@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{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}
}
@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{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{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}
}
@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}
}
@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{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{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}
}
@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}
}