\"o}nnte. NM k{\"o}nnte also der Ausl{\"o}ser f{\"u}r einen autoimmunen Pathomechanismus in der parkinsonschen Krankheit sein.}, subject = {Parkinson-Krankheit}, language = {de} } @article{HeddergottKruegerBabuetal.2012, author = {Heddergott, Nico and Kr{\"u}ger, Timothy and Babu, Sujin B. and Wei, Ai and Stellamanns, Erik and Uppaluri, Sravanti and Pfohl, Thomas and Stark, Holger and Engstler, Markus}, title = {Trypanosome Motion Represents an Adaptation to the Crowded Environment ofthe Vertebrate Bloodstream}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-78421}, year = {2012}, abstract = {Blood is a remarkable habitat: it is highly viscous, contains a dense packaging of cells and perpetually flows at velocities varying over three orders of magnitude. Only few pathogens endure the harsh physical conditions within the vertebrate bloodstream and prosper despite being constantly attacked by host antibodies. African trypanosomes are strictly extracellular blood parasites, which evade the immune response through a system of antigenic variation and incessant motility. How the flagellates actually swim in blood remains to be elucidated. Here, we show that the mode and dynamics of trypanosome locomotion are a trait of life within a crowded environment. Using high-speed fluorescence microscopy and ordered micro-pillar arrays we show that the parasites mode of motility is adapted to the density of cells in blood. Trypanosomes are pulled forward by the planar beat of the single flagellum. Hydrodynamic flow across the asymmetrically shaped cell body translates into its rotational movement. Importantly, the presence of particles with the shape, size and spacing of blood cells is required and sufficient for trypanosomes to reach maximum forward velocity. If the density of obstacles, however, is further increased to resemble collagen networks or tissue spaces, the parasites reverse their flagellar beat and consequently swim backwards, in this way avoiding getting trapped. In the absence of obstacles, this flagellar beat reversal occurs randomly resulting in irregular waveforms and apparent cell tumbling. Thus, the swimming behavior of trypanosomes is a surprising example of micro-adaptation to life at low Reynolds numbers. For a precise physical interpretation, we compare our high-resolution microscopic data to results from a simulation technique that combines the method of multi-particle collision dynamics with a triangulated surface model. The simulation produces a rotating cell body and a helical swimming path, providing a functioning simulation method for a microorganism with a complex swimming strategy}, subject = {Biologie}, language = {en} } @article{HeddergottKruegerBabuetal.2012, author = {Heddergott, Niko and Kr{\"u}ger, Timothy and Babu, Sujin B. and Wei, Ai and Stellamanns, Erik and Uppaluri, Sravanti and Pfohl, Thomas and Stark, Holger and Engstler, Markus}, title = {Trypanosome Motion Represents an Adaptation to the Crowded Environment of the Vertebrate Bloodstream}, series = {PLoS Pathogens}, volume = {8}, journal = {PLoS Pathogens}, number = {11}, doi = {10.1371/journal.ppat.1003023}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-134595}, pages = {e1003023}, year = {2012}, abstract = {Blood is a remarkable habitat: it is highly viscous, contains a dense packaging of cells and perpetually flows at velocities varying over three orders of magnitude. Only few pathogens endure the harsh physical conditions within the vertebrate bloodstream and prosper despite being constantly attacked by host antibodies. African trypanosomes are strictly extracellular blood parasites, which evade the immune response through a system of antigenic variation and incessant motility. How the flagellates actually swim in blood remains to be elucidated. Here, we show that the mode and dynamics of trypanosome locomotion are a trait of life within a crowded environment. Using high-speed fluorescence microscopy and ordered micro-pillar arrays we show that the parasites mode of motility is adapted to the density of cells in blood. Trypanosomes are pulled forward by the planar beat of the single flagellum. Hydrodynamic flow across the asymmetrically shaped cell body translates into its rotational movement. Importantly, the presence of particles with the shape, size and spacing of blood cells is required and sufficient for trypanosomes to reach maximum forward velocity. If the density of obstacles, however, is further increased to resemble collagen networks or tissue spaces, the parasites reverse their flagellar beat and consequently swim backwards, in this way avoiding getting trapped. In the absence of obstacles, this flagellar beat reversal occurs randomly resulting in irregular waveforms and apparent cell tumbling. Thus, the swimming behavior of trypanosomes is a surprising example of micro-adaptation to life at low Reynolds numbers. For a precise physical interpretation, we compare our high-resolution microscopic data to results from a simulation technique that combines the method of multi-particle collision dynamics with a triangulated surface model. The simulation produces a rotating cell body and a helical swimming path, providing a functioning simulation method for a microorganism with a complex swimming strategy.}, language = {en} } @article{SchmittBackesNourkamiTutdibietal.2012, author = {Schmitt, Jana and Backes, Christina and Nourkami-Tutdibi, Nasenien and Leidinger, Petra and Deutscher, Stephanie and Beier, Markus and Gessler, Manfred and Graf, Norbert and Lenhof, Hans-Peter and Keller, Andreas and Meese, Eckart}, title = {Treatment-independent miRNA signature in blood of wilms tumor patients}, series = {BMC Genomics}, volume = {13}, journal = {BMC Genomics}, number = {379}, doi = {10.1186/1471-2164-13-379}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-124034}, year = {2012}, abstract = {Background Blood-born miRNA signatures have recently been reported for various tumor diseases. Here, we compared the miRNA signature in Wilms tumor patients prior and after preoperative chemotherapy according to SIOP protocol 2001. Results We did not find a significant difference between miRNA signature of both groups. However both, Wilms tumor patients prior and after chemotherapy showed a miRNA signature different from healthy controls. The signature of Wilms tumor patients prior to chemotherapy showed an accuracy of 97.5\% and of patients after chemotherapy an accuracy of 97.0\%, each as compared to healthy controls. Conclusion Our results provide evidence for a blood-born Wilms tumor miRNA signature largely independent of four weeks preoperative chemotherapy treatment.}, language = {en} } @article{FoersterBeisserGrohmeetal.2012, author = {F{\"o}rster, Frank and Beisser, Daniela and Grohme, Markus A. and Liang, Chunguang and Mali, Brahim and Siegl, Alexander Matthias and Engelmann, Julia C. and Shkumatov, Alexander V. and Schokraie, Elham and M{\"u}ller, Tobias and Schn{\"o}lzer, Martina and Schill, Ralph O. and Frohme, Marcus and Dandekar, Thomas}, title = {Transcriptome analysis in tardigrade species reveals specific molecular pathways for stress adaptations}, series = {Bioinformatics and biology insights}, volume = {6}, journal = {Bioinformatics and biology insights}, doi = {10.4137/BBI.S9150}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-123089}, pages = {69-96}, year = {2012}, abstract = {Tardigrades have unique stress-adaptations that allow them to survive extremes of cold, heat, radiation and vacuum. To study this, encoded protein clusters and pathways from an ongoing transcriptome study on the tardigrade \(Milnesium\) \(tardigradum\) were analyzed using bioinformatics tools and compared to expressed sequence tags (ESTs) from \(Hypsibius\) \(dujardini\), revealing major pathways involved in resistance against extreme environmental conditions. ESTs are available on the Tardigrade Workbench along with software and databank updates. Our analysis reveals that RNA stability motifs for \(M.\) \(tardigradum\) are different from typical motifs known from higher animals. \(M.\) \(tardigradum\) and \(H.\) \(dujardini\) protein clusters and conserved domains imply metabolic storage pathways for glycogen, glycolipids and specific secondary metabolism as well as stress response pathways (including heat shock proteins, bmh2, and specific repair pathways). Redox-, DNA-, stress- and protein protection pathways complement specific repair capabilities to achieve the strong robustness of \(M.\) \(tardigradum\). These pathways are partly conserved in other animals and their manipulation could boost stress adaptation even in human cells. However, the unique combination of resistance and repair pathways make tardigrades and \(M.\) \(tardigradum\) in particular so highly stress resistant.}, language = {en} } @article{NanguneriFlottmannHorstmannetal.2012, author = {Nanguneri, Siddharth and Flottmann, Benjamin and Horstmann, Heinz and Heilemann, Mike and Kuner, Thomas}, title = {Three-Dimensional, Tomographic Super-Resolution Fluorescence Imaging of Serially Sectioned Thick Samples}, series = {PLoS One}, volume = {7}, journal = {PLoS One}, number = {5}, doi = {10.1371/journal.pone.0038098}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-134434}, pages = {e38098}, year = {2012}, abstract = {Three-dimensional fluorescence imaging of thick tissue samples with near-molecular resolution remains a fundamental challenge in the life sciences. To tackle this, we developed tomoSTORM, an approach combining single-molecule localization-based super-resolution microscopy with array tomography of structurally intact brain tissue. Consecutive sections organized in a ribbon were serially imaged with a lateral resolution of 28 nm and an axial resolution of 40 nm in tissue volumes of up to 50 \(\mu\)mx50\(\mu\)mx2.5\(\mu\)m. Using targeted expression of membrane bound (m)GFP and immunohistochemistry at the calyx of Held, a model synapse for central glutamatergic neurotransmission, we delineated the course of the membrane and fine-structure of mitochondria. This method allows multiplexed super-resolution imaging in large tissue volumes with a resolution three orders of magnitude better than confocal microscopy.}, language = {en} } @article{AsoHerbOguetaetal.2012, author = {Aso, Yoshinori and Herb, Andrea and Ogueta, Maite and Siwanowicz, Igor and Templier, Thomas and Friedrich, Anja B. and Ito, Kei and Scholz, Henrike and Tanimoto, Hiromu}, title = {Three Dopamine Pathways Induce Aversive Odor Memories with Different Stability}, series = {PLoS Genetics}, volume = {8}, journal = {PLoS Genetics}, number = {7}, doi = {10.1371/journal.pgen.1002768}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-130631}, pages = {e1002768}, year = {2012}, abstract = {Animals acquire predictive values of sensory stimuli through reinforcement. In the brain of Drosophila melanogaster, activation of two types of dopamine neurons in the PAM and PPL1 clusters has been shown to induce aversive odor memory. Here, we identified the third cell type and characterized aversive memories induced by these dopamine neurons. These three dopamine pathways all project to the mushroom body but terminate in the spatially segregated subdomains. To understand the functional difference of these dopamine pathways in electric shock reinforcement, we blocked each one of them during memory acquisition. We found that all three pathways partially contribute to electric shock memory. Notably, the memories mediated by these neurons differed in temporal stability. Furthermore, combinatorial activation of two of these pathways revealed significant interaction of individual memory components rather than their simple summation. These results cast light on a cellular mechanism by which a noxious event induces different dopamine signals to a single brain structure to synthesize an aversive memory.}, language = {en} } @article{HuserRohwedderApostolopoulouetal.2012, author = {Huser, Annina and Rohwedder, Astrid and Apostolopoulou, Anthi A. and Widmann, Annekathrin and Pfitzenmaier, Johanna E. and Maiolo, Elena M. and Selcho, Mareike and Pauls, Dennis and von Essen, Alina and Gupta, Tript and Sprecher, Simon G. and Birman, Serge and Riemensperger, Thomas and Stocker, Reinhard F. and Thum, Andreas S.}, title = {The Serotonergic Central Nervous System of the Drosophila Larva: Anatomy and Behavioral Function}, series = {PLoS One}, volume = {7}, journal = {PLoS One}, number = {10}, doi = {10.1371/journal.pone.0047518}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-130437}, pages = {e47518}, year = {2012}, abstract = {The Drosophila larva has turned into a particularly simple model system for studying the neuronal basis of innate behaviors and higher brain functions. Neuronal networks involved in olfaction, gustation, vision and learning and memory have been described during the last decade, often up to the single-cell level. Thus, most of these sensory networks are substantially defined, from the sensory level up to third-order neurons. This is especially true for the olfactory system of the larva. Given the wealth of genetic tools in Drosophila it is now possible to address the question how modulatory systems interfere with sensory systems and affect learning and memory. Here we focus on the serotonergic system that was shown to be involved in mammalian and insect sensory perception as well as learning and memory. Larval studies suggested that the serotonergic system is involved in the modulation of olfaction, feeding, vision and heart rate regulation. In a dual anatomical and behavioral approach we describe the basic anatomy of the larval serotonergic system, down to the single-cell level. In parallel, by expressing apoptosis-inducing genes during embryonic and larval development, we ablate most of the serotonergic neurons within the larval central nervous system. When testing these animals for naive odor, sugar, salt and light perception, no profound phenotype was detectable; even appetitive and aversive learning was normal. Our results provide the first comprehensive description of the neuronal network of the larval serotonergic system. Moreover, they suggest that serotonin per se is not necessary for any of the behaviors tested. However, our data do not exclude that this system may modulate or fine-tune a wide set of behaviors, similar to its reported function in other insect species or in mammals. Based on our observations and the availability of a wide variety of genetic tools, this issue can now be addressed.}, language = {en} } @article{NaseemDandekar2012, author = {Naseem, Muhammad and Dandekar, Thomas}, title = {The Role of Auxin-Cytokinin Antagonism in Plant-Pathogen Interactions}, series = {PLOS Pathogens}, volume = {8}, journal = {PLOS Pathogens}, number = {11}, doi = {10.1371/journal.ppat.1003026}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-131901}, pages = {e1003026}, year = {2012}, abstract = {No abstract available.}, language = {en} } @article{RhiemEngelGraeseretal.2012, author = {Rhiem, Kerstin and Engel, Christoph and Graeser, Monika and Zachariae, Silke and Kast, Karin and Kiechle, Marion and Ditsch, Nina and Janni, Wolfgang and Mundhenke, Christoph and Golatta, Michael and Varga, Dominic and Preisler-Adams, Sabine and Heinrich, Tilman and Bick, Ulrich and Gadzicki, Dorothea and Briest, Susanne and Meindl, Alfons and Schmutzler, Rita K.}, title = {The risk of contralateral breast cancer in patients from BRCA1/2 negative high risk families as compared to patients from BRCA1 or BRCA2 positive families: a retrospective cohort study}, series = {Breast Cancer Research}, volume = {14}, journal = {Breast Cancer Research}, number = {6}, doi = {10.1186/bcr3369}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-135715}, year = {2012}, abstract = {Introduction: While it has been reported that the risk of contralateral breast cancer in patients from BRCA1 or BRCA2 positive families is elevated, little is known about contralateral breast cancer risk in patients from high risk families that tested negative for BRCA1/2 mutations. Methods: A retrospective, multicenter cohort study was performed from 1996 to 2011 and comprised 6,235 women with unilateral breast cancer from 6,230 high risk families that had tested positive for BRCA1 (n = 1,154) or BRCA2 (n = 575) mutations or tested negative (n = 4,501). Cumulative contralateral breast cancer risks were calculated using the Kaplan-Meier product-limit method and were compared between groups using the log-rank test. Cox regression analysis was applied to assess the impact of the age at first breast cancer and the familial history stratified by mutation status. Results: The cumulative risk of contralateral breast cancer 25 years after first breast cancer was 44.1\% (95\%CI, 37.6\% to 50.6\%) for patients from BRCA1 positive families, 33.5\% (95\%CI, 22.4\% to 44.7\%) for patients from BRCA2 positive families and 17.2\% (95\%CI, 14.5\% to 19.9\%) for patients from families that tested negative for BRCA1/2 mutations. Younger age at first breast cancer was associated with a higher risk of contralateral breast cancer. For women who had their first breast cancer before the age of 40 years, the cumulative risk of contralateral breast cancer after 25 years was 55.1\% for BRCA1, 38.4\% for BRCA2, and 28.4\% for patients from BRCA1/2 negative families. If the first breast cancer was diagnosed at the age of 50 or later, 25-year cumulative risks were 21.6\% for BRCA1, 15.5\% for BRCA2, and 12.9\% for BRCA1/2 negative families. Conclusions: Contralateral breast cancer risk in patients from high risk families that tested negative for BRCA1/2 mutations is similar to the risk in patients with sporadic breast cancer. Thus, the mutation status should guide decision making for contralateral mastectomy.}, language = {en} } @phdthesis{Cook2012, author = {Cook, Mandy}, title = {The neurodegenerative Drosophila melanogaster AMPK mutant loechrig}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72027}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {In dieser Doktorarbeit wird die Drosophila Mutante loechrig (loe), die progressive Degeneration des Nervensystems aufweist, weiter beschrieben. In der loe Mutante fehlt eine neuronale Isoform der γ- Untereinheit der Proteinkinase AMPK (AMP-activated protein kinase). Die heterotrimere AMPK (auch als SNF4Aγ bekannt) kontrolliert das Energieniveau der Zelle, was st{\"a}ndiges Beobachten des ATP/AMP- Verh{\"a}ltnis erfordert. AMPK wird durch niedrige Energiekonzentrationen und Beeintr{\"a}chtigungen im Metabolismus, wie zum Beispiel Sauerstoffmangel, aktiviert und reguliert mehrere wichtige Signaltransduktionswege, die den Zellmetabolismus kontrollieren. Jedoch ist die Rolle von AMPK im neuronalen {\"U}berleben noch unklar. Eines der Proteine, dass von AMPK reguliert wird, ist HMGR (hydroxymethylglutaryl-CoA- reductase), ein Schl{\"u}sselenzym in der Cholesterin- und Isoprenoidsynthese. Es wurde gezeigt, dass wenn die Konzentration von HMGR manipuliert wird, auch der Schweregrad des neurodegenerativen Ph{\"a}notyps in loe beeinflusst wird. Obwohl die regulatorische Rolle von AMPK auf HMGR in Drosophila konserviert ist, k{\"o}nnen Insekten Cholesterin nicht de novo synthetisieren. Dennoch ist der Syntheseweg von Isoprenoiden zwischen Vertebraten und Insekten evolution{\"a}r konserviert. Isoprenylierung von Proteinen, wie zum Beispiel von kleinen G-Proteinen, stellt den Proteinen einen hydophobischen Anker bereit, mit denen sie sich an die Zellmembran binden k{\"o}nnen, was in anschließender Aktivierung resultieren kann. In dieser Doktorarbeit wird gezeigt, dass die loe Mutation die Prenylierung von Rho1 und den LIM-Kinasesignalweg beeinflusst, was eine wichtige Rolle im Umsatz von Aktin und axonalem Auswachsen spielt. Die Ergebnisse weisen darauf hin, dass die Mutation in LOE, Hyperaktivit{\"a}t des Isoprenoidsynthesewegs verursacht, was zur erh{\"o}hten Farnesylierung von Rho1 und einer dementsprechend h{\"o}heren Konzentration von Phospho- Cofilin f{\"u}hrt. Eine Mutation in Rho1 verbessert den neurodegenerativen Ph{\"a}notyp und die Lebenserwartung von loe. Der Anstieg vom inaktiven Cofilin in loe f{\"u}hrt zu einer Zunahme von filament{\"o}sen Aktin. Aktin ist am Auswachen von Neuronen beteiligt und Experimente in denen loe Neurone analysiert wurden, gaben wertvolle Einblicke in eine m{\"o}gliche Rolle die AMPK, und dementsprechend Aktin, im Neuronenwachstum spielt. Des Weiteren wurde demonstriert, dass Neurone, die von der loe Mutante stamen, einen verlangsamten axonalen Transport aufweisen, was darauf hinweist dass Ver{\"a}nderungen, die durch den Einfluss von loe auf den Rho1 Signalweg im Zytoskelettnetzwerk hervorgerufen wurden, zur St{\"o}rung des axonalen Transports und anschließenden neuronalen Tod f{\"u}hren. Es zeigte außerdem, dass Aktin nicht nur am neuronalen Auswachsen beteiligt ist, sondern auch wichtig f{\"u}r die Aufrechterhaltung von Neuronen ist. Das bedeutet, dass {\"A}nderungen der Aktindynamik zur progressiven Degeneration von Neuronen f{\"u}hren kann. Zusammenfassend unterstreichen diese Ergebnisse die wichtige Bedeutung von AMPK in den Funktionen und im {\"U}berleben von Neuronen und er{\"o}ffnen einen neuartigen funktionellen Mechanismus in dem {\"A}nderungen in AMPK neuronale Degeneration hervorrufen kann.}, subject = {Taufliege}, language = {en} } @article{MergetKoetschanHackletal.2012, author = {Merget, Benjamin and Koetschan, Christian and Hackl, Thomas and F{\"o}rster, Frank and Dandekar, Thomas and M{\"u}ller, Tobias and Schultz, J{\"o}rg and Wolf, Matthias}, title = {The ITS2 Database}, series = {Journal of Visual Expression}, volume = {61}, journal = {Journal of Visual Expression}, number = {e3806}, doi = {10.3791/3806}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-124600}, year = {2012}, abstract = {The internal transcribed spacer 2 (ITS2) has been used as a phylogenetic marker for more than two decades. As ITS2 research mainly focused on the very variable ITS2 sequence, it confined this marker to low-level phylogenetics only. However, the combination of the ITS2 sequence and its highly conserved secondary structure improves the phylogenetic resolution1 and allows phylogenetic inference at multiple taxonomic ranks, including species delimitation. The ITS2 Database presents an exhaustive dataset of internal transcribed spacer 2 sequences from NCBI GenBank accurately reannotated. Following an annotation by profile Hidden Markov Models (HMMs), the secondary structure of each sequence is predicted. First, it is tested whether a minimum energy based fold (direct fold) results in a correct, four helix conformation. If this is not the case, the structure is predicted by homology modeling. In homology modeling, an already known secondary structure is transferred to another ITS2 sequence, whose secondary structure was not able to fold correctly in a direct fold. The ITS2 Database is not only a database for storage and retrieval of ITS2 sequence-structures. It also provides several tools to process your own ITS2 sequences, including annotation, structural prediction, motif detection and BLAST search on the combined sequence-structure information. Moreover, it integrates trimmed versions of 4SALE and ProfDistS for multiple sequence-structure alignment calculation and Neighbor Joining tree reconstruction. Together they form a coherent analysis pipeline from an initial set of sequences to a phylogeny based on sequence and secondary structure. In a nutshell, this workbench simplifies first phylogenetic analyses to only a few mouse-clicks, while additionally providing tools and data for comprehensive large-scale analyses.}, language = {en} } @phdthesis{Seida2012, author = {Seida, Ahmed Adel}, title = {The Immunomodulatory Role of Endogenous Glucocorticoids in Ovarian Cancer}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73901}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Ovarian cancer currently causes ~6,000 deaths per year in Germany alone. Since only palliative treatment is available for ovarian carcinomas that have developed resistance against platinum-based chemotherapy and paclitaxel, there is a pressing medical need for the development of new therapeutic approaches. As survival is strongly influenced by immunological parameters, immunotherapeutic strategies appear promising. The research of our group thus aims at overcoming tumour immune escape by counteracting immunosuppressive mechanisms in the tumour microenvironment. In this context, we found that tumour-infiltrating myeloid-derived suppressor cells (MDSC) or tumour associated macrophages (TAM) which are abundant in ovarian cancer express high levels of the enzyme 11β-hydroxysteroid dehydrogenase1 (11-HSD1). This oxido-reductase enzyme is essential for the conversion of biologically inactive cortisone into active cortisol. In line with this observation, high endogenous cortisol levels could be detected in serum, ascitic fluid and tumour exudates from ovarian cancer patients. Considering that cortisol exerts strong anti-inflammatory and immunosuppressive effects on immune cells, it appears likely that high endogenous cortisol levels contribute to immune escape in ovarian cancer. We thus hypothesised that local activation of endogenous glucocorticoids could suppress beneficial immune responses in the tumour microenvironment and thereby prevent a successful immunotherapy. To investigate the in vivo relevance of this postulated immune escape mechanism, irradiated PTENloxP/loxP loxP-Stop-loxP-krasG12D mice were reconstituted with hematopoietic stem cells from either glucocorticoid receptor (GR) expressing mice (GRloxP/loxP) or from mice with a T cell-specific glucocorticoid receptor knock-out (lck-Cre GRloxP/loxP) mice. In the host mice, the combination of a conditional PTEN knock-out with a latent oncogenic kras leads to tumour development when a Cre-encoding adenovirus is injected into the ovarian bursa. Using this model, mice that had been reconstituted with GC-insensitive T cells showed better intratumoural T cell infiltration than control mice that had received functionally unaltered GRloxP/loxP cells via adoptive transfer. However, tumour-infiltrating T cells mostly assumed a Foxp3+ (regulatory) phenotype and survival was even shortened in mice with cortisol-insensitive T cells. Thus, endogenous cortisol seems to inhibit immune cell infiltration in ovarian cancer, but productive anti-tumour immune responses might still be prevented by further factors from the tumour microenvironment. Thus, our data did not provide a sufficiently strong rationale to further pursue the antagonisation of glucocorticoid signalling in ovarian cancer patients, Moreover, glucocorticoids are frequently administered to cancer patients to reduce inflammation and swelling and to prevent chemotherapy-related toxic side effects like nausea or hypersensitivity reactions associated with paclitaxel therapy. Thus, we decided to address the question whether specific signalling pathways in innate immune cells, preferentially in NK cells, could still be activated even in the presence of GC. A careful investigation of the various activating NK cell receptors (i.e. NKp30, NKp44, NKp46), DNAM-1 and NKG2D) was thus performed which revealed that NKp30, NKp44 and NKG2D are all down-regulated by cortisol whereas NKp46 is actually induced by cortisol. Interestingly, NKp46 is the only known receptor that is strictly confined to NK cells. Its activation via crosslinking leads to cytokine release and activation of cytotoxic activity. Stimulation of NK cells via NKp46 may contribute to immune-mediated tumour destruction by triggering the lysis of tumour cells and by altering the cytokine pattern in the tumour microenvironment, thereby generating more favourable conditions for the recruitment of antigen-specific immune cells. Accordingly, our observation that even cortisol-treated NK cells can still be activated via NKp46 and CD2 might become valuable for the design of immunotherapies that can still be applied in the presence of endogenous or therapeutically administered glucocorticoids.}, subject = {Cortison}, 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} } @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{Knaf2012, author = {Knaf, Tobias}, title = {Spezifische Bindung von Aluminium und Eisen an den kationenselektiven Kanal MppA von Microthrix parvicella}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-77011}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Schwermetallsalze wie beispielsweise Aluminium- oder Eisensalze werden in der Abwasserbehandlung zur Pr{\"a}vention und Bek{\"a}mpfung von Bl{\"a}hschlamm, Schwimmschlamm und Schaumbildung verwendet. Dadurch kann eine Verbesserung der Schlammabsetzeigenschaften im Nachkl{\"a}rbecken erreicht werden. {\"U}berm{\"a}ßiges Wachstum des grampositiven Bakteriums Microthrix parvicella gilt dabei als Hauptursache von Schlammabsetzproblemen und kann ebenfalls durch die Dosierung von schwermetallhaltigen Flockungs- und F{\"a}llungsmitteln vermieden werden. Da diese Verbindungen in Wasser gel{\"o}st sind, m{\"u}ssen sie die Außenmembran bestimmter Bakterien passieren. Nur der Einbau von wassergef{\"u}llten Kan{\"a}len erlaubt den gel{\"o}sten Salzen das Passieren der durch hydrophobe Fetts{\"a}uren aufgebauten zus{\"a}tzlichen Permeabilit{\"a}tsbarriere. In dieser Arbeit wurden wassergef{\"u}llten Kan{\"a}le von Microthrix parvicella isoliert, aufgereinigt und mit Hilfe der Black-Lipid-Bilayer-Technik charakterisiert. Erg{\"a}nzend wurde der Einfluss und der Durchlass der Flockungs- und F{\"a}llungsmittel in Titrationsexperimenten untersucht. Dabei konnte ein wassergef{\"u}llter Kanal, der die Bezeichnung MppA erhielt, gefunden werden, welcher eine Leitf{\"a}higkeit von 600 pS in 1 M Kaliumchlorid und eine Bindestelle f{\"u}r mehrwertige Kationen wie Eisen oder Aluminium zeigte. Die Bindung dieser mehrwertigen Kationen f{\"u}hrte zu einer {\"A}nderung der Ionenselektivit{\"a}t. Ohne Bindung mehrwertiger Kationen zeigte der Kanal eine leichte Kationenselektivit{\"a}t. Nach der Bindung wechselte die Ionenselektivit{\"a}t zu einer Anionenselektivit{\"a}t, was auf eine spezifische Ladungsverteilung im Kanal hinweist. Der Kanal MppA zeigte gleichwertige Bindekonstanten f{\"u}r Aluminium und Eisen. Beide Metalle werden als F{\"a}llungs- und Flockungsmittel in Kl{\"a}ranlagen zum Verhindern von Schwimm- und Bl{\"a}hschlamm verwendet. Fr{\"u}here Arbeiten offenbarten bereits, dass haupts{\"a}chlich der Aluminiumanteil entscheidend f{\"u}r die Wirkung dieser Mittel ist. Diese Beobachtungen in Verbindung mit den Ergebnissen dieser Arbeit f{\"u}hrten zu der Annahme, dass Eisen und Aluminium eine kompetitive Bindung an der Bindestelle im Kanalinneren zeigen k{\"o}nnten. So k{\"o}nnte in manchen F{\"a}llen Aluminium anstelle des sonst als Spurenelement ben{\"o}tigten Eisens durch den Kanal transportiert werden und in Enzym-Substrat-Komplexen eingebaut werden. Dadurch k{\"o}nnten toxische Effekte auftreten, die letztlich ein Absterben des Organismus zur Folge h{\"a}tten. F{\"u}r die Bindung der Metallsalze konnte zus{\"a}tzlich eine pH-Abh{\"a}ngigkeit beobachtet werden. Nur eine Zugabe von Metalll{\"o}sungen mit einem pH-Wert kleiner 6 f{\"u}hrte zu einer Bindung im Kanal. Die Zugabe von Metalll{\"o}sungen mit einem pH-Wert gr{\"o}ßer 6 zeigte keinen Effekt auf die Leitf{\"a}higkeit des Kanals. Diese Ergebnisse best{\"a}tigen die auf Kl{\"a}ranlagen und in vorherigen Arbeiten get{\"a}tigte Beobachtung, dass der pH-Wert f{\"u}r die Wirksamkeit der Verbindungen entscheidend ist. In dieser Arbeit konnte jedoch erstmals gezeigt werden, dass der pH-Wert direkt die Bindung der Metallsalze beeinflusst.}, subject = {Aluminium}, language = {de} } @article{HsiehLinsenmair2012, author = {Hsieh, Yu-Lung and Linsenmair, Karl Eduard}, title = {Seasonal dynamics of arboreal spider diversity in a temperate forest}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-75158}, year = {2012}, abstract = {Measuring and estimating biodiversity patterns is a fundamental task of the scientist working to support conservation and informmanagement decisions.Most biodiversity studies in temperate regions were often carried out over a very short period of time (e.g., a single season) and it is often—at least tacitly—assumed that these short-termfindings are representative of long-termgeneral patterns.However, should the studied biodiversity pattern in fact contain significant temporal dynamics, perhaps leading to contradictory conclusions. Here, we studied the seasonal diversity dynamics of arboreal spider communities dwelling in 216 European beeches (Fagus sylvatica L.) to assess the spider community composition in the following seasons: two cold seasons (I:November 2005-January 2006; II: February-April) and two warm seasons (III: May-July; IV: August-October). We show that the usually measured diversity of the warmseason community (IV: 58 estimated species) alone did not deliver a reliable image of the overall diversity present in these trees, and therefore, we recommend it should not be used for sampling protocols aimed at providing a full picture of a forest's biodiversity in the temperate zones. In particular, when the additional samplings of other seasons (I, II, III) were included, the estimated species richness nearly doubled (108). Community I possessed the lowest diversity and evenness due to the harsh winter conditions: this community was comprised of one dominant species together with several species low in abundance. Similarity was lowest (38.6\%) between seasonal communities I and III, indicating a significant species turnover due to recolonization, so that community III had the highest diversity. Finally, using nonparametric estimators, we found that further sampling in late winter (February-April) is most needed to complete our inventory. Our study clearly demonstrates that seasonal dynamics of communities should be taken into account when studying biodiversity patterns of spiders, and probably forest arthropods in general.}, subject = {Biologie}, language = {en} } @article{DandekarFieselmannPoppetal.2012, author = {Dandekar, Thomas and Fieselmann, Astrid and Popp, Jasmin and Hensel, Michael}, title = {Salmonella enterica: a surprisingly well-adapted intracellular lifestyle}, series = {Frontiers in Microbiology}, journal = {Frontiers in Microbiology}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-123135}, year = {2012}, abstract = {The infectious intracellular lifestyle of Salmonella enterica relies on the adaptation to nutritional conditions within the Salmonella-containing vacuole (SCV) in host cells. We summarize latest results on metabolic requirements for Salmonella during infection. This includes intracellular phenotypes of mutant strains based on metabolic modeling and experimental tests, isotopolog profiling using (13)C-compounds in intracellular Salmonella, and complementation of metabolic defects for attenuated mutant strains towards a comprehensive understanding of the metabolic requirements of the intracellular lifestyle of Salmonella. Helpful for this are also genomic comparisons. We outline further recent studies and which analyses of intracellular phenotypes and improved metabolic simulations were done and comment on technical required steps as well as progress involved in the iterative refinement of metabolic flux models, analyses of mutant phenotypes, and isotopolog analyses. Salmonella lifestyle is well-adapted to the SCV and its specific metabolic requirements. Salmonella metabolism adapts rapidly to SCV conditions, the metabolic generalist Salmonella is quite successful in host infection.}, 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{Kistenpfennig2012, author = {Kistenpfennig, Christa}, title = {Rhodopsin 7 and Cryptochrome - circadian photoreception in Drosophila}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72209}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Many organisms evolved an endogenous clock to adapt to the daily environmental changes caused by the earth's rotation. Light is the primary time cue ("Zeitgeber") for entrainment of circadian clocks to the external 24-h day. In Drosophila, several visual pigments are known to mediate synchronization to light: The blue-light photopigment Cryptochrome (CRY) and six well-described rhodopsins (Rh1-Rh6). CRY is present in the majority of clock neurons as well as in the compound eyes, whereas the location of rhodopsins is restricted to the photoreceptive organs - the compound eyes, the ocelli and the HB-eyelets. CRY is thought to represent the key photoreceptor of Drosophila's circadian clock. Nevertheless, mutant flies lacking CRY (cry01) are able to synchronize their locomotor activity rhythms to light-dark (LD) cycles, but need significantly longer than wild-type flies. In this behavior, cry01 mutants strongly resemble mammalian species that do not possess any internal photoreceptors and perceive light information exclusively through their photoreceptive organs (eyes). Thus, a mammalian-like phase-shifting behavior would be expected in cry01 flies. We investigated this issue by monitoring a phase response curve (PRC) of cry01 and wild-type flies to 1-h light pulses of 1000 lux irradiance. Indeed, cry01 mutants produced a mammalian-similar so called type 1 PRC of comparatively low amplitude (< 25\% of wild-type) with phase delays to light pulses during the early subjective night and phase advances to light pulses during the late subjective night (~1 h each). Despite the predominant role of CRY, the visual system contributes to the light sensitivity of the fly's circadian clock, mainly around dawn and dusk. Furthermore, this phase shifting allows for the slow re-entrainment which we observed in cry01 mutants to 8-h phase delays of the LD 12 h:12 h cycle. However, cry01 also showed surprising differences in their shifting ability: First of all, their PRC was characterized by a second dead zone in the middle of the subjective night (ZT17-ZT19) in addition to the usual unresponsiveness during the subjective day. Second, in contrast to wild-type flies, cry01 mutants did not increase their shift of activity rhythms neither in response to longer stimuli nor to light pulses of higher irradiance. In contrast, both 6-h light pulses of 1000 lux and 1-h light pulses of 10,000 lux light intensity during the early subjective night even resulted in phase advances instead of the expected delays. Thus, CRY seems to be not only responsible for the high light sensitivity of the wild-type circadian clock, but is apparently also involved in integrating and processing light information. Rhodopsin 7 (Rh7) is a yet uncharacterized protein, but became a good photoreceptor candidate due to sequence similarities to the six known Drosophila Rhs. The second part of this thesis investigated the expression pattern of Rh7 and its possible functions, especially in circadian photoreception. Furthermore, we were interested in a potential interaction with CRY and thus, tested cry01 and rh70 cry01 mutants as well. Rh1 is the main visual pigment of the Drosophila compound eye and expressed in six out of eight photoreceptors cells (R1-R6) in each of the ~800 ommatidia. Motion vision depends exclusively on Rh1 function but, moreover, Rh1 plays an important structural role and assures proper photoreceptor cell development and maintenance. In order to investigate its possible photoreceptive function, we expressed Rh7 in place of Rh1. Rh7 was indeed able to overtake the role of Rh1 in both aspects: It prevented retinal degeneration and mediated the optomotor response (OR), a motion vision-dependent behavior. At the transcriptional level, rh7 is expressed at approximately equal amounts in adult fly brains and retinas. Due to a reduced specificity of anti-Rh7 antibodies, we could not verify this result at the protein level. However, analysis of rh7 null mutants (rh70) suggested different Rh7 functions in vivo. Previous experiments strongly indicated an increased sensitivity of the compound eyes in the absence of Rh7 and suggested impaired light adaptation. We aimed to test this hypothesis at the levels of circadian photoreception. Locomotor activity rhythms are a reliable output of the circadian clock. Rh70 mutant flies generally displayed a wild-type similar bimodal activity pattern comprising morning (M) and evening (E) activity bouts. Activity monitoring supported the proposed "shielding" function, since rh70 mutants behaved like wild-type flies experiencing high irradiances. Under all investigated conditions, their activity peaks lay further apart resulting in a prolonged midday break. The behavior of cry01 mutants was mainly characterized by an unexpectedly high flexibility in the timing of M and E activity bouts which allowed tracking of lights-on and lights-off even under extreme photoperiods. Activity profiles of the corresponding rh70 cry01 double mutants reflected neither synergistic nor antagonistic effects of Rh7 and CRY and were dominated by a broad E activity peak. In the future, the different circadian phenotypes will be further investigated on the molecular level by analysis of clock protein cycling in the underlying pacemaker neurons. The work of this thesis confirmed that Rh7 is indeed able to work as a photoreceptor and to initiate the classical phototransduction cascade. On the other hand, it provided further evidence at the levels of circadian photoreception that Rh7 might serve as a shielding pigment for Rh1 in vivo, thereby mediating proper light adaptation.}, language = {en} } @article{SchneiderTautzGruenewaldetal.2012, author = {Schneider, Christof W. and Tautz, J{\"u}rgen and Gr{\"u}newald, Bernd and Fuchs, Stefan}, title = {RFID Tracking of Sublethal Effects of Two Neonicotinoid Insecticides on the Foraging Behavior of Apis mellifera}, series = {PLoS One}, volume = {7}, journal = {PLoS One}, number = {1}, doi = {10.1371/journal.pone.0030023}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-131753}, pages = {e30023}, year = {2012}, abstract = {The development of insecticides requires valid risk assessment procedures to avoid causing harm to beneficial insects and especially to pollinators such as the honeybee Apis mellifera. In addition to testing according to current guidelines designed to detect bee mortality, tests are needed to determine possible sublethal effects interfering with the animal's vitality and behavioral performance. Several methods have been used to detect sublethal effects of different insecticides under laboratory conditions using olfactory conditioning. Furthermore, studies have been conducted on the influence insecticides have on foraging activity and homing ability which require time-consuming visual observation. We tested an experimental design using the radiofrequency identification (RFID) method to monitor the influence of sublethal doses of insecticides on individual honeybee foragers on an automated basis. With electronic readers positioned at the hive entrance and at an artificial food source, we obtained quantifiable data on honeybee foraging behavior. This enabled us to efficiently retrieve detailed information on flight parameters. We compared several groups of bees, fed simultaneously with different dosages of a tested substance. With this experimental approach we monitored the acute effects of sublethal doses of the neonicotinoids imidacloprid (0.15-6 ng/bee) and clothianidin (0.05-2 ng/bee) under field-like circumstances. At field-relevant doses for nectar and pollen no adverse effects were observed for either substance. Both substances led to a significant reduction of foraging activity and to longer foraging flights at doses of >= 0.5 ng/bee (clothianidin) and >= 1.5 ng/bee (imidacloprid) during the first three hours after treatment. This study demonstrates that the RFID-method is an effective way to record short-term alterations in foraging activity after insecticides have been administered once, orally, to individual bees. We contribute further information on the understanding of how honeybees are affected by sublethal doses of insecticides.}, language = {en} } @phdthesis{Haydn2012, author = {Haydn, Johannes}, title = {Regulation of ERK1/2 signaling in melanoma}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85727}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Die Mechanismen in einer Zelle, die die Genexpression und somit den Stoffwechsel, das Wachstum und das gesamte Zellverhalten steuern, sind ebenso bedeutsam f{\"u}r das Verst{\"a}ndnis der grundlegenden Biologie einer lebenden Zelle wie f{\"u}r die Vorg{\"a}nge der Krebsentstehung. Dabei bilden hochvernetzte, und strikt regulierte Signaltransduktionswege die Basis f{\"u}r ein belastbares und zugleich hochflexibles regulatorisches Netzwerk. Die St{\"o}rung solcher Signalkaskaden kann zum einen urs{\"a}chlich aber auch modifizierend auf die Bildung von Tumoren wirken. Die von Rezeptortyrosinkinasen (RTK) und RAS abh{\"a}ngigen Signalwege, die zur Aktivierung von AKT und ERK1/2 f{\"u}hren, sind hierbei von besonderem Interesse f{\"u}r die Entstehung des malignen Melanoms. Mutationen in Komponenten dieser Wege (z.B. NRAS, BRAF oder PTEN), die die Signalst{\"a}rke erh{\"o}hen kommen in Melanomen sehr h{\"a}ufig vor. Im ersten Teil dieser Arbeit wurden die unterschiedlichen und vielf{\"a}ltigen Funktionen von MKP2, einem Feedbackregulator des ERK1/2-Weges, unter verschiedenen zellul{\"a}ren Rahmenbedingungen, untersucht. Des Weiteren wird eine Funktion des zum AP1-Komplex geh{\"o}renden FOSL1, einem unter transkriptioneller Kontrolle des ERK1/2-Weges stehendem Transkriptionsfaktors, hinsichtlich der Steuerung der Zell-Proliferation gezeigt. Weiterhin habe ich Aspekte der direkten pharmakologischen Inhibition des ERK1/2-Weges hinsichtlich ihres Effekts auf die Ausl{\"o}sung von Apoptose untersucht. Aufgrund der H{\"a}ufigkeit von Mutationen in Genen, die f{\"u}r Proteine des ERK1/2-Weges kodieren (z.B. NRASQ61K, BRAFV600E), gilt die Inhibition dieses Signalwegs als vielversprechende Strategie zur Behandlung des Melanoms. Auch wenn klinische Studien, die Inhibitoren f{\"u}r MEK oder RAF als Einzelmedikamente verwenden, bei mehrmonatiger Behandlung sehr erfolgreich sind, konnten so keine langfristigen Erfolge erzielt werden. Aus diesem Grund werden nun Kombinationstherapien, die einen Inhibitor des ERK1/2-Weges und eine weitere Form der Therapie kombinieren, untersucht. Der zweite Teil dieser Arbeit beschreibt, dass der spezifische MEK Inhibitor PD184352 Melanomzellen vor der Apoptosewirkung von Cisplatin sch{\"u}tzen kann. Einzelbehandlung mit Cisplatin f{\"u}hrt hierbei zur Akkumulation von DNA Sch{\"a}den, die wiederum Caspase-abh{\"a}ngig Apoptose induzieren. Zus{\"a}tzliche Anwendung des MEK Inhibitors verringerte jedoch in einigen Zelllinien das Potential von Cisplatin, Apoptose auszul{\"o}sen. Diese Zellen zeigten eine verst{\"a}rkte Aktivierung der Serin/Threonin-KInase AKT nach MEK Inhibition. Diese AKT Aktivierung f{\"u}hrte zur Inaktivierung der FOXO Transkriptionsfaktoren, was wiederum die Expression des pro-apoptotischen BH3-only Proteins PUMA verringerte. PUMA selbst ist ein wichtiger Bestandteil der Apoptose Maschinerie, die durch Cisplatin aktiviert wird. Die im Rahmen dieser Arbeit erhaltenen Befunde deuten darauf hin, dass RTKs, im besonderen EGFR, bei diesem Crosstalk eine Rolle spielen. Diese Ergebnisse zeigen, dass die Inhibition des RAS/RAF/MEK/ERK Signalweges im Melanom nicht zwangsl{\"a}ufig von Vorteil sein muss, falls die Zellen gleichzeitig mit einem genotoxischen Medikament behandelt werden. Hier kann sie sogar die {\"U}berlebensf{\"a}higkeit von Melanomzellen unter Apoptose induzierenden Bedingungen verbessern.}, subject = {Melanom}, language = {en} } @phdthesis{Kubisch2012, author = {Kubisch, Alexander}, title = {Range border formation in the light of dispersal evolution}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-70639}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Understanding the emergence of species' ranges is one of the most fundamental challenges in ecology. Early on, geographical barriers were identified as obvious natural constraints to the spread of species. However, many range borders occur along gradually changing landscapes, where no sharp barriers are obvious. Mechanistic explanations for this seeming contradiction incorporate environmental gradients that either affect the spatio-temporal variability of conditions or the increasing fragmentation of habitat. Additionally, biological mechanisms like Allee effects (i.e. decreased growth rates at low population sizes or densities), condition-dependent dispersal, and biological interactions with other species have been shown to severely affect the location of range margins. The role of dispersal has been in the focus of many studies dealing with range border formation. Dispersal is known to be highly plastic and evolvable, even over short ecological time-scales. However, only few studies concentrated on the impact of evolving dispersal on range dynamics. This thesis aims at filling this gap. I study the influence of evolving dispersal rates on the persistence of spatially structured populations in environmental gradients and its consequences for the establishment of range borders. More specially I investigate scenarios of range formation in equilibrium, periods of range expansion, and range shifts under global climate change ...}, subject = {Areal}, language = {en} } @article{SangesScheuermannZahedietal.2012, author = {Sanges, C. and Scheuermann, C. and Zahedi, R. P. and Sickmann, A. and Lamberti, A. and Migliaccio, N. and Baljuls, A. and Marra, M. and Zappavigna, S. and Rapp, U. and Abbruzzese, A. and Caraglia, M. and Arcari, P.}, title = {Raf kinases mediate the phosphorylation of eukaryotic translation elongation factor 1A and regulate its stability in eukaryotic cells}, series = {Cell Death \& Disease}, volume = {3}, journal = {Cell Death \& Disease}, number = {e276}, doi = {10.1038/cddis.2012.16}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-134673}, year = {2012}, abstract = {We identified eukaryotic translation elongation factor 1A (eEF1A) Raf-mediated phosphorylation sites and defined their role in the regulation of eEF1A half-life and of apoptosis of human cancer cells. Mass spectrometry identified in vitro S21 and T88 as phosphorylation sites mediated by B-Raf but not C-Raf on eEF1A1 whereas S21 was phosphorylated on eEF1A2 by both B-and C-Raf. Interestingly, S21 belongs to the first eEF1A GTP/GDP-binding consensus sequence. Phosphorylation of S21 was strongly enhanced when both eEF1A isoforms were preincubated prior the assay with C-Raf, suggesting that the eEF1A isoforms can heterodimerize thus increasing the accessibility of S21 to the phosphate. Overexpression of eEF1A1 in COS 7 cells confirmed the phosphorylation of T88 also in vivo. Compared with wt, in COS 7 cells overexpressed phosphodeficient (A) and phospho-mimicking (D) mutants of eEF1A1 (S21A/D and T88A/D) and of eEF1A2 (S21A/D), resulted less stable and more rapidly proteasome degraded. Transfection of S21 A/D eEF1A mutants in H1355 cells increased apoptosis in comparison with the wt isoforms. It indicates that the blockage of S21 interferes with or even supports C-Raf induced apoptosis rather than cell survival. Raf-mediated regulation of this site could be a crucial mechanism involved in the functional switching of eEF1A between its role in protein biosynthesis and its participation in other cellular processes.}, language = {en} } @article{LandoEndesfelderBergeretal.2012, author = {Lando, David and Endesfelder, Ulrike and Berger, Harald and Subramanian, Lakxmi and Dunne, Paul D. and McColl, James and Klenerman, David and Carr, Antony M. and Sauer, Markus and Allshire, Robin C. and Heilemann, Mike and Laue, Ernest D.}, title = {Quantitative single-molecule microscopy reveals that CENP-A\(^{Cnp1}\) deposition occurs during G2 in fission yeast}, series = {Open Biology}, volume = {2}, journal = {Open Biology}, number = {120078}, doi = {10.1098/rsob.120078}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-134682}, year = {2012}, abstract = {The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand the process of epigenetic inheritance, or propagation of histones and histone variants, as nucleosomes are disassembled and reassembled in living eukaryotic cells, we have explored the feasibility of exploiting photo-activated localization microscopy (PALM). PALM of single molecules in living cells has the potential to reveal new concepts in cell biology, providing insights into stochastic variation in cellular states. However, thus far, its use has been limited to studies in bacteria or to processes occurring near the surface of eukaryotic cells. With PALM, one literally observes and 'counts' individual molecules in cells one-by-one and this allows the recording of images with a resolution higher than that determined by the diffraction of light (the so-called super-resolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-A\(^{Cnp1}\) with single-molecule sensitivity in fission yeast (Schizosaccharomyces pombe). The results obtained are validated by and compared with ChIP-seq analyses. Using this approach, CENP-A\(^{Cnp1}\) levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-A(Cnp1) is deposited solely during the G2 phase of the cell cycle.}, language = {en} } @phdthesis{Cook2012, author = {Cook, Vanessa Janine}, title = {Protection of healthy tissues from infection with systemically administered vaccinia virus strains}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-69654}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Oncolytic virotherapy using recombinant vaccinia virus strains is a promising approach for the treatment of cancer. To further improve the safety of oncolytic vaccinia viruses, the cellular microRNA machinery can be applied as the host's own security mechanism to avoid unwanted viral replication in healthy tissues. MicroRNAs are a class of small single-stranded RNAs which due to their ability to mediate post-transcriptional gene-silencing, play a crucial role in almost every regulatory process in cellular metabolism. Different cancers display unique microRNA expression patterns, showing significant up- or downregulation of endogenously expressed microRNAs. Furthermore, the behavior of cancer cells can be altered by either adding microRNAs known to inhibit cancer cell spread and proliferation or suppressing cancer promoting microRNAs (oncomirs) making microRNAs promising targets for cancer gene therapy. The cell's own RNAi machinery can also be utilized to control viral replication due to the virus dependence on the host cell replication machinery, a process controlled by microRNAs. GLV-1h68 is a replication-competent recombinant oncolytic vaccinia virus constructed and generated by Genelux Corp., San Diego, CA, USA which carries insertions of three reporter gene cassettes for detection and attenuation purposes and is currently being evaluated for cancer treatment in clinical trials. Though there are hardly any side effects found in GLV-1h68 mediated oncolytic therapy an increased tropism for replication exclusively in cancer cells is desirable. Therefore it was investigated whether or not further cancer cell specificity of a recombinant vaccinia virus strain could be obtained without compromising its oncolytic activity using microRNA interference. Let-7a is a well characterized microRNA known to be expressed in high levels in healthy tissues and strongly downregulated in most cancers. To control vaccinia virus replication rates, four copies of the mature human microRNA let-7a target sequence were cloned behind the stop codon in the 3'end of the vaccinia virus D4R gene, using a GLV-1h68 derivative, GLV-1h190, as parental strain yielding the new recombinant virus strain GLV-1h250. The D4R gene belongs to the group of early transcribed vaccinia genes and encodes an essential enzyme, uracil DNA glycosylase, which catalyzes the removal of uracil residues from double-stranded DNA. A defect in D4R prevents vaccinia virus from entering into the intermediate and late phase of replication, leading to an aborted virus replication. After expression of the microRNA target sequence from the vaccinia virus genome, the endogenously expressed microRNA-let-7a should recognize its target structure within the viral mRNA transcript, thereby binding and degrading the viral mRNA which should lead to a strong inhibition of the virus replication in healthy cells. GLV-1h250 replication rates in cancerous A549 lung adenocarcinoma cells, which show a strong down-regulation of microRNA let-7a, was comparable to the replication rates of its parental strain GLV-1h190 and the control strain GLV-1h68. In contrast, GLV-1h250 displayed a 10-fold decrease in viral replication in non-cancerous ERC cells when compared to GLV-1h190 and GLV-1h68. In A549 tumor bearing nude mice GLV-1h250 replicated exclusively in the tumorous tissue and resulted in efficient tumor regression without adverse effects leading to the conclusion that GLV-1h250 replicates preferentially in cancerous cells and tissues, which display low endogenous let-7a expression levels.}, subject = {Vaccinia-Virus}, language = {en} } @article{TuChenLimetal.2012, author = {Tu, Xiaolin and Chen, Jianquan and Lim, Joohyun and Karner, Courtney M. and Lee, Seung-Yon and Heisig, Julia and Wiese, Cornelia and Surendran, Kameswaran and Kopan, Raphael and Gessler, Manfred and Long, Fanxin}, title = {Physiological Notch Signaling Maintains Bone Homeostasis via RBPjk and Hey Upstream of NFATc1}, series = {PLoS Genetics}, volume = {8}, journal = {PLoS Genetics}, number = {3}, doi = {10.1371/journal.pgen.1002577}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-133490}, pages = {e1002577}, year = {2012}, abstract = {Notch signaling between neighboring cells controls many cell fate decisions in metazoans both during embryogenesis and in postnatal life. Previously, we uncovered a critical role for physiological Notch signaling in suppressing osteoblast differentiation in vivo. However, the contribution of individual Notch receptors and the downstream signaling mechanism have not been elucidated. Here we report that removal of Notch2, but not Notch1, from the embryonic limb mesenchyme markedly increased trabecular bone mass in adolescent mice. Deletion of the transcription factor RBPjk, a mediator of all canonical Notch signaling, in the mesenchymal progenitors but not the more mature osteoblast-lineage cells, caused a dramatic high-bone-mass phenotype characterized by increased osteoblast numbers, diminished bone marrow mesenchymal progenitor pool, and rapid age-dependent bone loss. Moreover, mice deficient in Hey1 and HeyL, two target genes of Notch-RBPjk signaling, exhibited high bone mass. Interestingly, Hey1 bound to and suppressed the NFATc1 promoter, and RBPjk deletion increased NFATc1 expression in bone. Finally, pharmacological inhibition of NFAT alleviated the high-bone-mass phenotype caused by RBPjk deletion. Thus, Notch-RBPjk signaling functions in part through Hey1-mediated inhibition of NFATc1 to suppress osteoblastogenesis, contributing to bone homeostasis in vivo.}, language = {en} } @article{CorneliusLeingaertnerHoissetal.2012, author = {Cornelius, C. and Leing{\"a}rtner, A. and Hoiss, B. and Krauss, J. and Steffan-Dewenter, I. and Menzel, A.}, title = {Phenological response of grassland species to manipulative snowmelt and drought along an altitudinal gradient}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-77969}, year = {2012}, abstract = {Plant communities in the European Alps are assumed to be highly affected by climate change since temperature rise in this region is above the global average. It is predicted that higher temperatures will lead to advanced snowmelt dates and that the number of extreme weather events will increase. The aims of this study were to determine the impacts of extreme climatic events on flower phenology and to assess whether those impacts differed between lower and higher altitudes. In 2010 an experiment simulating advanced and delayed snowmelt as well as drought event was conducted along an altitudinal transect ca. every 250m (600-2000 m a.s.l.) in the Berchtesgaden National Park, Germany. The study showed that flower phenology is strongly affected by altitude; however there were few effects of the manipulative treatments on flowering. The effects of advanced snowmelt were significantly greater at higher than at lower sites, but no significant difference was found between both altitudinal bands for the other treatments. The response of flower phenology to temperature declined through the season and the length of flowering duration was not significantly influenced by treatments. The stronger effect of advanced snowmelt at higher altitudes might be a response to differences in treatment intensity across the gradient. Consequently, shifts in the date of snowmelt due to global warming may affect species more at higher than at lower altitudes since changes may be more pronounced at higher altitudes. Our data indicate a rather low risk of drought events on flowering phenology in the Bavarian Alps.}, subject = {Biologie}, language = {en} } @article{ZoephelReiherRexeretal.2012, author = {Zoephel, Judith and Reiher, Wencke and Rexer, Karl-Heinz and Kahnt, J{\"o}rg and Wegener, Christian}, title = {Peptidomics of the Agriculturally Damaging Larval Stage of the Cabbage Root Fly Delia radicum (Diptera: Anthomyiidae)}, series = {PLoS One}, volume = {7}, journal = {PLoS One}, number = {7}, doi = {10.1371/journal.pone.0041543}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-131727}, pages = {e41543}, year = {2012}, abstract = {The larvae of the cabbage root fly induce serious damage to cultivated crops of the family Brassicaceae. We here report the biochemical characterisation of neuropeptides from the central nervous system and neurohemal organs, as well as regulatory peptides from enteroendocrine midgut cells of the cabbage maggot. By LC-MALDI-TOF/TOF and chemical labelling with 4-sulfophenyl isothiocyanate, 38 peptides could be identified, representing major insect peptide families: allatostatin A, allatostatin C, FMRFamide-like peptides, kinin, CAPA peptides, pyrokinins, sNPF, myosuppressin, corazonin, SIFamide, sulfakinins, tachykinins, NPLP1-peptides, adipokinetic hormone and CCHamide 1. We also report a new peptide (Yamide) which appears to be homolog to an amidated eclosion hormone-associated peptide in several Drosophila species. Immunocytochemical characterisation of the distribution of several classes of peptide-immunoreactive neurons and enteroendocrine cells shows a very similar but not identical peptide distribution to Drosophila. Since peptides regulate many vital physiological and behavioural processes such as moulting or feeding, our data may initiate the pharmacological testing and development of new specific peptide-based protection methods against the cabbage root fly and its larva.}, language = {en} } @article{PatilGentschevNolteetal.2012, author = {Patil, Sandeep S. and Gentschev, Ivaylo and Nolte, Ingo and Ogilvie, Gregory and Szalay, Aladar A.}, title = {Oncolytic virotherapy in veterinary medicine: current status and future prospects for canine patients}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-75128}, year = {2012}, abstract = {Oncolytic viruses refer to those that are able to eliminate malignancies by direct targeting and lysis of cancer cells, leaving non-cancerous tissues unharmed. Several oncolytic viruses including adenovirus strains, canine distemper virus and vaccinia virus strains have been used for canine cancer therapy in preclinical studies. However, in contrast to human studies, clinical trials with oncolytic viruses for canine cancer patients have not been reported. An 'ideal' virus has yet to be identified. This review is focused on the prospective use of oncolytic viruses in the treatment of canine tumors - a knowledge that will undoubtedly contribute to the development of oncolytic viral agents for canine cancer therapy in the future.}, subject = {Medizin}, language = {en} } @article{HarringtonScelsiHarteletal.2012, author = {Harrington, John M. and Scelsi, Chris and Hartel, Andreas and Jones, Nicola G. and Engstler, Markus and Capewell, Paul and MacLeod, Annette and Hajduk, Stephen}, title = {Novel African Trypanocidal Agents: Membrane Rigidifying Peptides}, series = {PLoS One}, volume = {7}, journal = {PLoS One}, number = {9}, doi = {10.1371/journal.pone.0044384}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-135179}, pages = {e44384}, year = {2012}, abstract = {The bloodstream developmental forms of pathogenic African trypanosomes are uniquely susceptible to killing by small hydrophobic peptides. Trypanocidal activity is conferred by peptide hydrophobicity and charge distribution and results from increased rigidity of the plasma membrane. Structural analysis of lipid-associated peptide suggests a mechanism of phospholipid clamping in which an internal hydrophobic bulge anchors the peptide in the membrane and positively charged moieties at the termini coordinate phosphates of the polar lipid headgroups. This mechanism reveals a necessary phenotype in bloodstream form African trypanosomes, high membrane fluidity, and we suggest that targeting the plasma membrane lipid bilayer as a whole may be a novel strategy for the development of new pharmaceutical agents. Additionally, the peptides we have described may be valuable tools for probing the biosynthetic machinery responsible for the unique composition and characteristics of African trypanosome plasma membranes.}, language = {en} } @article{GrafePreiningerSztatecsnyetal.2012, author = {Grafe, T. Ulmar and Preininger, Doris and Sztatecsny, Marc and Kasah, Rosli and Dehling, J. Maximilian and Proksch, Sebastian and H{\"o}dl, Walter}, title = {Multimodal Communication in a Noisy Environment: A Case Study of the Bornean Rock Frog Staurois parvus}, series = {PLoS One}, volume = {7}, journal = {PLoS One}, number = {5}, doi = {10.1371/journal.pone.0037965}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-133718}, year = {2012}, abstract = {High background noise is an impediment to signal detection and perception. We report the use of multiple solutions to improve signal perception in the acoustic and visual modality by the Bornean rock frog, Staurois parvus. We discovered that vocal communication was not impaired by continuous abiotic background noise characterised by fast-flowing water. Males modified amplitude, pitch, repetition rate and duration of notes within their advertisement call. The difference in sound pressure between advertisement calls and background noise at the call dominant frequency of 5578 Hz was 8 dB, a difference sufficient for receiver detection. In addition, males used several visual signals to communicate with conspecifics with foot flagging and foot flashing being the most common and conspicuous visual displays, followed by arm waving, upright posture, crouching, and an open-mouth display. We used acoustic playback experiments to test the efficacy-based alerting signal hypothesis of multimodal communication. In support of the alerting hypothesis, we found that acoustic signals and foot flagging are functionally linked with advertisement calling preceding foot flagging. We conclude that S. parvus has solved the problem of continuous broadband low-frequency noise by both modifying its advertisement call in multiple ways and by using numerous visual signals. This is the first example of a frog using multiple acoustic and visual solutions to communicate in an environment characterised by continuous noise.}, language = {en} } @phdthesis{Pleiser2012, author = {Pleiser, Sandra}, title = {Mouse genetic analyses of Spir functions}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73634}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Das Aktin-Zytoskelett ist f{\"u}r viele zellul{\"a}re Funktionen unerl{\"a}sslich, dazu geh{\"o}ren der strukturelle Aufbau von Zellen, die Zellwanderung und Vesikeltransportprozesse. Die funktionelle Vielfalt der Aktinstrukturen spiegelt sich in einer Vielzahl verschiedener molekularer Mechanismen wieder, welche die Polymerisierung von Aktinfilamenten regulieren. Die sponante Aktinpolymerisierung wird jedoch verhindert aufgrund der Instabilit{\"a}t von kleinen Aktin Oligomeren und durch Aktin Monomer bindende Proteine, welche die Bildung solcher Oligomere unterbinden. Aktinnukleationsfaktoren helfen diese kinetische Barriere der Filamentbildung zu {\"u}berwinden und sind wesentlich f{\"u}r die Erzeugung von neuen Aktinfilamenten an bestimmten subzellul{\"a}ren Kompartimenten. Spir Proteine sind die ersten beschriebenen Mitglieder der neuen Klasse von WH2 Dom{\"a}nen Aktinnukleationsfaktoren. Sie leiten die Polymerisierung von Aktin ein, indem sie Aktinmonomere an die vier WH2 Dom{\"a}nen im Zentrum des Proteins binden. Trotz ihrer Eigenschaft Aktinpolymerisation in vitro selber zu nukleieren, bilden Spir Proteine einen regulatorischen Komplex mit anderen Aktinnukleatoren der formin Untergruppe von forminen. Spir hat eine Funktion bei der Regulierung von vesikul{\"a}r erzeugten filament{\"o}sen Aktinstrukturen, Vesikeltransportprozessen und der Bildung der Teilungsfurche w{\"a}hrend der asymmetrischen meiotischen Zellteilung. Das S{\"a}ugetiergenom kodiert zwei spir Gene, spir-1 und spir-2. Die entsprechenden Proteine haben einen identischen strukturellen Aufbau und sind zu einem großen Teil homolog zueinander. Um die Spir Funktion im sich entwickelnden und adulten Nervensystem zu untersuchen, wurde die bisher unbekannte Expression des Maus spir-2 Gens analysiert. Real-time PCR Analysen haben ergeben, dass spir-2 in adulten M{\"a}usen in Oozyten, dem Gehirn, im Gastrointestinaltrakt, den Hoden und der Niere exprimiert wird. In situ Hybridisierungen wurden durchgef{\"u}hrt um die zellul{\"a}re Natur der spir Expression nachzuweisen. W{\"a}hrend der Embryogenese haben in situ Hybridisierungen gezeigt, dass spir-2 im sich entwickelnden Nervensytem und Darmtrakt exprimiert wird. In adulten Mausgeweben, wurde die h{\"o}chste Expression von spir-2 in Epithelzellen des Verdauungstraktes, in neuronalen Zellen des Nervensystems und in Spermatocyten gefunden. Im Gegensatz zur eher begrenzten Expression des Maus spir-1 Gens, welches {\"u}berwiegend im Nervensystem, den Oozyten und Hoden zu finden ist, zeigen die hier aufgef{\"u}hrten Daten ein breiteres Expressionsmuster des spir-2 Gens und unterst{\"u}tzen damit eine allgemeinere zellbiologische Funktion der neuen Aktinnukleatoren. Um die Funktion des Spir Proteins im sich entwickelnden und adulten Nervensystem zu untersuchen, wurden Spir-1 defiziente M{\"a}use mit Hilfe der gene trap Methode generiert. Spir-1 defiziente M{\"a}use sind lebensf{\"a}hig und eignen sich daher perfekt um die Neurobiologie des Spir-1 Aktinnukleators zu untersuchen. Die Analyse von prim{\"a}ren kortikalen Neuronen von Spir-1 defizienten M{\"a}usen zeigte eine Reduktion dendritischer Verzweigungen und ist die erste Beschreibung einer neuronalen Funktion von Spir-1. Desweiteren wurde eine transgene Mauslinie (thy1-GFP-M) eingesetzt, die das gr{\"u}ne Fluoreszenzprotein (GFP) unter der Kontrolle von Neuronen-spezifischen Elementen des thy1 Promoters exprimiert. GFP ist dabei nur in einer Teilmenge von Neuronen exprimiert, f{\"a}rbt diese Neuronen jedoch in ihrer Gesamtheit an. Spir-1 defiziente M{\"a}use, die das GFP Transgen exprimieren wurden generiert und analysiert. Es wurde herausgefunden, dass Spir-1 defiziente M{\"a}use eine reduzierte Anzahl an dendritischen Dornen im entorhinalen Kortex im Vergleich zu Wildtyp- Geschwistertieren aufweisen. Zusammengefasst gibt diese Studie neue Erkenntnisse {\"u}ber die zellbiologische Funktion von Spir und liefert Einsichten wie das neuronale Netzwerk sturkturiert wird.}, subject = {Actin-bindende Proteine}, language = {en} } @phdthesis{Schriefer2012, author = {Schriefer, Eva-Maria}, title = {Molekulare und biochemische Charakterisierung der β-Laktamasen von Yersinia enterocolitica und deren Sekretionsverhalten}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-69580}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {In dieser Arbeit wurden zwei Aspekte der Yersinia β-Laktamasen bearbeitet: (1) Charakterisierung der β-Laktamasen hinsichtlich β-Laktam-Antibiotikaresistenz, Sekretion und Thermostabilit{\"a}t. (2) Untersuchung der Sekretionsf{\"a}higkeit von verschiedenen thermostabilen β Laktamasen {\"u}ber das Yersinia T3SS. Im ersten Teil wurden β Laktamase-Deletionsmutanten im Y. enterocolitica Serotyp O:8 Stamm WA-314 hergestellt, um den Einfluss der chromosomalen β Laktamasen auf die in vitro-Resistenz zu untersuchen. Es konnte gezeigt werden, dass WA-314 konstitutiv BlaA produziert und BlaA somit - unter nicht-induzierbaren Bedingungen - der dominante Faktor in der in vitro-Resistenz gegen{\"u}ber Penicillinen mit erweitertem Wirkungsspektrum (z.B. Ampicillin) und Cephalosporinen der 1. Generation (z.B. Cefazolin) ist. Weiterhin konnte gezeigt werden, dass die zweite chromosomale β Laktamase AmpC (BlaB) unter Zugabe von subinhibitorischen Konzentrationen von Imipenem stark induziert wird. Keine der β Laktamasen ist in der Lage, in vitro-Resistenz gegen{\"u}ber Carbapenemen und Monobactamen zu vermitteln. Die Konstruktion und Bestimmung der in vitro Antibiotika-Empfindlichkeit der β Laktamase-Deletionsmutanten dient als Grundlage f{\"u}r nachfolgende Untersuchungen im Mausinfektionsmodell. Weiterhin wurden die Transporteigenschaften beider β Laktamasen untersucht. In Gram-negativen Bakterien sind reife β Laktamasen im Periplasma lokalisiert und m{\"u}ssen somit nach der Synthese im Cytosol {\"u}ber die Cytoplasmamembran transportiert werden. Bis auf drei Ausnahmen (β Laktamasen aus Mycobacterium smegmatis, M. tuberculosis und Stenotrophomonas maltophila) sind bisher nur Sec-abh{\"a}ngige β Laktamasen beschrieben worden. Mittels Fusionsproteinen bestehend aus β Laktamase-Signalpeptiden und GFP konnte in dieser Arbeit eindeutig gezeigt werden, dass es sich bei Yersinia BlaA um ein Tat-Substrat handelt, bei Yersinia AmpC hingegen um ein Sec-Substrat. Somit konnte im Rahmen dieser Arbeit zum ersten Mal eine Tat-abh{\"a}ngige β Laktamase bei einer Bakterienart aus der Familie der Enterobacteriaceae nachgewiesen werden. Außerdem konnte gezeigt werden, dass die β Laktamase BlaA nicht diffus im Periplasma, sondern auf bestimmte Bereiche im Periplasma lokalisiert verteilt ist. Allerdings konnte die Art der Lokalisierung bisher nicht genau spezifiziert werden. Die cytosolische Faltung und die Tat-abh{\"a}ngige Translokation von BlaA lassen vermuten, dass eine besondere Thermostabilit{\"a}t von BlaA vorliegt. Deshalb wurde das BlaA-Enzym hinsichtlich seiner Thermostabilit{\"a}t und temperaturabh{\"a}ngigen enzymatischen Aktivit{\"a}t untersucht. Im Vergleich zur E. coli β Laktamase TEM-1 und der hitzestabilen TEM-1-Variante MEGA zeigte BlaA eine erh{\"o}hte Thermostabilit{\"a}t und einen starken Anstieg der Aktivit{\"a}t in einem Temperaturbereich zwischen 30 °C und 45 °C. Im zweiten Teil dieser Arbeit wurde gepr{\"u}ft, ob die charakterisierten Yersinia β Laktamasen als Reporterkonstrukte zur Untersuchung des Typ III Sekretionssystems (T3SS) geeignet sind. Y. enterocolitica besitzt ein pYV Virulenzplasmid, auf dem der vollst{\"a}ndige Satz der Gene f{\"u}r das Ysc-T3SS und die Effektor-Yops (Yersinia outer protein) lokalisiert sind. Injektion der Yops in eukaryotische Zielzellen erm{\"o}glicht das extrazellul{\"a}re {\"U}berleben der Yersinien im Wirtsorganismus. Bei YopE handelt es sich um ein gut charakterisiertes Effektor-Yop, dessen N Terminus fusioniert an den reifen Teil der β Laktamase TEM-1 bereits vielfach als Reporterkonstrukt eingesetzt wurde. Unter Verwendung des fluoreszierenden β Laktamase-Substrats CCF4-AM kann die Translokation von YopEi-TEM-1 in Zielzellen in Zellkultur-Experimenten und im Mausinfektionsmodell visualisiert werden. In dieser Arbeit sollte deshalb die T3SS-Sekretionsf{\"a}higkeit von YopE-β Laktamase-Fusionsproteinen in Abh{\"a}ngigkeit von der „Schmelztemperatur" (temperaturabh{\"a}ngige Stabilit{\"a}t, TM) untersucht werden. Yop-Substrate werden im ungefalteten Zustand (YscN wirkt dabei vermutlich als ATP-abh{\"a}ngige „Unfoldase") {\"u}ber das Ysc-„Injektisom" transloziert. YopEi-TEM-1 wird effizient sekretiert und transloziert (TM (TEM-1) = 50,8 °C). YopE-Fusionsproteine mit thermostabilen TEM-1 Varianten, YopEi-RLT bzw. YopEi-MEGA (TM (RLT) = 60,4 °C; TM (MEGA) = 69,2 °C) werden hingegen nur schwach bzw. nicht sekretiert. Weiterhin konnte gezeigt werden, dass die Sec-abh{\"a}ngige β Laktamase AmpC als YopE-Fusionsprotein (YopEi-AmpC) effizient T3SS-abh{\"a}ngig sekretiert und transloziert werden kann; das native Tat-Substrat BlaA (YopEi-BlaA) kann jedoch weder sekretiert noch transloziert wird. Eine m{\"o}gliche Erkl{\"a}rung w{\"a}re, dass die ATPase YscN nicht in der Lage ist, BlaA und die thermostabilen TEM-1-Varianten zu entfalten und {\"u}ber das T3SS zu sekretieren und zu translozieren. RLT und MEGA k{\"o}nnen hingegen mithilfe ihrer nativen Signalsequenz {\"u}ber das Sec-System (und somit im ungefalteten Zustand) transloziert werden.}, subject = {Yersinia enterocolitica}, language = {de} } @phdthesis{Schlegelmilch2012, author = {Schlegelmilch, Katrin}, title = {Molecular function of WISP1/CCN4 in the musculoskeletal system with special reference to apoptosis and cell survival}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73430}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Human adult cartilage is an aneural and avascular type of connective tissue, which consequently reflects reduced growth and repair rates. The main cell type of cartilage are chondrocytes, previously derived from human mesenchymal stem cells (hMSCs). They are responsible for the production and maintainance of the cartilaginous extracellular matrix (ECM), which consists mainly of collagen and proteoglycans. Signal transmission to or from chondrocytes, generally occurs via interaction with signalling factors connected to the cartilaginous ECM. In this context, proteins of the CCN family were identified as important matricellular and multifunctional regulators with high significance during skeletal development and fracture repair. In this thesis, main focus lies on WISP1/CCN4, which is known as a general survival factor in a variety of cell types and seems to be crucial during lineage progression of hMSCs into chondrocytes. We intend to counter the lack of knowledge about the general importance of WISP1-signalling within the musculoskeletal system and especially regarding cell death and survival by a variety of molecular and cell biology methods. First, we established a successful down-regulation of endogenous WISP1 transcripts within different cell types of the human musculoskeletal system through gene-silencing. Interestingly, WISP1 seems to be crucial to the survival of all examined cell lines and primary hMSCs, since a loss of WISP1 resulted in cell death. Bioinformatical analyses of subsequent performed microarrays (WISP1 down-regulated vs. control samples) confirmed this observation in primary hMSCs and the chondrocyte cell line Tc28a2. Distinct clusters of regulated genes, closely related to apoptosis induction, could be identified. In this context, TRAIL induced apoptosis as well as p53 mediated cell death seem to play a crucial role during the absence of WISP1 in hMSCs. By contrast, microarray analysis of WISP1 down-regulated chondrocytes indicated rather apoptosis induction via MAPK-signalling. Despite apoptosis relevant gene regulations, microarray analyses also identified clusters of differentially expressed genes of other important cellular activities, e.g. a huge cluster of interferon-inducible genes in hMSCs or gene regulations affecting cartilage homeostasis in chondrocytes. Results of this thesis emphasize the importance of regulatory mechanisms that influence cell survival of primary hMSCs and chondrocytes in the enforced absence of WISP1. Moreover, findings intensified the assumed importance for WISP1-signalling in cartilage homeostasis. Thus, this thesis generated an essential fundament for further examinations to investigate the role of WISP1-signalling in cartilage homeostasis and cell death.}, subject = {Knorpelzelle}, language = {en} } @article{RatzkaFoersterLiangetal.2012, author = {Ratzka, Carolin and F{\"o}rster, Frank and Liang, Chunguang and Kupper, Maria and Dandekar, Thomas and Feldhaar, Heike and Gross, Roy}, title = {Molecular characterization of antimicrobial peptide genes of the carpenter ant Camponotus floridanus}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-75985}, year = {2012}, abstract = {The production of antimicrobial peptides (AMPs) is a major defense mechanism against pathogen infestation and of particular importance for insects relying exclusively on an innate immune system. Here, we report on the characterization of three AMPs from the carpenter ant Camponotus floridanus. Due to sequence similarities and amino acid composition these peptides can be classified into the cysteine-rich (e.g. defensin) and glycine-rich (e.g. hymenoptaecin) AMP groups, respectively. The gene and cDNA sequences of these AMPs were established and their expression was shown to be induced by microbial challenge. We characterized two different defensin genes. The defensin-2 gene has a single intron, whereas the defensin-1 gene has two introns. The deduced amino acid sequence of the C. floridanus defensins is very similar to other known ant defensins with the exception of a short C-terminal extension of defensin-1. The hymenoptaecin gene has a single intron and a very peculiar domain structure. The corresponding precursor protein consists of a signal- and a pro-sequence followed by a hymenoptaecin-like domain and six directly repeated hymenoptaecin domains. Each of the hymenoptaecin domains is flanked by an EAEP-spacer sequence and a RR-site known to be a proteolytic processing site. Thus, proteolytic processing of the multipeptide precursor may generate several mature AMPs leading to an amplification of the immune response. Bioinformatical analyses revealed the presence of hymenoptaecin genes with similar multipeptide precursor structure in genomes of other ant species suggesting an evolutionary conserved important role of this gene in ant immunity.}, subject = {Biologie}, language = {en} } @article{RuczyńskiBartoń2012, author = {Ruczyński, Ireneusz and Bartoń, Kamil A.}, title = {Modelling Sensory Limitation: The Role of Tree Selection, Memory and Information Transfer in Bats' Roost Searching Strategies}, series = {PLoS One}, volume = {7}, journal = {PLoS One}, number = {9}, doi = {10.1371/journal.pone.0044897}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-133963}, pages = {e44897}, year = {2012}, abstract = {Sensory limitation plays an important role in the evolution of animal behaviour. Animals have to find objects of interest (e.g. food, shelters, predators). When sensory abilities are strongly limited, animals adjust their behaviour to maximize chances for success. Bats are nocturnal, live in complex environments, are capable of flight and must confront numerous perceptual challenges (e.g. limited sensory range, interfering clutter echoes). This makes them an excellent model for studying the role of compensating behaviours to decrease costs of finding resources. Cavity roosting bats are especially interesting because the availability of tree cavities is often limited, and their quality is vital for bats during the breeding season. From a bat's sensory point of view, cavities are difficult to detect and finding them requires time and energy. However, tree cavities are also long lasting, allowing information transfer among conspecifics. Here, we use a simple simulation model to explore the benefits of tree selection, memory and eavesdropping (compensation behaviours) to searches for tree cavities by bats with short and long perception range. Our model suggests that memory and correct discrimination of tree suitability are the basic strategies decreasing the cost of roost finding, whereas perceptual range plays a minor role in this process. Additionally, eavesdropping constitutes a buffer that reduces the costs of finding new resources (such as roosts), especially when they occur in low density. We conclude that natural selection may promote different strategies of roost finding in relation to habitat conditions and cognitive skills of animals.}, language = {en} } @article{HoennemannSanzMorenoWolfetal.2012, author = {H{\"o}nnemann, Jan and Sanz-Moreno, Adrian and Wolf, Elmar and Eilers, Martin and Els{\"a}sser, Hans-Peter}, title = {Miz1 Is a Critical Repressor of cdkn1a during Skin Tumorigenesis}, series = {PLoS One}, volume = {7}, journal = {PLoS One}, number = {4}, doi = {10.1371/journal.pone.0034885}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-133285}, pages = {e34885}, year = {2012}, abstract = {The transcription factor Miz1 forms repressive DNA-binding complexes with the Myc, Gfi-1 and Bcl-6 oncoproteins. Known target genes of these complexes encode the cyclin-dependent kinase inhibitors (CKIs) cdkn2b (p15\(^{Ink4}\)), cdkn1a (p21\(^{Cip1}\)), and cdkn1c (p57\(^{Kip2}\)). Whether Miz1-mediated repression is important for control of cell proliferation in vivo and for tumor formation is unknown. Here we show that deletion of the Miz1 POZ domain, which is critical for Miz1 function, restrains the development of skin tumors in a model of chemically-induced, Ras-dependent tumorigenesis. While the stem cell compartment appears unaffected, interfollicular keratinocytes lacking functional Miz1 exhibit a reduced proliferation and an accelerated differentiation of the epidermis in response to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Tumorigenesis, proliferation and normal differentiation are restored in animals lacking cdkn1a, but not in those lacking cdkn2b. Our data demonstrate that Miz1-mediated attenuation of cell cycle arrest pathways via repression of cdkn1a has a critical role during tumorigenesis in the skin.}, language = {en} } @article{JazbutyteFiedlerKneitzetal.2012, author = {Jazbutyte, Virginija and Fiedler, Jan and Kneitz, Susanne and Galuppo, Paolo and Just, Annette and Holzmann, Angelika and Bauersachs, Johann and Thum, Thomas}, title = {MicroRNA-22 increases senescence and activates cardiac fibroblasts in the aging heart}, series = {AGE}, volume = {35}, journal = {AGE}, number = {3}, doi = {10.1007/s11357-012-9407-9}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-126745}, pages = {747-762}, year = {2012}, abstract = {MicroRNAs (miRs) are small non- coding RNA molecules controlling a plethora of biological processes such as development, cellular survival and senescence. We here determined miRs differentially regulated during cardiac postnatal development and aging. Cardiac function, morphology and miR expression profiles were determined in neonatal, 4 weeks, 6 months and 19 months old normotensive male healthy C57/Bl6N mice. MiR-22 was most prominently upregulated during cardiac aging. Cardiac expression of its bioinformatically predicted target mimecan (osteoglycin, OGN) was gradually decreased with advanced age. Luciferase reporter assays validated mimecan as a bona fide miR-22 target. Both, miR-22 and its target mimecan were co- expressed in cardiac fibroblasts and smooth muscle cells. Functionally, miR-22 overexpression induced cellular senescence and promoted migratory activity of cardiac fibroblasts. Small interference RNA-mediated silencing of mimecan in cardiac fibroblasts mimicked the miR-22-mediated effects. Rescue experiments revealed that the effects of miR-22 on cardiac fibroblasts were only partially mediated by mimecan. In conclusion, miR-22 upregulation in the aging heart contributed at least partly to accelerated cardiac fibroblast senescence and increased migratory activity. Our results suggest an involvement of miR-22 in age-associated cardiac changes, such as cardiac fibrosis.}, language = {en} } @phdthesis{Cecil2012, author = {Cecil, Alexander [geb. Schmid]}, title = {Metabolische Netzwerkanalysen f{\"u}r den Weg von xenobiotischen zu vertr{\"a}glichen antibiotischen Substanzen}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-71866}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Durch das Auftreten neuer St{\"a}mme resistenter Krankheitserreger ist die Suche nach neuartigen Wirkstoffen gegen diese, sich st{\"a}ndig weiter ausbreitende Bedrohung, dringend notwendig. Der interdisziplin{\"a}re Sonderforschungsbereich 630 der Universit{\"a}t W{\"u}rzburg stellt sich dieser Aufgabe, indem hier neuartige Xenobiotika synthetisiert und auf ihre Wirksamkeit getestet werden. Die hier vorgelegte Dissertation f{\"u}gt sich hierbei nahtlos in die verschiedenen Fachbereiche des SFB630 ein: Sie stellt eine Schnittstelle zwischen Synthese und Analyse der Effekte der im Rahmen des SFB630 synthetisierten Isochinolinalkaloid-Derivaten. Mit den hier angewandten bioinformatischen Methoden wurden zun{\"a}chst die wichtigsten Stoffwechselwege von S. epidermidis R62A, S. aureus USA300 und menschlicher Zellen in sogenannten metabolischen Netzwerkmodellen nachgestellt. Basierend auf diesen Modellen konnten Enzymaktivit{\"a}ten f{\"u}r verschiedene Szenarien an zugesetzten Xenobiotika berechnet werden. Die hierf{\"u}r ben{\"o}tigten Daten wurden direkt aus Genexpressionsanalysen gewonnen. Die Validierung dieser Methode erfolgte durch Metabolommessungen. Hierf{\"u}r wurde S. aureus USA300 mit verschiedenen Konzentrationen von IQ-143 behandelt und gem{\"a}ß dem in dieser Dissertation vorgelegten Ernteprotokoll aufgearbeitet. Die Ergebnisse hieraus lassen darauf schließen, dass IQ-143 starke Effekte auf den Komplex 1 der Atmungskette aus{\"u}bt - diese Resultate decken sich mit denen der metabolischen Netzwerkanalyse. F{\"u}r den Wirkstoff IQ-238 ergaben sich trotz der strukturellen {\"A}hnlichkeiten zu IQ-143 deutlich verschiedene Wirkeffekte: Dieser Stoff verursacht einen direkten Abfall der Enzymaktivit{\"a}ten in der Glykolyse. Dadurch konnte eine unspezifische Toxizit{\"a}t dieser Stoffe basierend auf ihrer chemischen Struktur ausgeschlossen werden. Weiterhin konnten die bereits f{\"u}r IQ-143 und IQ-238 auf Bakterien angewandten Methoden erfolgreich zur Modellierung der Effekte von Methylenblau auf verschiedene resistente St{\"a}mme von P. falciparum 3D7 angewandt werden. Dadurch konnte gezeigt werden, dass Methylenblau in einer Kombination mit anderen Pr{\"a}paraten gegen diesen Parasiten zum einen die Wirkung des Prim{\"a}rpr{\"a}parates verst{\"a}rkt, zum anderen aber auch in gewissem Maße vorhandene Resistenzen gegen das Prim{\"a}rpr{\"a}parat zu verringern vermag. Somit konnte durch die vorgelegte Arbeit eine Pipeline zur Identifizierung der metabolischen Effekte verschiedener Wirkstoffe auf unterschiedliche Krankheitserreger erstellt werden. Diese Pipeline kann jederzeit auf andere Organismen ausgeweitet werden und stellt somit einen wichtigen Ansatz um Netzwerkeffekte verschiedener, potentieller Medikamente aufzukl{\"a}ren.}, subject = {Stoffwechsel}, language = {de} } @phdthesis{Karunakaran2012, author = {Karunakaran, Karthika}, title = {Mechanisms of apoptosis regulation in human cells infected with Simkania negevensis}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72098}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Chlamydiales are obligate intracellular gram-negative bacteria that have gained high medical relevance. These important human pathogens cause diverse diseases including trachoma and wide spread sexually transmitted diseases. Chlamydia establishes membrane bound inclusions in the host cell and loots the host for nutritional requirements. Infections are usually recognized by the host immune system and eliminated systematically, by triggering apoptosis. However, the pathogen Chlamydia has evolved various strategies to prevent the detection as well as protect the invaded cell against apoptosis or any other form of cell death. The evolutionary conservation of cell death regulation has not been investigated in the order Chlamydiales, which also includes Chlamydia-like organisms with a broader host spectrum. The present study was aimed at investigating the apoptotic response of human cells infected with the Chlamydia-like organism Simkania negevensis (Sn). Simkania infected cells exhibited strong resistance to apoptosis induced by intrinsic stress or by the activation of cell death receptors. Apoptotic signaling was blocked upstream of mitochondria since Bax translocation, Bax and Bak oligomerisation and cytochrome c release were absent in these cells. Caspases were differentially regulated upon Sn infection. Caspase-3 and -9 were not activated upon Sn infection and apoptosis induction; whereas caspases-8 was activated in Sn infected cells even without apoptosis induction. This indicates that, Sn utilizes death receptor association independent caspase activation for thriving in the host environment. Infected cells turned on pro-survival pathways like cellular Inhibitor of Apoptosis Proteins (IAP-1/2 and XIAP) and the Akt/PI3K pathway. Sn infection also 20 activated the pro-survival transcription factor NF-кB. Blocking any of these survival pathways sensitized the infected host cell towards apoptosis induction, demonstrating their role in infection-induced apoptosis resistance. The NF-кB mutant cells also showed reduced infectivity of Sn, which indicated an essential role of NF-кB in Sn infection. It was interesting to observe that, Acanthamoeba castellanii, a natural host of Sn, survived maintaining its trophozoite forms after infection with Sn upon starvation. The metacaspases, responsible for encystment could be regulated by Sn upon infection. This suggests an early level of gene regulation indicating how the pathogen evolved its ability to inhibit apoptosis in higher organisms. The resistance to apoptosis pathways subverted in Sn-infected cells was similar but not identical to those modulated by Chlamydia. Together, the data supports the hypothesis of evolutionary conserved signaling pathways to apoptosis resistance as common denominators in the order Chlamydiales.}, subject = {Apoptosis}, language = {en} } @article{MenescalSchmidtLiedtkeetal.2012, author = {Menescal, Luciana and Schmidt, Cornelia and Liedtke, Daniel and Schartl, Manfred}, title = {Liver hyperplasia after tamoxifen induction of Myc in a transgenic medaka model}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-75316}, year = {2012}, abstract = {Myc is a global transcriptional regulator and one of the most frequently overexpressed oncoproteins in human tumors. It is well established that activation of Myc leads to enhanced cell proliferation but can also lead to increased apoptosis. The use of animal models expressing deregulated levels of Myc has helped to both elucidate its function in normal cells and give insight into how Myc initiates and maintains tumorigenesis. Analyses of the medaka (Oryzias latipes) genome uncovered the unexpected presence of two Myc gene copies in this teleost species. Comparison of these Myc versions to other vertebrate species revealed that one gene, myc17, differs by the loss of some conserved regulatory protein motifs present in all other known Myc genes. To investigate how such differences might affect the basic biological functions of Myc, we generated a tamoxifeninducible in vivo model utilizing a natural, fish-specific Myc gene. Using this model we show that, when activated, Myc17 leads to increased proliferation and to apoptosis in a dose-dependent manner, similar to human Myc. We have also shown that long-term Myc17 activation triggers liver hyperplasia in adult fish, allowing this newly established transgenic medaka model to be used to study the transition from hyperplasia to liver cancer and to identify Myc-induced tumorigenesis modifiers.}, subject = {Biologie}, language = {en} } @article{RoierLeitnerIwashkiwetal.2012, author = {Roier, Sandro and Leitner, Deborah R. and Iwashkiw, Jeremy and Schild-Pr{\"u}fert, Kristina and Feldman, Mario F. and Krohne, Georg and Reidl, Joachim and Schild, Stefan}, title = {Intranasal Immunization with Nontypeable Haemophilus influenzae Outer Membrane Vesicles Induces Cross-Protective Immunity in Mice}, series = {PLoS One}, volume = {7}, journal = {PLoS One}, number = {8}, doi = {10.1371/journal.pone.0042664}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-135201}, pages = {e42664}, year = {2012}, abstract = {Haemophilus influenzae is a Gram-negative human-restricted bacterium that can act as a commensal and a pathogen of the respiratory tract. Especially nontypeable H. influenzae (NTHi) is a major threat to public health and is responsible for several infectious diseases in humans, such as pneumonia, sinusitis, and otitis media. Additionally, NTHi strains are highly associated with exacerbations in patients suffering from chronic obstructive pulmonary disease. Currently, there is no licensed vaccine against NTHi commercially available. Thus, this study investigated the utilization of outer membrane vesicles (OMVs) as a potential vaccine candidate against NTHi infections. We analyzed the immunogenic and protective properties of OMVs derived from various NTHi strains by means of nasopharyngeal immunization and colonization studies with BALB/c mice. The results presented herein demonstrate that an intranasal immunization with NTHi OMVs results in a robust and complex humoral and mucosal immune response. Immunoprecipitation revealed the most important immunogenic proteins, such as the heme utilization protein, protective surface antigen D15, heme binding protein A, and the outer membrane proteins P1, P2, P5 and P6. The induced immune response conferred not only protection against colonization with a homologous NTHi strain, which served as an OMV donor for the immunization mixtures, but also against a heterologous NTHi strain, whose OMVs were not part of the immunization mixtures. These findings indicate that OMVs derived from NTHi strains have a high potential to act as a vaccine against NTHi infections.}, language = {en} } @article{FraunholzSinha2012, author = {Fraunholz, Martin and Sinha, Bhanu}, title = {Intracellular staphylococcus aureus: Live-in and let die}, series = {Frontiers in Cellular and Infection Microbiology}, volume = {2}, journal = {Frontiers in Cellular and Infection Microbiology}, number = {43}, doi = {10.3389/fcimb.2012.00043}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-123374}, year = {2012}, abstract = {Staphylococcus aureus uses a plethora of virulence factors to accommodate a diversity of niches in its human host. Aside from the classical manifestations of S. aureus-induced diseases, the pathogen also invades and survives within mammalian host cells. The survival strategies of the pathogen are as diverse as strains or host cell types used. S. aureus is able to replicate in the phagosome or freely in the cytoplasm of its host cells. It escapes the phagosome of professional and non-professional phagocytes, subverts autophagy, induces cell death mechanisms such as apoptosis and pyronecrosis, and even can induce anti-apoptotic programs in phagocytes. The focus of this review is to present a guide to recent research outlining the variety of intracellular fates of S. aureus.}, language = {en} } @phdthesis{Sbiera2012, author = {Sbiera, Silviu}, title = {Interaction of Human Polyomavirus JC with cells of the hematopoietic system in the periphery}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-74183}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Primary contact with human polyomaviruses is followed by lifelong asymptomatic persistence of viral DNA. Under severe immunosuppression JCV activation may lead to unrestricted virus growth in the CNS followed by development of progressive multifocal leukoencephalopathy (PML). Besides the kidney and the brain, target cells of persistent infection were also found in the hematopoietic system. This included the presence of JCV genomes in peripheral blood cells (PBCs). In the attempt to understand the role of PBCs for the JCV infection in humans, we asked for the type of cells affected as well as for virus interaction with PBCs. Analysis of separated subpopulations by highly sensitive and specific polymerase chain reaction and Southern blot hybridization revealed the presence of JCV DNA mostly in circulating granulocytes. These cells have important functions in innate immunity and are professional phagocytes. This suggested that PCR amplified DNA might be the result of an extranuclear association of the virus due to membrane attachment or phagocytosis rather than JCV infection with presence of viral DNA in the nucleus. In the attempt to answer this question JCV DNA was subcellularly localized in the blood of 22 healthy donors by JCV specific fluorescence in situ hybridization (FISH). Granulocytes and peripheral blood mononuclear cells (PBMCs) were separated by Percoll gradient centrifugation. Intracellular JCV DNA was hybridized with Digoxigenin-labeled JCV specific DNA probes covering half of the viral genome. As the sensitivity of the anti-digoxigenin antibody system was lower than the PCR detection level, a chemical amplification step was included consisting of peroxidase labeled secondary antibody precipitating biotinylated tyramide followed by detection with streptavidin-Texas-Red and fluorescence microscopy. Comparison of the number of cells affected in healthy individuals with 15 HIV-1 infected patients with and without PML revealed that the rate of affected PBMCs was comparable in both groups (2.5±0.4 and 14.5±0.9 per 1000). In contrast, the rate of JCV positive granulocytes in the immunosuppressed group was 92.6±1.7\% compared to 4±1.4\% in healthy donors thus confirming that granulocytes are the major group of circulating cells affected by JCV and that HIV-1 associated immune impairment has an important effect on the virus-cell association. Localization revealed that JCV DNA was predominantly located within the cytoplasm, although hybridizing signals occasionally covered the nuclear compartment. The fluorescent glow of chemical amplification combined with classical fluorescence microscopy did not allow an unequivocal localization of viral DNA. However, confocal microscopy of 24 sections through single cells combined with FISH without chemical amplification confirmed cytoplasmic localization of JCV DNA in a large number of cells. Additionally, it clearly demonstrated that JCV DNA was also located in the nucleus and nuclear localization directly correlated with the number of cells affected. Calculation of the virus load in subcellular compartments revealed that up to 50\% of the JCV genomes were located in the nucleus thus pointing to viral infection at least in the granulocytes of HIV-1 infected patients. This may contribute to the distribution of the virus from sites of peripheral infection to the CNS and may promote the development of active PML in the severely immune impaired patients.}, subject = {Polyomaviren}, language = {en} } @phdthesis{Krueger2012, author = {Kr{\"u}ger, Beate}, title = {Integration und Kombination bioinformatischer Methoden in Biotechnologie, synthetischer Biologie und Pharmaindustrie}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-70702}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Die Bioinformatik ist eine interdisziplin{\"a}re Wissenschaft, welche Probleme aus allen Lebenswissenschaften mit Hilfe computergest{\"u}tzter Methoden bearbeitet. Ihr Ziel ist es, die Verarbeitung und Interpretation großer Datenmengen zu erm{\"o}glichen. Zudem unterst{\"u}tzt sie den Designprozess von Experimenten in der Synthetischen Biologie. Die synthetische Biologie besch{\"a}ftigt sich mit der Generierung neuer Komponenten und deren Eigenschaften, welche durch die Behandlung und Manipulation lebender Organismen oder Teilen daraus entstehen. Ein besonders interessantes Themengebiet hierbei sind Zweikomponenten-Systeme (Two-Component System, TCS). TCS sind wichtige Signalkaskaden in Bakterien, welche in der Lage sind Informationen aus der Umgebung in eine Zelle zu {\"u}bertragen und darauf zu reagieren. Die vorliegende Dissertation besch{\"a}ftigt sich mit der Beurteilung, Nutzung und Weiterentwicklung von bioinformatischen Methoden zur Untersuchung von Proteininteraktionen und biologischen Systemen. Der wissenschaftliche Beitrag der vorliegenden Arbeit kann in drei Aspekte unterteilt werden: - Untersuchung und Beurteilung von bioinformatischen Methoden und Weiterf{\"u}hrung der Ergebnisse aus der vorhergehenden Diplomarbeit zum Thema Protein-Protein-Interaktionsvorhersagen. - Analyse genereller evolution{\"a}rer Modifikationsm{\"o}glichkeiten von TCS sowie deren Design und spezifische Unterschiede. - Abstraktion bzw. Transfer der gewonnenen Erkenntnisse auf technische und biologische Zusammenh{\"a}nge. Mit dem Ziel das Design neuer Experimente in der synthetischen Biologie zu vereinfachen und die Vergleichbarkeit von technischen und biologischen Prozessen sowie zwischen Organismen zu erm{\"o}glichen. Das Ergebnis der durchgef{\"u}hrten Studie zeigte, dass Zweikomponenten-Systeme in ihrem Aufbau sehr konserviert sind. Nichtsdestotrotz konnten viele spezifische Eigenschaften und drei generelle Modifikationsm{\"o}glichkeiten entdeckt werden. Die Untersuchungen erm{\"o}glichten die Identifikation neuer Promotorstellen, erlaubten aber auch die Beschreibung der Beschaffenheit unterschiedlicher Signalbindestellen. Zudem konnten bisher fehlende Komponenten aus TCS entdeckt werden, ebenso wie neue divergierte TCS-Dom{\"a}nen im Organismus Mycoplasma. Eine Kombination aus technischen Ans{\"a}tzen und synthetischer Biologie vereinfachte die gezielte Manipulation von TCS oder anderen modularen Systemen. Die Etablierung der vorgestellten zweistufigen Modul-Klassifikation erm{\"o}glichte eine effizientere Analyse modular aufgebauter Prozesse und erlaubte somit das molekulare Design synthetischer, biologischer Anwendungen. Zur einfachen Nutzung dieses Ansatzes wurde eine frei zug{\"a}ngliche Software GoSynthetic entwickelt. Konkrete Beispiele demonstrierten die praktische Anwendbarkeit dieser Analysesoftware. Die vorgestellte Klassifikation der synthetisch-biologischen und technischen Einheiten soll die Planung zuk{\"u}nftiger Designexperimente vereinfachen und neue Wege f{\"u}r sinnverwandte Bereiche aufzeigen. Es ist nicht die Hauptaufgabe der Bioinformatik, Experimente zu ersetzen, sondern resultierende große Datenmengen sinnvoll und effizient auszuwerten. Daraus sollen neue Ideen f{\"u}r weitere Analysen und alternative Anwendungen gewonnen werden, um fehlerhafte oder falsche Ans{\"a}tze fr{\"u}hzeitig zu erkennen. Die Bioinformatik bietet moderne, technische Verfahren, um vertraute, aber oft m{\"u}hsame experimentelle Wege durch neue, vielversprechende Ans{\"a}tze zur Datenstrukturierung und Auswertung großer Datenmengen zu erg{\"a}nzen. Neue Sichtweisen werden durch die Erleichterung des Testprozederes gef{\"o}rdert. Die resultierende Zeitersparnis f{\"u}hrt zudem zu einer Kostenreduktion.}, subject = {Biotechnologie}, language = {de} } @phdthesis{Schleyer2012, author = {Schleyer, Michael}, title = {Integrating past, present and future: mechanisms of a simple decision in larval Drosophila}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-78923}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Is behaviour response or action? In this Thesis I study this question regarding a rather simple organism, the larva of the fruit fly Drosophila melanogaster. Despite its numerically simple brain and limited behavioural repertoire, it is nevertheless capable to accomplish surprisingly complex tasks. After association of an odour and a rewarding or punishing reinforcement signal, the learnt odour is able to retrieve the formed memory trace. However, the activated memory trace is not automatically turned into learned behaviour: Appetitive memory traces are behaviourally expressed only in absence of the rewarding tastant whereas aversive memory traces are behaviourally expressed in the presence of the punishing tastant. The 'decision' whether to behaviourally express a memory trace or not relies on a quantitive comparison between memory trace and current situation: only if the memory trace (after odour-sugar training) predicts a stronger sugar reward than currently present, animals show appetitive conditioned behaviour. Learned appetitive behaviour is best seen as active search for food - being pointless in the presence of (enough) food. Learned aversive behaviour, in turn, can be seen as escape from a punishment - being pointless in absence of punishment. Importantly, appetitive and aversive memory traces can be formed and retrieved independent from each other but also can, under appriate circumstances, summate to jointly organise conditioned behaviour. In contrast to learned behaviour, innate olfactory behaviour is not influenced by gustatory processing and vice versa. Thus, innate olfactory and gustatory behaviour is rather rigid and reflexive in nature, being executed almost regardless of other environmental cues. I suggest a behavioural circuit-model of chemosensory behaviour and the 'decision' process whether to behaviourally express a memory trace or not. This model reflects known components of the larval chemobehavioural circuit and provides clear hypotheses about the kinds of architecture to look for in the currently unknown parts of this circuit. The second chapter deals with gustatory perception and processing (especially of bitter substances). Quinine, the bitter tastant in tonic water and bitter lemon, is aversive for larvae, suppresses feeding behaviour and can act as aversive reinforcer in learning experiments. However, all three examined behaviours differ in their dose-effect dynamics, suggesting different molecular and cellular processing streams at some level. Innate choice behaviour, thought to be relatively reflexive and hard-wired, nevertheless can be influenced by the gustatory context. That is, attraction toward sweet tastants is decreased in presence of bitter tastants. The extent of this inhibitory effect depends on the concentration of both sweet and bitter tastant. Importantly, sweet tastants differ in their sensitivity to bitter interference, indicating a stimulus-specific mechanism. The molecular and cellular processes underlying the inhibitory effect of bitter tastants are unknown, but the behavioural results presented here provide a framework to further investigate interactions of gustatory processing streams.}, subject = {Lernen}, language = {en} } @article{BeisserGrohmeKopkaetal.2012, author = {Beisser, Daniela and Grohme, Markus A. and Kopka, Joachim and Frohme, Marcus and Schill, Ralph O. and Hengherr, Steffen and Dandekar, Thomas and Klau, Gunnar W. and Dittrich, Marcus and M{\"u}ller, Tobias}, title = {Integrated pathway modules using time-course metabolic profiles and EST data from Milnesium tardigradum}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-75241}, year = {2012}, abstract = {Background: Tardigrades are multicellular organisms, resistant to extreme environmental changes such as heat, drought, radiation and freezing. They outlast these conditions in an inactive form (tun) to escape damage to cellular structures and cell death. Tardigrades are apparently able to prevent or repair such damage and are therefore a crucial model organism for stress tolerance. Cultures of the tardigrade Milnesium tardigradum were dehydrated by removing the surrounding water to induce tun formation. During this process and the subsequent rehydration, metabolites were measured in a time series by GC-MS. Additionally expressed sequence tags are available, especially libraries generated from the active and inactive state. The aim of this integrated analysis is to trace changes in tardigrade metabolism and identify pathways responsible for their extreme resistance against physical stress. Results: In this study we propose a novel integrative approach for the analysis of metabolic networks to identify modules of joint shifts on the transcriptomic and metabolic levels. We derive a tardigrade-specific metabolic network represented as an undirected graph with 3,658 nodes (metabolites) and 4,378 edges (reactions). Time course metabolite profiles are used to score the network nodes showing a significant change over time. The edges are scored according to information on enzymes from the EST data. Using this combined information, we identify a key subnetwork (functional module) of concerted changes in metabolic pathways, specific for de- and rehydration. The module is enriched in reactions showing significant changes in metabolite levels and enzyme abundance during the transition. It resembles the cessation of a measurablemetabolism (e.g. glycolysis and amino acid anabolism) during the tun formation, the production of storage metabolites and bioprotectants, such as DNA stabilizers, and the generation of amino acids and cellular components from monosaccharides as carbon and energy source during rehydration. Conclusions: The functional module identifies relationships among changed metabolites (e.g. spermidine) and reactions and provides first insights into important altered metabolic pathways. With sparse and diverse data available, the presented integrated metabolite network approach is suitable to integrate all existing data and analyse it in a combined manner.}, subject = {Milnesium tardigradum}, language = {en} } @phdthesis{Hancock2012, author = {Hancock, Christine [geb. Herbst]}, title = {Influence of land use on Plantago lanceolata L. and its higher trophic levels at different spatial scales and in three geographic regions}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73877}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Heutzutage pr{\"a}gen landwirtschaftlich genutzte Fl{\"a}chen einen großen Teil der deutschen Landschaft. Die Umwandlung von nat{\"u}rlichen Lebensr{\"a}umen zu bewirtschaftetem Gr{\"u}nland beeinflusst grundlegend die Vielfalt von Pflanzen und Tieren. Zwar erh{\"o}ht die intensive Nutzung dieser Fl{\"a}chen die Produktivit{\"a}t der Pflanzen oder die Biomasse als Viehfutter auf den Wiesen. Wie diese Einfl{\"u}sse auf die Artenvielfalt, {\"O}kosysteme und trophische Interaktionen, im Laufe der Jahre wirken ist jedoch immer noch nicht vollst{\"a}ndig verstanden. Um die Funktionen der Biodiversit{\"a}t in einer landwirtschaftlich genutzten Fl{\"a}che zu verstehen konzentrierte sich meine Arbeit auf den Einfluss der Landnutzung (D{\"u}ngung, Beweidung und Mahd) auf ein Herbivor-Parasitoid-System von Plantago lanceolata. Der Spitzwegerich ist ein generalistisches Kraut mit kosmopolitischem Vorkommen. Er kann in einem sehr breiten Spektrum von Bodenverh{\"a}ltnissen (sowohl in nassen und auch in trockenen Lebensr{\"a}umen) vorkommen und ist daher ein ideales Modellsystem zur Untersuchung tritrophischer Systeme in einem Landnutzungs-intensit{\"a}tsgradienten. Die R{\"u}sselk{\"a}fer Mecinus labilis und M. pascuorum ern{\"a}hren sich von P. lanceolata und legen dort ihre Eier ab. Mesopolobus incultus ist ein generalistisch lebender Parasitoid, der verschiedenen Insektenordnungen parasitiert. Die einzigen Wirte auf P. lanceolata sind jedoch die beiden erw{\"a}hnten R{\"u}sselk{\"a}ferarten. Das Ziel meiner Studie war es, den Einfluss der Landnutzung auf ein tritrophisches System und seiner umgebenden Vegetation (Struktur, Dichte und Artenreichtum) auf unterschiedlichen r{\"a}umlichen Skalen wie Subplot, Plot und Landschaftebene in drei verschiedenen Regionen (Nord-, Mittel- und S{\"u}ddeutschland) zu untersuchen. Ich untersuchte den Einfluss der Nutzungsintensit{\"a}t nicht nur korrelativ, sondern auch experimentell. Zus{\"a}tzlich zielte ich darauf ab, aufzuzeigen wie die Vegetationszusammensetzung die Metabolite der Wirtspflanze ver{\"a}ndert und ob diese Ver{\"a}nderungen Auswirkungen auf h{\"o}here trophische Ebenen im Feld haben.}, subject = {Landnutzung}, language = {en} } @article{AzzamiRitterTautzetal.2012, author = {Azzami, Klara and Ritter, Wolfgang and Tautz, J{\"u}rgen and Beier, Hildburg}, title = {Infection of honey bees with acute bee paralysis virus does not trigger humoral or cellular immune responses}, series = {Archives of Virology}, volume = {157}, journal = {Archives of Virology}, number = {4}, doi = {10.1007/s00705-012-1223-0}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-126863}, pages = {689-702}, year = {2012}, abstract = {We have studied the responses of honey bees at different life stages (Apis mellifera) to controlled infection with acute bee paralysis virus and have identified the haemolymph of infected larvae and adult worker bees as the compartment where massive propagation of ABPV occurs. Insects respond with a broad spectrum of induced innate immune reactions to bacterial infections, whereas defence mechanisms based on RNA interference play a major role in antiviral immunity. In this study, we have determined that honey bee larvae and adult workers do not produce a humoral immune reaction upon artificial infection with ABPV, in contrast to control individuals challenged with Escherichia coli. ABPV-infected bees produced neither elevated levels of specific antimicrobial peptides (AMPs), such as hymenoptaecin and defensin, nor any general antimicrobial activity, as revealed by inhibition-zone assays. Additionally, adult bees did not generate melanised nodules upon ABPV infection, an important cellular immune function activated by bacteria and viruses in some insects. Challenge of bees with both ABPV and E. coli showed that innate humoral and cellular immune reactions are induced in mixed infections, albeit at a reduced level.}, language = {en} } @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} } @article{DossoYeoKonateetal.2012, author = {Dosso, Kanvaly and Yeo, Kolo and Konate, Souleymane and Linsenmair, Karl Eduard}, title = {Importance of protected areas for biodiversity conservation in central Cote d'Ivoire: Comparison of termite assemblages between two neighboring areas under differing levels of disturbance}, series = {Journal of Insect Science}, volume = {12}, journal = {Journal of Insect Science}, number = {131}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-133218}, year = {2012}, abstract = {To highlight human impact on biodiversity in the Lamto region, termites were studied with regard to their use as bio-indicators of habitat change in the tropics. Using a standardized method, termites were sampled in the three most common habitat types, i.e., in semi-deciduous forest, savanna woodland, and annually burned savanna, all inside Lamto Reserve and its surrounding rural domain. Termite species richness fell from 25 species in the Lamto forest to 13 species in the rural area, involving strong modification in the species composition (species turnover = 59 \%). In contrast, no significant change in diversity was found between the Lamto savannas and the rural ones. In addition, the relative abundance of termites showed a significantly greater decline in the rural domain, even in the species Ancistrotermes cavithorax (Sjostedt) (Isoptera: Termitidae), which is known to be ecologically especially versatile. Overall, the findings of this study suggest further investigation around Lamto Reserve on the impact of human activities on biodiversity, focusing on forest conversion to land uses (e.g. agricultural and silvicultural systems).}, language = {en} } @phdthesis{Bartl2012, author = {Bartl, Jasmin}, title = {Impairment of insulin signaling pathway in Alzheimer's disease}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-74197}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {The neurodegenerative disorder Alzheimer's disease (AD) is the cause of approximately 60\% of the world's 35 million patients suffering from dementia. Current research focuses here are on association with other diseases such as diabetes type 2 (T2DM), possible genetic markers, specific signal transduction pathways within the brain and potential protein modification, because the pathogenesis and etiology of AD are still not fully understood. Specifically association of T2DM with AD came to the focus with the so-called "Rotterdam study" in 1999, indicating that T2DM doubles the risk of developing AD. In the meantime, it is known that the prevalence rate in patients with T2DM is 30\%. Drugs commonly used in the treatment of T2DM such as peroxisome proliferator-activated receptors gamma (PPARγ) agonists show improvement of the cognitive abilities in patients with early stage of dementia, with potential therapeutically relevance. Therefore it is important not only to investigate a link between these diseases, but also to investigate the insulin signaling pathway in the brain of AD patients. In order to investigate this complex issue in more details and demonstrate additional links between T2DM and AD, the present study used several basic biological methods to clarify the question: "Is impaired insulin signaling pathway within the brain crucial for the development of AD?" from several points of view. The methods used in this work have been i) an analysis of single nucleotide (SNP) polymorphism of the insulin-degrading enzyme gene (IDE) in relation to risk of AD and / or of T2DM, ii) post-mortem histochemical studies of brain tissue of patients with only AD, with AD combined with T2DM and with only T2DM compared with an age-matched control group, and iii.) investigations of neurochemical pathways and gene/protein expression changes of a human cell culture as a consequences of amyloid β (Aβ) treatment. After analysis of the IDE SNP polymorphism in the selected VITA (Vienna Trans Danube Aging) cohort disease-specific effects were discovered. The upstream polymorphism (IDE2) was found to influence AD risk in a protective manner, while the downstream polymorphism (IDE7) modified the T2DM risk. Based on the SNP results, the presented study delineate the model that IDE promoter and 3‟ untranslated region/downstream variation can have different effects on IDE expression, maybe a relevant endophenotype with disorder-specific effects on AD and T2DM susceptibility. Furthermore, the human post-mortem studies could show that both AD as well as T2DM patients had a significantly lower density of the insulin receptor (IR) in the hippocampus, whereas a significantly increased density of inactive phosphorylated PPARγ has been found and this persisted even in patients with both diseases. Summarizing the histological study, it was possible to reveal common histological features of AD and T2DM, but no direct connection between the two diseases. Although AD is nowadays not only characterized by amyloid-containing plaque deposits and by the hyperphosphorylation of tau protein, the excessive Aβ42 presence in the brains of AD patients is still playing a key role. Up to date it is still not entirely clear which physical form of Aβ42 is responsible for the development of AD. The present work investigated, what impact has the state of aggregation of Aβ42 on genes and proteins of the insulin signaling pathway and the amyloid cascade. It could be shown that the oligomeric variant enhanced specifically the gene and protein expression of glycogen synthase kinase (GSK) 3β and also the enzyme activity was significantly increased, but has in turn strongly inhibited the IR gene and protein expression. Additionally, the effect of Aβ42 on monoamine oxidase B (MAO-B) was examined. An effect of both aggregated forms of Aβ42 had on enzyme activity was discovered. However, the fibrillar variants led to significantly increased activity of MAO-B while the oligomeric variants inhibited the enzyme activity. Several previous studies have demonstrated the involvement of increased MAO-B activity in AD, but the present work provides for the first time a direct link between the states of aggregation of Aβ42 to enzyme activity. Finally the results of the presented thesis can be summarized to following conclusion: Although AD and T2DM sharing some degrees of common features, still there is a lack of direct association, and therefore the diseases must be considered more independent rather than linked. But the impaired cerebral insulin signaling pathway seems to be another manifested hallmark of AD.}, subject = {Alzheimer-Krankheit}, language = {en} } @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{Ratzka2012, author = {Ratzka, Carolin}, title = {Immune responses of the ant Camponotus floridanus towards pathogens and its obligate mutualistic endosymbiont Blochmannia floridanus}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-69350}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Ants of the species Camponotus floridanus live in huge colonies composed of genetically identical or closely related animals, which should predispose them to an increased vulnerability towards infection by pathogens (Cremer et al. 2007). Therefore the question is how ants (or social insects in general) can nevertheless efficiently combat infections. In order to investigate the immune response of the ant C. floridanus, the present study initially focused on the identification of possible immune factors, encoded by the ant´s genome. By using the method "suppression subtractive hybridization" as well as by Illumnia sequencing technology, several immune-related genes could be identified. Among these were genes encoding proteins involved in pathogen recognition, signal transduction, antimicrobial activity, or general stress response. In accordance with the ant´s genome sequence (Bonasio et al. 2010), only three antimicrobial peptide (AMP) genes could be identified in C. floridanus. The gene and cDNA sequences of these AMPs were established and their expression was shown to be induced by microbial challenge. Two different defensin genes (type 1 and 2) were characterized. A detailed characterization of the mRNA and gene sequence of the other AMP, a hymenoptaecin, revealed a special repeat structure. The C. floridanus hymenoptaecin has a signal and a pro-sequence followed by a hymenoptaecin-like domain and six directly repeated hymenoptaecin domains (HDs). Since each HD is flanked by two known processing sites, proteolytic processing of the precursor protein may generate several mature AMPs. Bioinformatical analyses revealed the presence of hymenoptaecin genes with similar multipeptide precursor structure in genomes of other ant species suggesting an evolutionary conserved important role of this gene in ant immunity. C. floridanus ants harbor the obligate intracellular bacterium, Blochmannia floridanus, in specialized cells (so-called bacteriocytes), which are intercalated between midgut cells as well as in ovaries of females (Blochmann 1882; Sauer et al. 2002; Schr{\"o}der et al. 1996). Ant hosts face the problem that on the one hand they have to maintain the beneficial symbiotic bacteria and on the other hand they need to raise an immune response against harmful pathogenic bacteria during an infection. It was investigated, if endosymbionts are actually detected by the host immune system. Injection of B. floridanus induced an immune response of its host C. floridanus, which was comparable to the one towards pathogens. This means that, despite the evolutionary established cooperation of the endosymbionts and their hosts, these bacteria are still recognized as „non-self" by the host immune system. This finding led to the question, if the ant immune system might be involved in regulation of the endosymbiont number in the midgut tissue in order to avoid their uncontrolled replication. During the holometabolous life cycle of the ant hosts the distribution of bacteriocytes and of Blochmannia endosymbionts is remarkably dynamic and peaks in late pupal stages, in which the entire midgut is transformed into a symbiotic organ (Stoll et al. 2010). It was hypothesized that hosts could regulate the number of endosymbionts present in their tissues via the innate immune system. A quantitative gene expression analysis of assumed symbiosis-relevant candidate genes revealed distinct expression patterns of some genes according to developmental stage and tissue. Moreover, the immune gene expression in response to bacterial challenge was investigated in the pupal stage. By an artificial immune-challenge of pupae it was confirmed that in fact the immune response of the endosymbiont-bearing midgut tissue differs from that of other body parts. The data support a key role for amidase peptidoglycan recognition proteins (PGRPs), especially PGRP-LB, in endosymbiont tolerance and suggest an involvement of the lysosomal system in control of Blochmannia endosymbionts. In sum, this thesis provides a first description of the immune response of the ant C. floridanus. A comprehensive set of immune-relevant genes was determined. Especially, the identification and molecular characterization of the hymenoptaecin gene delivered new insights into the immune competence of ants in general. Moreover, first indications could be gathered for the involvement of the immune system in controlling the endosymbiont B. floridanus.}, subject = {Humorale Immunit{\"a}t}, language = {en} } @article{PrustyBoehmeBergmannetal.2012, author = {Prusty, Bhupesh K. and B{\"o}hme, Linda and Bergmann, Birgit and Siegl, Christine and Krause, Eva and Mehlitz, Adrian and Rudel, Thomas}, title = {Imbalanced oxidative stress causes chlamydial persistence during non-productive Human Herpes Virus co-infection}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-76215}, year = {2012}, abstract = {Both human herpes viruses and Chlamydia are highly prevalent in the human population and are detected together in different human disorders. Here, we demonstrate that co-infection with human herpes virus 6 (HHV6) interferes with the developmental cycle of C. trachomatis and induces persistence. Induction of chlamydial persistence by HHV6 is independent of productive virus infection, but requires the interaction and uptake of the virus by the host cell. On the other hand, viral uptake is strongly promoted under co-infection conditions. Host cell glutathione reductase activity was suppressed by HHV6 causing NADPH accumulation, decreased formation of reduced glutathione and increased oxidative stress. Prevention of oxidative stress restored infectivity of Chlamydia after HHV6-induced persistence. We show that co-infection with Herpes simplex virus 1 or human Cytomegalovirus also induces chlamydial persistence by a similar mechanism suggesting that Chlamydia -human herpes virus co-infections are evolutionary shaped interactions with a thus far unrecognized broad significance.}, subject = {Biologie}, language = {en} } @phdthesis{Mihlan2012, author = {Mihlan, Sabrina [geb. Jasper]}, title = {Identifikation von Zonula Occludens 2 (ZO-2) als neuen LASP-1 Interaktionspartner und Aufkl{\"a}rung der LASP-1/ZO-2 Kern-Zytosol Translokation}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73442}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {LASP-1 (LIM und SH3 Dom{\"a}nen Protein) ist ein in Zellen ubiquit{\"a}r vorkommendes Protein, welches in verschiedenen Tumorgeweben eine pathophysiologische {\"U}berexpression aufweist. Das Protein besitzt eine LIM Dom{\"a}ne, zwei Aktinbindungsregionen sowie eine SH3 Dom{\"a}ne und bindet einerseits an dynamischen Aktinstrukturen wie den fokalen Kontakten, Lamellopodien und Membranforts{\"a}tzen, kann andererseits aber auch in den Zellkern translokalisieren. F{\"u}r Aktinstrukturen wirkt LASP-1 als Ger{\"u}stprotein und ist wichtig f{\"u}r die Migration und Proliferation der Zellen. Die Funktion von LASP-1 im Zellkern ist noch nicht bekannt, da aber in Tumorzellen eine erh{\"o}hte nukleare Akkumulation von LASP-1 beobachtet werden konnte, deren Intensit{\"a}t mit der Tumorgr{\"o}ße sowie dem Langzeit{\"u}berleben der Patientinnen korreliert, ist LASP-1, zus{\"a}tzlich zu seiner Funktion als Strukturprotein, vermutlich auch ein Transkriptionsfaktor oder ein transkriptioneller Kofaktor. Eine Herunterregulation von LASP-1 in verschiedenen Tumorentit{\"a}ten f{\"u}hrt zur Inhibition der Proliferation und Migration. In dieser Arbeit konnte der bisher unbekannte Zellkernimport und -export von LASP-1 aufgekl{\"a}rt werden. Maßgeblich daran beteiligt ist ein durch Pulldown Experimente neu identifizierter LASP-1 Bindungspartner: das Zonula Occludens 2 Protein (ZO-2). Mittels Immunpr{\"a}zipitationen und Immunfluoreszenzen wurde diese Interaktion best{\"a}tigt. Nach Phosphorylierung von LASP-1 an Ser-146 durch Aktivierung der cAMP-abh{\"a}ngigen Proteinkinase (PKA) kommt es zu einer partiellen Abl{\"o}sung des LASP-1/ZO-2 Komplexes aus den fokalen Kontakten hin zu einer vermehrten Kernlokalisation beider Proteine. Dies l{\"a}sst sich durch Kern/Zytosol Trennungen belegen. Dabei ist die Bindung von LASP-1 an ZO-2 essentiell f{\"u}r die Translokation in den Zellkern, da bei einem ZO-2 Knockdown auch nach PKA Aktivierung LASP-1 zytosolisch lokalisiert bleibt. Wie Mutationsanalysen zeigen, findet die Interaktion zwischen der C-terminalen SH3 Dom{\"a}ne im LASP-1 und der Prolin-reichen SH3-Bindungssequenz im Bereich der Aminos{\"a}uren 1103-1121 am C-Terminus im ZO-2 statt. Die Translokation des Komplexes in den Kern erfolgt dabei {\"u}ber das Kernlokalisationssignal im ZO-2, da die LASP-1 Sequenz selbst keine nukleare Importsequenz aufweist. Im Zellkern konnte die direkte Interaktion von LASP-1 und ZO-2 mittels Duolink® Proximity Ligation Assay sichtbar gemacht werden. Der Export der Proteine erfolgt {\"u}ber das Protein CRM1. Eine Inhibition der Kernexportmaschinerie mit Leptomycin B erh{\"o}ht die Konzentration beider Proteine im Zellkern. Das nukleare Exportsignal (NES) im LASP-1 konnte durch Punktmutationen N-terminal der Leucin-reichen Aminos{\"a}uresequenz 70-77 zugeordnet werden (NLRLKQQS). Im letzten Schritt dieses Zyklus erfolgt die Relokalisation von LASP-1 zur{\"u}ck an die Zellmembranstrukturen. Der neu gefundene Signalweg dient wahrscheinlich zur Weiterleitung von externen Stimuli in den Kern und zur Genregulation - mit LASP-1 als Transkriptionsfaktor oder transkriptionellen Kofaktor.}, subject = {Tumorzelle}, language = {de} } @article{BugaScholzKumaretal.2012, author = {Buga, Ana-Maria and Scholz, Claus J{\"u}rgen and Kumar, Senthil and Herndon, James G. and Alexandru, Dragos and Cojocaru, Gabriel Radu and Dandekar, Thomas and Popa-Wagner, Aurel}, title = {Identification of New Therapeutic Targets by Genome-Wide Analysis of Gene Expression in the Ipsilateral Cortex of Aged Rats after Stroke}, series = {PLoS One}, volume = {7}, journal = {PLoS One}, number = {12}, doi = {10.1371/journal.pone.0050985}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-130657}, pages = {e50985}, year = {2012}, abstract = {Background: Because most human stroke victims are elderly, studies of experimental stroke in the aged rather than the young rat model may be optimal for identifying clinically relevant cellular responses, as well for pinpointing beneficial interventions. Methodology/Principal Findings: We employed the Affymetrix platform to analyze the whole-gene transcriptome following temporary ligation of the middle cerebral artery in aged and young rats. The correspondence, heat map, and dendrogram analyses independently suggest a differential, age-group-specific behaviour of major gene clusters after stroke. Overall, the pattern of gene expression strongly suggests that the response of the aged rat brain is qualitatively rather than quantitatively different from the young, i.e. the total number of regulated genes is comparable in the two age groups, but the aged rats had great difficulty in mounting a timely response to stroke. Our study indicates that four genes related to neuropathic syndrome, stress, anxiety disorders and depression (Acvr1c, Cort, Htr2b and Pnoc) may have impaired response to stroke in aged rats. New therapeutic options in aged rats may also include Calcrl, Cyp11b1, Prcp, Cebpa, Cfd, Gpnmb, Fcgr2b, Fcgr3a, Tnfrsf26, Adam 17 and Mmp14. An unexpected target is the enzyme 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 in aged rats, a key enzyme in the cholesterol synthesis pathway. Post-stroke axonal growth was compromised in both age groups. Conclusion/Significance: We suggest that a multi-stage, multimodal treatment in aged animals may be more likely to produce positive results. Such a therapeutic approach should be focused on tissue restoration but should also address other aspects of patient post-stroke therapy such as neuropathic syndrome, stress, anxiety disorders, depression, neurotransmission and blood pressure.}, language = {en} } @article{VieiraJonesDanonetal.2012, author = {Vieira, Jacqueline and Jones, Alex R. and Danon, Antoine and Sakuma, Michiyo and Hoang, Nathalie and Robles, David and Tait, Shirley and Heyes, Derren J. and Picot, Marie and Yoshii, Taishi and Helfrich-F{\"o}rster, Charlotte and Soubigou, Guillaume and Coppee, Jean-Yves and Klarsfeld, Andr{\´e} and Rouyer, Francois and Scrutton, Nigel S. and Ahmad, Margaret}, title = {Human Cryptochrome-1 Confers Light Independent Biological Activity in Transgenic Drosophila Correlated with Flavin Radical Stability}, series = {PLoS One}, volume = {7}, journal = {PLoS One}, number = {3}, doi = {10.1371/journal.pone.0031867}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-134513}, pages = {e31867}, year = {2012}, abstract = {Cryptochromes are conserved flavoprotein receptors found throughout the biological kingdom with diversified roles in plant development and entrainment of the circadian clock in animals. Light perception is proposed to occur through flavin radical formation that correlates with biological activity in vivo in both plants and Drosophila. By contrast, mammalian (Type II) cryptochromes regulate the circadian clock independently of light, raising the fundamental question of whether mammalian cryptochromes have evolved entirely distinct signaling mechanisms. Here we show by developmental and transcriptome analysis that Homo sapiens cryptochrome - 1 (HsCRY1) confers biological activity in transgenic expressing Drosophila in darkness, that can in some cases be further stimulated by light. In contrast to all other cryptochromes, purified recombinant HsCRY1 protein was stably isolated in the anionic radical flavin state, containing only a small proportion of oxidized flavin which could be reduced by illumination. We conclude that animal Type I and Type II cryptochromes may both have signaling mechanisms involving formation of a flavin radical signaling state, and that light independent activity of Type II cryptochromes is a consequence of dark accumulation of this redox form in vivo rather than of a fundamental difference in signaling mechanism.}, language = {en} } @phdthesis{Schuster2012, author = {Schuster, Beatrice}, title = {Genotyping Fanconi Anemia : From Known to Novel Genes -From Classical Genetic Approaches to Next Generation Sequencing}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85515}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Fanconi anemia (FA) is an autosomal recessive or X-chromosomal inherited disorder, which is not only phenotypically but also genotypically very heterogeneous. While its hallmark feature is progressive bone marrow failure, many yet not all patients suffer additionally from typical congenital malformations like radial ray defects and growth retardation. In young adulthood the cumulative risk for developing hematological or other malignancies is compared to the general population several hundred-fold increased. The underlying molecular defect is the deficiency of DNA interstrand crosslink (ICL) repair. ICLs are deleterious lesions, which interfere with crucial cellular processes like transcription and replication and thereby can lead to malignant transformation, premature senescence or cell death. To overcome this threat evolution developed a highly complex network of interacting DNA repair pathways, which is conserved completely only in vertebrates. The so called FA/BRCA DNA damage response pathway is able to recognize ICLs on stalled replication forks and promotes their repair through homologous recombination (HR). Today we know 15 FA genes (FANCA, -B, -C, -D1, -D2, -E, -F, -G, -I, -J, -L, -M, -N, -O and -P) whose products are involved in this pathway. Although more than 80\% of FA patients carry biallelic mutations in either FANCA, FANCC or FANCG, there are still some who cannot be assigned to any of the known complementation groups. This work aimed to indentify the di¬sease causing mutations in a cohort of those unassigned patients. Initial screens of the candidate genes FAN1, MHF1 and MHF2 did not reveal any pathogenic alterations. Moreover, FAN1 could be excluded as FA candidate gene because patients carrying a homozygous microdeletion including the FAN1 locus did not show a phenotype comparable to FA patients. In the case of MHF1 and MHF2 the reason for the negative screening result is not clear. Mutation carriers might be rare or, regarding the diverse and also FA pathway independent protein functions, phenotypically not comparable to FA patients. Nevertheless, this study contri¬buted to the identification and characterization of the most recent members of the FA pathway - RAD51C (FANCO), SLX4 (FANCP) and XPF (FANCQ). FANCO is one of the RAD51 paralogs and is involved in crucial steps of HR. But since the only reported FA-O patient has so far not developed any hematological anomalies, FANCO is tentatively designated as gene underlying an FA-like disorder. In contrast, patients carrying biallelic mutations in FANCP do not only show hematological anomalies, but as well congenital malformations typical for FA. The distinct role of FANCP in the FA pathway could not be determined, but it is most likely the coordination of structure-specific nucleases during ICL excision. One of these nucleases is the heterodimer XPF/ERCC1. XPF is probably disease causing in the complementation group FA-Q and is the first FA gene, which was identified by Next Generation Sequencing (NGS). Extraordinarily is that mutations in this gene had previously been reported to cause two other disorders, xeroderma pigmentosum and segmental progeria. Despite some overlaps, it was shown that the divergent phenotypes could clearly be distinguished and are caused by distinct functional defects of XPF. Additionally, this work aimed to improve and accelerate the genotyping process of FA patients in general. Therefore, classical approaches should be complemented or fully replaced by approa¬ches using NGS. Massively parallel sequencing of the whole exome proved to be most appro¬priate and the establishment of an FA-specific analysis pipeline facilitated improved molecular diagnostics by combining complementation group assignment and mutation analysis in one step. Consequently two NGS studies revealed the pathogenic defect in several previously unassigned FA patients and thereby added another patient to one of the most recent subtypes, FA-P. In summary, this work contributed not only to further completion of the FA/BRCA DNA repair network by adding three novel genes, it also showed that classical molecular approaches for re¬search as well as for diagnostics could be replaced by NGS.}, subject = {Fanconi An{\"a}mie}, language = {en} } @article{AgostonLiHaslingeretal.2012, author = {Agoston, Zsuzsa and Li, Naixin and Haslinger, Anja and Wizenmann, Andrea and Schulte, Dorothea}, title = {Genetic and physical interaction of Meis2, Pax3 and Pax7 during dorsal midbrain development}, series = {BMC Developmental Biology}, volume = {12}, journal = {BMC Developmental Biology}, number = {10}, doi = {10.1186/1471-213X-12-10}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-132626}, year = {2012}, abstract = {Background: During early stages of brain development, secreted molecules, components of intracellular signaling pathways and transcriptional regulators act in positive and negative feed-back or feed-forward loops at the mid-hindbrain boundary. These genetic interactions are of central importance for the specification and subsequent development of the adjacent mid-and hindbrain. Much less, however, is known about the regulatory relationship and functional interaction of molecules that are expressed in the tectal anlage after tectal fate specification has taken place and tectal development has commenced. Results: Here, we provide experimental evidence for reciprocal regulation and subsequent cooperation of the paired-type transcription factors Pax3, Pax7 and the TALE-homeodomain protein Meis2 in the tectal anlage. Using in ovo electroporation of the mesencephalic vesicle of chick embryos we show that (i) Pax3 and Pax7 mutually regulate each other's expression in the mesencephalic vesicle, (ii) Meis2 acts downstream of Pax3/7 and requires balanced expression levels of both proteins, and (iii) Meis2 physically interacts with Pax3 and Pax7. These results extend our previous observation that Meis2 cooperates with Otx2 in tectal development to include Pax3 and Pax7 as Meis2 interacting proteins in the tectal anlage. Conclusion: The results described here suggest a model in which interdependent regulatory loops involving Pax3 and Pax7 in the dorsal mesencephalic vesicle modulate Meis2 expression. Physical interaction with Meis2 may then confer tectal specificity to a wide range of otherwise broadly expressed transcriptional regulators, including Otx2, Pax3 and Pax7.}, language = {en} } @phdthesis{Englberger2012, author = {Englberger, Eva}, title = {Gene regulation in hearts of Hey-mutant mouse embryos and monitoring of sub-cellular Hey1 distribution}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73395}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Hey-mutant mouse hearts at embryonic day E14.5 were shown to react to the knock out of Hey2 with several up-regualted genes. This up-regulation is due to the lack of Hey2 and cannot be explained by the structural changes in heart morphology as shown using control animals. Part of the gene regulation was further validated using in situ hybridization. Hey1 was located to the nucleus in immunofluorescence experiments. However, experiments on protein level showed also amount of Hey1 within the cytoplasm. The nuclear localization of Hey1 was unchanged during all cell cycle phases as well as when CaMKII was co-expressed or other cellular pathways were inhibited or stimulated. Hey1 does not seem to interact with the nuclear transport proteins importin-alpha and -beta, therefore it still needs to be elucidated how Hey1 is transported into the nucleus.}, subject = {Maus}, language = {en} } @article{SturmHessWeibeletal.2012, author = {Sturm, Julia B. and Hess, Michael and Weibel, Stephanie and Chen, Nanhei G. and Yu, Yong A. and Zhang, Quian and Donat, Ulrike and Reiss, Cora and Gambaryan, Stepan and Krohne, Georg and Stritzker, Jochen and Szalay, Aladar A.}, title = {Functional hyper-IL-6 from vaccinia virus-colonized tumors triggers platelet formation and helps to alleviate toxicity of mitomycin C enhanced virus therapy}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-75224}, year = {2012}, abstract = {Background: Combination of oncolytic vaccinia virus therapy with conventional chemotherapy has shown promise for tumor therapy. However, side effects of chemotherapy including thrombocytopenia, still remain problematic. Methods: Here, we describe a novel approach to optimize combination therapy of oncolytic virus and chemotherapy utilizing virus-encoding hyper-IL-6, GLV-1h90, to reduce chemotherapy-associated side effects. Results: We showed that the hyper-IL-6 cytokine was successfully produced by GLV-1h90 and was functional both in cell culture as well as in tumor-bearing animals, in which the cytokine-producing vaccinia virus strain was well tolerated. When combined with the chemotherapeutic mitomycin C, the anti-tumor effect of the oncolytic virotherapy was significantly enhanced. Moreover, hyper-IL-6 expression greatly reduced the time interval during which the mice suffered from chemotherapy-induced thrombocytopenia. Conclusion: Therefore, future clinical application would benefit from careful investigation of additional cytokine treatment to reduce chemotherapy-induced side effects.}, subject = {Biologie}, language = {en} } @phdthesis{Ulrich2012, author = {Ulrich, Tanja}, title = {Function of Lin9 in vivo and MAP3K4-p38 signaling regulates p53 mediated cell cycle arrest after defective mitosis}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73975}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Eine genaue Kontrolle des Verlaufs durch die Mitose ist entscheidend f{\"u}r die Gew{\"a}hrleistung genomischer Stabilit{\"a}t und f{\"u}r die Vermeidung von Aneuploidy. Der DREAM Komplex ist ein wichtiger Regulator der Expression von mitotischen Genen. Die Depletion der DREAM-Untereinheit Lin9, f{\"u}hrt zu einer verminderten Expression von G2/M Genen und beeintr{\"a}chtigt die Proliferation. In konditionellen knockout Mauszellen (MEFs) verursacht das Ausschalten von Lin9 Defekte in Mitose und Zytokinese und l{\"o}st vorzeitige Seneszenz aus, um eine weitere Zellproliferation zu verhindern. In dieser Arbeit konnte gezeigt werden, dass der seneszente Ph{\"a}notyp in Lin9 knockout MEFs unabh{\"a}ngig von den beiden Tumorsuppressor-Signalwegen p53-p21 und p16-pRB induziert wird. Untersuchungen mit dem konditionellen Lin9 knockout Mausmodell verdeutlichten die wichtige Funktion von Lin9 in der Regulierung der mitotischen Genexpression und der Proliferation in vivo. Das Fehlen von Lin9 f{\"u}hrte zu einer verringerten Proliferation in den Krypten des D{\"u}nndarms und verursachte eine Atrophie des Darmepithels und einen schnell eintretenden Tod der Tiere. Im zweiten Teil der Arbeit wurden Signalwege untersucht, die nach fehlerhafter Zytokinese zu einem p53 vermittelten G1-Arrest f{\"u}hren. Hierf{\"u}r wurde ein chemischer Inhibitor der mitotischen Kinase Aurora B verwendet. Mit Hilfe eines Hochdurchsatz siRNA Screens wurde die MAP Kinase MAP3K4 als Aktivator des p53 Signalwegs identifiziert. Es konnte gezeigt werden, dass MAP3K4 die Stresskinase p38b aktiviert, um den p53 vermittelten Zellzyklusarrest in tetraploiden Zellen auszul{\"o}sen. Dabei wurde p38b nach Hemmung von Aurora B f{\"u}r die transkriptionelle Aktivierung des p53 Zielgens p21 ben{\"o}tigt. Im Gegenteil dazu erfolgte die Phosphorylierung, Stabilisierung und die Rekrutierung von p53 an den p21 Promoter unabh{\"a}ngig von p38. Die teilweise Hemmung von Aurora B zeigte, dass fehlerhafte Segregation von Chromosomen auch den MAP3K4-p38-p53 Signalweg aktiviert und l{\"a}sst darauf schließen, dass subtile Defekte in der Mitose ausreichen diesen Stress-Signalweg zu induzieren. Obwohl p38 f{\"u}r den G1 Zellzyklusarrest nach mitotischen Sch{\"a}den erforderlich war, f{\"u}hrte die gleichzeitige Inhibierung von p38 und Aurora B {\"u}ber einen l{\"a}ngeren Zeitraum zu einer verringerten Proliferation, vermutlich aufgrund verst{\"a}rkter Apoptose. Es ist anzunehmen, dass der MAP3K4-p38-p53 Signalweg generell nach Defekten in der Mitose oder Zytokinese aktiviert wird um Zellen in G1 zu arretieren und um chromosomale Instabilit{\"a}t zu vermeiden.}, subject = {Mitose}, language = {en} } @article{OwenSauerGaus2012, author = {Owen, Dylan M. and Sauer, Markus and Gaus, Katharina}, title = {Fluorescence localization microscopy}, series = {Communicative \& Integrative Biology}, volume = {5}, journal = {Communicative \& Integrative Biology}, number = {4}, doi = {10.4161/cib.20348}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-124416}, pages = {345-349}, year = {2012}, abstract = {Localization microscopy techniques are super-resolution fluorescence imaging methods based on the detection of individual molecules. Despite the relative simplicity of the microscope setups and the availability of commercial instruments, localization microscopy faces unique challenges. While achieving super-resolution is now routine, issues concerning data analysis and interpretation mean that revealing novel biological insights is not. Here, we outline why data analysis and the design of robust test samples may hold the key to harness the full potential of localization microscopy.}, language = {en} } @article{RakosyStreinzerPaulusetal.2012, author = {Rakosy, Demetra and Streinzer, Martin and Paulus, Hannes F. and Spaethe, Johannes}, title = {Floral visual signal increases reproductive success in a sexually deceptive orchid}, series = {Arthropod-Plant Interactions}, volume = {6}, journal = {Arthropod-Plant Interactions}, number = {4}, doi = {10.1007/s11829-012-9217-0}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-127209}, pages = {671-681}, year = {2012}, abstract = {Sexually deceptive orchids mimic signals emitted by female insects in order to attract mate-searching males. Specific attraction of the targeted pollinator is achieved by sex pheromone mimicry, which constitutes the major attraction channel. In close vicinity of the flower, visual signals may enhance attraction, as was shown recently in the sexually deceptive orchid Ophrys heldreichii. Here, we conducted an in situ manipulation experiment in two populations of O. heldreichii on Crete to investigate whether the presence/absence of the conspicuous pink perianth affects reproductive success in two natural orchid populations. We estimated reproductive success of three treatment groups (with intact, removed and artificial perianth) throughout the flowering period as pollinaria removal (male reproductive success) and massulae deposition (female reproductive success). Reproductive success was significantly increased by the presence of a strong visual signal—the conspicuous perianth—in one study population, however, not in the second, most likely due to the low pollinator abundance in the latter population. This study provides further evidence that the coloured perianth in O. heldreichii is adaptive and thus adds to the olfactory signal to maximise pollinator attraction and reproductive success.}, language = {en} } @phdthesis{Chen2012, author = {Chen, Yi-chun}, title = {Experimental access to the content of an olfactory memory trace in larval Drosophila}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-83705}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Animals need to evaluate their experiences in order to cope with new situations they encounter. This requires the ability of learning and memory. Drosophila melanogaster lends itself as an animal model for such research because elaborate genetic techniques are available. Drosphila larva even saves cellular redundancy in parts of its nervous system. My Thesis has two parts dealing with associative olfactory learning in larval Drosophila. Firstly, I tackle the question of odour processing in respect to odour quality and intensity. Secondly, by focusing on the evolutionarily conserved presynaptic protein Synapsin, olfactory learning on the cellular and molecular level is investigated. Part I.1. provides a behaviour-based estimate of odour similarity in larval Drosophila by using four recognition-type experiments to result in a combined, task-independent estimate of perceived difference between odour-pairs. A further comparison of these combined perceived differences to published calculations of physico-chemical difference reveals a weak correlation between perceptual and physico-chemical similarity. Part I.2. focuses on how odour intensity is interpreted in the process of olfactory learning in larval Drosophila. First, the dose-effect curves of learnability across odour intensities are described in order to choose odour intensities such that larvae are trained at intermediate odour intensity, but tested for retention either with that trained intermediate odour intensity, or with respectively HIGHer or LOWer intensities. A specificity of retention for the trained intensity is observed for all the odours used. Such intensity specificity of learning adds to appreciate the richness in 'content' of olfactory memory traces, and to define the demands on computational models of associative olfactory memory trace formation. In part II.1. of the thesis, the cellular site and molecular mode of Synapsin function is investigated- an evolutionarily conserved, presynaptic vesicular phosphoprotein. On the cellular level, the study shows a Synapsin-dependent memory trace in the mushroom bodies, a third-order "cortical" brain region of the insects; on the molecular level, Synapsin engages as a downstream element of the AC-cAMP-PKA signalling cascade.}, subject = {Taufliege}, language = {en} } @phdthesis{Chaianunporn2012, author = {Chaianunporn, Thotsapol}, title = {Evolution of dispersal and specialization in systems of interacting species}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-76779}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {A metacommunity approach will be a useful framework to assess and predict changes in biodiversity in spatially structured landscapes and changing environments. However, the relationship between two core elements of metacommunity dynamics, dispersal and species interaction are not well understood. Most theoretical studies on dispersal evolution assume that target species are in isolation and do not interact with other species although the species interactions and community structure should have strong interdependence with dispersal. On the one hand, a species interaction can change the cost and benefit structure of dispersing in relation to non-dispersing individuals. On the other hand, with dispersal, an individual can follow respectively avoid species partners. Moreover, it is also important to explore the interdependence between dispersal and species interaction with spatial and temporal heterogeneity of environment because it would allow us to gain more understanding about responses of community to disturbances such as habitat destruction or global climate change, and this aspect is up to now not well-studied. In this thesis, I focus on the interactive and evolutionary feedback effects between dispersal and various types of interspecific interactions in different environmental settings. More specifically, I contrast dispersal evolution in scenarios with different types of interactions (chapter 2), explore the concurrent evolution of dispersal and habitat niche width (specialization) in spatial heterogeneous landscape (chapter 3) and consider (potential) multidimensional evolutionary responses under climate change (chapter 4). Moreover, I investigate consequences of different dispersal probability and group tolerance on group formation respectively group composition and the coexistence of 'marker types' (chapter 5). For all studies, I utilize individual-based models of single or multiple species within spatially explicit (grid-based) landscapes. In chapter 5, I also use an analytical model in addition to an individual-based model to predict phenomenon in group recognition and group formation. ...}, subject = {Tiergesellschaft}, language = {en} } @phdthesis{Buckel2012, author = {Buckel, Lisa}, title = {Evaluating the combination of oncolytic vaccinia virus and ionizing radiation in therapy of preclinical glioma models}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85309}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Glioblastoma multiforme (GBM) represents the most aggressive form of malignant brain tumors and remains a therapeutically challenge. Intense research in the field has lead to the testing of oncolytic viruses to improve tumor control. Currently, a variety of different oncolytic viruses are being evaluated for their ability to be used in anti-cancer therapy and a few have entered clinical trials. Vaccinia virus, is one of the viruses being studied. GLV-1h68, an oncolytic vaccinia virus engineered by Genelux Corporation, was constructed by insertion of three gene cassettes, RUC-GFP fusion, β-galactosidase and β- glucuronidase into the genome of the LIVP strain. Since focal tumor radiotherapy is a mainstay for cancer treatment, including glioma therapy, it is of clinical relevance to assess how systemically administered oncolytic vaccinia virus could be combined with targeted ionizing radiation for therapeutic gain. In this work we show how focal ionizing radiation (IR) can be combined with multiple systemically delivered oncolytic vaccinia virus strains in murine models of human U-87 glioma. After initial experiments which confirmed that ionizing radiation does not damage viral DNA or alter viral tropism, animal studies were carried out to analyze the interaction of vaccinia virus and ionizing radiation in the in vivo setting. We found that irradiation of the tumor target, prior to systemic administration of oncolytic vaccinia virus GLV-1h68, increased viral replication within the U-87 xenografts as measured by viral reporter gene expression and viral titers. Importantly, while GLV-1h68 alone had minimal effect on U-87 tumor growth delay, IR enhanced GLV-1h68 replication, which translated to increased tumor growth delay and mouse survival in subcutaneous and orthotopic U-87 glioma murine models compared to monotherapy with IR or GLV-1h68. The ability of IR to enhance vaccinia replication was not restricted to the multi-mutated GLV-1h68, but was also seen with the less attenuated oncolytic vaccinia, LIVP 1.1.1. We have demonstrated that in animals treated with combination of ionizing radiation and LIVP 1.1.1 a strong pro-inflammatory tissue response was induced. When IR was given in a more clinically relevant fractionated scheme, we found oncolytic vaccinia virus replication also increased. This indicates that vaccinia virus could be incorporated into either larger hypo-fraction or more conventionally fractionated radiotherapy schemes. The ability of focal IR to mediate selective replication of systemically injected oncolytic vaccinia was demonstrated in a bilateral glioma model. In mice with bilateral U-87 tumors in both hindlimbs, systemically administered oncolytic vaccinia replicated preferentially in the focally irradiated tumor compared to the shielded non- irradiated tumor in the same mouse We demonstrated that tumor control could be further improved when fractionated focal ionizing radiation was combined with a vaccinia virus caring an anti-angiogenic payload targeting vascular endothelial growth factor (VEGF). Our studies showed that following ionizing radiation expression of VEGF is upregulated in U-87 glioma cells in culture. We further showed a concentration dependent increase in radioresistance of human endothelial cells in presence of VEGF. Interestingly, we found effects of vascular endothelial growth factor on endothelial cells were reversible by adding purified GLAF-1 to the cells. GLAF-1 is a single- chain antibody targeting human and murine VEGF and is expressed by oncolytic vaccinia virus GLV-109. In U-87 glioma xenograft murine models the combination of fractionated ionizing radiation with GLV-1h164, a vaccinia virus also targeting VEGF, resulted in the best volumetric tumor response and a drastic decrease in vascular endothelial growth factor. Histological analysis of embedded tumor sections 14 days after viral administration confirmed that blocking VEGF translated into a decrease in vessel number to 30\% of vessel number found in control tumors in animals treated with GLV-164 and fractionated IR which was lower than for all other treatment groups. Our experiments with GLV-1h164 and fractionated radiotherapy have shown that in addition to ionizing radiation and viral induced tumor cell destruction we were able to effectively target the tumor vasculature. This was achieved by enhanced viral replication translating in increased levels of GLAF-2 disrupting tumor vessels as well as the radiosensitization of tumor vasculature to IR by blocking VEGF. Our preclinical results have important clinical implications of how focal radiotherapy can be combined with systemic oncolytic viral administration for highly aggressive, locally advanced tumors with the potential, by using a vaccinia virus targeting human vascular endothelial growth factor, to further increase tumor radiation sensitivity by engaging the vascular component in addition to cancer cells.}, subject = {Gliom}, language = {en} } @phdthesis{Schmidt2012, author = {Schmidt, Traudel}, title = {Establishment of Hey-triple-KO-ES cells and characterisation of Bre, a Hey binding partner}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85459}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Hey1, Hey2 and HeyL are downstream effectors of the Notch signalling pathway. Hey genes play decisive roles during embryonic development for example in cardiovascular development. However, the precise transcriptional programmes and genes, which are affected by each single Hey gene, are still poorly understood. One drawback for the analysis of Hey1, Hey2 or HeyL single gene function is that these genes are co-expressed in many tissues and share a high degree of functional redundancy. Thus, it was necessary to establish a system, which is either devoid of Hey expression, or just comprises one single Hey gene family member. For this, Hey1(fl/fl)/Hey2(-/-)/HeyL(-/-)- as well as Hey-triple- knock out (KO)-ES cells (embryonic stem cells) were generated in this work, because ES cells and their differentiation as EBs (embryoid bodies) represent a valuable tool for the in vitro analysis of embryonic developmental processes. After the establishment of Hey1(fl/fl)/Hey2(-/-)/HeyL(-/-)- and Hey-triple- KO-ES cells, it could be seen by ALP staining and pluripotency marker expression that loss of Hey expression did not affect ES cell pluripotency features. Thus, these ES cells represent bona fide ES cells and could be further used for the differentiation as EBs. Here, differences in gene expression between Hey1(fl/fl)/Hey2(-/-)/HeyL(-/-)- and Hey-triple- KO-ES cells (after the loss of Hey1) could be observed in realtime-RT-PCR analysis for the endodermal marker AFP as well as for neural and myogenic markers in d10 EBs. However, the establishment of inducible Hey1, Hey2 or HeyL ES cell lines will be essential to confirm these findings and to search for novel Hey target genes. To get further insight into the mode of Hey action, the analysis of Hey interaction partners is necessary. One such binding partner, the Bre protein, has previously been found in a yeast-two-hybrid screen. Bre has been described to be a member of two distinct complexes (i.e. the nuclear BRCA1-A complex with a function in DNA damage response and the cytoplasmic BRISC complex), to directly interact with the TNF-receptor and Fas and to interfere with apoptotic signalling. The Hey-Bre interaction could be further corroborated in this work; yet, it was not possible to narrow down the interaction site of Bre with Hey1. It rather seems that non-overlapping parts of the Bre protein may bind to Hey. This interaction may be direct- pointing to more than one interaction site inside the Bre protein - or via a common binding partner such as the endogenous Bre protein itself. Besides the interaction studies, functional assays were performed for a more detailed characterisation of Hey1 and Bre interaction. Here, it could be shown that Hey1 over-expression did not have any influence on Bre sub-cellular localisation. Interestingly, it could be demonstrated that Bre positively interfered with Hey1 repressive function in luciferase assays at three of four promoters analysed. Moreover, interaction with Bre seems to lead to a stabilisation of Hey1. As Bre has been described to modulate the E3-ligase activity intrinsic to the BRCC complex it was analysed whether Bre over-expression results in an ubiquitination of Hey1. Yet, this could not be observed in the present work. Furthermore, an interaction of Bre with ubiquitinated proteins could not be demonstrated in an ubiquitin binding assay. To obtain a better insight into Bre function, Bre LacZ gene trap-ES cells and animals were generated. However, realtime-RT-analyses revealed that these cells and mice did not show a loss of Bre expression on mRNA level indicating that insertion mutagenesis did not occur as expected. However, embryos derived from these mice could nevertheless be used for the detection of tissues with Bre expression by β-galactosidase staining. Bre deficiency on mRNA levels was only achieved after the deletion of the floxed exon 3 resulting in the generation of Bre del-mice. Bre del-mice were fertile and without any obvious phenotype and they were used for the generation of Bre del- and wt-MEFs (murine embryonic fibroblasts). Characterisation of these cells showed that proliferation was not affected after loss of Bre (neither under normal nor under stress conditions). However, loss of Bre notably resulted in a reduction in the BRCA1 DNA damage response, in a slightly increased sensitivity towards apoptosis induction by FasL treatment and in an increase in the K63-poly-ubiquitin content in Bre del-cytoplasmic fractions, probably linked to a change in the BRISC de-ubiquitinase activity. Even though these results have the same tendencies as observed in former studies, the effects in the present work are less striking. Further studies as well as intercrossing of Bre del- to Hey KO-animals will be necessary to further understand the functional relevance of Hey and Bre interaction.}, subject = {Embryonale Stammzelle}, language = {en} } @phdthesis{CardosoeCastro2012, author = {Cardoso e Castro, In{\^e}s Sofia}, title = {Epigenetic switch induced by MYC in Non-Small-Cell Lung Cancer}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-76713}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Non-Small-Cell Lung Cancer (NSCLC) is the most frequent human lung cancer and a major cause of death due to its high rate of metastasis1. These facts emphasize the urgent need for the investigation of new targets for anti-metastatic therapy. Up to now a number of genes and gene products have been identified that positively or negatively affect the probability of established human tumor cell lines to metastasize2. Previously, together with the group of Professor Ulf Rapp, we have described the first conditional mouse model for metastasis of NSCLC and identified a gene, c-MYC, that is able to orchestrate all steps of this process. We could identify potential markers for detection of metastasis and highlighted GATA4, which is exclusively expressed during lung development, as a target for future therapeutic intervention2. However, the mechanism underlying this metastatic conversion remained to be identified, and was therefore the focus of the present work. Here, GATA4 is identified as a MYC target in the development of metastasis and epigenetic alterations at the GATA4 promoter level are shown after MYC expression in NSCLC in vivo and in vitro. Such alterations include site-specific demethylation that accompanies the displacement of the MYC-associated zinc finger protein (MAZ) from the GATA4 promoter, which leads to GATA4 expression. Histone modification analysis of the GATA4 promoter revealed a switch from repressive histone marks to active histone marks after MYC binding, which corresponds to active GATA4 expression. This work identifies a novel epigenetic mechanism by which MYC activates GATA4 leading to metastasis in NSCLC, suggesting novel potential targets for the development of anti-metastatic therapy.}, subject = {Nicht-kleinzelliges Bronchialkarzinom}, language = {en} } @article{GassenBrechtefeldSchandryetal.2012, author = {Gassen, Alwine and Brechtefeld, Doris and Schandry, Niklas and Arteaga-Salas, J. Manuel and Israel, Lars and Imhof, Axel and Janzen, Christian J.}, title = {DOT1A-dependent H3K76 methylation is required for replication regulation in Trypanosoma brucei}, series = {Nucleic Acids Research}, volume = {40}, journal = {Nucleic Acids Research}, number = {20}, doi = {10.1093/nar/gks801}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-131449}, pages = {10302 - 10311}, year = {2012}, abstract = {Cell-cycle progression requires careful regulation to ensure accurate propagation of genetic material to the daughter cells. Although many cell-cycle regulators are evolutionarily conserved in the protozoan parasite Trypanosoma brucei, novel regulatory mechanisms seem to have evolved. Here, we analyse the function of the histone methyltransferase DOT1A during cell-cycle progression. Over-expression of DOT1A generates a population of cells with aneuploid nuclei as well as enucleated cells. Detailed analysis shows that DOT1A over-expression causes continuous replication of the nuclear DNA. In contrast, depletion of DOT1A by RNAi abolishes replication but does not prevent karyokinesis. As histone H3K76 methylation has never been associated with replication control in eukaryotes before, we have discovered a novel function of DOT1 enzymes, which might not be unique to trypanosomes.}, language = {en} } @article{KruegerFriedrichFoersteretal.2012, author = {Krueger, Beate and Friedrich, Torben and F{\"o}rster, Frank and Bernhardt, J{\"o}rg and Gross, Roy and Dandekar, Thomas}, title = {Different evolutionary modifications as a guide to rewire two-component systems}, series = {Bioinformatics and Biology Insights}, volume = {6}, journal = {Bioinformatics and Biology Insights}, doi = {10.4137/BBI.S9356}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-123647}, pages = {97-128}, year = {2012}, abstract = {Two-component systems (TCS) are short signalling pathways generally occurring in prokaryotes. They frequently regulate prokaryotic stimulus responses and thus are also of interest for engineering in biotechnology and synthetic biology. The aim of this study is to better understand and describe rewiring of TCS while investigating different evolutionary scenarios. Based on large-scale screens of TCS in different organisms, this study gives detailed data, concrete alignments, and structure analysis on three general modification scenarios, where TCS were rewired for new responses and functions: (i) exchanges in the sequence within single TCS domains, (ii) exchange of whole TCS domains; (iii) addition of new components modulating TCS function. As a result, the replacement of stimulus and promotor cassettes to rewire TCS is well defined exploiting the alignments given here. The diverged TCS examples are non-trivial and the design is challenging. Designed connector proteins may also be useful to modify TCS in selected cases.}, language = {en} } @phdthesis{Gaetschenberger2012, author = {G{\"a}tschenberger, Heike}, title = {Die Expression humoraler und zellul{\"a}rer Immunreaktionen bei Drohnenlarven und adulten Drohnen der Honigbiene (Apis mellifera)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-71960}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Soziale Insekten wie die Honigbiene (Apis mellifera) besitzen ein breites Spektrum an Abwehrmechanismen gegen Pathogenbefall, sowohl auf der Ebene der Kolonie (soziale Immunit{\"a}t) als auch auf der Stufe des Individuums (angeborenes Immunsystem). Die Hauptaufgabe der relativ kurzlebigen Drohnen besteht in der Begattung von Jungk{\"o}niginnen. Daher stellte sich die Frage, ob auch die Drohnen {\"a}hnlich den Arbeiterinnen mit energieaufwendigen Immunreaktionen auf Infektionen reagieren. Wie im Folgenden beschrieben, konnte ich nachweisen, dass Drohnen eine ausgepr{\"a}gte Immunkompetenz besitzen. Das angeborene Immunsystem setzt sich aus humoralen und zellul{\"a}ren Abwehrreaktionen zusammen. Bei der humoralen Immunantwort werden bestimmte evolution{\"a}r konservierte Signalkaskaden aktiviert, an deren Ende die Expression einer Vielzahl von antimikrobiellen Peptiden (AMPs) und immunspezifischen Proteinen (IRPs) steht. Zur Analyse der humoralen Immunantwort wurden von mir zum einen Hemmhoftests durchgef{\"u}hrt, um die gesamte antimikrobielle Aktivit{\"a}t der Haemolymphe nach artifizieller Infektion zu ermitteln und zum anderen spezifische AMPs bzw. IRPs identifiziert. Hierzu wurden die Haemolymphproteine in ein- oder zwei-dimensionalen Polyacrylamidgelen aufgetrennt und ausgew{\"a}hlte Proteinbanden bzw. -spots mittels nano HPLC/Massenspektrometrie analysiert. Die Hauptkomponenten des zellul{\"a}ren Immunsystems sind Wundheilung, Phagozytose, Einkapselung und Nodulation. In meiner Arbeit habe ich zum ersten Mal Noduli bei infizierten Drohnen nachweisen k{\"o}nnen. Frisch geschl{\"u}pfte adulte Drohnen (1d) weisen ein breites Spektrum an Immunreaktionen auf, das sowohl humorale als auch zellul{\"a}re Immunantworten umfasst. Nach Infektion mit dem Gram-negativen Bakterium E.coli und verschiedenen bakteriellen Zellwandbestandteilen wie Lipopolysaccharid (LPS), Peptidoglycan (PGN) und 1,3ß-Glucan (Bestandteil von Pilzzellw{\"a}nden), werden die AMPs Hymenoptaecin, Defensin 1 und Abaecin induziert. Desweiteren exprimieren junge adulte Drohnen eine Reihe hochmolekularer immunspezifischer Proteine (IRPs) wie z.B. Carboxylesterase (CE 1), eine Serinprotease, die m{\"o}glicherweise an der Prozessierung der Prophenoloxidase beteiligt ist, ein Peptidoglycan-interagierendes Protein (PGRP-S2) und zwei Proteine unbekannter Funktion, IRp42 und IRp30. Parallel zu bekannten bienenspezifischen AMPs wurde ein animales Peptidtoxin (APT) in Drohnenlarven, adulten Drohnen und adulten Hummeln nach E.coli Infektion in der Haemolymphe nachgewiesen. Von dem als OCLP 1 (ω-conotoxin-like protein 1) benannten Peptid war bereits bekannt, dass es in Fischen paralytische und damit toxische Effekte ausl{\"o}st. Meine Beobachtungen lassen vermuten, dass es sich bei OCLP 1 um ein Peptidtoxin mit antimikrobiellen Eigenschaften und damit um eine neue Klasse von AMPs handelt. Die allgemeine humorale Immunkompetenz scheint w{\"a}hrend der gesamten Lebensspanne adulter Drohnen (~ 7 Wochen) konstant zu bleiben, wie durch die gleichbleibende antimikrobielle Aktivit{\"a}t im Hemmhoftest gezeigt wurde. Junge Drohnen reagieren auf eine E.coli Infektion mit der Bildung zahlreicher Noduli (~1000 Noduli/Drohn), die vor allem entlang des Herzschlauches zu finden sind. Diese zellul{\"a}re Immunantwort nimmt mit dem Alter der Drohnen ab, so dass bei 18 d alten Drohnen nur noch rund 10 Noduli/Drohn gefunden werden. Auf der anderen Seite nimmt die phagozytotische Aktivit{\"a}t bei {\"a}lteren Drohnen scheinbar zu. In einer Reihe von parallel laufenden Versuchsreihen konnte ich eindrucksvoll zeigen, dass zellul{\"a}re Immunreaktionen wie Phagozytose und Nodulation unmittelbar nach bakterieller Infektion einsetzen. Hierbei erreicht die Nodulibildung 8-10 h p.i. eine Plateauphase, wohingegen die humorale Immunantwort erst 6 h p.i. schwach einsetzt, danach stetig zunimmt und noch 72 h p.i. nachweisbar ist. Es ist mir gelungen, eine Methode zur k{\"u}nstlichen Aufzucht von Drohnenlarven zu etablieren. Diese erm{\"o}glichte konstante und sterile Versuchsbedingungen zur Untersuchung der Immunreaktionen von Larven. Nach Infektion mit E.coli reagieren Drohnenlarven mit einer starken Aktivierung ihrer humoralen Immunantwort durch die Expression von AMPs, jedoch werden keine hochmolekularen IRPs wie in adulten Drohnen hochreguliert. Zudem ist die Nodulibildung in Larven nur schwach ausgepr{\"a}gt. V{\"o}llig unerwartete Beobachtungen wurden beim Studium der Immunkompetenz von Drohnenpuppen gemacht. Nach Injektion lebender E.coli Zellen in Drohnenpuppen stellte ich eine dramatische Ver{\"a}nderung im Aussehen der Puppen fest. Die Puppen verf{\"a}rbten sich gr{\"a}ulich schwarz. Genauere Untersuchungen haben dann gezeigt, dass die Drohnenpuppen, wie auch die der Arbeiterinnen, offensichtlich keine zellul{\"a}re Abwehrreaktion aktivieren k{\"o}nnen und die humorale Immunantwort nur sehr schwach ausf{\"a}llt und viel zu sp{\"a}t einsetzt.}, subject = {Humorale Immunit{\"a}t}, language = {de} } @phdthesis{Jahn2012, author = {Jahn, Daniel}, title = {Die Bedeutung von verk{\"u}rzten Spleißvarianten des Lamin A-Gens f{\"u}r die Meiose und f{\"u}r die Pathogenese von Laminopathien}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-74123}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Die Lamina ist ein dichtes Netzwerk aus Intermedi{\"a}r-Filamenten, den Laminen, an der nucleoplasmatischen Seite der inneren Kernmembran. Hier interagieren Lamine sowohl mit Transmembran-Proteinen der Kernh{\"u}lle als auch mit dem Chromatin. Diese Wechselwirkungen mit Interaktionspartnern verschiedener zellul{\"a}rer Kompartimente macht die Lamina, neben einer Ger{\"u}ststruktur mit wichtigen mechanische Aufgaben, auch zu einer zentralen Schnittstelle von Signalwegen, die eine intrazellul{\"a}re Kommunikation zwischen Nucleus und Cytoplasma erm{\"o}glichen. Die Lamina ist somit ein entscheidender Regulator der funktionellen Organisation des Chromatins und der differentiellen Genexpression. Das Expressionsmuster der Lamine w{\"a}hrend der Spermatogenese von S{\"a}ugern unterscheidet erheblich von der Lamin-Expression somatischer Zellen und weist einige Besonderheiten auf. Dies schließt unter anderem die spezifische Expression der verk{\"u}rzten A-Typ Lamin-Spleißvariante C2 w{\"a}hrend der meiotischen Phase der Spermatogenese ein. Diese und andere Beobachtungen deuteten bereits l{\"a}nger darauf hin, dass der speziellen Zusammensetzung der Lamina und vor allem dem meiosespezifischen Lamin C2 w{\"a}hrend der Gametogenese im m{\"a}nnlichen Organismus eine entscheidende Rolle zukommen k{\"o}nnte. Neuere Studien im Mausmodell bekr{\"a}ftigen diese Hypothese und leisten dar{\"u}ber hinaus einen entscheidenden Betrag dazu, die Funktion der Lamina w{\"a}hrend der Meiose auf molekularer Ebene pr{\"a}zise zu definieren. Im deutlichen Gegensatz zu den weitreichenden Kenntnissen zur Situation in M{\"a}nnchen lagen zu Beginn der vorliegenden Arbeit keine Daten {\"u}ber die Zusammensetzung der Lamina in weiblichen Keimzellen vor. Konsequenterweise existierten auch keine funktionellen Untersuchungen zur Relevanz der Lamina f{\"u}r die Oogenese. In der vorliegenden Arbeit wurden diese reproduktionsbiologisch hoch interessanten Fragestellungen detailliert untersucht. Dabei zeigte sich unter anderem, dass Lamin C2 auch in weiblichen Keimzellen spezifisch w{\"a}hrend der Meiose exprimiert wird. Durch Studien an einer Lamin C2-defizienten Mauslinie wurde die Funktion von Lamin C2 in der Meiose in Weibchen genau untersucht. Dabei wurde eine erhebliche Beeintr{\"a}chtigung der strukturellen Paarung der homologen Chromosomen und der homologen Rekombination in Lamin C2-defizienten Weibchen festgestellt. Da die genannten Prozesse Schl{\"u}sselereignisse f{\"u}r die korrekte Segregation der Homologen in sp{\"a}teren Stadien der Meiose sind, deuten die erzielten Ergebnisse auf eine erhebliche qualitative Beeintr{\"a}chtigung der reifen Gameten in Lamin C2-defizienten Weibchen hin. Ein weiterer zentraler Aspekt der Arbeit war die Analyse der molekularen Eigenschaften des meiosespezifischen Lamin C2 in vitro. Diese Experimente definieren wichtige Unterschiede hinsichtlich seiner Polymerisationseigenschaften im Vergleich zu Laminen somatischer Zellen und tragen, zusammen mit anderen Studien, dadurch erheblich dazu bei, die Funktion von Lamin C2 in der Meiose im mechanistischen Sinne besser zu verstehen. Zudem deckt die vorliegende Arbeit erstmals einen funktionellen Zusammenhang zwischen der Lamina-Zusammensetzung und der Qualit{\"a}t der Keimzellen weiblicher S{\"a}uger auf und erm{\"o}glicht dadurch zuk{\"u}nftige Studien zur Rolle der Lamine in der Oogenese, die m{\"o}glicherweise auch f{\"u}r die menschliche Fertilit{\"a}t sehr interessant sein k{\"o}nnte. Der zweite Teil der Dissertation besch{\"a}ftigt sich mit der Beschreibung einer trunkierten A-Typ Lamin-Spleißvariante in einer Mauslinie, die bislang als A-Typ Lamin-defizient angesehen wurde (Lmna-/-). Die durchgef{\"u}hrten Untersuchungen besitzen vor allem dadurch hohe Relevanz, dass die untersuchte Lmna-/- Mauslinie seit Jahren als das wichtigste Modell zur funktionellen Untersuchung der A-Typ Lamine gilt und bereits in einer Vielzahl von Publikationen eingesetzt wurde. In den hierzu durchgef{\"u}hrten Versuchen konnte das in der Lmna-/- Mauslinie persistierende A-Typ Lamin mittels diverser methodischer Ans{\"a}tze als C-terminale Deletionsmutante definiert werden, der die Exons 8-11 der insgesamt 12 Exons des Lmna-Gens fehlen. Daher wurde diese Lamin A-Mutante als Lamin AΔ8-11 bezeichnet. Die Konsequenzen der C-terminalen Deletion f{\"u}r die physiologischen Eigenschaften des Lamin Adelta8-11 sowie die Auswirkungen seiner Expression in der Lmna-/- Mauslinie auf aktuelle Modellvorstellungen zur Funktion der A-Typ Lamine und zur Entstehung Lamin-assoziierter, humaner Erkrankungen (Laminopathien) werden in der Arbeit ausf{\"u}hrlich diskutiert.}, subject = {Meiose}, language = {de} } @article{SchartlKneitzWildeetal.2012, author = {Schartl, Manfred and Kneitz, Susanne and Wilde, Brigitta and Wagner, Toni and Henkel, Christiaan V. and Spaink, Hermann P. and Meierjohann, Svenja}, title = {Conserved expression signatures between medaka and human pigment cell tumors}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-75848}, year = {2012}, abstract = {Aberrations in gene expression are a hallmark of cancer cells. Differential tumor-specific transcript levels of single genes or whole sets of genes may be critical for the neoplastic phenotype and important for therapeutic considerations or useful as biomarkers. As an approach to filter out such relevant expression differences from the plethora of changes noted in global expression profiling studies, we searched for changes of gene expression levels that are conserved. Transcriptomes from massive parallel sequencing of different types of melanoma from medaka were generated and compared to microarray datasets from zebrafish and human melanoma. This revealed molecular conservation at various levels between fish models and human tumors providing a useful strategy for identifying expression signatures strongly associated with disease phenotypes and uncovering new melanoma molecules.}, subject = {Biologie}, language = {en} } @article{SchokraieWarnkenHotzWagenblattetal.2012, author = {Schokraie, Elham and Warnken, Uwe and Hotz-Wagenblatt, Agnes and Grohme, Markus A. and Hengherr, Steffen and F{\"o}rster, Frank and Schill, Ralph O. and Frohme, Marcus and Dandekar, Thomas and Schn{\"o}lzer, Martina}, title = {Comparative proteome analysis of Milnesium tardigradum in early embryonic state versus adults in active and anhydrobiotic state}, series = {PLoS One}, volume = {7}, journal = {PLoS One}, number = {9}, doi = {10.1371/journal.pone.0045682}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-134447}, pages = {e45682}, year = {2012}, abstract = {Tardigrades have fascinated researchers for more than 300 years because of their extraordinary capability to undergo cryptobiosis and survive extreme environmental conditions. However, the survival mechanisms of tardigrades are still poorly understood mainly due to the absence of detailed knowledge about the proteome and genome of these organisms. Our study was intended to provide a basis for the functional characterization of expressed proteins in different states of tardigrades. High-throughput, high-accuracy proteomics in combination with a newly developed tardigrade specific protein database resulted in the identification of more than 3000 proteins in three different states: early embryonic state and adult animals in active and anhydrobiotic state. This comprehensive proteome resource includes protein families such as chaperones, antioxidants, ribosomal proteins, cytoskeletal proteins, transporters, protein channels, nutrient reservoirs, and developmental proteins. A comparative analysis of protein families in the different states was performed by calculating the exponentially modified protein abundance index which classifies proteins in major and minor components. This is the first step to analyzing the proteins involved in early embryonic development, and furthermore proteins which might play an important role in the transition into the anhydrobiotic state.}, language = {en} } @phdthesis{Scheller2012, author = {Scheller, Katharina}, title = {Charakterisierung und Anwendung von humanen, prim{\"a}ren mikrovaskul{\"a}ren Endothelzellen mit erweiterter Proliferationsf{\"a}higkeit}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-76577}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Das Arbeitsgebiet Tissue Engineering befasst sich mit der Kl{\"a}rung der Mechanismen, die der Funktionen verschiedener Gewebearten zu Grunde liegen sowie mit der Entwicklung alternativer Strategien zur Behandlung von Organversagen bzw. Organverlusten. Einer der kritischsten Punkte im Tissue Engineering ist die ausreichende Versorgung der Zellen mit N{\"a}hrstoffen und Sauerstoff. Bioartifizielle Gewebe mit einer Dicke von bis zu 200 µm k{\"o}nnen mittels Diffusion ausreichend versorgt werden. F{\"u}r dickere Transplantate ist die Versorgung der Zellen alleine durch Diffusion jedoch nicht gegeben. Hierf{\"u}r m{\"u}ssen Mechanismen und Strategien zur Pr{\"a}vaskularisierung der artifiziellen Gewebekonstrukte entwickelt werden, damit die N{\"a}hrstoff- und Sauerstoffversorgung aller Zellen, auch im Inneren des Transplantates, von Anfang an gew{\"a}hrleistet ist. Eine wichtige Rolle bei der Pr{\"a}vaskularisierung spielt die Angiogenese. Dabei ist die Wahl einer geeigneten Zellquelle entscheidend, da die Zellen die Basis f{\"u}r die Angiogenese darstellen. Mikrovaskul{\"a}re Endothelzellen (mvEZ) sind maßgeblich an der Angiogenese beteiligt. Das Problem bei der Verwendung von humanen prim{\"a}ren mvEZ ist ihre geringe Verf{\"u}gbarkeit, ihre limitierte Proliferationskapazit{\"a}t und der schnelle Verlust ihrer typischen Endothelzellmarker in-vitro. Der Aufbau standardisierter in-vitro Testsysteme ist durch die geringe Zellausbeute auch nicht m{\"o}glich. Die upcyte® Technologie bietet hierf{\"u}r einen L{\"o}sungsansatz. In der vorliegenden Arbeit konnten upcyte® mvEZ als Alternative zu prim{\"a}ren mvEZ generiert werden. Es konnte gezeigt werden, dass die Zellen eine erweiterte Proliferationsf{\"a}higkeit aufweisen und im Vergleich zu prim{\"a}ren mvEZ durchschnittlich 15 zus{\"a}tzliche Populationsverdopplungen leisten k{\"o}nnen. Dadurch ist es m{\"o}glich 3x104-fach mehr upcyte® mvEZ eines Spenders zu generieren verglichen mit den korrespondierenden Prim{\"a}rzellen. Die gute und ausreichende Verf{\"u}gbarkeit der Zellen macht sie interessant f{\"u}r die Standardisierung von in-vitro Testsystemen, ebenso k{\"o}nnen die Zellen zur Pr{\"a}vaskularisierung von Transplantaten eingesetzt werden. Upcyte® mvEZ zeigen zahlreiche Prim{\"a}rzellmerkmale, die in der Literatur beschrieben sind. Im konfluenten Zustand zeigen sie die f{\"u}r prim{\"a}re mvEZ spezifische pflastersteinartige Morphologie. Dar{\"u}ber hinaus exprimieren upcyte® mvEZ typische Endothelzellmarker wie CD31, vWF, eNOS, CD105, CD146 und VEGFR-2 vergleichbar zu prim{\"a}ren mvEZ. Eine weitere endothelzellspezifische Eigenschaft ist die Bindung von Ulex europaeus agglutinin I Lektin an die alpha-L-Fucose enthaltene Kohlenhydratstrukturen von mvEZs. Auch hier wurden upcyte® Zellen mit prim{\"a}ren mvEZ verglichen und zeigten die hierf{\"u}r charkteristischen Strukturen. Zus{\"a}tzlich zu Morphologie, Proliferationskapazit{\"a}t und endothelzellspezifischen Markern, zeigen upcyte® mvEZ auch mehrere funktionelle Eigenschaften, welche in prim{\"a}ren mvEZ beobachtet werden k{\"o}nnen, wie beispielsweise die Aufnahme von Dil-markiertem acetyliertem Low Density Lipoprotein (Dil-Ac-LDL) oder die F{\"a}higkeit den Prozess der Angiognese zu unterst{\"u}tzen. Zus{\"a}tzlich bilden Sph{\"a}roide aus upcyte® mvEZ dreidimensionale lumin{\"a}re Zellformationen in einer Kollagenmatrix aus. Diese Charakteristika zeigen den quasi-prim{\"a}ren Ph{\"a}notyp der upcyte® mvEZs. Upcyte® mvEZ stellen dar{\"u}ber hinaus eine neuartige m{\"o}gliche Zellquelle f{\"u}r die Generierung pr{\"a}vaskularisierter Tr{\"a}germaterialien im Tissue Engineering dar. In der vorliegenden Arbeit konnte die Wiederbesiedlung der biologisch vaskularisierte Matrix (BioVaSc) mit upcyte® mvEZ vergleichbar zu prim{\"a}ren mvEZ gezeigt werden. Der Einsatz von upcyte® mvEZ in der BioVaSc stellt einen neuen, vielversprechenden Ansatz zur Herstellung eines vaskularisierten Modells f{\"u}r Gewebekonstrukte dar, wie beispielsweise einem Leberkonstrukt. Zusammenfassend konnte in der vorliegenden Arbeit gezeigt werden, dass upcyte® mvEZ vergleichbar zu prim{\"a}ren mvEZs sind und somit eine geeignete Alternative f{\"u}r die Generierung pr{\"a}vaskulierter Tr{\"a}germaterialien und Aufbau von in-vitro Testsystemen darstellen. Dar{\"u}ber hinaus wurde ein neues, innovatives System f{\"u}r die Generierung einer perfundierten, mit Endothelzellen wiederbesiedelten Matrix f{\"u}r k{\"u}nstliches Gewebe in-vitro entwickelt.}, subject = {Tissue Engineering}, language = {de} } @phdthesis{Kronhardt2012, author = {Kronhardt, Angelika}, title = {Channel Formation, Binding and Translocation Properties of Anthrax, CDT and Related Toxins of the AB7 type}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-71559}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {The ability to produce toxins is spread among a huge variety of bacterial strains. A very prominent class of bacterial protein toxins is the family of binary AB toxins sharing a common mode of intoxication. A pore forming component B binds and translocates an enzymatic component A into the cytosol of target cells exhibiting a fatal mode of action. These components are supposed to be not toxic themselves but both required for cell toxicity. Anthrax toxin produced by the Gram-positive bacteria Bacillus anthracis is the best studied binary toxin especially since its use as a biological weapon in the context of the attacks of 9/11 in 2001. In contrast to other binary toxins, Anthrax toxin possesses two different enzymatic components, edema factor (EF), a calcium- and calmodulin-dependent adenylat-cyclase and lethal factor (LF), a zinc-dependent metalloprotease. Protective antigen (PA) is the pore-forming component responsible for binding and translocation. Clostridium botulinum possesses in addition to the well known botulinum toxin (Botox) a variety of other toxins, such as the binary C2 toxin. C2 toxin is composed of the binding and translocation moiety C2II and the enzymatic moiety C2I acting as an actin-ADP-ribosyltransferase. In this study, the mode of translocation and the binding kinetics to the enzymatic component were studied in a biophysical experimental setup. In chapter 2, the binding of the N-terminal fractions EFN and LFN to the PA channel are analyzed in artificial bilayer membranes revealing lower binding affinity compared to full-length EF and LF. Other biophysical properties like voltage-dependency and ionic-strength dependency are not influenced. The results suggest that additional forces are involved in the binding process, than those concerning the N-terminus exclusively, as it was supposed previously. As the treatment of an Anthrax infection with antibiotics is often medicated very late due to the lack of early symptoms, tools to prevent intoxication are required. 4-aminoquinolones like chloroquine are known to block the PA channel, thereby inhibiting intoxication but they also lead to severe side-effects. In chapter 3 new promising agents are described that bind to PA in artificial bilayer systems, elucidating common motives and features which are necessary for binding to PA in general. The possible interaction of Anthrax and C2 toxin is investigated by measuring the binding of one enzymatic component to the respective other toxin's pore (chapter 4). Interestingly, in vitro experiments using the black lipid bilayer assay show that PA is able to bind to C2I resulting in half saturation constants in the nanomolar range. Furthermore, in vivo this combination of toxin components exhibits cell toxicity in human cell lines. This is first-time evidence that a heterologous toxin combination is functional in in vitro and in vivo systems. In contrast, C2II is able to bind to EF as well as to LF in vitro, whereas in in vivo studies almost no toxic effect is detected. In the case of PA, an N-terminal His6-tag attached to the enzymatic subunit increased the binding affinity (chapter 5). A His6-tag attached to not related proteins also led to high binding affinities, providing the possibility to establish PA as a general cargo protein. In chapter 6 a set of different molecules and proteins is summarized, which are either related or not related to binary toxins, PA is able to bind. In first line, the presence of positive charges is found to be responsible for binding to PA which is in accordance to the fact that PA is highly cation selective. Furthermore, we present evidence that different cationic electrolytes serve as a binding partner to the PA channel. In the last decade another toxin has aroused public attention as it was found to be responsible for a rising number of nosocomial infections: Clostridium difficile CDT toxin. The mode of action of the enzymatic subunit CDTa is similar to C2I of C2 toxin, acting as an ADP-ribosylating toxin. The channel forming and binding properties of CDT toxin are studied in artificial bilayer membranes (chapter 7). We found that two different types of channels are formed by the B component CDTb. The first channel is similar to that of iota toxin's Ib of Clostridium perfringens with comparable single channel conductance, selectivity and binding properties to the enzymatic subunit CDTa. The formation of this type of channel is cholesterol-dependent, whereas in the absence of cholesterol another kind of channel is observed. This channel has a single channel conductance which is rather high compared to all other binary toxin channels known so far, it is anion selective and does not show any binding affinity to the enzymatic component CDTa. The results reveal completely new insights in channel formation properties and the flexibility of a pore-forming component. Additionally, these findings suggest further possibilities of toxicity of the pore forming component itself which is not known for any other binary toxin yet. Therefore, the pathogenic role of this feature has to be studied in detail.}, subject = {Bacillus anthracis}, language = {en} } @phdthesis{Reinboth2012, author = {Reinboth, Jennifer}, title = {Cellular Factors Contributing to Host Cell Permissiveness in Support of Oncolytic Vaccinia Virus Replication}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85392}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {In initial experiments, the well characterized VACV strain GLV-1h68 and three wild-type LIVP isolates were utilized to analyze gene expression in a pair of autologous human melanoma cell lines (888-MEL and 1936 MEL) after infection. Microarray analyses, followed by sequential statistical approaches, characterized human genes whose transcription is affected specifically by VACV infection. In accordance with the literature, those genes were involved in broad cellular functions, such as cell death, protein synthesis and folding, as well as DNA replication, recombination, and repair. In parallel to host gene expression, viral gene expression was evaluated with help of customized VACV array platforms to get better insight over the interplay between VACV and its host. Our main focus was to compare host and viral early events, since virus genome replication occurs early after infection. We observed that viral transcripts segregated in a characteristic time-specific pattern, consistent with the three temporal expression classes of VACV genes, including a group of genes which could be classified as early-stage genes. In this work, comparison of VACV early replication and respective early gene transcription led to the identification of seven viral genes whose expression correlated strictly with replication. We considered the early expression of those seven genes to be representative for VACV replication and we therefore referred to them as viral replication indicators (VRIs). To explore the relationship between host cell transcription and viral replication, we correlated viral (VRI) and human early gene expression. Correlation analysis revealed a subset of 114 human transcripts whose early expression tightly correlated with early VRI expression and thus early viral replication. These 114 human molecules represented an involvement in broad cellular functions. We found at least six out of 114 correlates to be involved in protein ubiquitination or proteasomal function. Another molecule of interest was the serine-threonine protein kinase WNK lysine-deficient protein kinase 1 (WNK1). We discovered that WNK1 features differences on several molecular biological levels associated with permissiveness to VACV infection. In addition to that, a set of human genes was identified with possible predictive value for viral replication in an independent dataset. A further objective of this work was to explore baseline molecular biological variances associated with permissiveness which could help identifying cellular components that contribute to the formation of a permissive phenotype. Therefore, in a subsequent approach, we screened a set of 15 melanoma cell lines (15-MEL) regarding their permissiveness to GLV-1h68, evaluated by GFP expression levels, and classified the top four and lowest four cell lines into high and low permissive group, respectively. Baseline gene transcriptional data, comparing low and highly permissive group, suggest that differences between the two groups are at least in part due to variances in global cellular functions, such as cell cycle, cell growth and proliferation, as well as cell death and survival. We also observed differences in the ubiquitination pathway, which is consistent with our previous results and underlines the importance of this pathway in VACV replication and permissiveness. Moreover, baseline microRNA (miRNA) expression between low and highly permissive group was considered to provide valuable information regarding virus-host co-existence. In our data set, we identified six miRNAs that featured varying baseline expression between low and highly permissive group. Finally, copy number variations (CNVs) between low and highly permissive group were evaluated. In this study, when investigating differences in the chromosomal aberration patterns between low and highly permissive group, we observed frequent segmental amplifications within the low permissive group, whereas the same regions were mostly unchanged in the high group. Taken together, our results highlight a probable correlation between viral replication, early gene expression, and the respective host response and thus a possible involvement of human host factors in viral early replication. Furthermore, we revealed the importance of cellular baseline composition for permissiveness to VACV infection on different molecular biological levels, including mRNA expression, miRNA expression, as well as copy number variations. The characterization of human target genes that influence viral replication could help answering the question of host cell response to oncolytic virotherapy and provide important information for the development of novel recombinant vaccinia viruses with improved features to enhance replication rate and hence trigger therapeutic outcome.}, subject = {Vaccinia-Virus}, language = {en} } @article{KesslerHertelJungkunstetal.2012, author = {Kessler, Michael and Hertel, Dietrich and Jungkunst, Hermann F. and Kluge, J{\"u}rgen and Abrahamczyk, Stefan and Bos, Merijn and Buchori, Damayanti and Gerold, Gerhard and Gradstein, S. Robbert and K{\"o}hler, Stefan and Leuschner, Christoph and Moser, Gerald and Pitopang, Ramadhanil and Saleh, Shahabuddin and Schulze, Christian H. and Sporn, Simone G. and Steffan-Dewenter, Ingolf and Tjitrosoedirdjo, Sri S. and Tscharntke, Teja}, title = {Can Joint Carbon and Biodiversity Management in Tropical Agroforestry Landscapes Be Optimized?}, series = {PLoS One}, volume = {7}, journal = {PLoS One}, number = {10}, doi = {10.1371/journal.pone.0047192}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-132016}, pages = {e47192}, year = {2012}, abstract = {Managing ecosystems for carbon storage may also benefit biodiversity conservation, but such a potential 'win-win' scenario has not yet been assessed for tropical agroforestry landscapes. We measured above-and below-ground carbon stocks as well as the species richness of four groups of plants and eight of animals on 14 representative plots in Sulawesi, Indonesia, ranging from natural rainforest to cacao agroforests that have replaced former natural forest. The conversion of natural forests with carbon stocks of 227-362 Mg C ha\(^{-1}\) to agroforests with 82-211 Mg C ha\(^{-1}\) showed no relationships to overall biodiversity but led to a significant loss of forest-related species richness. We conclude that the conservation of the forest-related biodiversity, and to a lesser degree of carbon stocks, mainly depends on the preservation of natural forest habitats. In the three most carbon-rich agroforestry systems, carbon stocks were about 60\% of those of natural forest, suggesting that 1.6 ha of optimally managed agroforest can contribute to the conservation of carbon stocks as much as 1 ha of natural forest. However, agroforestry systems had comparatively low biodiversity, and we found no evidence for a tight link between carbon storage and biodiversity. Yet, potential win-win agroforestry management solutions include combining high shade-tree quality which favours biodiversity with cacao-yield adapted shade levels.}, language = {en} } @phdthesis{Zeeshan2012, author = {Zeeshan, Ahmed}, title = {Bioinformatics Software for Metabolic and Health Care Data Management}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73926}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Computer Science approaches (software, database, management systems) are powerful tools to boost research. Here they are applied to metabolic modelling in infections as well as health care management. Starting from a comparative analysis this thesis shows own steps and examples towards improvement in metabolic modelling software and health data management. In section 2, new experimental data on metabolites and enzymes induce high interest in metabolic modelling including metabolic flux calculations. Data analysis of metabolites, calculation of metabolic fluxes, pathways and their condition-specific strengths is now possible by an advantageous combination of specific software. How can available software for metabolic modelling be improved from a computational point of view? A number of available and well established software solutions are first discussed individually. This includes information on software origin, capabilities, development and used methodology. Performance information is obtained for the compared software using provided example data sets. A feature based comparison shows limitations and advantages of the compared software for specific tasks in metabolic modeling. Often found limitations include third party software dependence, no comprehensive database management and no standard format for data input and output. Graphical visualization can be improved for complex data visualization and at the web based graphical interface. Other areas for development are platform independency, product line architecture, data standardization, open source movement and new methodologies. The comparison shows clearly space for further software application development including steps towards an optimal user friendly graphical user interface, platform independence, database management system and third party independence especially in the case of desktop applications. The found limitations are not limited to the software compared and are of course also actively tackled in some of the most recent developments. Other improvements should aim at generality and standard data input formats, improved visualization of not only the input data set but also analyzed results. We hope, with the implementation of these suggestions, metabolic software applications will become more professional, cheap, reliable and attractive for the user. Nevertheless, keeping these inherent limitations in mind, we are confident that the tools compared can be recommended for metabolic modeling for instance to model metabolic fluxes in bacteria or metabolic data analysis and studies in infection biology. ...}, subject = {Stoffwechsel}, language = {en} } @article{ElKeredySchleyerKoenigetal.2012, author = {El-Keredy, Amira and Schleyer, Michael and K{\"o}nig, Christian and Ekim, Aslihan and Gerber, Bertram}, title = {Behavioural Analyses of Quinine Processing in Choice, Feeding and Learning of Larval Drosophila}, series = {PLoS One}, volume = {7}, journal = {PLoS One}, number = {7}, doi = {10.1371/journal.pone.0040525}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-130811}, pages = {e40525}, year = {2012}, abstract = {Gustatory stimuli can support both immediate reflexive behaviour, such as choice and feeding, and can drive internal reinforcement in associative learning. For larval Drosophila, we here provide a first systematic behavioural analysis of these functions with respect to quinine as a study case of a substance which humans report as "tasting bitter". We describe the dose-effect functions for these different kinds of behaviour and find that a half-maximal effect of quinine to suppress feeding needs substantially higher quinine concentrations (2.0 mM) than is the case for internal reinforcement (0.6 mM). Interestingly, in previous studies (Niewalda et al. 2008, Schipanski et al 2008) we had found the reverse for sodium chloride and fructose/sucrose, such that dose-effect functions for those tastants were shifted towards lower concentrations for feeding as compared to reinforcement, arguing that the differences in dose-effect function between these behaviours do not reflect artefacts of the types of assay used. The current results regarding quinine thus provide a starting point to investigate how the gustatory system is organized on the cellular and/or molecular level to result in different behavioural tuning curves towards a bitter tastant.}, language = {en} } @article{TomeiAdamsUccellinietal.2012, author = {Tomei, Sara and Adams, Sharon and Uccellini, Lorenzo and Bedognetti, Davide and De Giorgi, Valeria and Erdenebileg, Narnygerel and Libera Ascierto, Maria and Reinboth, Jennifer and Liu, Qiuzhen and Bevilacqua, Generoso and Wang, Ena and Mazzanti, Chiara and Marincola, Francesco M.}, title = {Association between HRAS rs12628 and rs112587690 polymorphisms with the risk of melanoma in the North American population}, series = {Medical Oncology}, volume = {29}, journal = {Medical Oncology}, number = {5}, doi = {dx.doi.org/10.1007/s12032-012-0255-3}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-126834}, pages = {3456-3461}, year = {2012}, abstract = {HRAS belongs to the RAS genes superfamily. RAS genes are important players in several human tumors and the single-nucleotide polymorphism rs12628 has been shown to contribute to the risk of bladder, colon, gastrointestinal, oral, and thyroid carcinoma. We hypothesized that this SNP may affect the risk of cutaneous melanoma as well. HRAS gene contains a polymorphic region (rs112587690), a repeated hexanucleotide -GGGCCT- located in intron 1. Three alleles of this region, P1, P2, and P3, have been identified that contain two, three, and four repeats of the hexanucleotide, respectively. We investigated the clinical impact of these polymorphisms in a case-control study. A total of 141 melanoma patients and 118 healthy donors from the North America Caucasian population were screened for rs12628 and rs112587690 polymorphisms. Genotypes were assessed by capillary sequencing or fragment analysis, respectively, and rs12628 CC and rs112587690 P1P1 genotypes significantly associated with increased melanoma risk (OR = 3.83, p = 0.003; OR = 11.3, p = 0.033, respectively), while rs112587690 P1P3 frequency resulted significantly higher in the control group (OR = 0.5, p = 0.017). These results suggest that rs12628 C homozygosis may be considered a potential risk factor for melanoma development in the North American population possibly through the linkage to rs112587690.}, language = {en} } @phdthesis{Heidbreder2012, author = {Heidbreder, Meike}, title = {Association and Activation of TNF-Receptor I Investigated with Single-Molecule Tracking and Super-Resolution Microscopy in Live Cells}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73191}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Cellular responses to outer stimuli are the basis for all biological processes. Signal integration is achieved by protein cascades, recognizing and processing molecules from the environment. Factors released by pathogens or inflammation usually induce an inflammatory response, a signal often transduced by Tumour Necrosis Factor alpha (TNF). TNFα receptors TNF-R1 and TNF-R2 can in turn lead to apoptosis or proliferation via NF-B. These processes are closely regulated by membrane compartimentalization, protein interactions and trafficking. Fluorescence microscopy offers a reliable and non-invasive method to probe these cellular events. However, some processes on a native membrane are not resolvable, as they are well below the diffraction limit of microscopy. The recent development of super-resolution fluorescence microscopy methods enables the observation of these cellular players well below this limit: by localizing, tracking and counting molecules with high spatial and temporal resolution, these new fluorescence microscopy methods offer a previously unknown insight into protein interactions at the near-molecular level. Direct stochastic optical reconstruction microscopy (dSTORM) utilizes the reversible, stochastic blinking events of small commercially available fluorescent dyes, while photoactivated localization microscopy (PALM) utilizes phototransformation of genetically encoded fluorescent proteins. By photoactivating only a small fraction of the present fluorophores in each observation interval, single emitters can be localized with high precision and a super-resolved image can be reconstructed. Quantum Dot Triexciton imaging (QDTI) utilizes the three-photon absorption (triexcitonic) properties of quantum dots (QD) and to achieve a twofold resolution increase using conventional confocal microscopes. In this thesis, experimental approaches were implemented to achieve super-resolution microscopy in fixed and live-cells to study the spatial and temporal dynamics of TNF and other cellular signaling events. We introduce QDTI to study the three-dimensional cellular distribution of biological targets, offering an easy method to achieve resolution enhancement in combination with optical sectioning, allowing the preliminary quantification of labeled proteins. As QDs are electron dense, QDTI can be used for correlative fluorescence and transmission electron microscopy, proving the versatility of QD probes. Utilizing the phototransformation properties of fluorescent proteins, single-receptor tracking on live cells was achieved, applying the concept of single particle tracking PALM (sptPALM) to track the dynamics of a TNF-R1-tdEos chimera on the membrane. Lateral receptor dynamics can be tracked with high precision and the influences of ligand addition or lipid disruption on TNF-R1 mobility was observed. The results reveal complex receptor dynamics, implying internalization processes in response to TNFα stimulation and a role for membrane domains with reduced fluidity, so-called lipid raft domains, in TNF-R1 compartimentalization prior or post ligand induction. Comparisons with previously published FCS data show a good accordance, but stressing the increased data depth available in sptPALM experiments. Additionally, the active transport of NF-κB-tdEos fusions was observed in live neurons under chemical stimulation and/or inhibition. Contrary to phototransformable proteins that need no special buffers to exhibit photoconversion or photoactivation, dSTORM has previously been unsuitable for in vivo applications, as organic dyes relied on introducing the probes via immunostaining in concert with a reductive, oxygen-free medium for proper photoswitching behaviour. ATTO655 had been previously shown to be suitable for live-cell applications, as its switching behavior can be catalyzed by the reductive environment of the cytoplasm. By introducing the cell-permeant organic dye via a chemical tag system, a high specificity and low background was achieved. Here, the labeled histone H2B complex and thus single nucleosome movements in a live cell can be observed over long time periods and with ~20 nm resolution. Implementing these new approaches for imaging biological processes with high temporal and spatial resolution provides new insights into the dynamics and spatial heterogeneities of proteins, further elucidating their function in the organism and revealing properties that are usually only detectable in vitro.  }, subject = {Fluoreszenzmikrosopie}, language = {en} } @phdthesis{Bruder2012, author = {Bruder, Jessica}, title = {Antigenerkennung bei autoaggressiven Lymphozyten}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73342}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Millionen Menschen weltweit leiden an den verschiedensten Autoimmunerkrankungen. Diese Krankheiten entstehen, wenn das Immunsystem gesundes k{\"o}rpereigenes Gewebe angreift und zerst{\"o}rt. An der Pathogenese sind sowohl Komponenten des angeborenen Immunsystems als auch Bestandteile des adaptiven Immunsystems, wie Lymphozyten und Antik{\"o}rper, beteiligt. Da die Ursachen und molekularen Mechanismen der Pathogenese dieser Erkrankungen bis heute weitgehend unbekannt sind, wurden in dieser Arbeit autoaggressive Lymphozyten bei den humanen Autoimmunerkrankungen Polymyositis und Multiple Sklerose n{\"a}her untersucht. Die Polymyositis ist eine chronisch entz{\"u}ndliche Erkrankung der Skelettmuskulatur. Die Muskelfasern werden dabei von zytotoxischen CD8+ gd-T-Lymphozyten infiltriert, attackiert und schließlich zerst{\"o}rt. In einem seltenen Fall der Polymyositis wurden die Muskelzellen hingegen in {\"a}hnlicher Weise von CD8- gd-T-Lymphozyten angegriffen. Die gd-T-Lymphozyten waren monoklonal expandiert und ihr Rezeptor, im Folgenden als M88 bezeichnet, wurde als Vg1.3+Vd2+ identifiziert. Fr{\"u}here Untersuchungen der Antigenspezifit{\"a}t dieser Zellen zeigten, dass M88 mehrere funktionell und strukturell verschiedene Proteine aus unterschiedlichen Spezies erkennt. Die Bindung erfolgt spezifisch durch die Antigenerkennungsregionen beider Rezeptorketten von M88. In dieser Arbeit wurden verschiedene bakterielle und humane Proteine des Translationsapparates als Antigene von M88 identifiziert. Weitere ausf{\"u}hrliche Untersuchungen eines paradigmatischen bakteriellen Antigens, dem Translationsinitiationsfaktor EcIF1, zeigten, dass M88 an Oberfl{\"a}chen-exponierte Konformationsepitope von Proteinen bindet. Interessanterweise erkennt M88 mehrere humane Aminoacyl-tRNA-Synthetasen, Antigene, die in anderen Formen der Myositis von Autoantik{\"o}rpern angegriffen werden. Diese Beobachtung ergibt eine bemerkenswerte Verbindung zwischen T-Zell- und Antik{\"o}rper-vermittelten B-Zell-Antworten bei der autoimmunen Myositis. Bei der Multiplen Sklerose ist das zentrale Nervensystem betroffen. Autoaggressive Lymphozyten greifen die Myelinschicht der Nervenzellen im Gehirn und R{\"u}ckenmark an und zerst{\"o}ren sie. Im Liquor cerebrospinalis von Patienten lassen sich klonal expandierte und affinit{\"a}tsgereifte B-Zellen sowie „oligoklonale Banden" (OKB) Antik{\"o}rper nachweisen. Obwohl diese Merkmale auf eine Antigen-induzierte Immunantwort hindeuten, sind die zugrundeliegenden Antigene und die Rolle der OKB bei der Pathogenese bis heute unbekannt. In dieser Arbeit wurde die Antigenspezifit{\"a}t von f{\"u}nf IgG OKB-Antik{\"o}rpern aus drei Patienten untersucht. Durch verschiedene proteinbiochemische Methoden konnten intrazellul{\"a}re Kandidatenantigene identifiziert werden. Interessanterweise sind darunter mehrere nukle{\"a}re Proteine, die an der Transkriptionsregulation oder der RNA-Prozessierung beteiligt sind. Reaktivit{\"a}ten gegen intrazellul{\"a}re Antigene treten auch bei anderen Autoimmunerkrankungen, wie beispielsweise dem systemischen Lupus erythematodes, auf. Diese Ergebnisse k{\"o}nnten auf einen allgemeinen Mechanismus der Entstehung und Funktion von Autoantik{\"o}rpern bei diesen humanen Autoimmunerkrankungen hindeuten.}, subject = {Multiple Sklerose}, language = {de} } @article{ZirkelCecilSchaeferetal.2012, author = {Zirkel, J. and Cecil, A. and Sch{\"a}fer, F. and Rahlfs, S. and Ouedraogo, A. and Xiao, K. and Sawadogo, S. and Coulibaly, B. and Becker, K. and Dandekar, T.}, title = {Analyzing Thiol-Dependent Redox Networks in the Presence of Methylene Blue and Other Antimalarial Agents with RT-PCR-Supported in silico Modeling}, series = {Bioinformatics and Biology Insights}, volume = {6}, journal = {Bioinformatics and Biology Insights}, doi = {10.4137/BBI.S10193}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-123751}, pages = {287-302}, year = {2012}, abstract = {BACKGROUND: In the face of growing resistance in malaria parasites to drugs, pharmacological combination therapies are important. There is accumulating evidence that methylene blue (MB) is an effective drug against malaria. Here we explore the biological effects of both MB alone and in combination therapy using modeling and experimental data. RESULTS: We built a model of the central metabolic pathways in P. falciparum. Metabolic flux modes and their changes under MB were calculated by integrating experimental data (RT-PCR data on mRNAs for redox enzymes) as constraints and results from the YANA software package for metabolic pathway calculations. Several different lines of MB attack on Plasmodium redox defense were identified by analysis of the network effects. Next, chloroquine resistance based on pfmdr/and pfcrt transporters, as well as pyrimethamine/sulfadoxine resistance (by mutations in DHF/DHPS), were modeled in silico. Further modeling shows that MB has a favorable synergism on antimalarial network effects with these commonly used antimalarial drugs. CONCLUSIONS: Theoretical and experimental results support that methylene blue should, because of its resistance-breaking potential, be further tested as a key component in drug combination therapy efforts in holoendemic areas.}, language = {en} } @phdthesis{Nilla2012, author = {Nilla, Jaya Santosh Chakravarthy}, title = {An Integrated Knowledgebase and Network Analysis Applied on Platelets and Other Cell Types}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85730}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Systems biology looks for emergent system effects from large scale assemblies of molecules and data, for instance in the human platelets. However, the computational efforts in all steps before such insights are possible can hardly be under estimated. In practice this involves numerous programming tasks, the establishment of new database systems but as well their maintenance, curation and data validation. Furthermore, network insights are only possible if strong algorithms decipher the interactions, decoding the hidden system effects. This thesis and my work are all about these challenges. To answer this requirement, an integrated platelet network, PlateletWeb, was assembled from different sources and further analyzed for signaling in a systems biological manner including multilevel data integration and visualization. PlateletWeb is an integrated network database and was established by combining the data from recent platelet proteome and transcriptome (SAGE) studies. The information on protein-protein interactions and kinase-substrate relationships extracted from bioinformatical databases as well as published literature were added to this resource. Moreover, the mass spectrometry-based platelet phosphoproteome was combined with site-specific phosphorylation/ dephosphorylation information and then enhanced with data from Phosphosite and complemented by bioinformatical sequence analysis for site-specific kinase predictions. The number of catalogued platelet proteins was increased by over 80\% as compared to the previous version. The integration of annotations on kinases, protein domains, transmembrane regions, Gene Ontology, disease associations and drug targets provides ample functional tools for platelet signaling analysis. The PlateletWeb resource provides a novel systems biological workbench for the analysis of platelet signaling in the functional context of protein networks. By comprehensive exploration, over 15000 phosphorylation sites were found, out of which 2500 have the corresponding kinase associations. The network motifs were also investigated in this anucleate cell and characterize signaling modules based on integrated information on phosphorylation and protein-protein interactions. Furthermore, many algorithmic approaches have been introduced, including an exact approach (heinz) based on integer linear programming. At the same time, the concept of semantic similarities between two genes using Gene Ontology (GO) annotations has become an important basis for many analytical approaches in bioinformatics. Assuming that a higher number of semantically similar gene functional annotations reflect biologically more relevant interactions, an edge score was devised for functional network analysis. Bringing these two approaches together, the edge score, based on the GO similarity, and the node score, based on the expression of the proteins in the analyzed cell type (e.g. data from proteomic studies), the functional module as a maximum-scoring sub network in large protein-protein interaction networks was identified. This method was applied to various proteome datasets (different types of blood cells, embryonic stem cells) to identify protein modules that functionally characterize the respective cell type. This scalable method allows a smooth integration of data from various sources and retrieves biologically relevant signaling modules.}, subject = {Systembiologie}, language = {en} } @article{JahnSchrammSchnoelzeretal.2012, author = {Jahn, Daniel and Schramm, Sabine and Schn{\"o}lzer, Martina and Heilmann, Clemens J. and de Koster, Chris G. and Sch{\"u}tz, Wolfgang and Benavente, Ricardo and Alsheimer, Manfred}, title = {A truncated lamin A in the Lmna\(^{-/-}\) mouse line: Implications for the understanding of laminopathies}, series = {Nucleus}, volume = {3}, journal = {Nucleus}, number = {5}, doi = {10.4161/nucl.21676}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-127281}, pages = {463-474}, year = {2012}, abstract = {During recent years a number of severe clinical syndromes, collectively termed laminopathies, turned out to be caused by various, distinct mutations in the human LMNA gene. Arising from this, remarkable progress has been made to unravel the molecular pathophysiology underlying these disorders. A great benefit in this context was the generation of an A-type lamin deficient mouse line (Lmna\(^{-/-}\)) by Sullivan and others,1 which has become one of the most frequently used models in the field and provided profound insights to many different aspects of A-type lamin function. Here, we report the unexpected finding that these mice express a truncated Lmna gene product on both transcriptional and protein level. Combining different approaches including mass spectrometry, we precisely define this product as a C-terminally truncated lamin A mutant that lacks domains important for protein interactions and post-translational processing. Based on our findings we discuss implications for the interpretation of previous studies using Lmna\(^{-/-}\) mice and the concept of human laminopathies.}, language = {en} } @article{WeisseHeddergottHeydtetal.2012, author = {Weiße, Sebastian and Heddergott, Niko and Heydt, Matthias and Pfl{\"a}sterer, Daniel and Maier, Timo and Haraszti, Tamas and Grunze, Michael and Engstler, Markus and Rosenhahn, Axel}, title = {A Quantitative 3D Motility Analysis of Trypanosoma brucei by Use of Digital In-line Holographic Microscopy}, series = {PLoS One}, volume = {7}, journal = {PLoS One}, number = {5}, doi = {10.1371/journal.pone.0037296}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-130666}, pages = {e37296}, year = {2012}, abstract = {We present a quantitative 3D analysis of the motility of the blood parasite Trypanosoma brucei. Digital in-line holographic microscopy has been used to track single cells with high temporal and spatial accuracy to obtain quantitative data on their behavior. Comparing bloodstream form and insect form trypanosomes as well as mutant and wildtype cells under varying external conditions we were able to derive a general two-state-run-and-tumble-model for trypanosome motility. Differences in the motility of distinct strains indicate that adaption of the trypanosomes to their natural environments involves a change in their mode of swimming.}, language = {en} } @article{StaigerCadotKooteretal.2012, author = {Staiger, Christine and Cadot, Sidney and Kooter, Raul and Dittrich, Marcus and M{\"u}ller, Tobias and Klau, Gunnar W. and Wessels, Lodewyk F. A.}, title = {A Critical Evaluation of Network and Pathway-Based Classifiers for Outcome Prediction in Breast Cancer}, series = {PLoS One}, volume = {7}, journal = {PLoS One}, number = {4}, doi = {10.1371/journal.pone.0034796}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-131323}, pages = {e34796}, year = {2012}, abstract = {Recently, several classifiers that combine primary tumor data, like gene expression data, and secondary data sources, such as protein-protein interaction networks, have been proposed for predicting outcome in breast cancer. In these approaches, new composite features are typically constructed by aggregating the expression levels of several genes. The secondary data sources are employed to guide this aggregation. Although many studies claim that these approaches improve classification performance over single genes classifiers, the gain in performance is difficult to assess. This stems mainly from the fact that different breast cancer data sets and validation procedures are employed to assess the performance. Here we address these issues by employing a large cohort of six breast cancer data sets as benchmark set and by performing an unbiased evaluation of the classification accuracies of the different approaches. Contrary to previous claims, we find that composite feature classifiers do not outperform simple single genes classifiers. We investigate the effect of (1) the number of selected features; (2) the specific gene set from which features are selected; (3) the size of the training set and (4) the heterogeneity of the data set on the performance of composite feature and single genes classifiers. Strikingly, we find that randomization of secondary data sources, which destroys all biological information in these sources, does not result in a deterioration in performance of composite feature classifiers. Finally, we show that when a proper correction for gene set size is performed, the stability of single genes sets is similar to the stability of composite feature sets. Based on these results there is currently no reason to prefer prognostic classifiers based on composite features over single genes classifiers for predicting outcome in breast cancer.}, language = {en} }