@article{HohenauerBerkingSchmidtetal.2013,
  author    = {Hohenauer, Tobias and Berking, Carola and Schmidt, Andreas and Haferkamp, Sebastian and Senft, Daniela and Kammerbauer, Claudia and Fraschka, Sabine and Graf, Saskia Anna and Irmler, Martin and Beckers, Johannes and Flaig, Michael and Aigner, Achim and H{\"o}bel, Sabrina and Hoffmann, Franziska and Hermeking, Heiko and Rothenfusser, Simon and Endres, Stefan and Ruzicka, Thomas and Besch, Robert},
  title     = {The neural crest transcription factor Brn3a is expressed in melanoma and required for cell cycle progression and survival},
  series = {EMBO Molecular Medicine},
  volume    = {5},
  journal   = {EMBO Molecular Medicine},
  issn      = {1757-4676},
  doi       = {10.1002/emmm.201201862},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-122193},
  pages     = {919-934},
  year      = {2013},
  abstract  = {Pigment cells and neuronal cells both are derived from the neural crest. Here, we describe the Pit-Oct-Unc (POU) domain transcription factor Brn3a, normally involved in neuronal development, to be frequently expressed in melanoma, but not in melanocytes and nevi. RNAi-mediated silencing of Brn3a strongly reduced the viability of melanoma cell lines and decreased tumour growth in vivo. In melanoma cell lines, inhibition of Brn3a caused DNA double-strand breaks as evidenced by Mre11/Rad50-containing nuclear foci. Activated DNA damage signalling caused stabilization of the tumour suppressor p53, which resulted in cell cycle arrest and apoptosis. When Brn3a was ectopically expressed in primary melanocytes and fibroblasts, anchorage-independent growth was increased. In tumourigenic melanocytes and fibroblasts, Brn3a accelerated tumour growth in vivo. Furthermore, Brn3a cooperated with proliferation pathways such as oncogenic BRAF, by reducing oncogene-induced senescence in non-malignant melanocytes. Together, these results identify Brn3a as a new factor in melanoma that is essential for melanoma cell survival and that promotes melanocytic transformation and tumourigenesis.},
  language  = {en}
}
@article{SalatWinklerUrlaubetal.2015,
  author    = {Salat, Daniela and Winkler, Anja and Urlaub, Henning and Gessler, Manfred},
  title     = {Hey bHLH Proteins Interact with a FBXO45 Containing SCF Ubiquitin Ligase Complex and Induce Its Translocation into the Nucleus},
  series = {PLoS One},
  volume    = {10},
  journal   = {PLoS One},
  number    = {6},
  doi       = {10.1371/journal.pone.0130288},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-125769},
  pages     = {e0130288},
  year      = {2015},
  abstract  = {The Hey protein family, comprising Hey1, Hey2 and HeyL in mammals, conveys Notch signals in many cell types. The helix-loop-helix (HLH) domain as well as the Orange domain, mediate homo- and heterodimerization of these transcription factors. Although distinct interaction partners have been identified so far, their physiological relevance for Hey functions is still largely unclear. Using a tandem affinity purification approach and mass spectrometry analysis we identified members of an ubiquitin E3-ligase complex consisting of FBXO45, PAM and SKP1 as novel Hey1 associated proteins. There is a direct interaction between Hey1 and FBXO45, whereas FBXO45 is needed to mediate indirect Hey1 binding to SKP1. Expression of Hey1 induces translocation of FBXO45 and PAM into the nucleus. Hey1 is a short-lived protein that is degraded by the proteasome, but there is no evidence for FBXO45-dependent ubiquitination of Hey1. On the contrary, Hey1 mediated nuclear translocation of FBXO45 and its associated ubiquitin ligase complex may extend its spectrum to additional nuclear targets triggering their ubiquitination. This suggests a novel mechanism of action for Hey bHLH factors.},
  language  = {en}
}
@article{NaseemKunzDandekar2014,
  author    = {Naseem, Muhammad and Kunz, Meik and Dandekar, Thomas},
  title     = {Probing the unknowns in cytokinin-mediated immune defense in Arabidopsis with systems biology approaches},
  series = {Bioinformatics and Biology Insights},
  volume    = {8},
  journal   = {Bioinformatics and Biology Insights},
  issn      = {1177-9322},
  doi       = {10.4137/bbi.s13462},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-120199},
  pages     = {35-44},
  year      = {2014},
  abstract  = {Plant hormones involving salicylic acid (SA), jasmonic acid (JA), ethylene (Et), and auxin, gibberellins, and abscisic acid (ABA) are known to regulate host immune responses. However, plant hormone cytokinin has the potential to modulate defense signaling including SA and JA. It promotes plant pathogen and herbivore resistance; underlying mechanisms are still unknown. Using systems biology approaches, we unravel hub points of immune interaction mediated by cytokinin signaling in Arabidopsis. High-confidence Arabidopsis protein-protein interactions (PPI) are coupled to changes in cytokinin-mediated gene expression. Nodes of the cellular interactome that are enriched in immune functions also reconstitute sub-networks. Topological analyses and their specific immunological relevance lead to the identification of functional hubs in cellular interactome. We discuss our identified immune hubs in light of an emerging model of cytokinin-mediated immune defense against pathogen infection in plants.},
  language  = {en}
}
@phdthesis{Bedenk2018,
  author    = {Bedenk, Kristina},
  title     = {Biochemische und strukturelle Charakterisierung der Genexpressionsmaschinerie des Vaccinia Virus},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-135538},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Die Familie der Pockenviren zeichnet sich durch ein komplexes DNA Genom aus und hat großes medizinisches Potential. Am eindrucksvollsten ist dies f{\"u}r das Vaccinia-Virus (VACV) belegt, welches nicht nur als Pocken-Impfstoff eingesetzt wird, sondern auch als onkolytisches Virus in der Tumorbiologie. VACV hat einen außergew{\"o}hnlichen Replikationszyklus, welcher ausschließlich im Zytoplasma der Wirtszelle stattfindet. Somit ist die gesamte virale Genexpressionsmaschinerie v{\"o}llig unabh{\"a}ngig von kernvermittelten Reaktionen des Wirts und somit auch aus Sicht der Grundlagenforschung von gr{\"o}ßtem Interesse. Die Schl{\"u}sselkomponente der viralen Genexpression ist die makromolekulare DNA-abh{\"a}ngige RNA Polymerase (vvRPO), deren Untereinheiten allesamt Virus-kodiert sind. Zwar wurden in den letzten Jahren Protokolle zur biochemischen und funktionellen Charakterisierung der vvRPO etabliert, ein detailliertes Wissen {\"u}ber deren Zusammenlagerung in vivo und die r{\"a}umlichen und zeitlichen Interaktionen mit den Transkriptions- bzw. Prozessierungsfaktoren sind aber weitgehend unbekannt. Diese Arbeit umfasst Untersuchungen zur strukturellen und funktionellen Charakterisierung der vvRPO und seiner assoziierten Faktoren. Grundlage hierf{\"u}r war die Etablierung eines Reinigungsprotokolls mithilfe eines neu konstruierten rekombinanten VACV (GLV-1h439). Diese Strategie erlaubte es hoch-molekulare native vvRPO Komplexe zu isolieren. Ein transkriptions-inaktiver Komplex (Komplex I) mit einer kalkulierten Masse von 575 kDa bestand aus den acht Untereinheiten des vvRPO Holoenzyms und den Polymerase-assoziierten Faktoren RAP94 und D6. Ein zweiter, transkriptionell aktiver Komplex (Komplex II) mit einer Masse von 803 kDa enthielt, neben dem Holoenzym der vvRPO, noch weitere Faktoren, die prim{\"a}r die Erkennung der DNA-Matrize und die Prozessierung der naszierenden RNA vermitteln. Hierbei handelt es sich um RAP94, das virale Capping Enzym bestehend aus den zwei Untereinheiten D1 und D12, A7 und dem Terminationsfaktor NPH I. Interessanterweise enthielt dieser Komplex zus{\"a}tzlich mit E11 eine bislang unbekannte weitere Protein-Komponente, sowie tRNAGln und tRNAArg. Der isolierte Kompelx II ist daher ein Ribonukleoprotein (RNP). Die Verf{\"u}gbarkeit von hoch-reinen vvRPO Komplexen erlaubte es erstmals deren strukturelle Architektur zu untersuchen. Hierf{\"u}r wurden drei experimentelle Ans{\"a}tze, die klassische R{\"o}ntgenstrukturanalyse, die Kryo-Elektronenmikroskopie (Kryo-EM) und Quervernetzungssstudien miteinander kombiniert. Die Strukturen der Komplexe I und II haben eine Aufl{\"o}sung von 11-12 {\AA}, wobei auff{\"a}llig war, dass beide eine markante strukturelle {\"A}hnlichkeit zur eukaryotischen RNA Polymerase II aufwiesen. Dar{\"u}ber hinaus gelang es zus{\"a}tzliche Bereiche im Komplex II zu definieren, welche die Polymerase-assoziierten Prozessierungsfaktoren beherbergen. Zudem konnte die atomare Struktur von E11, mittels R{\"o}ntgenstrukturanalyse bei einer Aufl{\"o}sung von 1,9 {\AA}, gel{\"o}st werden. Das E11 Protein besitzt ein neuartiges Faltungsmuster und weist einen intensiven Dimerisierungskontakt auf, welcher sich {\"u}ber vier ß-Faltbl{\"a}tter ausbildet. Die im Rahmen dieser Arbeit erhaltenen Daten legen die Grundlage f{\"u}r ein detailliertes Verst{\"a}ndnis der r{\"a}umlichen Organisation der viralen Transkriptonsmaschinerie. Dar{\"u}ber hinaus werden sie funktionelle Studien erm{\"o}glichen, welche die Rolle der einzelnen Proteine, sowie der tRNAs bei der mRNA Synthese kl{\"a}ren helfen.},
  subject      = {Vaccinia-Virus},
  language  = {de}
}
@article{StepniakKaestnerPoggietal.2015,
  author    = {Stepniak, Beata and K{\"a}stner, Anne and Poggi, Giulia and Mitjans, Marina and Begemann, Martin and Hartmann, Annette and Van der Auwera, Sandra and Sananbenesi, Farahnaz and Kr{\"u}ger-Burg, Dilja and Matuszko, Gabriela and Brosi, Cornelia and Homuth, Georg and V{\"o}lzke, Henry and Benseler, Fritz and Bagni, Claudia and Fischer, Utz and Dityatev, Alexander and Grabe, Hans-J{\"o}rgen and Rujescu, Dan and Fischer, Andre and Ehrenreich, Hannelore},
  title     = {Accumulated common variants in the broader fragile X gene family modulate autistic phenotypes},
  series = {EMBO Molecular Medicine},
  volume    = {7},
  journal   = {EMBO Molecular Medicine},
  number    = {12},
  doi       = {10.15252/emmm.201505696},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-136893},
  pages     = {1565-1579},
  year      = {2015},
  abstract  = {Fragile X syndrome (FXS) is mostly caused by a CGG triplet expansion in the fragile X mental retardation 1 gene (FMR1). Up to 60\% of affected males fulfill criteria for autism spectrum disorder (ASD), making FXS the most frequent monogenetic cause of syndromic ASD. It is unknown, however, whether normal variants (independent of mutations) in the fragile X gene family (FMR1, FXR1, FXR2) and in FMR2 modulate autistic features. Here, we report an accumulation model of 8 SNPs in these genes, associated with autistic traits in a discovery sample of male patients with schizophrenia (N = 692) and three independent replicate samples: patients with schizophrenia (N = 626), patients with other psychiatric diagnoses (N = 111) and a general population sample (N = 2005). For first mechanistic insight, we contrasted microRNA expression in peripheral blood mononuclear cells of selected extreme group subjects with high-versus low-risk constellation regarding the accumulation model. Thereby, the brain-expressed miR-181 species emerged as potential "umbrella regulator", with several seed matches across the fragile X gene family and FMR2. To conclude, normal variation in these genes contributes to the continuum of autistic phenotypes.},
  language  = {en}
}
@article{SpiveyDeGiorgiZhaoetal.2012,
  author    = {Spivey, Tara L. and De Giorgi, Valeria and Zhao, Yingdong and Bedognetti, Davide and Pos, Zoltan and Liu, Qiuzhen and Tomei, Sara and Ascierto, Maria Libera and Uccellini, Lorenzo and Reinboth, Jennifer and Chouchane, Lotfi and Stroncek, David F. and Wang, Ena and Marincola, Francesco M.},
  title     = {The stable traits of melanoma genetics: an alternate approach to target discovery},
  series = {BMC Genomics},
  volume    = {13},
  journal   = {BMC Genomics},
  number    = {156},
  doi       = {10.1186/1471-2164-13-156},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-131992},
  year      = {2012},
  abstract  = {Background: The weight that gene copy number plays in transcription remains controversial; although in specific cases gene expression correlates with copy number, the relationship cannot be inferred at the global level. We hypothesized that genes steadily expressed by 15 melanoma cell lines (CMs) and their parental tissues (TMs) should be critical for oncogenesis and their expression most frequently influenced by their respective copy number. Results: Functional interpretation of 3,030 transcripts concordantly expressed (Pearson's correlation coefficient p-value < 0.05) by CMs and TMs confirmed an enrichment of functions crucial to oncogenesis. Among them, 968 were expressed according to the transcriptional efficiency predicted by copy number analysis (Pearson's correlation coefficient p-value < 0.05). We named these genes, "genomic delegates" as they represent at the transcriptional level the genetic footprint of individual cancers. We then tested whether the genes could categorize 112 melanoma metastases. Two divergent phenotypes were observed: one with prevalent expression of cancer testis antigens, enhanced cyclin activity, WNT signaling, and a Th17 immune phenotype (Class A). This phenotype expressed, therefore, transcripts previously associated to more aggressive cancer. The second class (B) prevalently expressed genes associated with melanoma signaling including MITF, melanoma differentiation antigens, and displayed a Th1 immune phenotype associated with better prognosis and likelihood to respond to immunotherapy. An intermediate third class (C) was further identified. The three phenotypes were confirmed by unsupervised principal component analysis. Conclusions: This study suggests that clinically relevant phenotypes of melanoma can be retraced to stable oncogenic properties of cancer cells linked to their genetic back bone, and offers a roadmap for uncovering novel targets for tailored anti-cancer therapy.},
  language  = {en}
}
@article{GrossHennardMasourisetal.2012,
  author    = {Gross, Henrik and Hennard, Christine and Masouris, Ilias and Cassel, Christian and Barth, Stephanie and Stober-Gr{\"a}sser, Ute and Mamiani, Alfredo and Moritz, Bodo and Ostareck, Dirk and Ostareck-Lederer, Antje and Neuenkirchen, Nils and Fischer, Utz and Deng, Wen and Leonhardt, Heinrich and Noessner, Elfriede and Kremmer, Elisabeth and Gr{\"a}sser, Friedrich A.},
  title     = {Binding of the Heterogeneous Ribonucleoprotein K (hnRNP K) to the Epstein-Barr Virus Nuclear Antigen 2 (EBNA2) Enhances Viral LMP2A Expression},
  series = {PLoS One},
  volume    = {7},
  journal   = {PLoS One},
  number    = {8},
  doi       = {10.1371/journal.pone.0042106},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-133707},
  year      = {2012},
  abstract  = {The Epstein-Barr Virus (EBV) -encoded EBNA2 protein, which is essential for the in vitro transformation of B-lymphocytes, interferes with cellular processes by binding to proteins via conserved sequence motifs. Its Arginine-Glycine (RG) repeat element contains either symmetrically or asymmetrically di-methylated arginine residues (SDMA and ADMA, respectively). EBNA2 binds via its SDMA-modified RG-repeat to the survival motor neurons protein (SMN) and via the ADMA-RG-repeat to the NP9 protein of the human endogenous retrovirus K (HERV-K (HML-2) Type 1). The hypothesis of this work was that the methylated RG-repeat mimics an epitope shared with cellular proteins that is used for interaction with target structures. With monoclonal antibodies against the modified RG-repeat, we indeed identified cellular homologues that apparently have the same surface structure as methylated EBNA2. With the SDMA-specific antibodies, we precipitated the Sm protein D3 (SmD3) which, like EBNA2, binds via its SDMA-modified RG-repeat to SMN. With the ADMA-specific antibodies, we precipitated the heterogeneous ribonucleoprotein K (hnRNP K). Specific binding of the ADMA-antibody to hnRNP K was demonstrated using E. coli expressed/ADMA-methylated hnRNP K. In addition, we show that EBNA2 and hnRNP K form a complex in EBV-infected B-cells. Finally, hnRNP K, when co-expressed with EBNA2, strongly enhances viral latent membrane protein 2A (LMP2A) expression by an unknown mechanism as we did not detect a direct association of hnRNP K with DNA-bound EBNA2 in gel shift experiments. Our data support the notion that the methylated surface of EBNA2 mimics the surface structure of cellular proteins to interfere with or co-opt their functional properties.},
  language  = {en}
}
@article{GowdaGodderKmieciaketal.2011,
  author    = {Gowda, Madhu and Godder, Kamar and Kmieciak, Maciej and Worschech, Andrea and Ascierto, Maria-Libera and Wang, Ena and Francesco M., Marincola and Manjili, Masoud H.},
  title     = {Distinct signatures of the immune responses in low risk versus high risk neuroblastoma},
  series = {Journal of Translational Medicine},
  volume    = {9},
  journal   = {Journal of Translational Medicine},
  number    = {170},
  doi       = {10.1186/1479-5876-9-170},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-135147},
  pages     = {1-8},
  year      = {2011},
  abstract  = {Background: Over 90\% of low risk (LR) neuroblastoma patients survive whereas less than 30\% of high risk (HR) patients are long term survivors. Age (children younger than 18 months old) is associated with LR disease. Considering that adaptive immune system is well developed in older children, and that T cells were shown to be involved in tumor escape and progression of cancers, we sought to determine whether HR patients may tend to show a signature of adaptive immune responses compared to LR patients who tend to have diminished T-cell responses but an intact innate immune response. Methods: We performed microarray analysis of RNA extracted from the tumor specimens of HR and LR patients. Flow cytometry was performed to determine the cellular constituents in the blood while multiplex cytokine array was used to detect the cytokine profile in patients' sera. A HR tumor cell line, SK-N-SH, was also used for detecting the response to IL-1 beta, a cytokines which is involved in the innate immune responses. Results: Distinct patterns of gene expression were detected in HR and LR patients indicating an active T-cell response and a diminished adaptive immune response, respectively. A diminished adaptive immune response in LR patients was evident by higher levels of IL-10 in the sera. In addition, HR patients had lower levels of circulating myeloid derived suppressor cells (MDSC) compared with a control LR patient. LR patients showed slightly higher levels of cytokines of the innate immune responses. Treatment of the HR tumor line with IL-1b induced expression of cytokines of the innate immune responses. Conclusions: This data suggests that adaptive immune responses may play an important role in the progression of HR disease whereas innate immune responses may be active in LR patients.},
  language  = {en}
}
@article{WeibelBasseLuesebrinkHessetal.2013,
  author    = {Weibel, Stephanie and Basse-Luesebrink, Thomas Christian and Hess, Michael and Hofmann, Elisabeth and Seubert, Carolin and Langbein-Laugwitz, Johanna and Gentschev, Ivaylo and Sturm, Volker J{\"o}rg Friedrich and Ye, Yuxiang and Kampf, Thomas and Jakob, Peter Michael and Szalay, Aladar A.},
  title     = {Imaging of Intratumoral Inflammation during Oncolytic Virotherapy of Tumors by \(^{19}\)F-Magnetic Resonance Imaging (MRI)},
  series = {PLoS ONE},
  volume    = {8},
  journal   = {PLoS ONE},
  number    = {3},
  doi       = {10.1371/journal.pone.0056317},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-130311},
  pages     = {e56317},
  year      = {2013},
  abstract  = {Background Oncolytic virotherapy of tumors is an up-coming, promising therapeutic modality of cancer therapy. Unfortunately, non-invasive techniques to evaluate the inflammatory host response to treatment are rare. Here, we evaluate \(^{19}\)F magnetic resonance imaging (MRI) which enables the non-invasive visualization of inflammatory processes in pathological conditions by the use of perfluorocarbon nanoemulsions (PFC) for monitoring of oncolytic virotherapy. Methodology/Principal Findings The Vaccinia virus strain GLV-1h68 was used as an oncolytic agent for the treatment of different tumor models. Systemic application of PFC emulsions followed by \(^1H\)/\(^{19}\)F MRI of mock-infected and GLV-1h68-infected tumor-bearing mice revealed a significant accumulation of the \(^{19}\)F signal in the tumor rim of virus-treated mice. Histological examination of tumors confirmed a similar spatial distribution of the \(^{19}\)F signal hot spots and \(CD68^+\)-macrophages. Thereby, the \(CD68^+\)-macrophages encapsulate the GFP-positive viral infection foci. In multiple tumor models, we specifically visualized early inflammatory cell recruitment in Vaccinia virus colonized tumors. Furthermore, we documented that the \(^{19}\)F signal correlated with the extent of viral spreading within tumors. Conclusions/Significance These results suggest \(^{19}\)F MRI as a non-invasive methodology to document the tumor-associated host immune response as well as the extent of intratumoral viral replication. Thus, \(^{19}\)F MRI represents a new platform to non-invasively investigate the role of the host immune response for therapeutic outcome of oncolytic virotherapy and individual patient response.},
  language  = {en}
}
@article{KrehanHeubeckMenzeletal.2012,
  author    = {Krehan, Mario and Heubeck, Christian and Menzel, Nicolas and Seibel, Peter and Sch{\"o}n, Astrid},
  title     = {RNase MRP RNA and RNase P activity in plants are associated with a Pop1p containing complex},
  series = {Nucleic Acids Research},
  volume    = {40},
  journal   = {Nucleic Acids Research},
  number    = {16},
  doi       = {10.1093/nar/gks476},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-130648},
  pages     = {7956- 7966},
  year      = {2012},
  abstract  = {RNase P processes the 5'-end of tRNAs. An essential catalytic RNA has been demonstrated in Bacteria, Archaea and the nuclei of most eukaryotes; an organism-specific number of proteins complement the holoenzyme. Nuclear RNase P from yeast and humans is well understood and contains an RNA, similar to the sister enzyme RNase MRP. In contrast, no protein subunits have yet been identified in the plant enzymes, and the presence of a nucleic acid in RNase P is still enigmatic. We have thus set out to identify and characterize the subunits of these enzymes in two plant model systems. Expression of the two known Arabidopsis MRP RNA genes in vivo was verified. The first wheat MRP RNA sequences are presented, leading to improved structure models for plant MRP RNAs. A novel mRNA encoding the central RNase P/MRP protein Pop1p was identified in Arabidopsis, suggesting the expression of distinct protein variants from this gene in vivo. Pop1p-specific antibodies precipitate RNase P activity and MRP RNAs from wheat extracts. Our results provide evidence that in plants, Pop1p is associated with MRP RNAs and with the catalytic subunit of RNase P, either separately or in a single large complex.},
  language  = {en}
}
@article{WangChenMinevetal.2013,
  author    = {Wang, Huiqiang and Chen, Nanhai G. and Minev, Boris R. and Zimmermann, Martina and Aguilar, Richard J. and Zhang, Qian and Sturm, Julia B. and Fend, Falko and Yu, Yong A. and Cappello, Joseph and Lauer, Ulrich M. and Szalay, Aladar A.},
  title     = {Optical Detection and Virotherapy of Live Metastatic Tumor Cells in Body Fluids with Vaccinia Strains},
  series = {PLoS ONE},
  volume    = {8},
  journal   = {PLoS ONE},
  number    = {9},
  doi       = {10.1371/journal.pone.0071105},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-130059},
  pages     = {e71105},
  year      = {2013},
  abstract  = {Metastatic tumor cells in body fluids are important targets for treatment, and critical surrogate markers for evaluating cancer prognosis and therapeutic response. Here we report, for the first time, that live metastatic tumor cells in blood samples from mice bearing human tumor xenografts and in blood and cerebrospinal fluid samples from patients with cancer were successfully detected using a tumor cell-specific recombinant vaccinia virus (VACV). In contrast to the FDA-approved CellSearch system, VACV detects circulating tumor cells (CTCs) in a cancer biomarker-independent manner, thus, free of any bias related to the use of antibodies, and can be potentially a universal system for detection of live CTCs of any tumor type, not limited to CTCs of epithelial origin. Furthermore, we demonstrate for the first time that VACV was effective in preventing and reducing circulating tumor cells in mice bearing human tumor xenografts. Importantly, a single intra-peritoneal delivery of VACV resulted in a dramatic decline in the number of tumor cells in the ascitic fluid from a patient with gastric cancer. Taken together, these results suggest VACV to be a useful tool for quantitative detection of live tumor cells in liquid biopsies as well as a potentially effective treatment for reducing or eliminating live tumor cells in body fluids of patients with metastatic disease.},
  language  = {en}
}
@phdthesis{Pelz2015,
  author    = {Pelz, Jann-Patrick},
  title     = {Strukturbiologische Untersuchungen zur Chaperone-vermittelten Zusammenlagerung spleißosomaler U-snRNPs},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-116973},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Durch die Spleißreaktion werden nicht-kodierende Sequenzelemente (Introns) aus eukaryotischen Vorl{\"a}ufer-mRNAs entfernt und die kodierenden Sequenzelemente (Exons) miteinander zu einem offenen Leserahmen verbunden. Dieser zentrale Prozessierungsschritt w{\"a}hrend der eukaryotischen Genexpression wird durch das Spleißosom katalysiert, das aus den vier kleinen nukle{\"a}ren Ribonucleoproteinpartikeln (snRNPs) U1, U2, U4/U6 und U5, sowie einer Vielzahl weiterer Proteinfaktoren gebildet wird. Alle snRNPs besitzen eine gemeinsame ringf{\"o}rmige Kernstruktur, die aus sieben gemeinsamen Sm-Proteinen (SmB/B'-D1-D2-D3-E-F-G) besteht, die ein einzelstr{\"a}ngiges Sequenzmotiv auf der snRNAs binden. W{\"a}hrend sich diese, als Sm-Core-Dom{\"a}ne bezeichnete Struktur in vitro spontan ausbilden kann, erfolgt die Zusammenlagerung in vivo in einem assistierten und hochregulierten Prozess. Dieser ist abh{\"a}ngig von insgesamt mindestens 12 trans-agierenden Faktoren, die in den PRMT5- und SMN-Komplexen organisiert sind. Der PRMT5-Komplex agiert in der fr{\"u}hen Phase der Zusammenlagerung, indem er die Sm-Proteine durch die Untereinheit pICln rekrutiert und die symmetrische Methylierung von Argininresten in den C terminalen Schw{\"a}nzen von SmB/B', SmD1 und SmD3 katalysiert. Als Resultat dieser fr{\"u}hen Phase befinden sich die Sm-Proteine SmD1-D2-E-F-G und SmB/B'-D3 in zwei getrennten und durch pICln organisierten Komplexen. W{\"a}hrend SmB/B'-D3-pICln am PRMT5-Komplex gebunden bleibt, existiert der zweite Komplex als freies Intermediat mit einem Sedimentationskoeffizienten von 6S. Diese Intermediate k{\"o}nnen nicht mit RNA assoziieren, sodass f{\"u}r die Fortsetzung des Zusammenlagerungsprozesses die Interaktion der Sm-Proteine mit pICln aufgel{\"o}st werden muss. Dies geschieht in der sp{\"a}ten Phase der Sm-Core-Zusammenlagerung, in der die Sm-Proteine vom SMN-Komplex (bestehend aus SMN, Gemin2-8 und unrip) {\"u}bernommen werden und pICln dissoziiert wird. Dadurch werden die Sm-Proteine f{\"u}r ihre Interaktion mit der snRNA aktiviert und k{\"o}nnen auf die Sm-Bindestelle transferiert werden, wodurch die Formierung des Sm-Core abgeschlossen wird. Im Rahmen dieser Arbeit konnten mit Hilfe einer Kombination r{\"o}ntgenkristallographischer und elektronenmikroskopischer Methoden zwei wichtige Intermediate dieses Zusammenlagerungs-prozesses strukturbiologisch charakterisiert werden. Bei diesen Intermediaten handelt es sich um den 6S-Komplex, sowie um ein Sm-Protein-Transferintermediat mit einem Sedimentations-koeffizienten von 8S. In diesem ist der 6S-Komplex an zwei zentrale Untereinheiten des SMN-Komplexes (SMN und Gemin2) gebunden, w{\"a}hrend pICln den Komplex noch nicht verlassen hat. Der 8S-Komplex stellt daher ein „gefangenes" Intermediat zwischen der fr{\"u}hen und sp{\"a}ten Phase der Zusammenlagerung dar. Zun{\"a}chst gelang es eine erste Kristallform des rekombinant hergestellten 8S-Komplexes zu erhalten, die jedoch keine Strukturl{\"o}sung erlaubte. Durch eine kombinierte Optimierung der Kristallisationsbedingung und der verwendeten Proteine wurde eine weitere {\"a}hnliche Kristallform erhalten, mit der die Kristallstruktur des 8S-Komplexes gel{\"o}st werden konnte. Die Kristallisation des 6S-Komplexes gelang im Anschluss auf Basis der Hypothese, dass Kristalle beider Komplexe aufgrund der kompositionellen Verwandtschaft zwischen 6S und 8S auch {\"A}hnlichkeiten in der Architektur ihrer Kristallgitter aufweisen k{\"o}nnten. Daher wurden innerhalb von pICln gezielt Aminos{\"a}uren substituiert, die sich innerhalb von Kristallkontakten der 8S-Kristalle befanden und konformationell eingeschr{\"a}nkt waren. Mit entsprechend rekonstituierten 6S-Pr{\"a}parationen konnten dann zwei Kristallformen erzeugt werden, die eine Strukturl{\"o}sung des 6S-Komplexes erm{\"o}glichten. Durch die Kristallstruktur des 6S-Komplexes konnte f{\"u}r pICln eine strukturelle Mimikry der Sm-Proteine identifiziert werden. Diese erm{\"o}glicht eine Bindung der Sm-Proteine und eine fr{\"u}hzeitige topologische Organisation des Sm-Pentamers D1-D2-F-E-G in einer geschlossenen hexameren Ringstruktur. Die Kristallstruktur des 8S-Komplexes zeigt, wie der SMN-Komplex {\"u}ber Gemin2 an das Sm-Pentamer bindet. In Kombination mit einer EM-Struktur des 8S-Komplexes gelang es weiterhin, einen plausiblen Mechanismus f{\"u}r die Elimination von pICln und die Aktivierung der Sm-Proteine f{\"u}r die snRNA-Bindung zu formulieren. Somit konnten diese Arbeiten zu einem besseren Verst{\"a}ndnis der Funktionen von trans-agierenden Faktoren bei Zusammenlagerung von RNA-Protein-Komplexen in vivo beitragen.},
  subject      = {Spleißosom},
  language  = {de}
}
@article{BenzMaierBaueretal.2014,
  author    = {Benz, Roland and Maier, Elke and Bauer, Susanne and Ludwig, Albrecht},
  title     = {The Deletion of Several Amino Acid Stretches of Escherichia coli Alpha-Hemolysin (HlyA) Suggests That the Channel-Forming Domain Contains Beta-Strands},
  series = {PLOS ONE},
  volume    = {9},
  journal   = {PLOS ONE},
  number    = {12},
  issn      = {1932-6203},
  doi       = {10.1371/journal.pone.0112248},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-118115},
  pages     = {e112248},
  year      = {2014},
  abstract  = {Escherichia coli α-hemolysin (HlyA) is a pore-forming protein of 110 kDa belonging to the family of RTX toxins. A hydrophobic region between the amino acid residues 238 and 410 in the N-terminal half of HlyA has previously been suggested to form hydrophobic and/or amphipathic α-helices and has been shown to be important for hemolytic activity and pore formation in biological and artificial membranes. The structure of the HlyA transmembrane channel is, however, largely unknown. For further investigation of the channel structure, we deleted in HlyA different stretches of amino acids that could form amphipathic β-strands according to secondary structure predictions (residues 71-110, 158-167, 180-203, and 264-286). These deletions resulted in HlyA mutants with strongly reduced hemolytic activity. Lipid bilayer measurements demonstrated that HlyAΔ71-110 and HlyAΔ264-286 formed channels with much smaller single-channel conductance than wildtype HlyA, whereas their channel-forming activity was virtually as high as that of the wildtype toxin. HlyAΔ158-167 and HlyAΔ180-203 were unable to form defined channels in lipid bilayers. Calculations based on the single-channel data indicated that the channels generated by HlyAΔ71-110 and HlyAΔ264-286 had a smaller size (diameter about 1.4 to 1.8 nm) than wildtype HlyA channels (diameter about 2.0 to 2.6 nm), suggesting that in these mutants part of the channel-forming domain was removed. Osmotic protection experiments with erythrocytes confirmed that HlyA, HlyAΔ71-110, and HlyAΔ264-286 form defined transmembrane pores and suggested channel diameters that largely agreed with those estimated from the single-channel data. Taken together, these results suggest that the channel-forming domain of HlyA might contain β-strands, possibly in addition to α-helical structures.},
  language  = {en}
}
@phdthesis{Karl2015,
  author    = {Karl, Ingolf},
  title     = {Die Bedeutung von TRAF2 bei TRAIL-induzierter Apoptose und Nekroptose},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-114506},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Die vorliegende Arbeit behandelt TRAIL-induzierte Apoptose und Nekroptose in verschiedenen Zelllinien. Im Speziellen wurden die verschiedenen Funktionen des TNF receptor-associated factor 2 (TRAF2) untersucht. Hierzu wurde ein transienter Knockdown etabliert und dessen Wirkung auf die Suszeptibilit{\"a}t der Zellen gegen{\"u}ber dem Zytokin TRAIL untersucht. Es konnte gezeigt werden, dass ein Knockdown von TRAF2 nicht nur zur Sensitivierung f{\"u}r Apoptose f{\"u}hrt, sondern auch in Nekroptose-kompetenten Zellen zu einer Verst{\"a}rkung der durch Caspaseinhibition mittels zVAD-fmk nach TRAIL-Stimulation induzierten Nekroptose f{\"u}hrt. Mittels des Zytokins Fc-TWEAK wurde Fn14-vermittelt TRAF2 aus dem Zytosol in ein Triton X100-unl{\"o}sliches Kompartiment rekrutiert und dadurch physiologisch depletiert. Dies f{\"u}hrte zwar kaum zu gesteigerter TRAIL-abh{\"a}ngiger Apoptose, sensitivierte jedoch analog zum TRAF2-Knockdown RIP3-exprimierende Zellen f{\"u}r Nekroptose. Durch Vergleich RIP3-negativer (HeLa-Leervektor) mit RIP3-exprimierenden Zellen (HeLa RIP3, HT29, HaCaT) konnte die Essentialit{\"a}t von RIP3 f{\"u}r die Nekroptose herausgestellt werden und Einsatz des RIP1-Kinase-Inhibitors Necrostatin-1 sowie des MLKL-Inhibitors Necrosulfonamide belegte die Beteiligung der Nekroptosomkomponenten RIP1 und MLKL. Antagonismus putativen autokrinen TNFs bewies, dass es sich bei dem durch Fc-TWEAK verst{\"a}rkten Zelltod um einen direkten TRAIL-Effekt handelte und Inhibition kanonischen NFkBs durch IKK2-Inhibitor TPCA-1, dass die TRAF2-Knockdown-vermittelte Sensitivierung gegen{\"u}ber TRAIL nicht auf ver{\"a}ndertes NFkB-Signalling zur{\"u}ckzuf{\"u}hren ist. Einsatz des SMAC-Mimetikums BV6 rekapitulierte zudem stark das im TRAF2-Knockdown Gesehene und unterstrich die Bedeutung der cIAPs. Immunpr{\"a}zipitation von Caspase 8 unter nekroptotischen Bedingungen zeigte bei TRAF2-Knockdown eine Depletion von TRAF2 und cIAP1/2 sowie RIP1 und RIP3 aus dem Komplex mit Caspase 8. Insgesamt wird deutlich, dass TRAF2 einerseits antiapoptotisch wirkt als K48-Ubiquitinligase, die die Halbwertszeit aktiver Caspase 8-Komplexe determiniert und andererseits eine antinekroptotische Funktion hat, da es durch Rekrutierung von cIAP1/2 an RIP1 die TRAIL-induzierte Nekroptose verhindert, wenn die Caspasen inhibiert sind.},
  subject      = {Nekrose},
  language  = {de}
}
@phdthesis{Sibilski2014,
  author    = {Sibilski, Claudia},
  title     = {Identification and characterization of the novel mKSR1 phosphorylation site Tyr728 and its role in MAPK signaling},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-114672},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {In mammals, KSR1 functions as an essential scaffold that coordinates the assembly of RAF/MEK/ERK complexes and regulates intracellular signal transduction upon extracellular stimulation. Aberrant activation of the equivalent MAPK signaling pathway has been implicated in multiple human cancers and some developmental disorders. The mechanism of KSR1 regulation is highly complex and involves several phosphorylation/dephosphorylation steps. In the present study, a number of novel in vivo phosphorylation sites were detected in mKSR1 by use of mass spectrometry analysis. Among others, Tyr728 was identified as a unique regulatory residue phosphorylated by LCK, a Src kinase family member. To understand how phosphorylation of Tyr728 may regulate the function of KSR1 in signal transduction and cellular processes, structural modeling and biochemical studies were integrated in this work. Computational modeling of the mKSR1(KD) protein structure revealed strong hydrogen bonding between phospho-Tyr728 and the residues surrounding Arg649. Remarkably, this pattern was altered when Tyr728 was non-phosphorylated or substituted. As confirmed by biochemical analysis, Arg649 may serve as a major anchor point for phospho-Tyr728 in order to stabilize internal structures of KSR1. In line with the protein modeling results, mutational studies revealed that substitution of Tyr728 by phenylalanine leads to a less compact interaction between KSR1 and MEK, a facilitated KSR1/B-RAF binding and an increased phosphorylation of MEK in complex with KSR1. From these findings it can be concluded that phospho-Tyr728 is involved in tightening the KSR1/MEK interaction interface and in regulating the phosphorylation of KSR1-bound MEK by either RAF or KSR1 kinases. Beside the Tyr728, Ser722 was identified as a novel regulatory phosphorylation site. Amino acid exchanges at the relevant position demonstrated that Ser722 regulates KSR1-bound MEK phosphorylation without affecting KSR1/MEK binding per se. Due to its localization, Ser722 might consequently control the catalytic activity of KSR1 by interfering with the access of substrate (possibly MEK) to the active site of KSR1 kinase. Together with Ser722, phosphorylated Tyr728 may further positively affect the kinase activity of KSR1 as a consequence of its vicinity to the activation and catalytic loop in the KSR1(KD). As revealed by structural modeling, phospho-Tyr728 builds a hydrogen bond with the highly conserved Lys685. Consequently, phospho-Tyr728 has a stabilizing effect on internal structures involved in the catalytic reaction and possibly enhances the phosphate transfer within the catalytic cleft in KSR1. Considering these facts, it seems very likely that the LCK-dependent phosphorylation of Tyr728 plays a crucial role in the regulation of KSR1 catalytic activity. Results of fractionation and morphology analyses revealed that KSR1 recruits LCK to cytoskeleton for its phosphorylation at Tyr728 suggesting that this residue may regulate cytoskeleton dynamics and, consequently, cell motility. Beside that, phosphorylation of Tyr728 is involved in the regulation of cell proliferation, as shown by a significantly reduced population doubling time of KSR1-Y728F cells compared to cells expressing wild type KSR1. Taken together, tyrosine phosphorylation in KSR1 uncovers a new link between Src family kinases and MAPK signaling. Tyr728, the novel regulatory phosphorylation site in murine KSR1, may coordinate the transition between the scaffolding and the catalytic function of KSR1 serving as a control point used to fine-tune cellular responses.},
  subject      = {MAP-Kinase},
  language  = {en}
}
@article{LinderHirmerGaletal.2014,
  author    = {Linder, Bastian and Hirmer, Anja and Gal, Andreas and R{\"u}ther, Klaus and Bolz, Hanno J{\"o}rn and Winkler, Christoph and Laggerbauer, Bernhard and Fischer, Utz},
  title     = {Identification of a PRPF4 Loss-of-Function Variant That Abrogates U4/U6.U5 Tri-snRNP Integration and Is Associated with Retinitis Pigmentosa},
  doi       = {10.1371/journal.pone.0111754},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-113663},
  year      = {2014},
  abstract  = {Pre-mRNA splicing by the spliceosome is an essential step in the maturation of nearly all human mRNAs. Mutations in six spliceosomal proteins, PRPF3, PRPF4, PRPF6, PRPF8, PRPF31 and SNRNP200, cause retinitis pigmentosa (RP), a disease characterized by progressive photoreceptor degeneration. All splicing factors linked to RP are constituents of the U4/U6.U5 tri-snRNP subunit of the spliceosome, suggesting that the compromised function of this particle may lead to RP. Here, we report the identification of the p.R192H variant of the tri-snRNP factor PRPF4 in a patient with RP. The mutation affects a highly conserved arginine residue that is crucial for PRPF4 function. Introduction of a corresponding mutation into the zebrafish homolog of PRPF4 resulted in a complete loss of function in vivo. A series of biochemical experiments suggested that p.R192H disrupts the binding interface between PRPF4 and its interactor PRPF3. This interferes with the ability of PRPF4 to integrate into the tri-snRNP, as shown in a human cell line and in zebrafish embryos. These data suggest that the p.R192H variant of PRPF4 represents a functional null allele. The resulting haploinsufficiency of PRPF4 compromises the function of the tri-snRNP, reinforcing the notion that this spliceosomal particle is of crucial importance in the physiology of the retina.},
  language  = {en}
}
@phdthesis{Schubert2015,
  author    = {Schubert, Andreas},
  title     = {Protein kinases as targets for the development of novel drugs against alveolar echinococcosis},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-113694},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {The metacestode larval stage of the fox tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis (AE), one of the most lethal zoonosis of the northern hemisphere. The development of metacestode vesicles by asexual multiplication and the almost unrestricted infiltrative growth within the host organs is ensured from a population of undifferentiated, proliferative cells, so-called germinative cells. AE treatment options include surgery, if possible, as well as Benzimidazole-based chemotherapy (BZ). Given that the cellular targets of BZs, the -tubulins, are highly conserved between cestodes and humans, the chemotherapy is associated with considerable side-effects. Therefore, BZ can only be applied in parasitostatic doses and has to be given lifelong. Furthermore, the current anti-AE chemotherapy is ineffective in eliminating the germinative cell population of the parasite, which leads to remission of parasite growth as soon as therapy is discontinued. This work focuses on protein kinases involved in the proliferation and development of the parasite with the intention of developing novel anti-AE therapies. Polo-like kinases (Plks) are important regulators of the eukaryotic cell cycle and are involved in the regulation and formation of the mitotic spindles during the M-phase of the cell cycle. Plks have already been shown to be associated with deregulated cellular growth in human cancers and have been investigated as novel drug targets in the flatworm parasite Schistosoma mansoni. In the first part of this work, the characterisation of a novel and druggable parasite enzyme, EmPlk1, which is homologous to the polo-like kinase 1 (Plk1) of humans and S. mansoni (SmPlk1), is presented. Through in situ hybridisation, it could be demonstrated that emplk1 is specifically expressed in the Echinococcus germinative cells. Upon heterologous expression in the Xenopus oocyte system, EmPlk1 induced germinal vesicle breakdown, thus indicating that it is an active kinase. Furthermore, BI 2536, a compound originally designed to inhibit the human ortholog of EmPlk1, inhibited the EmPlk1 activity at a concentration of 25 nM. In vitro treatment of parasite vesicles with similar concentrations of BI 2536 led to the elimination of the germinative cells from Echinococcus larvae, thus preventing the growth and further development of the parasite. In in vitro cultivation systems for parasite primary cells, BI 2536 effectively inhibited the formation of new metacestode vesicles from germinative cells. Thus, BI 2536 has profound anti-parasitic activities in vitro at concentrations well within the range of plasma levels measured after the administration of safe dosages to patients (50 nM after 24 h). This implies that EmPlk1 is a promising new drug target for the development of novel anti-AE drugs that would specifically affect the parasite's stem cell population, namely the only parasite cells capable of proliferation. In addition to the chemotherapeutic aspects of this work, the inhibitor BI 2536 could be further used to study the function of stem cells in this model organism, utilising a method of injection of parasite stem cells into metacestode vesicles, for instance, as has been developed in this work. In the second part of this work, a novel receptor tyrosine kinase, the Venus flytrap kinase receptor (EmVKR) of E. multilocularis has been characterised. Members of this class of single-pass transmembrane receptors have recently been discovered in the related trematode S. mansoni and are associated with the growth and differentiation of sporocyst germinal cells and ovocytes. The ortholog receptor in EmVKR is characterised by an unusual domain composition of an extracellular Venus flytrap module (VFT), which shows significant similarity to GABA receptors, such as the GABAB receptor (γ-amino butyric acid type B) and is linked through a single transmembrane domain to an intracellular tyrosine kinase domain with similarities to the kinase domains of human insulin receptors. Based upon the size (5112bp) of emvkr and nucleotide sequence specificities, efforts have been made to isolate the gene from cell culture samples to study the ligand for the activation of this receptor type in Xenopus oocytes. To date, this type of receptor has only been described in invertebrates, thus making it an attractive target for drug screening. In a first trial, the ATP competitive inhibitor AG 1024 was tested in our in vitro cell culture. In conclusion, the EmVKR represents a novel receptor tyrosine kinase in E. multilocularis. Further efforts have to be made to identify the activating ligand of the receptor and its cellular function, which might strengthen the case for EmVKR as a potential drug target. The successful depletion of stem cells in the metacestode vesicle by the Plk1 inhibitor BI 2536 gives rise to optimising the chemical component for EmPlk1 as a new potential drug target. Furthermore, this inhibitor opens a new cell culture technique with high potential to study the cellular behaviour and influencing factors of stem cells in vitro.},
  subject      = {Chemotherapie},
  language  = {en}
}
@article{HofmannWeibelSzalay2014,
  author    = {Hofmann, Elisabeth and Weibel, Stephanie and Szalay, Aladar A.},
  title     = {Combination treatment with oncolytic Vaccinia virus and cyclophosphamide results in synergistic antitumor effects in human lung adenocarcinoma bearing mice},
  doi       = {10.1186/1479-5876-12-197},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-110168},
  year      = {2014},
  abstract  = {Background The capacity of the recombinant Vaccinia virus GLV-1h68 as a single agent to efficiently treat different human or canine cancers has been shown in several preclinical studies. Currently, its human safety and efficacy are investigated in phase I/II clinical trials. In this study we set out to evaluate the oncolytic activity of GLV-1h68 in the human lung adenocarcinoma cell line PC14PE6-RFP in cell cultures and analyzed the antitumor potency of a combined treatment strategy consisting of GLV-1h68 and cyclophosphamide (CPA) in a mouse model of PC14PE6-RFP lung adenocarcinoma. Methods PC14PE6-RFP cells were treated in cell culture with GLV-1h68. Viral replication and cell survival were determined by plaque assays and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, respectively. Subcutaneously implanted PC14PE6-RFP xenografts were treated by systemic injection of GLV-1h68, CPA or a combination of both. Tumor growth and viral biodistribution were monitored and immune-related antigen profiling of tumor lysates was performed. Results GLV-1h68 efficiently infected, replicated in and lysed human PC14PE6-RFP cells in cell cultures. PC14PE6-RFP tumors were efficiently colonized by GLV-1h68 leading to much delayed tumor growth in PC14PE6-RFP tumor-bearing nude mice. Combination treatment with GLV-1h68 and CPA significantly improved the antitumor efficacy of GLV-1h68 and led to an increased viral distribution within the tumors. Pro-inflammatory cytokines and chemokines were distinctly elevated in tumors of GLV-1h68-treated mice. Factors expressed by endothelial cells or present in the blood were decreased after combination treatment. A complete loss in the hemorrhagic phenotype of the PC14PE6-RFP tumors and a decrease in the number of blood vessels after combination treatment could be observed. Conclusions CPA and GLV-1h68 have synergistic antitumor effects on PC14PE6-RFP xenografts. We strongly suppose that in the PC14PE6-RFP model the enhanced tumor growth inhibition achieved by combining GLV-1h68 with CPA is due to an effect on the vasculature rather than an immunosuppressive action of CPA. These results provide evidence to support further preclinical studies of combining GLV-1h68 and CPA in other highly angiogenic tumor models. Moreover, data presented here demonstrate that CPA can be combined successfully with GLV-1h68 based oncolytic virus therapy and therefore might be promising as combination therapy in human clinical trials.},
  language  = {en}
}
@article{MayerLoefflerLozaValdesetal.2019,
  author    = {Mayer, Alexander E. and L{\"o}ffler, Mona C. and Loza Vald{\´e}s, Angel E. and Schmitz, Werner and El-Merahbi, Rabih and Trujillo-Viera, Jonathan and Erk, Manuela and Zhang, Thianzhou and Braun, Ursula and Heikenwalder, Mathias and Leitges, Michael and Schulze, Almut and Sumara, Grzegorz},
  title     = {The kinase PKD3 provides negative feedback on cholesterol and triglyceride synthesis by suppressing insulin signaling},
  series = {Science Signaling},
  journal   = {Science Signaling},
  edition   = {accepted manuscript},
  doi       = {10.1126/scisignal.aav9150},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-250025},
  year      = {2019},
  abstract  = {Hepatic activation of protein kinase C (PKC) isoforms by diacylglycerol (DAG) promotes insulin resistance and contributes to the development of type 2 diabetes (T2D). The closely related protein kinase D (PKD) isoforms act as effectors for DAG and PKC. Here, we showed that PKD3 was the predominant PKD isoform expressed in hepatocytes and was activated by lipid overload. PKD3 suppressed the activity of downstream insulin effectors including the kinase AKT and mechanistic target of rapamycin complex 1 and 2 (mTORC1 and mTORC2). Hepatic deletion of PKD3 in mice improved insulin-induced glucose tolerance. However, increased insulin signaling in the absence of PKD3 promoted lipogenesis mediated by SREBP (sterol regulatory element-binding protein) and consequently increased triglyceride and cholesterol content in the livers of PKD3-deficient mice fed a high-fat diet. Conversely, hepatic-specific overexpression of a constitutively active PKD3 mutant suppressed insulin-induced signaling and caused insulin resistance. Our results indicate that PKD3 provides feedback on hepatic lipid production and suppresses insulin signaling. Therefore, manipulation of PKD3 activity could be used to decrease hepatic lipid content or improve hepatic insulin sensitivity.},
  language  = {en}
}
@phdthesis{Schaefer2014,
  author    = {Sch{\"a}fer, Christin Marliese},
  title     = {Approaching antimicrobial resistance - Structural and functional characterization of the fungal transcription factor Mrr1 from Candida albicans and the bacterial ß-ketoacyl-CoA thiolase FadA5 from Mycobacterium tuberculosis},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-108400},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {The number of fungal infections is rising in Germany and worldwide. These infections are mainly caused by the opportunistic fungal pathogen C. albicans, which especially harms immunocompromised people. With increasing numbers of fungal infections, more frequent and longer lasting treatments are necessary and lead to an increase of drug resistances, for example against the clinically applied therapeutic fluconazole. Drug resistance in C. albicans can be mediated by the Multidrug resistance pump 1 (Mdr1), a membrane transporter belonging to the major facilitator family. However, Mdr1-mediated fluconazole drug resistance is caused by the pump's regulator, the transcription factor Mrr1 (Multidrug resistance regulator 1). It was shown that Mrr1 is hyperactive without stimulation or further activation in resistant strains which is due to so called gain of function mutations in the MRR1 gene. To understand the mechanism that lays behind this constitutive activity of Mrr1, the transcription factor should be structurally and functionally (in vitro) characterized which could provide a basis for successful drug development to target Mdr1-mediated drug resistance caused by Mrr1. Therefore, the entire 1108 amino acid protein was successfully expressed in Escherichia coli. However, further purification was compromised as the protein tended to form aggregates, unsuitable for crystallization trials or further characterization experiments. Expression trials in the eukaryote Pichia pastoris neither yielded full length nor truncated Mrr1 protein. In order to overcome the aggregation problem, a shortened variant, missing the N-terminal 249 amino acids named Mrr1 '250', was successfully expressed in E. coli and could be purified without aggregation. Similar to the wild type Mrr1 '250', selected gain of function variants were successfully cloned, expressed and purified with varying yields and with varying purity. The Mrr1 `250' construct contains most of the described regulatory domains of Mrr1. It was used for crystallization and an initial comparative analysis between the wild type protein and the variants. The proposed dimeric form of the transcription factor, necessary for DNA binding, could be verified for both, the wild type and the mutant proteins. Secondary structure analysis by circular dichroism measurements revealed no significant differences in the overall fold of the wild type and variant proteins. In vitro, the gain of function variants seem to be less stable compared to the wild type protein, as they were more prone to degradation. Whether this observation holds true for the full length protein's stability in vitro and in vivo remains to be determined. The crystallization experiments, performed with the Mrr1 '250' constructs, led to few small needle shaped or cubic crystals, which did not diffract very well and were hardly reproducible. Therefore no structural information of the transcription factor could be gained so far. Infections with M. tuberculosis, the causative agent of tuberculosis, are the leading cause of mortality among bacterial diseases. Especially long treatment times, an increasing number of resistant strains and the prevalence of for decades persisting bacteria create the necessity for new drugs against this disease. The cholesterol import and metabolism pathways were discovered as promising new targets and interestingly they seem to play an important role for the chronic stage of the tuberculosis infection and for persisting bacteria. In this thesis, the 3-ketoacyl-CoA thiolase FadA5 from M. tuberculosis was characterized and the potential for specifically targeting this enzyme was investigated. FadA5 catalyzes the last step of the β-oxidation reaction in the side-chain degradation pathway of cholesterol. We solved the three dimensional structure of this enzyme by X-ray crystallography and obtained two different apo structures and three structures in complex with acetyl-CoA, CoA and a hydrolyzed steroid-CoA, which is the natural product of FadA5. Analysis of the FadA5 apo structures revealed a typical thiolase fold as it is common for biosynthetic and degradative enzymes of this class for one of the structures. The second apo structure showed deviations from the typical thiolase fold. All obtained structures show the enzyme as a dimer, which is consistent with the observed dimer formation in solution. Thus the dimer is likely to be the catalytically active form of the enzyme. Besides the characteristic structural fold, the catalytic triad, comprising two cysteines and one histidine, as well as the typical coenzyme A binding site of enzymes belonging to the thiolase class could be identified. The two obtained apo structures differed significantly from each other. One apo structure is in agreement with the characteristic thiolase fold and the well-known dimer interface could be identified in our structure. The same characteristics were observed in all complex structures. In contrast, the second apo structure followed the thiolase fold only partially. One subdomain, spanning 30 amino acids, was in a different orientation. This reorientation was caused by the formation of two disulfide bonds, including the active site cysteines, which rendered the enzyme inactive. The disulfide bonds together with the resulting domain swap still permitted dimer formation, yet with a significantly shifted dimer interface. The comparison of the apo structures together with the preliminary activity analysis performed by our collaborator suggest, that FadA5 can be inactivated by oxidation and reactivated by reduction. If this redox switch is of biological importance requires further evaluation, however, this would be the first reported example of a bacterial thiolase employing redox regulation. Our obtained complex structures represent different stages of the thiolase reaction cycle. In some complex structures, FadA5 was found to be acetylated at the catalytic cysteine and it was in complex with acetyl-CoA or CoA. These structures, together with the FadA5 structure in complex with a hydrolyzed steroid-CoA, revealed important insights into enzyme dynamics upon ligand binding and release. The steroid-bound structure is as yet a unique example of a thiolase enzyme interacting with a complex ligand. The characterized enzyme was used as platform for modeling studies and for comparison with human thiolases. These studies permitted initial conclusions regarding the specific targetability of FadA5 as a drug target against M. tuberculosis infection, taking the closely related human enzymes into account. Additional analyses led to the proposal of a specific lead compound based on the steroid and ligand interactions within the active site of FadA5.},
  subject      = {Multidrug-Resistenz},
  language  = {en}
}
@phdthesis{Weber2014,
  author    = {Weber, David},
  title     = {Hey target gene regulation in embryonic stem cells and cardiomyocytes},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-101663},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {The Notch signaling pathway is crucial for mammalian heart development. It controls cell-fate decisions, coordinates patterning processes and regulates proliferation and differentiation. Critical Notch effectors are Hey bHLH transcription factors (TF) that are expressed in atrial (Hey1) and ventricular (Hey2) cardiomyocytes (CM) and in the developing endocardium (Hey1/2/L). The importance of Hey proteins for cardiac development is demonstrated by knockout (KO) mice, which suffer from lethal cardiac defects, such as ventricular septum defects (VSD), valve defects and cardiomyopathy. Despite this clear functional relevance, little is known about Hey downstream targets in the heart and the molecular mechanism by which they are regulated. Here, I use a cell culture system with inducible Hey1, Hey2 or HeyL expression to study Hey target gene regulation in HEK293 cells, in murine embryonic stem cells (ESC) and in ESC derived CM. In HEK293 cells, I could show that genome wide binding sites largely overlap between all three Hey proteins, but HeyL has many additional binding sites that are not bound by Hey1 or Hey2. Shared binding sites are located close to transcription start sites (TSS) where Hey proteins preferentially bind to canonical E boxes, although more loosely defined modes of binding exist. Additional sites only bound by HeyL are more scattered across the genome. The ability of HeyL to bind these sites depends on the C-terminal part of the protein. Although there are genes which are differently regulated by HeyL, it is unclear whether this regulation results from binding of additional sites by HeyL. Additionally, Hey target gene regulation was studied in ESC and differentiated CM, which are more relevant for the observed cardiac phenotypes. ESC derived CM contract in culture and are positive for typical cardiac markers by qRT PCR and staining. According to these markers differentiation is unaffected by prolonged Hey1 or Hey2 overexpression. Regulated genes are largely redundant between Hey1 and Hey2. These are mainly other TF involved in e.g. developmental processes, apoptosis, cell migration and cell cycle. Many target genes are cell type specifically regulated causing a shift in Hey repression of genes involved in cell migration in ESC to repression of genes involved in cell cycle in CM. The number of Hey binding sites is reduced in CM and HEK293 cells compared to ESC, most likely due to more regions of dense chromatin in differentiated cells. Binding sites are enriched at the proximal promoters of down-regulated genes, compared to up-or non-regulated genes. This indicates that up-regulation primarily results from indirect effects, while down-regulation is the direct results of Hey binding to target promoters. The extent of repression generally correlates with the amount of Hey binding and subsequent recruitment of histone deacetylases (Hdac) to target promoters resulting in histone H3 deacetylation. However, in CM the repressive effect of Hey binding on a subset of genes can be annulled, likely due to binding of cardiac specific activators like Srf, Nkx2-5 and Gata4. These factors seem not to interfere with Hey binding in CM, but they recruit histone acetylases such as p300 that may counteract Hey mediated histone H3 deacetylation. Such a scenario explains differential regulation of Hey target genes between ESC and CM resulting in gene and cell-type specific regulation.},
  subject      = {Transkriptionsfaktor},
  language  = {en}
}
@phdthesis{Nube2013,
  author    = {Nube, Jacqueline Sui Lin},
  title     = {Comparative Analysis of Vaccinia Virus-Encoded Markers Reflecting Actual Viral Titres in Oncolytic Virotherapy},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85689},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2013},
  abstract  = {Using viruses to treat cancer is a novel approach to an age-old disease. Oncolytic viruses are native or recombinant viruses that have the innate or enhanced capability to infect tumour cells, replicate within the tumour microenvironment and subsequently lyse those cells. One representative, the vaccinia virus (VACV), belongs to the orthopoxvirus genus of the Poxviridae family. GLV-1h68, a recombinant and attenuated vaccinia virus devel- oped by the Genelux Corporation, is a member of this family currently being tested in various phase I/II clinical trials under the name GL-ONC1. It has been shown to specif- ically replicate in tumour cells while sparing healthy tissue and to metabolise prodrug at or transport immunological payloads to the site of affliction. Since imaging modalities offer little insight into viral replication deep within the body, and because oncolytic virotherapy is dependent on replication within the target tissue, the need for a monitoring system is evident. Pharmacokinetic analysis of this oncolytic agent was to give insight into the dynamics present in tumours during treatment. This, in turn, would give clinicians the opportunity to monitor the efficacy as early as possible after the onset of treatment, to observe treatment progression and possibly to gauge prognosis, without resorting to invasive procedures, e.g. biopsies. A criteria for viable biomarkers was that it had to be directly dependent on viral replica- tion. Ideally, a marker for treatment efficacy would be specific to the treatment modality, not necessarily the treatment type. Such a marker would be highly detectable (high sen- sitivity), specific for the treatment (high specificity), and present in an easily obtained specimen (blood). Taking this into consideration, the biomarkers were chosen for their potential to be indicators of viral replication. Thus, the biomarkers analysed in this thesis are: the native proteins expressed by the viral genes A27L and B5R, the virally encoded recombinant proteins β-galactosidase, β-glucuronidase, green fluorescent protein (GFP), carboxypeptidase G2 (CPG2) and carcinoembryonic antigen (CEA). Each marker is under the control of one of five different promoters present. All recombinant viruses used in this thesis express A27L, B5R, GFP and β-glucuronidase and all are derived from the parental virus GLV-1h68. In addition to these markers, GLV-1h68 expresses β-galactosidase; GLV-1h181 expresses CPG2. [...]},
  subject      = {Onkolyse},
  language  = {en}
}
@phdthesis{Flegler2022,
  author    = {Flegler, Vanessa Judith},
  title     = {Application of electron cryomicroscopy for structural and functional studies on the mechanosensitive channels of small conductance},
  doi       = {10.25972/OPUS-26897},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-268979},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Bacteria thrive and survive in many different environments, and as a result, they have developed robust mechanisms to adapt rapidly to alterations in their surroundings. The protection against osmotic forces is provided by mechanosensitive channels: their primary function is to maintain the integrity of the cell upon a hypoosmotic shock. The mechanosensitive channel of small conductance (MscS) is not only the smallest common structural unit of a diverse family that allows for a tailored response in osmoregulation; it is also the most intensively studied homologue. Mechanosensitive channels directly sense elevated membrane tension levels generated by increased pressure within the cell and open transiently. Escherichia coli has six paralogues that differ in their gating properties and the number of additional transmembrane (TM) helices. These TM helices, termed sensor paddles, are essential for sensing, as they directly contact the surrounding membrane; however, the role of the additional TM helices is still unclear. Furthermore, lipids occupy hydrophobic pockets far away from the membrane plane. A recent gating model for MscS states that increased membrane tension triggers the expulsion of lipids out of those pockets, modulating different conformational states of MscS. This model focuses on bound lipids, but it is still unclear to what extent the direct interaction with the membrane influences sensing and how relevant it is for the larger paralogues. In the herein described work, structural studies on two larger paralogues, the medium-sized channel YnaI and the large channel YbiO were realised using electron cryomicroscopy (cryo-EM). Lipids were identified in YnaI in the pockets in a similar position and orientation as in MscS, suggesting a conserved sensing mechanism. Moreover, the copolymer diisobutylene/maleic acid (DIBMA) allowed the extraction of artificially activated YnaI from plasma membranes, leading to an open-like form of this channel. This novel conformation indicated that the pore helices bend at a GGxGG motif during gating, which is unique among the Escherichia coli paralogues, concomitant with a structural reorganisation of the sensor paddles. Thus, despite a high similarity of their closed states, the gating mechanisms of MscS and YnaI are surprisingly different. Furthermore, the comparison of MscS, YnaI, and YbiO accentuates variations and similarities between the differently sized family members, implying fine-tuning of channel properties in the pore regions and the cytosolic lateral entry sides into the channel. Structural analyses of MscS reconstituted into different systems showed the advantages and disadvantages of certain polymers and detergents. The novel DIBMA copolymer and the more conventional amphiphilic polymers, so-called Amphipols, perturb contacting transmembrane helices or lead to their denaturation. Due to this observation, the obtained structures of YnaI must also be cautiously considered. The structures obtained in detergents resulted in unaffected channels; however, the applicability of detergents for MscS-like channels is limited by the increased required sample concentration. The role of lipids for gating MscS in the absence of a membrane was examined by deliberately removing coordinated lipid molecules from MscS using different amounts and kinds of detergent. The effects on the channel were inspected by cryo-EM. These experiments showed that closed MscS adopts the open conformation when it is enough delipidated by incubation with the detergent n-dodecyl-β-D-maltoside, and adding lipids to the open channel reverses this process. The results agree with the state-of-the-art model that the amount of lipid molecules in the pockets and grooves is responsible for the conformational state of MscS. Furthermore, incubation with the detergent lauryl maltose neopentyl glycol, which has stabilising and delipidating characteristics, resulted in a high-resolution structure of open MscS exhibiting an intricate network of ligands. Based on this structure, an updated gating model is proposed, which states that upon opening, lipids from the pockets migrate into the cytosolic membrane leaflet, while lipids from the periplasmic leaflet enter the grooves that arise between the sensor paddles.},
  language  = {en}
}
@phdthesis{Orth2021,
  author    = {Orth, Barbara},
  title     = {Identification of an atypical peptide binding mode of the BTB domain of the transcription factor MIZ1 with a HUWE1-derived peptide},
  doi       = {10.25972/OPUS-25044},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-250447},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Ubiquitination is a posttranslational modification with immense impact on a wide range of cellular processes, including proteasomal degradation, membrane dynamics, transcription, translation, cell cycle, apoptosis, DNA repair and immunity. These diverse functions stem from the various ubiquitin chain types, topologies, and attachment sites on substrate proteins. Substrate recruitment and modification on lysine, serine or threonine residues is catalyzed by ubiquitin ligases (E3s). An important E3 that decides about the fate of numerous substrates is the HECT-type ubiquitin ligase HUWE1. Depending on the substrate, HUWE1 is involved in different processes, such as cell proliferation and differentiation, DNA repair, and transcription. One of the transcription factors that is ubiquitinated by HUWE1 is the MYC interacting zinc finger protein 1 (MIZ1). MIZ1 is a BTB/POZ (Bric-{\`a}-brac, Tramtrack and Broad-Complex/Pox virus and zinc finger) zinc finger (ZF) protein that binds to DNA through its 13 C2H2-type zinc fingers and either activates or represses the transcription of target genes, including genes involved in cell cycle arrest, such as P21CIP1 (CDKN1A). The precise functions of MIZ1 depend on its interactions with the MYC-MAX heterodimer, but also its heterodimerization with other BTB-ZF proteins, such as BCL6 or NAC1. How MIZ1 interacts with HUWE1 has not been studied and, as a consequence, it has not been possible to rationally develop tools to manipulate this interaction with specificity in order to better understand the effects of the interaction on the transcriptional function of MIZ1 on target genes or processes downstream. One aspect of my research, therefore, aimed at characterizing the MIZ1-HUWE1 interaction at a structural level. I determined a crystal structure of the MIZ1-BTB-domain in complex with a peptide, referred to as ASC, derived from a C terminal region of HUWE1, previously named 'activation segment'. The binding mode observed in this crystal structure could be validated by binding and activity assays in vitro and by cell-based co-IP experiments in the context of N-terminally truncated HUWE1 constructs. I was not able to provide unambiguous evidence for the identified binding mode in the context of full-length HUWE1, indicating that MIZ1 recognition by HUWE1 requires yet unknown regions in the cell. While the structural details of the MIZ1-HUWE1 interaction remains to be elucidated in the context of the full-length proteins, the binding mode between MIZ1BTB and ASC revealed an interesting, atypical structural feature of the BTB domain of MIZ1 that, to my knowledge, has not been described for other BTB-ZF proteins: The B3 region in MIZ1BTB is conformationally malleable, which allows for a HUWE1-ASC-peptide-mediated β-sheet extension of the upper B1/B2-strands, resulting in a mixed, 3 stranded β-sheet. Such β-sheet extension does not appear to occur in other homo- or heterodimeric BTB-ZF proteins, including MIZ1-heterodimers, since these proteins typically possess a pre-formed B3-strand in at least one subunit. Instead, BCL6 co repressor-derived peptides (SMRT and BCOR) were found to extend the lower β-sheet in BCL6BTB by binding to an adjacent 'lateral groove'. This interaction follows a 1:1 stoichiometry, whereas the MIZ1BTB-ASC-complex shows a 2:1 stoichiometry. The crystal structure of the MIZ1BTB-ASC-complex I determined, along with comparative binding studies of ASC with monomeric, homodimeric, and heterodimeric MIZ1BTB variants, respectively, suggests that ASC selects for MIZ1BTB homodimers. The structural data I generated may serve as an entry point for the prediction of additional interaction partners of MIZ1 that also have the ability to extend the upper β-sheet of MIZ1BTB. If successful, such interaction partners and structures thereof might aid the design of peptidomimetics or small-molecule inhibitors of MIZ1 signaling. Proof-of-principle for such a structure-guided approach targeting BTB domains has been provided by small-molecule inhibitors of BCL6BTB co-repressors interactions. If a similar approach led to molecules that interfere with specific interactions of MIZ1, they would provide intriguing probes to study MIZ1 biology and may eventually allow for the development of MIZ1-directed cancer therapeutics.},
  subject      = {Ubiquitin},
  language  = {en}
}
@phdthesis{Nordblom2023,
  author    = {Nordblom, Noah Frieder},
  title     = {Synthese und Evaluation von Gephyrinsonden f{\"u}r hochaufl{\"o}sende Mikroskopieverfahren},
  doi       = {10.25972/OPUS-30230},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-302300},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {This decade saw the development of new high-end light microscopy approaches. These technologies are increasingly used to expand our understanding of cellular function and the molecular mechanisms of life and disease. The precision of state-of-the-art super resolution microscopy is limited by the properties of the applied fluorescent label. Here I describe the synthesis and evaluation of new functional fluorescent probes that specifically stain gephyrin, universal marker of the neuronal inhibitory post-synapse. Selected probe precursor peptides were synthesised using solid phase peptide synthesis and conjugated with selected super resolution capable fluorescent dyes. Identity and purity were defined using chromatography and mass spectrometric methods. To probe the target specificity of the resulting probe variants in cellular context, a high-throughput assay was established. The established semi-automated and parallel workflow was used for the evaluation of three selected probes by defining their co-localization with the expressed fluorescent target protein. My work provided NN1Dc and established the probe as a visualisation tool for essentially background-free visualisation of the synaptic marker protein gephyrin in a cellular context. Furthermore, NN1DA became part of a toolbox for studying the inhibitory synapse ultrastructure and brain connectivity and turned out useful for the development of a label-free, high-throughput protein interaction quantification assay.},
  subject      = {Fluoreszenzmikroskopie},
  language  = {en}
}
@article{TsonevaMinevFrentzenetal.2017,
  author    = {Tsoneva, Desislava and Minev, Boris and Frentzen, Alexa and Zhang, Qian and Wege, Anja K. and Szalay, Aladar A.},
  title     = {Humanized Mice with Subcutaneous Human Solid Tumors for Immune Response Analysis of Vaccinia Virus-Mediated Oncolysis},
  series = {Molecular Therapy Oncolytics},
  volume    = {5},
  journal   = {Molecular Therapy Oncolytics},
  doi       = {10.1016/j.omto.2017.03.001},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170786},
  pages     = {41-61},
  year      = {2017},
  abstract  = {Oncolytic vaccinia virus (VACV) therapy is an alternative cancer treatment modality that mediates targeted tumor destruction through a tumor-selective replication and an induction of anti-tumor immunity. We developed a humanized tumor mouse model with subcutaneous human tumors to analyze the interactions of VACV with the developing tumors and human immune system. A successful systemic reconstitution with human immune cells including functional T cells as well as development of tumors infiltrated with human T and natural killer (NK) cells was observed. We also demonstrated successful in vivo colonization of such tumors with systemically administered VACVs. Further, a new recombinant GLV-1h376 VACV encoding for a secreted human CTLA4-blocking single-chain antibody (CTLA4 scAb) was tested. Surprisingly, although proving CTLA4 scAb's in vitro binding ability and functionality in cell culture, beside the significant increase of CD56\(^{bright}\) NK cell subset, GLV-1h376 was not able to increase cytotoxic T or overall NK cell levels at the tumor site. Importantly, the virus-encoded β-glucuronidase as a measure of viral titer and CTLA4 scAb amount was demonstrated. Therefore, studies in our "patient-like" humanized tumor mouse model allow the exploration of newly designed therapy strategies considering the complex relationships between the developing tumor, the oncolytic virus, and the human immune system.},
  language  = {en}
}
@phdthesis{Amelingmeier2022,
  author    = {Amelingmeier, Florian},
  title     = {Identifizierung und Untersuchung TOP-mRNA - bindender Faktoren},
  doi       = {10.25972/OPUS-28923},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-289231},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Im Zellkern eukaryotischer Zellen werden Gene in mRNAs transkribiert, welche umfangreich prozessiert und aus dem Zellkern exportiert werden. Im Zytoplasma erfolgt die Translation der mRNAs in Proteine, ein Prozess, welcher viel Energie ben{\"o}tigt und daher mittels vielf{\"a}ltiger Mechanismen streng reguliert wird. Ein Beispiel hierf{\"u}r stellt die Klasse der TOP-mRNAs dar, eine RNA-Spezies, welche haupts{\"a}chlich Transkripte von Genen umfasst, die selbst in die Translation involviert sind. Die prominentesten Vertreter dieser Klasse sind die Proteine der kleinen und großen ribosomalen Untereinheiten. TOP-mRNAs zeichnen sich durch ein gemeinsames Sequenz-Motiv am Anfang Ihrer 5'-UTR aus, welches aus einem Pyrimidinstrang besteht und unmittelbar nach dem Cap mit einem Cytosin beginnt. Dieses allen TOP-RNAs gemeinsame Motiv erm{\"o}glicht die zeitgleiche Translationskontrolle dieser RNA-Klasse. So kann die Translation der TOP-mRNAs unter Stressbedingungen wie z.B. N{\"a}hrstoffmangel koordiniert inhibiert werden, wodurch Energie eingespart wird. Bereits lange wird nach einem Regulator gesucht, der an dieses TOP-Motiv bindet und die koordinierte Regulation erm{\"o}glicht. Man kann sich hier einen Inhibitor oder auch einen Aktivator vorstellen. Verschiedene Proteine wurden bereits in Erw{\"a}gung gezogen. In dieser Arbeit wurde das Protein TIAR mittels Massenspektrometrie als TOP-interagierender Faktor identifiziert und dessen Bindungseigenschaften mit dem TOP-Motiv durch Shift Assays untersucht. Hierbei konnten Minimalkonstrukte verschiedener Organismen sowie RNA-TOP - Sequenzen identifiziert werden, welche sich f{\"u}r Strukturanalysen eignen w{\"u}rden. Als weiterer TOP-interagierender Faktor wurde {\"u}ber verschiedene sequenzielle Reinigungsschritte das Protein 14-3-3ε identifiziert. Weiterhin wurden die TOP-Motiv-bindenden Proteine LARP1 und LARP7 auf Ihre Bindungseigenschaften mit Ihren Zielsequenzen untersucht. W{\"a}hrend gezeigt werden konnte, dass LARP1 einen inhibierenden Einfluss auf TOP-RNAs hat, wurde in weiteren Shift-Assays die Bindungseigenschaften von LARP7 mit 7SK untersucht, wobei ebenfalls ein minimales LARP7-Konstrukt sowie 7SK-Konstrukte f{\"u}r Strukturanalysen identifiziert werden konnten. Weiterhin konnte gezeigt werden, dass verschiedene Substanzen wie tRNA und Arginin einen starken Einfluss auf die LARP7-7SK - Interaktion aus{\"u}ben, welcher in weiteren Studien ber{\"u}cksichtigt werden sollte.},
  subject      = {Proteinbiosynthese},
  language  = {de}
}
@article{HaddadSocciChenetal.2016,
  author    = {Haddad, Dana and Socci, Nicholas and Chen, Chun-Hao and Chen, Nanhai G and Zhang, Qian and Carpenter, Susanne G and Mittra, Arjun and Szalay, Aladar A and Fong, Yuman},
  title     = {Molecular network, pathway, and functional analysis of-time dependent gene changes associated with pancreatic cancer susceptibility to oncolytic vaccinia virotherapy},
  series = {Molecular Therapy — Oncolytics},
  volume    = {3},
  journal   = {Molecular Therapy — Oncolytics},
  doi       = {10.1038/mto.2016.8},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-165855},
  pages     = {16008},
  year      = {2016},
  abstract  = {Background: Pancreatic cancer is a fatal disease associated with resistance to conventional therapies. This study aimed to determine changes in gene expression patterns associated with infection and susceptibility of pancreatic cancer cells to an oncolyticvaccinia virus, GLV-1h153, carrying the human sodium iodide symporter for deep tissue imaging of virotherapy. Methods: Replication and susceptibility of pancreatic adenocarcinoma PANC-1 cells to GLV-1h153 was confirmed with replication and cytotoxicity assays. PANC-1 cells were then infected with GLV-1h153 and near-synchronous infection confirmed via flow cytometry of viral-induced green fluorescent protein (GFP) expression. Six and 24 hours after infection, three samples of each time point were harvested, and gene expression patterns assessed using HG-U133A cDNA microarray chips as compared to uninfected control. Differentially expressed genes were identified using Bioconductor LIMMA statistical analysis package. A fold change of 2.0 or above was used as a cutoff, with a P value of 0.01. The gene list was then analyzed using Ingenuity Pathways Analysis software. Results: Differential gene analysis revealed a total of 12,412 up- and 11,065 downregulated genes at 6 and 24 hours postinfection with GLV-1h153 as compared to control. At 6 hours postinfection. A total of 139 genes were either up or downregulated >twofold (false discovery rate < 0.05), of which 124 were mapped by Ingenuity Pathway Analysis (IPA). By 24 hours postinfection, a total of 5,698 genes were identified and 5,563 mapped by IPA. Microarray revealed gene expression changes, with gene networks demonstrating downregulation of processes such as cell death, cell cycle, and DNA repair, and upregulation of infection mechanisms (P < 0.01). Six hours after infection, gene changes involved pathways such as HMGB-1, interleukin (IL)-2, IL-6, IL-8, janus kinase/signal tranducer and activator of transcription (JAK/STAT), interferon, and ERK 5 signaling (P < 0.01). By 24 hours, prominent pathways included P53- and Myc-induced apoptotic processes, pancreatic adenocarcinoma signaling, and phosphoinositide 3-kinase/v-akt murine thymoma vial oncogene homolog 1 (PI3/AKT) pathways. Conclusions: Our study reveals the ability to assess time-dependent changes in gene expression patterns in pancreatic cancer cells associated with infection and susceptibility to vaccinia viruses. This suggests that molecular assays may be useful to develop safer and more efficacious oncolyticvirotherapies and support the idea that these treatments may target pathways implicated in pancreatic cancer resistance to conventional therapies.},
  language  = {en}
}
@phdthesis{Schmid2020,
  author    = {Schmid, Benedikt},
  title     = {Molecular Signaling Mechanisms at the µ-Opioid Receptor},
  doi       = {10.25972/OPUS-17685},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-176850},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {To this day, opioids represent the most effective class of drugs for the treatment of severe pain. On a molecular level, all opioids in use today are agonists at the μ-opioid receptor (μ receptor). The μ receptor is a class A G protein-coupled receptor (GPCR). GPCRs are among the biological structures most frequently targeted by pharmaceuticals. They are membrane bound receptors, which confer their signals into the cell primarily by activating a variety of GTPases called G proteins. In the course of the signaling process, the μ receptor will be phosphorylated by GRKs, increasing its affinity for another entity of signaling proteins called β-arrestins (β-arrs). The binding of a β-arr to the activated μ receptor will end the G protein signal and cause the receptor to be internalized into the cell. Past research showed that the μ receptor's G protein signal puts into effect the desired pain relieving properties of opioid drugs, whereas β-arr recruitment is more often linked to adverse effects like obstipation, tolerance, and respiratory depression. Recent work in academic and industrial research picked up on these findings and looked into the possibility of enhancing G protein signaling while suppressing β-arr recruitment. The conceptual groundwork of such approaches is the phenomenon of biased agonism. It appreciates the fact that different ligands can change the relative contribution of any given pathway to the overall downstream signaling, thus enabling not only receptor-specific but even pathway-specific signaling. This work examined the ability of a variety of common opioid drugs to specifically activate the different signaling pathways and quantify it by means of resonance energy transfer and protein complementation experiments in living cells. Phosphorylation of the activated receptor is a central step in the canonical GPCR signaling process. Therefore, in a second step, expression levels of the phosphorylating GRKs were enhanced in search for possible effects on receptor signaling and ligand bias. In short, detailed pharmacological profiles of 17 opioid ligands were recorded. Comparison with known clinical properties of the compounds showed robust correlation of G protein activation efficacy and analgesic potency. Ligand bias (i.e. significant preference of any path- way over another by a given agonist) was found for a number of opioids in native HEK293 cells overexpressing μ receptor and β-arrs. Furthermore, overexpression of GRK2 was shown to fundamentally change β-arr pharmacodynamics of nearly all opioids. As a consequence, any ligand bias as detected earlier was abolished with GRK2 overexpression, with the exception of buprenorhin. In summary, the following key findings stand out: (1) Common opioid drugs exert biased agonism at the μ receptor to a small extent. (2) Ligand bias is influenced by expression levels of GRK2, which may vary between individuals, target tissues or even over time. (3) One of the opioids, buprenorhin, did not change its signaling properties with the overexpression of GRK2. This might serve as a starting point for the development of new opioids which could lack the ability of β-arr recruitment altogether and thus might help reduce adverse side effects in the treatment of severe pain.},
  subject      = {Opiatrezeptor},
  language  = {en}
}
@article{FischerHelfrichFoersterPeschel2016,
  author    = {Fischer, Robin and Helfrich-F{\"o}rster, Charlotte and Peschel, Nicolai},
  title     = {GSK-3 Beta Does Not Stabilize Cryptochrome in the Circadian Clock of Drosophila},
  series = {PLoS ONE},
  volume    = {11},
  journal   = {PLoS ONE},
  number    = {1},
  doi       = {10.1371/journal.pone.0146571},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-180370},
  year      = {2016},
  abstract  = {Cryptochrome (CRY) is the primary photoreceptor of Drosophila's circadian clock. It resets the circadian clock by promoting light-induced degradation of the clock protein Timeless (TIM) in the proteasome. Under constant light, the clock stops because TIM is absent, and the flies become arrhythmic. In addition to TIM degradation, light also induces CRY degradation. This depends on the interaction of CRY with several proteins such as the E3 ubiquitin ligases Jetlag (JET) and Ramshackle (BRWD3). However, CRY can seemingly also be stabilized by interaction with the kinase Shaggy (SGG), the GSK-3 beta fly orthologue. Consequently, flies with SGG overexpression in certain dorsal clock neurons are reported to remain rhythmic under constant light. We were interested in the interaction between CRY, Ramshackle and SGG and started to perform protein interaction studies in S2 cells. To our surprise, we were not able to replicate the results, that SGG overexpression does stabilize CRY, neither in S2 cells nor in the relevant clock neurons. SGG rather does the contrary. Furthermore, flies with SGG overexpression in the dorsal clock neurons became arrhythmic as did wild-type flies. Nevertheless, we could reproduce the published interaction of SGG with TIM, since flies with SGG overexpression in the lateral clock neurons shortened their free-running period. We conclude that SGG does not directly interact with CRY but rather with TIM. Furthermore we could demonstrate, that an unspecific antibody explains the observed stabilization effects on CRY.},
  language  = {en}
}
@article{SchmidtDenkWiegering2020,
  author    = {Schmidt, Stefanie and Denk, Sarah and Wiegering, Armin},
  title     = {Targeting protein synthesis in colorectal cancer},
  series = {Cancers},
  volume    = {12},
  journal   = {Cancers},
  number    = {5},
  issn      = {2072-6694},
  doi       = {10.3390/cancers12051298},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-206014},
  year      = {2020},
  abstract  = {Under physiological conditions, protein synthesis controls cell growth and survival and is strictly regulated. Deregulation of protein synthesis is a frequent event in cancer. The majority of mutations found in colorectal cancer (CRC), including alterations in the WNT pathway as well as activation of RAS/MAPK and PI3K/AKT and, subsequently, mTOR signaling, lead to deregulation of the translational machinery. Besides mutations in upstream signaling pathways, deregulation of global protein synthesis occurs through additional mechanisms including altered expression or activity of initiation and elongation factors (e.g., eIF4F, eIF2α/eIF2B, eEF2) as well as upregulation of components involved in ribosome biogenesis and factors that control the adaptation of translation in response to stress (e.g., GCN2). Therefore, influencing mechanisms that control mRNA translation may open a therapeutic window for CRC. Over the last decade, several potential therapeutic strategies targeting these alterations have been investigated and have shown promising results in cell lines, intestinal organoids, and mouse models. Despite these encouraging in vitro results, patients have not clinically benefited from those advances so far. In this review, we outline the mechanisms that lead to deregulated mRNA translation in CRC and highlight recent progress that has been made in developing therapeutic strategies that target these mechanisms for tumor therapy.},
  language  = {en}
}
@phdthesis{Steinmetzger2020,
  author    = {Steinmetzger, Christian},
  title     = {Fluorogenic Aptamers and Fluorescent Nucleoside Analogs as Probes for RNA Structure and Function},
  doi       = {10.25972/OPUS-20760},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-207604},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {RNA plays a key role in numerous cellular processes beyond the central dogma of molecular biology. Observing and understanding this wealth of functions, discovering new ones and engineering them into purpose-built tools requires a sensitive means of observation. Over the past decade, fluorogenic aptamers have emerged to fill this niche. These short oligonucleotides are generated by in vitro selection to specifically interact with small organic fluorophores and can be utilized as genetically encoded tags for RNAs of interest. The most versatile class of fluorogenic aptamers is based on derivatives of hydroxybenzylidene imidazolone (HBI), a conditional fluorophore mimicking the chromophore structure found in green and red fluorescent proteins. The respective aptamers are well-known by the "vegetable" nomenclature, including Spinach, Broccoli and Corn, and have found numerous applications for studying RNA function in vitro and in cells. Their success, however, is somewhat overshadowed by individual shortcomings such as a propensity for misfolding, dependence on unphysiologically high concentrations of magnesium ions or, in the case of Corn, dimerization that might affect the function of the tagged RNA. Moreover, most fluorogenic aptamers exhibit limited ligand promiscuity by design, thereby restricting their potential for spectral tuning to a narrow window of wavelengths. This thesis details the characterization of a new fluorogenic aptamer system nicknamed Chili. Chili is derived from an aptamer that was originally selected to bind 4-hydroxy-3,5-dimethoxy¬hydroxy-benzylidene imidazolone (DMHBI), resulting in a green fluorescent complex. Unlike other aptamers of its kind, Chili engages in a proton transfer cycle with the bound ligand, resulting in a remarkably large Stokes shift of more than 130 nm. By means of an empirical ligand optimization approach, several new DMHBI derivatives were found that bind to Chili with high affinity, furnishing complexes up to 7.5 times brighter compared to the parent ligand. In addition, Chili binds to π-extended DMHBI derivatives that confer fluorescence in the yellow-red region of the visible spectrum. The highest affinity and degree of fluorescence turn-on for both green and red fluorogenic ligands were achieved by the incorporation of a unique, positively charged substituent into the HBI scaffold. Supplemented by NMR spectroscopy, kinetic and thermodynamic studies showed that the binding site of Chili is loosely preorganized in the absence of ligand and likely forms a G-quadruplex upon ligand binding. To showcase future applications, Chili was incorporated into a FRET sensor for monitoring the cleavage of an RNA substrate by a 10-23 DNAzyme. Besides aptamers as macromolecular fluorescent complexes, fluorescent nucleobase analogs are powerful small isomorphic components of RNA suitable for studying structure and folding. Here, the highly emissive nucleobase analog 4-cyanoindole (4CI) was developed into a ribonucleoside (r4CI) for this purpose. A new phosphoramidite building block was synthesized to enable site-specific incorporation of 4CI into RNA. Thermal denaturation experiments confirmed that 4CI behaves as a universal nucleobase, i.e. without bias towards any particular hybridization partner. Photophysical characterization established r4CI as a generally useful fluorescent ribonucleoside analog. In this work, it was employed to gain further insight into the structure of the Chili aptamer. Using several 4CI-modified Chili-HBI complexes, a novel base-ligand FRET assay was established to obtain a set of combined distance and orientation restraints for the tertiary structure of the aptamer. In addition to their utility for interrogating structure and binding, supramolecular FRET pairs comprising a fluorescent nucleobase analog donor and an innately fluorogenic acceptor hold great promise for the construction of color-switchable RNA aptamer sensor devices.},
  subject      = {Aptamer},
  language  = {en}
}
@phdthesis{Kauk2018,
  author    = {Kauk, Michael},
  title     = {Investigating the Molecular Mechanism of Receptor Activation at Muscarinic Receptors by Means of Pathway-Specific Dualsteric Ligands and Partial Agonists},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-173729},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {G protein-coupled receptors (GPCRs) form the biggest receptor family that is encoded in the human genome and represent the most druggable target structure for modern therapeutics respectively future drug development. Belonging to aminergic class A GPCRs muscarinic Acetylcholine receptors (mAChRs) are already now of clinical relevance and are also seen as promising future drug targets for treating neurodegenerative diseases like Alzheimer or Parkinson. The mAChR family consist of five subtypes showing high sequence identity for the endogenous ligand binding region and thus it is challenging until now to selectively activate a single receptor subtype. A well accepted method to study ligand binding, dynamic receptor activation and downstream signaling is the fluorescence resonance energy transfer (FRET) application. Here, there relative distance between two fluorophores in close proximity (<10 nm) can be monitored in a dynamic manner. The perquisite for that is the spectral overlap of the emission spectrum of the first fluorophore with the excitation spectrum of the second fluorophore. By inserting two fluorophores into the molecular receptor structure receptor FRET sensors can serve as a powerful tool to study dynamic receptor pharmacology. Dualsteric Ligands consist of two different pharmacophoric entities and are regarded as a promising ligand design for future drug development. The orthosteric part interacts with high affinity with the endogenous ligand binding region whereas the allosteric part binds to a different receptor region mostly located in the extracellular vestibule. Both moieties are covalently linked. Dualsteric ligands exhibit a dynamic ligand binding. The dualsteric binding position is characterized by a simultaneous binding of the orthosteric and allosteric moiety to the receptor and thus by receptor activation. In the purely allosteric binding position no receptor activation can be monitored. In the present work the first receptor FRET sensor for the muscarinic subtype 1 (M1) was generated and characterized. The M1-I3N-CFP sensor showed an unaltered physiological behavior as well as ligand and concentration dependent responses. The sensor was used to characterize different sets of dualsteric ligands concerning their pharmacological properties like receptor activation. It was shown that the hybrids consisting of the synthetic full agonist iperoxo and the positive allosteric modulator of BQCA type is very promising. Furthermore, it was shown for orthosteric as well as dualsteric ligands that the degree of receptor activation is highly dependent on the length of and the chemical properties of the linker moiety. For dualsteric ligands a bell-shaped activation characteristic was reported for the first time, suggesting that there is an optimal linker length for dualsteric ligands. The gained knowledge about hybrid design was then used to generate and characterize the first photo-switchable dualsteric ligand. The resulting hybrids were characterized with the M1-I3N-CFP sensor and were described as photo-inactivatable and dimmable. In addition to the ligand characterization the ligand application methodology was further developed and improved. Thus, a fragment-based screening approach for dualsteric ligands was reported in this study for the first time. With this approach it is possible to investigate dualsteric ligands in greater detail by applying either single ligand fragments alone or in a mixture of building blocks. These studies revealed the insights that the effect of dualsteric ligands on a GPCR can be rebuild by applying the single building blocks simultaneously. The fragment-based screening provides high potential for the molecular understanding of dualsteric ligands and for future screening approaches. Next, a further development of the standard procedure for measuring FRET by sensitized emission was performed. Under normal conditions single cell FRET is measured on glass coverslips. After coating the coverslips surface with a 20 nm thick gold layer an increased FRET efficiency up to 60 \% could be reported. This finding was validated in different approaches und in different configurations. This FRET enhancement by plasmonic surfaces was until yet unreported in the literature for physiological systems and make FRET for future projects even more powerful.},
  subject      = {G-Protein gekoppelte Rezeptoren},
  language  = {en}
}
@phdthesis{Rydzek2019,
  author    = {Rydzek, Julian},
  title     = {NF-κB/NFAT Reporter Cell Platform for Chimeric Antigen Receptor (CAR)-Library Screening},
  doi       = {10.25972/OPUS-17918},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-179187},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Immunotherapy with engineered T cells expressing a tumor-specific chimeric antigen receptor (CAR) is under intense preclinical and clinical investigation. This involves a rapidly increasing portfolio of novel target antigens and CAR designs that need to be tested in time- and work-intensive screening campaigns in primary T cells. Therefore, we anticipated that a standardized screening platform, similar as in pharmaceutical small molecule and antibody discovery, would facilitate the analysis of CARs by pre-selecting lead candidates from a large pool of constructs that differ in their extracellular and intracellular modules. Because CARs integrate structural elements of the T cell receptor (TCR) complex and engage TCR-associated signaling molecules upon stimulation, we reasoned that the transcription factors nuclear factor-κB (NF-κB) and nuclear factor of activated T cells (NFAT) could serve as surrogate markers for primary T cell function. The nuclear translocation of both transcription factors in primary T cells, which we observed following CAR stimulation, supported our rationale to use NF-κB and NFAT as indicators of CAR-mediated activation in a screening platform. To enable standardized and convenient analyses, we have established a CAR-screening platform based on the human T cell lymphoma line Jurkat that has been modified to provide rapid detection of NF-κB and NFAT activation. For this purpose, Jurkat cells contained NF-κB and NFAT-inducible reporter genes that generate a duplex output of cyan fluorescent protein (CFP) and green fluorescent protein (GFP), respectively. Upon stimulation of NF-κB/NFAT reporter cells, the expression of both fluorophores could be readily quantified in high-throughput screening campaigns by flow cytometry. We modified the reporter cells with CD19-specific and ROR1-specific CARs, and we co-cultured them with antigen-positive stimulator cells to analyze NF-κB and NFAT activation. CAR-induced reporter signals could already be detected after 6 hours. The optimal readout window with high-level reporter activation was set to 24 hours, allowing the CAR-screening platform to deliver results in a rapid turnaround time. A reporter cell-screening campaign of a spacer library with CARs comprising a short, intermediate or long IgG4-Fc domain allowed distinguishing functional from non-functional constructs. Similarly, reporter cell-based analyses identified a ROR1-CAR with 4-1BB domain from a library with different intracellular signal modules due to its ability to confer high NF-κB activation, consistent with data from in vitro and in vivo studies with primary T cells. The results of both CAR screening campaigns were highly reproducible, and the time required for completing each testing campaign was substantially shorter with reporter cells (6 days) compared to primary T cells (21 days). We further challenged the reporter cells in a large-scale screening campaign with a ROR1 CAR library comprising mutations in the VH CDR3 sequence of the R11 scFv. This region is crucial for binding the R11 epitope of ROR1, and we anticipated that mutations here would cause a loss of specificity and affinity for most of the CAR variants. This provided the opportunity to determine whether the CAR screening platform was able to retrieve functional constructs from a large pool of CAR variants. Indeed, using a customized pre enrichment and screening strategy, the reporter cells identified a functional CAR variant that was present with a frequency of only 6 in 1.05x10^6. As our CAR-screening platform enabled the analysis of activating signal modules, it encouraged us to also evaluate inhibitory signal modules that change the CAR mode of action. Such an inhibitory CAR (iCAR) can be used in logic gates with an activating CAR to interfere with T cell stimulation. By selecting appropriate target antigens for iCAR and CAR, this novel application aims to improve the selectivity towards tumor cells, and it could readily be studied using our screening platform. Accordingly, we tested CD19-specific iCARs with inhibitory PD-1 signal module for their suppressive effect on reporter gene activation. In logic gates with CAR or TCR stimulation, a decrease of NF-κB and NFAT signals was only observed when activating and inhibitory receptors were forced into spatial proximity. These results were further verified by experiments with primary T cells. In conclusion, our reporter cell system is attractive as a platform technology because it is independent of testing in primary T cells, exportable between laboratories, and scalable to enable small- to large-scale screening campaigns of CAR libraries. The pre-selection of appropriate lead candidates with optimal extracellular and intracellular modules can reduce the number of CAR constructs to be investigated in further in vitro and in vivo studies with primary T cells. We are therefore confident that our CAR-screening platform based on NF-κB/NFAT reporter cells will be useful to accelerate translational research by facilitating the evaluation of CARs with novel design parameters.},
  subject      = {Antigenrezeptor},
  language  = {en}
}
@phdthesis{Kaiser2020,
  author    = {Kaiser, Sebastian},
  title     = {A RecQ helicase in disguise: Characterization of the unconventional Structure and Function of the human Genome Caretaker RecQ4},
  doi       = {10.25972/OPUS-16041},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-160414},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {From the simplest single-cellular organism to the most complex multicellular life forms, genetic information in form of DNA represents the universal basis for all biological processes and thus for life itself. Maintaining the structural and functional integrity of the genome is therefore of paramount importance for every single cell. DNA itself, as an active and complex macromolecular structure, is both substrate and product of many of these biochemical processes. A cornerstone of DNA maintenance is thus established by the tight regulation of the multitude of reactions in DNA metabolism, repressing adverse side reactions and ensuring the integrity of DNA in sequence and function. The family of RecQ helicases has emerged as a vital class of enzymes that facilitate genomic integrity by operating in a versatile spectrum of nucleic acid metabolism processes, such as DNA replication, repair, recombination, transcription and telomere stability. RecQ helicases are ubiquitously expressed and conserved in all kingdoms of life. Human cells express five different RecQ enzymes, RecQ1, BLM, WRN, RecQ4 and RecQ5, which all exhibit individual as well as overlapping functions in the maintenance of genomic integrity. Dysfunction of three human RecQ helicases, BLM, WRN and RecQ4, causes different heritable cancer susceptibility syndromes, supporting the theory that genomic instability is a molecular driving force for cancer development. However, based on their inherent DNA protective nature, RecQ helicases represent a double-edged sword in the maintenance of genomic integrity. While their activity in normal cells is essential to prevent cancerogenesis and cellular aging, cancer cells may exploit this DNA protective function by the overexpression of many RecQ helicases, aiding to overcome the disadvantageous results of unchecked DNA replication and simultaneously gaining resistance against chemotherapeutic drugs. Therefore, detailed knowledge how RecQ helicases warrant genomic integrity is required to understand their implication in cancerogenesis and aging, thus setting the stage to develop new strategies towards the treatment of cancer. The current study presents and discusses the first high-resolution X-ray structure of the human RecQ4 helicase. The structure encompasses the conserved RecQ4 helicase core, including a large fraction of its unique C- terminus. Our structural analysis of the RecQ4 model highlights distinctive differences and unexpected similarities to other, structurally conserved, RecQ helicases and permits to draw conclusions about the functional implications of the unique domains within the RecQ4 C-terminus. The biochemical characterization of various RecQ4 variants provides functional insights into the RecQ4 helicase mechanism, suggesting that RecQ4 might utilize an alternative DNA strand separation technique, compared to other human RecQ family members. Finally, the RecQ4 model permits for the first time the analysis of multiple documented RecQ4 patient mutations at the atomic level and thus provides the possibility for an advanced interpretation of particular structure-function relationships in RecQ4 pathogenesis.},
  subject      = {Helikasen},
  language  = {en}
}
@phdthesis{PrietoGarcia2022,
  author    = {Prieto Garc{\´i}a, Cristian},
  title     = {USP28 regulates Squamous cell oncogenesis and DNA repair via ΔNp63 deubiquitination},
  doi       = {10.25972/OPUS-27033},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-270332},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {∆Np63 is a master regulator of squamous cell identity and regulates several signaling pathways that crucially contribute to the development of squamous cell carcinoma (SCC) tumors. Its contribution to coordinating the expression of genes involved in oncogenesis, epithelial identity, DNA repair, and genome stability has been extensively studied and characterized. For SCC, the expression of ∆Np63 is an essential requirement to maintain the malignant phenotype. Additionally, ∆Np63 functionally contributes to the development of cancer resistance toward therapies inducing DNA damage. SCC patients are currently treated with the same conventional Cisplatin therapy as they would have been treated 30 years ago. In contrast to patients with other tumor entities, the survival of SCC patients is limited, and the efficacy of the current therapies is rather low. Considering the rising incidences of these tumor entities, the development of novel SCC therapies is urgently required. Targeting ∆Np63, the transcription factor, is a potential alternative to improve the therapeutic response and clinical outcomes of SCC patients. However, ∆Np63 is considered "undruggable." As is commonly observed in transcription factors, ∆Np63 does not provide any suitable domains for the binding of small molecule inhibitors. ∆Np63 regulates a plethora of different pathways and cellular processes, making it difficult to counteract its function by targeting downstream effectors. As ∆Np63 is strongly regulated by the ubiquitin-proteasome system (UPS), the development of deubiquitinating enzyme inhibitors has emerged as a promising therapeutic strategy to target ∆Np63 in SCC treatment. This work involved identifying the first deubiquitinating enzyme that regulates ∆Np63 protein stability. Stateof-the-art SCC models were used to prove that USP28 deubiquitinates ∆Np63, regulates its protein stability, and affects squamous transcriptional profiles in vivo and ex vivo. Accordingly, SCC depends on USP28 to maintain essential levels of ∆Np63 protein abundance in tumor formation and maintenance. For the first time, ∆Np63, the transcription factor, was targeted in vivo using a small molecule inhibitor targeting the activity of USP28. The pharmacological inhibition of USP28 was sufficient to hinder the growth of SCC tumors in preclinical mouse models. Finally, this work demonstrated that the combination of Cisplatin with USP28 inhibitors as a novel therapeutic alternative could expand the limited available portfolio of SCC therapeutics. Collectively, the data presented within this dissertation demonstrates that the inhibition of USP28 in SCC decreases ∆Np63 protein abundance, thus downregulating the Fanconi anemia (FA) pathway and recombinational DNA repair. Accordingly, USP28 inhibition reduces the DNA damage response, thereby sensitizing SCC tumors to DNA damage therapies, such as Cisplatin.},
  language  = {en}
}
@phdthesis{Nelke2022,
  author    = {Nelke, Johannes},
  title     = {Entwicklung multi-funktioneller TNFRSF Rezeptorspezifischer Antik{\"o}rper-Fusionsproteine mit FcγR-unabh{\"a}ngiger Aktivit{\"a}t},
  doi       = {10.25972/OPUS-27985},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-279855},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Antik{\"o}rper, die gegen eine klinisch relevante Gruppe von Rezeptoren innerhalb der Tumornekrosefaktor-Rezeptor-Superfamilie (TNFRSF) gerichtet sind, darunter CD40 und CD95 (Fas/Apo-1), ben{\"o}tigen ebenfalls eine Bindung an Fc-Gamma-Rezeptoren (FcγRs), um eine starke agonistische Wirkung zu entfalten. Diese FcγR-Abh{\"a}ngigkeit beruht weitgehend auf der bloßen zellul{\"a}ren Verankerung durch die Fc-Dom{\"a}ne des Antik{\"o}rpers und ben{\"o}tigt dabei kein FcγR-Signalling. Ziel dieser Doktorarbeit war es, das agonistische Potenzial von αCD40- und αCD95-Antik{\"o}rpern unabh{\"a}ngig von der Bindung an FcγRs durch die Verankerung an Myelomzellen zu entfalten. Zu diesem Zweck wurden verschiedene Antik{\"o}rpervarianten (IgG1, IgG1-N297A, Fab2) gegen die TNFRSF-Mitglieder CD40 und CD95 genetisch mit einem einzelkettig kodierten B-Zell-aktivierenden Faktor (scBaff) Trimer als C-terminale myelom-spezifische Verankerungsdom{\"a}ne fusioniert, welche die Fc-Dom{\"a}ne-vermittelte FcγR-Bindung ersetzt. Diese bispezifischen Antik{\"o}rper-scBaff-Fusionsproteine wurden in Bindungsstudien und funktionellen Assays mit Tumorzelllinien untersucht, die einen oder mehrere der drei Baff-Rezeptoren exprimieren: BaffR, Transmembran-Aktivator und CAML-Interaktor (TACI) und B-Zell-Reifungsantigen (BCMA). Zellul{\"a}re Bindungsstudien zeigten, dass die Bindungseigenschaften der verschiedenen Dom{\"a}nen innerhalb der Antik{\"o}rper-scBaff-Fusionen gegen{\"u}ber der Zielantigene vollst{\"a}ndig intakt blieben. In Ko-Kulturversuchen von CD40- und CD95-responsiven Zellen mit BaffR-, BCMA- oder TACI-exprimierenden Verankerungszellen zeigten die Antik{\"o}rper-Fusionsproteine einen starken Agonismus, w{\"a}hrend in Ko-Kulturen mit Zellen ohne Expression von Baff-interagierenden Rezeptoren nur eine geringe Rezeptorstimulation beobachtet wurde. Die hier vorgestellten αCD40- und αCD95-Antik{\"o}rper-scBaff-Fusionsproteine zeigen also Myelom-spezifische Aktivit{\"a}t und versprechen im Vergleich zu herk{\"o}mmlichen CD40- und CD95-Agonisten geringere systemische Nebenwirkungen.},
  subject      = {Antigen CD40},
  language  = {de}
}
@phdthesis{Ries2020,
  author    = {Ries, Lena Kerstin},
  title     = {From recognition to reaction: Mechanistic analysis of the interactions of the HECT ligase E6AP with ubiquitin},
  doi       = {10.25972/OPUS-17960},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-179609},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {The ubiquitination of proteins controls a multitude of physiological processes. This versatility of ubiquitin as a molecular signal arises from the diverse ways by which it can be attached to target proteins. Different ubiquitination patterns are then translated into different downstream consequences. Due to the enormous complexity of possible ubiquitin modifications, the ubiquitination machinery must be highly specific and tightly controlled. Ubiquitination proceeds through an enzymatic cascade, the last step of which is catalyzed by the E3 enzyme family. E3 enzymes are the crucial regulators since they dictate the specificity of substrate selection and modification. Deregulation of the HECT-type ubiquitin ligase E6AP (UBE3A) is implicated in human papilloma virus-induced cervical tumorigenesis and several neurodevelopmental disorders. Yet the structural underpinnings of activity, regulation and specificity in this crucial ligase are incompletely understood. One aim of this study was to unravel the role of the a1'-helix N-terminal to the HECT domain that was found to be a key element mediating regulation and oligomerization in other HECT ligases. I found that most N-terminally extended HECT domain constructs were insoluble when expressed in E. coli, indicating that additional regions N-terminal to the tested fragments may be essential to protect this highly hydrophobic helix from causing aggregation. Another question addressed in this study was how E6AP builds ubiquitin chains. Using single-turnover experiments, I showed that ubiquitin-loaded E6AP is unable to transfer an additional ubiquitin molecule onto a stably linked ubiquitin-E6AP complex. This indicates that E6AP cannot assemble chains on its active site and may instead follow a sequential addition mechanism in which one ubiquitin molecule is transferred at a time to the target protein. Using NMR spectroscopy and extensive mutational analyses, the determinants of ubiquitin recognition by the C-lobe of E6AP were unraveled and assigned to particular steps in the catalytic cycle. A functionally critical interface was identified that is specifically required during thioester formation between the C-terminus of ubiquitin and the ligase active site. This interface resembles the one utilized by NEDD4-type enzymes, suggesting a conserved ubiquitin binding mode across HECT ligases, independent of their linkage specificities. Moreover, I identified critical surface patches on ubiquitin and in the N- and C-terminal portions of the catalytic domain of E6AP that are important for the subsequent step of isopeptide bond formation. I also uncovered key determinants of the Lys48-linkage specificity of E6AP, both in the E6AP HECT domain and ubiquitin itself. This includes the C-terminal tail of E6AP and a hydrophilic surface region of ubiquitin in proximity to the acceptor site, Lys48. It is thus tempting to speculate that ubiquitin linkage formation by E6AP is substrate-assisted. Taken together, my results improve our mechanistic understanding of the structure-function relationship between E6AP and ubiquitin, thus providing a basis for ultimately manipulating the functions of this HECT ligase for therapeutic applications.},
  subject      = {Ubiquitin},
  language  = {en}
}
@phdthesis{Koelmel2020,
  author    = {K{\"o}lmel, Wolfgang},
  title     = {Structural and functional characterization of TFIIH from \(Chaetomium\) \(thermophilum\)},
  doi       = {10.25972/OPUS-16176},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-161769},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Gene expression and transfer of the genetic information to the next generation forms the basis of cellular life. These processes crucially rely on DNA, thus the preservation, transcription and translation of DNA is of fundamental importance for any living being. The general transcription factor TFIIH is a ten subunit protein complex, which consists of two subcomplexes: XPB, p62, p52, p44, p34, and p8 constitute the TFIIH core, CDK7, CyclinH, and MAT1 constitute the CAK. These two subcomplexes are connected via XPD. TFIIH is a crucial factor involved in both, DNA repair and transcription. The central role of TFIIH is underlined by three severe disorders linked to failure of TFIIH in these processes: xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. Only limited structural and functional data of TFIIH are available so far. Here, the model organism Chaetomium thermophilum was utilized with the aim to structurally and functionally characterize TFIIH. By combining the expression and purification of single TFIIH subunits with the co-expression and co-purification of dual complexes, a unique and powerful modular system of the TFIIH core subunits could be established, encompassing all proteins in high quality and fully functional. This system permits the step-wise assembly of TFIIH core, thereby making it possible to assess the influence of the intricate interaction network within TFIIH core on the overall enzymatic activities of TFIIH, which has not been possible so far. Utilizing the single subunits and dual complexes, a detailed interaction network of TFIIH core was established, revealing the crucial role of the p34 subunit as a central scaffold of TFIIH by linking the two proteins p44 and p52. Our studies also suggest that p62 constitutes the central interface of TFIIH to the environment rather than acting as a scaffold. TFIIH core complexes were assembled and investigated via electron microscopy. Preliminary data indicate that TFIIH adopts different conformational states, which are important to fulfill its functions in transcription and DNA repair. Additionally, a shortened construct of p62 was used to develop an easy-to-use, low cost strategy to overcome the crystallographic phase problem via cesium derivatization.},
  subject      = {Transkriptionsfaktor},
  language  = {en}
}
@article{WanzekSchwindtCapraetal.2017,
  author    = {Wanzek, Katharina and Schwindt, Eike and Capra, John A. and Paeschke, Katrin},
  title     = {Mms1 binds to G-rich regions in Saccharomyces cerevisiae and influences replication and genome stability},
  series = {Nucleic Acids Research},
  volume    = {45},
  journal   = {Nucleic Acids Research},
  number    = {13},
  doi       = {10.1093/nar/gkx467},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170577},
  pages     = {7796-7806},
  year      = {2017},
  abstract  = {The regulation of replication is essential to preserve genome integrity. Mms1 is part of the E3 ubiquitin ligase complex that is linked to replication fork progression. By identifying Mms1 binding sites genome-wide in Saccharomyces cerevisiae we connected Mms1 function to genome integrity and replication fork progression at particular G-rich motifs. This motif can form G-quadruplex (G4) structures in vitro. G4 are stable DNA structures that are known to impede replication fork progression. In the absence of Mms1, genome stability is at risk at these G-rich/G4 regions as demonstrated by gross chromosomal rearrangement assays. Mms1 binds throughout the cell cycle to these G-rich/G4 regions and supports the binding of Pif1 DNA helicase. Based on these data we propose a mechanistic model in which Mms1 binds to specific G-rich/G4 motif located on the lagging strand template for DNA replication and supports Pif1 function, DNA replication and genome integrity.},
  language  = {en}
}
@phdthesis{Kalb2021,
  author    = {Kalb, Jacqueline},
  title     = {The role of BRCA1 and DCP1A in the coordination of transcription and replication in neuroblastoma},
  doi       = {10.25972/OPUS-24871},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-248711},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {The deregulation of the MYC oncoprotein family plays a major role in tumorigenesis and tumour maintenance of many human tumours. Because of their structure and nuclear localisation, they are defined as undruggable targets which makes it difficult to find direct therapeutic approaches. An alternative approach for targeting MYC-driven tumours is the identification and targeting of partner proteins which score as essential in a synthetic lethality screen. Neuroblastoma, an aggressive entity of MYCN-driven tumours coming along with a bad prognosis, are dependent on the tumour suppressor protein BRCA1 as synthetic lethal data showed. BRCA1 is recruited to promoter regions in a MYCN-dependent manner. The aim of this study was to characterise the role of BRCA1 in neuroblastoma with molecular biological methods. BRCA1 prevents the accumulation of RNA Polymerase II (RNAPII) at the promoter region. Its absence results in the formation of DNA/RNA-hybrids, so called R-loops, and DNA damage. To prevent the accumulation of RNAPII, the cell uses DCP1A, a decapping factor known for its cytoplasmatic and nuclear role in mRNA decay. It is the priming factor in the removal of the protective 5'CAP of mRNA, which leads to degradation by exonucleases. BRCA1 is necessary for the chromatin recruitment of DCP1A and its proximity to RNAPII. Cells showed upon acute activation of MYCN a higher dependency on DCP1A. Its activity prevents the deregulation of transcription and leads to proper coordination of transcription and replication. The deregulation of transcription in the absence of DCP1A results in replication fork stalling and leads to activation of the Ataxia telangiectasia and Rad3 related (ATR) kinase. The result is a disturbed cell proliferation to the point of increased apoptosis. The activation of the ATR kinase pathway in the situation where DCP1A is knocked down and MYCN is activated, makes those cells more vulnerable for the treatment with ATR inhibitors. In summary, the tumour suppressor protein BRCA1 and the decapping factor DCP1A, mainly known for its function in the cytoplasm, have a new nuclear role in a MYCN-dependent context. This study shows their essentiality in the coordination of transcription and replication which leads to an unrestrained growth of tumour cells if uncontrolled.},
  subject      = {Neuroblastom},
  language  = {en}
}
@article{VeepaschitViswanathanBordonneetal.2021,
  author    = {Veepaschit, Jyotishman and Viswanathan, Aravindan and Bordonne, Remy and Grimm, Clemens and Fischer, Utz},
  title     = {Identification and structural analysis of the Schizosaccharomyces pombe SMN complex},
  series = {Nucleic Acids Research},
  volume    = {49},
  journal   = {Nucleic Acids Research},
  number    = {13},
  doi       = {10.1093/nar/gkab158},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259880},
  pages     = {7207-7223},
  year      = {2021},
  abstract  = {The macromolecular SMN complex facilitates the formation of Sm-class ribonucleoproteins involved in mRNA processing (UsnRNPs). While biochemical studies have revealed key activities of the SMN complex, its structural investigation is lagging behind. Here we report on the identification and structural determination of the SMN complex from the lower eukaryote Schizosaccharomyces pombe, consisting of SMN, Gemin2, 6, 7, 8 and Sm proteins. The core of the SMN complex is formed by several copies of SMN tethered through its C-terminal alpha-helices arranged with alternating polarity. This creates a central platform onto which Gemin8 binds and recruits Gemins 6 and 7. The N-terminal parts of the SMN molecules extrude via flexible linkers from the core and enable binding of Gemin2 and Sm proteins. Our data identify the SMN complex as a multivalent hub where Sm proteins are collected in its periphery to allow their joining with UsnRNA.},
  language  = {en}
}
@article{AndelovicWinterJakobetal.2021,
  author    = {Andelovic, Kristina and Winter, Patrick and Jakob, Peter Michael and Bauer, Wolfgang Rudolf and Herold, Volker and Zernecke, Alma},
  title     = {Evaluation of plaque characteristics and inflammation using magnetic resonance imaging},
  series = {Biomedicines},
  volume    = {9},
  journal   = {Biomedicines},
  number    = {2},
  issn      = {2227-9059},
  doi       = {10.3390/biomedicines9020185},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-228839},
  year      = {2021},
  abstract  = {Atherosclerosis is an inflammatory disease of large and medium-sized arteries, characterized by the growth of atherosclerotic lesions (plaques). These plaques often develop at inner curvatures of arteries, branchpoints, and bifurcations, where the endothelial wall shear stress is low and oscillatory. In conjunction with other processes such as lipid deposition, biomechanical factors lead to local vascular inflammation and plaque growth. There is also evidence that low and oscillatory shear stress contribute to arterial remodeling, entailing a loss in arterial elasticity and, therefore, an increased pulse-wave velocity. Although altered shear stress profiles, elasticity and inflammation are closely intertwined and critical for plaque growth, preclinical and clinical investigations for atherosclerosis mostly focus on the investigation of one of these parameters only due to the experimental limitations. However, cardiovascular magnetic resonance imaging (MRI) has been demonstrated to be a potent tool which can be used to provide insights into a large range of biological parameters in one experimental session. It enables the evaluation of the dynamic process of atherosclerotic lesion formation without the need for harmful radiation. Flow-sensitive MRI provides the assessment of hemodynamic parameters such as wall shear stress and pulse wave velocity which may replace invasive and radiation-based techniques for imaging of the vascular function and the characterization of early plaque development. In combination with inflammation imaging, the analyses and correlations of these parameters could not only significantly advance basic preclinical investigations of atherosclerotic lesion formation and progression, but also the diagnostic clinical evaluation for early identification of high-risk plaques, which are prone to rupture. In this review, we summarize the key applications of magnetic resonance imaging for the evaluation of plaque characteristics through flow sensitive and morphological measurements. The simultaneous measurements of functional and structural parameters will further preclinical research on atherosclerosis and has the potential to fundamentally improve the detection of inflammation and vulnerable plaques in patients.},
  language  = {en}
}
@article{TolayBuchberger2022,
  author    = {Tolay, Nazife and Buchberger, Alexander},
  title     = {Role of the ubiquitin system in stress granule metabolism},
  series = {International Journal of Molecular Sciences},
  volume    = {23},
  journal   = {International Journal of Molecular Sciences},
  number    = {7},
  issn      = {1422-0067},
  doi       = {10.3390/ijms23073624},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-284061},
  year      = {2022},
  abstract  = {Eukaryotic cells react to various stress conditions with the rapid formation of membrane-less organelles called stress granules (SGs). SGs form by multivalent interactions between RNAs and RNA-binding proteins and are believed to protect stalled translation initiation complexes from stress-induced degradation. SGs contain hundreds of different mRNAs and proteins, and their assembly and disassembly are tightly controlled by post-translational modifications. The ubiquitin system, which mediates the covalent modification of target proteins with the small protein ubiquitin ('ubiquitylation'), has been implicated in different aspects of SG metabolism, but specific functions in SG turnover have only recently emerged. Here, we summarize the evidence for the presence of ubiquitylated proteins at SGs, review the functions of different components of the ubiquitin system in SG formation and clearance, and discuss the link between perturbed SG clearance and the pathogenesis of neurodegenerative disorders. We conclude that the ubiquitin system plays an important, medically relevant role in SG biology.},
  language  = {en}
}
@article{TolayBuchberger2021,
  author    = {Tolay, Nazife and Buchberger, Alexander},
  title     = {Comparative profiling of stress granule clearance reveals differential contributions of the ubiquitin system},
  series = {Life Science Alliance},
  volume    = {4},
  journal   = {Life Science Alliance},
  number    = {5},
  doi       = {10.26508/lsa.202000927},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259810},
  pages     = {e202000927},
  year      = {2021},
  abstract  = {Stress granules (SGs) are cytoplasmic condensates containing untranslated mRNP complexes. They are induced by various proteotoxic conditions such as heat, oxidative, and osmotic stress. SGs are believed to protect mRNPs from degradation and to enable cells to rapidly resume translation when stress conditions subside. SG dynamics are controlled by various posttranslationalmodifications, but the role of the ubiquitin system has remained controversial. Here, we present a comparative analysis addressing the involvement of the ubiquitin system in SG clearance. Using high-resolution immuno-fluorescence microscopy, we found that ubiquitin associated to varying extent with SGs induced by heat, arsenite, H2O2, sorbitol, or combined puromycin and Hsp70 inhibitor treatment. SG-associated ubiquitin species included K48- and K63-linked conjugates, whereas free ubiquitin was not significantly enriched. Inhibition of the ubiquitin activating enzyme, deubiquitylating enzymes, the 26S proteasome and p97/VCP impaired the clearance of arsenite- and heat-induced SGs, whereas SGs induced by other stress conditions were little affected. Our data underline the differential involvement of the ubiquitin system in SG clearance, a process important to prevent the formation of disease-linked aberrant SGs.},
  language  = {en}
}
@phdthesis{Reil2023,
  author    = {Reil, Lucy Honor},
  title     = {The role of WASH complex subunit Strumpellin in platelet function},
  doi       = {10.25972/OPUS-24207},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-242077},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Strumpellin is a member of the highly conserved pentameric WASH complex, which stimulates the Arp2/3 complex on endosomes and induces the formation of a branched actin network. The WASH complex is involved in the formation and stabilisation of endosomal retrieval subdomains and transport carriers, into which selected proteins are packaged and subsequently transported to their respective cellular destination, e.g. the plasma membrane. Up until now, the role of Strumpellin in platelet function and endosomal trafficking has not been researched. In order to examine its role, a conditional knockout mouse line was generated, which specifically lacked Strumpellin in megakaryocytes and platelets. Conditional knockout of Strumpellin resulted in only a mild platelet phenotype. Loss of Strumpellin led to a decreased abundance of the αIIbβ3 integrin in platelets, including a reduced αIIbβ3 surface expression by approximately 20\% and an impaired αIIbβ3 activation after platelet activation. The reduced surface expression of αIIbβ3 was also detected in megakaryocytes. The expression of other platelet surface glycoproteins was not affected. Platelet count, size and morphology remained unaltered. The reduction of αIIbβ3 expression in platelets resulted in a reduced fibrinogen binding capacity after platelet activation. However, fibrinogen uptake under resting conditions, although slightly delayed, as well as overall fibrinogen content in Strumpellin-deficient platelets were comparable to controls. Most notably, reduced αIIbβ3 expression did not lead to any platelet spreading and aggregation defects in vitro. Furthermore, reduced WASH1 protein levels were detected in the absence of Strumpellin. In conclusion, loss of Strumpellin does not impair platelet function, at least not in vitro. However, the data demonstrates that Strumpellin plays a role in selectively regulating αIIbβ3 surface expression. As a member of the WASH complex, Strumpellin may regulate αIIbβ3 recycling back to the platelet surface. Furthermore, residual WASH complex subunits may still assemble and partially function in the absence of Strumpellin, which could explain the only 20\% decrease in αIIbβ3 surface expression. Nonetheless, the exact mechanism still remains unclear.},
  language  = {en}
}
@phdthesis{Klingler2023,
  author    = {Klingler, Philipp},
  title     = {Exploration of proteasome interactions with human platelet function},
  doi       = {10.25972/OPUS-32108},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-321089},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Platelets are anucleated cell fragments derived from megakaryocytes. They play a fundamental role in hemostasis, but there is rising evidence that they are also involved in immunological processes. Despite absence of a nucleus, human platelets are capable of de novo protein synthesis and contain a fully functional proteasome system, which is, in nucleated cells, involved in processes like cell cycle progression or apoptosis by its ability of protein degradation. The physiological significance of the proteasome system in human platelets is not yet fully understood and subject of ongoing research. Therefore, this study was conducted with the intention to outline the role of the proteasome system for functional characteristics of human platelets. For experimentation, citrated whole blood from healthy donors was obtained and preincubated with proteasome inhibitors. In addition to the commonly used bortezomib, the potent and selective proteasome inhibitor carfilzomib was selected as a second inhibitor to rule out agent-specific effects and to confirm that observed changes are related to proteasome inhibition. Irreversibly induced platelet activation and aggregation were not affected by proteasome blockade with bortezomib up to 24 hours. Conversely, proteasome inhibition led to enhanced threshold aggregation and agglutination up to 25 \%, accompanied by partial alleviation of induced VASP phosphorylation of approximately 10-15 \%. Expression of different receptors were almost unaffected. Instead, a significant increase of PP2A activity was observable in platelets after proteasome blockade, accompanied by facilitated platelet adhesion to coated surfaces in static experiments or flow chamber experiments. Carfilzomib, used for the first time in functional experimentation with human platelets in vitro, led to a dose-dependent decrease of proteasome activity with accumulation of poly ubiquitylated proteins. Like bortezomib, carfilzomib treatment resulted in enhanced threshold aggregation with attenuated VASP phosphorylation. As the main conclusion of this thesis, proteasome inhibition enhances the responsiveness of human platelets, provided by an alleviation of platelet inhibitory pathways and by an additional increase of PP2A activity, resulting in facilitated platelet adhesion under static and flow conditions. The proteasome system appears to be involved in the promotion of inhibitory counterregulation in platelets. The potential of proteasome inhibitors for triggering thromboembolic adverse events in patients must be clarified in further studies, in addition to their possible use for targeting platelet function to improve the hemostatic reactivity of platelets.},
  subject      = {Thrombozyt},
  language  = {en}
}
@phdthesis{Huber2023,
  author    = {Huber, Hannes},
  title     = {Biochemical and functional characterization of DHX30, an RNA helicase linked to neurodevelopmental disorder},
  doi       = {10.25972/OPUS-28050},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-280505},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {RNA helicases are key players in the regulation of gene expression. They act by remodeling local RNA secondary structures as well as RNA-protein interactions to enable the dynamic association of RNA binding proteins to their targets. The putative RNA helicase DHX30 is a member of the family of DEAH-box helicases with a putative role in the ATP-dependent unwinding of RNA secondary structures. Mutations in the DHX30 gene causes the autosomal dominant neuronal disease "Neurodevelopmental Disorder with severe Motor Impairment and Absent Language" (NEDMIAL;OMIM\#617804). In this thesis, a strategy was established that enabled the large-scale purification of enzymatically active DHX30. Through enzymatic studies performed in vitro, DHX30 was shown to act as an ATP-dependent 3' → 5' RNA helicase that catalyzes the unwinding of RNA:RNA and RNA:DNA substrates. Using recombinant DHX30, it could be shown that disease-causing missense mutations in the conserved helicase core caused the disruption of its ATPase and helicase activity. The protein interactome of DHX30 however, was unchanged indicating that the pathogenic missense-mutations do not cause misfolding of DHX30, but rather specifically affect its catalytic activity. DHX30 localizes predominantly in the cytoplasm where it forms a complex with ribosomes and polysomes. Using a cross-linking mass spectrometry approach, a direct interaction of the N-terminal double strand RNA binding domain of DHX30 with sites next to the ribosome's mRNA entry channel and the subunit interface was uncovered. RNA sequencing of DHX30 knockout cells revealed a strong de-regulation of mRNAs involved in neurogenesis and nervous system development, which is in line with the NEDMIAL disease phenotype. The knockdown of DHX30 results in a decreased 80S peak in polysome gradients, indicating that DHX30 has an effect on the translation machinery. Sequencing of the pool of active translating mRNAs revealed that upon DHX30 knockout mainly 5'TOP mRNAs are downregulated. These mRNAs are coding for proteins of the translational machinery and translation initiation factors. This study identified DHX30 as a factor of the translation machinery that selectively impacts the expression of a subset of proteins and provides insight on the etiology of NEDMIAL.},
  language  = {en}
}
@article{BenhalevyGuptaDananetal.2017,
  author    = {Benhalevy, Daniel and Gupta, Sanjay K. and Danan, Charles H. and Ghosal, Suman and Sun, Hong-Wei and Kazemeier, Hinke G. and Paeschke, Katrin and Hafner, Markus and Juranek, Stefan A.},
  title     = {The Human CCHC-type Zinc Finger Nucleic Acid-Binding Protein Binds G-Rich Elements in Target mRNA Coding Sequences and Promotes Translation},
  series = {Cell Reports},
  volume    = {18},
  journal   = {Cell Reports},
  number    = {12},
  doi       = {10.1016/j.celrep.2017.02.080},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-171122},
  pages     = {2979-2990},
  year      = {2017},
  abstract  = {The CCHC-type zinc finger nucleic acid-binding protein (CNBP/ZNF9) is conserved in eukaryotes and is essential for embryonic development in mammals. It has been implicated in transcriptional, as well as post-transcriptional, gene regulation; however, its nucleic acid ligands and molecular function remain elusive. Here, we use multiple systems-wide approaches to identify CNBP targets and function. We used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) to identify 8,420 CNBP binding sites on 4,178 mRNAs. CNBP preferentially bound G-rich elements in the target mRNA coding sequences, most of which were previously found to form G-quadruplex and other stable structures in vitro. Functional analyses, including RNA sequencing, ribosome profiling, and quantitative mass spectrometry, revealed that CNBP binding did not influence target mRNA abundance but rather increased their translational efficiency. Considering that CNBP binding prevented G-quadruplex structure formation in vitro, we hypothesize that CNBP is supporting translation by resolving stable structures on mRNAs.},
  language  = {en}
}
@article{OthmanFathyBekhitetal.2021,
  author    = {Othman, Eman M. and Fathy, Moustafa and Bekhit, Amany Abdlrehim and Abdel-Razik, Abdel-Razik H. and Jamal, Arshad and Nazzal, Yousef and Shams, Shabana and Dandekar, Thomas and Naseem, Muhammad},
  title     = {Modulatory and toxicological perspectives on the effects of the small molecule kinetin},
  series = {Molecules},
  volume    = {26},
  journal   = {Molecules},
  number    = {3},
  issn      = {1420-3049},
  doi       = {10.3390/molecules26030670},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-223064},
  year      = {2021},
  abstract  = {Plant hormones are small regulatory molecules that exert pharmacological actions in mammalian cells such as anti-oxidative and pro-metabolic effects. Kinetin belongs to the group of plant hormones cytokinin and has been associated with modulatory functions in mammalian cells. The mammalian adenosine receptor (A2a-R) is known to modulate multiple physiological responses in animal cells. Here, we describe that kinetin binds to the adenosine receptor (A2a-R) through the Asn253 residue in an adenosine dependent manner. To harness the beneficial effects of kinetin for future human use, we assess its acute toxicity by analyzing different biochemical and histological markers in rats. Kinetin at a dose below 1 mg/kg had no adverse effects on the serum level of glucose or on the activity of serum alanine transaminase (ALT) or aspartate aminotransferase (AST) enzymes in the kinetin treated rats. Whereas, creatinine levels increased after a kinetin treatment at a dose of 0.5 mg/kg. Furthermore, 5 mg/kg treated kinetin rats showed normal renal corpuscles, but a mild degeneration was observed in the renal glomeruli and renal tubules, as well as few degenerated hepatocytes were also observed in the liver. Kinetin doses below 5 mg/kg did not show any localized toxicity in the liver and kidney tissues. In addition to unraveling the binding interaction between kinetin and A2a-R, our findings suggest safe dose limits for the future use of kinetin as a therapeutic and modulatory agent against various pathophysiological conditions.},
  language  = {en}
}