@phdthesis{Dill2012,
  author    = {Dill, Holger},
  title     = {Functional characterization of the microRNA-26 family in zebrafish neurogenesis},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-70757},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {Formation oft the central nervous system (CNS) from multipotent neuronal stem cells (NSCs) requires a tightly controlled, step-wise activation of the neuronal gene expression program. Expression of neuronal genes at the transition from neural stem cell to mature neuron (i. e. neuronal cell differentiation) is controlled by the Repressor element 1 (RE1) silencing transcription factor (REST) complex. As a master transcriptional regulator, the REST-complex specifically inhibits expression of neuronal genes in non-neuronal tissues and neuronal progenitor cells. Differentiation of NSCs to mature neurons requires the activation of genes controlled by the REST-complex, but how abrogation of REST-complex mediated repression is achieved during neurogenesis is only poorly understood. MicroRNAs (miRNAs) are a class of small regulatory RNAs that posttranscriptionally control target gene expression. Binding of miRNAs to target sequences in the 3'UTR of mRNAs, leads either to degradation or translational inhibition of the mRNA. Distinct neuronal miRNAs (e.g. miR-124) were shown to modulate REST-complex activity by silencing expression of REST-complex components. Interestingly, these miRNAs are also under transcriptional control of the REST-complex and inactivation of the REST-complex precedes their expression. Hence, additional factors are required for derepression of neuronal genes at the onset of neurogenesis. In this study function of the miR-26 family during neurogenesis of the zebrafish (Danio rerio) was analyzed. Computational target prediction revealed a number of REST-complex components as putative miR-26 targets. One of these predicted target genes, the C-terminal domain small phosphatase 2 (Ctdsp2) was validated as an in vivo target for miR-26b. Ctdsps are important cofactors of REST and suppress neuronal gene expression by dephosphorylating the C-terminal domain (CTD) of RNA polymerase II (Pol II). Interestingly, miR-26b is encoded in an intron of the ctdsp2 primary transcript and is cotranscribed together with its host gene. Hence, miR-26b modulates expression of its host gene ctdsp2 in an intrinsic negative autoregulatory loop. This negative autoregulatory loop is inactive in NSCs because miR-26b biogenesis is inhibited at the precursor level. Generation of mature miR-26b is activated during neurogenesis, where it suppresses Ctdsp2 protein expression and is required for neuronal cell differentiation in vivo. Strikingly, miR-26b is expressed prior to miR-124 during neuronal cell differentiation. Thus, it is reasonable to speculate about a function of miR-26b in early events of neurogenesis. In line with this assumption, knockdown of miR-26b in zebrafish embryos results in downregulation of REST-complex controlled neuronal genes and a block in neuronal cell differentiation, most likely due to aberrant regulation of Ctdsp2 expression. This is evident by reduced numbers of secondary motor neurons compared to control siblings. In contrast, motor neuron progenitor cells and glia cells were not affected by depletion of miR-26b.This study identifies the ctdsp2/miR-26b autoregulatory loop as the first experimentally validated interaction between an intronic miRNA and its host gene transcript. Silencing of ctdsp2 by miR-26b in neurons is possible because biogenesis of the ctdsp2 mRNA and mature mir-26b is uncoupled at the posttranscriptional level. Furthermore the obtained data indicate a cell type specific role for miR-26b in vertebrate neurogenesis and CNS development.},
  subject      = {Zebrab{\"a}rbling},
  language  = {en}
}
@article{DonatRotherSchaeferetal.2014,
  author    = {Donat, Ulrike and Rother, Juliane and Sch{\"a}fer, Simon and Hess, Michael and H{\"a}rtl, Barbara and Kober, Christina and Langbein-Laugwitz, Johanna and Stritzker, Jochen and Chen, Nanhai G. and Aguilar, Richard J. and Weibel, Stephanie and Szalay, Alandar A.},
  title     = {Characterization of Metastasis Formation and Virotherapy in the Human C33A Cervical Cancer Model},
  series = {PLoS ONE},
  volume    = {9},
  journal   = {PLoS ONE},
  number    = {6},
  issn      = {1932-6203},
  doi       = {10.1371/journal.pone.0098533},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-119674},
  pages     = {e98533},
  year      = {2014},
  abstract  = {More than 90\% of cancer mortalities are due to cancer that has metastasized. Therefore, it is crucial to intensify research on metastasis formation and therapy. Here, we describe for the first time the metastasizing ability of the human cervical cancer cell line C33A in athymic nude mice after subcutaneous implantation of tumor cells. In this model, we demonstrated a steady progression of lumbar and renal lymph node metastases during tumor development. Besides predominantly occurring lymphatic metastases, we visualized the formation of hematogenous metastases utilizing red fluorescent protein (RFP) expressing C33A-RFP cells. RFP positive cancer cells were found migrating in blood vessels and forming micrometastases in lungs of tumor-bearing mice. Next, we set out to analyze the influence of oncolytic virotherapy in the C33A-RFP model and demonstrated an efficient virus-mediated reduction of tumor size and metastatic burden. These results suggest the C33A-RFP cervical cancer model as a new platform to analyze cancer metastases as well as to test novel treatment options to combat metastases.},
  language  = {en}
}
@article{DraganovSantidrianMinevetal.2019,
  author    = {Draganov, Dobrin D. and Santidrian, Antonio F. and Minev, Ivelina and Duong, Nguyen and Kilinc, Mehmet Okyay and Petrov, Ivan and Vyalkova, Anna and Lander, Elliot and Berman, Mark and Minev, Boris and Szalay, Aladar A.},
  title     = {Delivery of oncolytic vaccinia virus by matched allogeneic stem cells overcomes critical innate and adaptive immune barriers},
  series = {Journal of Translational Medicine},
  volume    = {17},
  journal   = {Journal of Translational Medicine},
  issn      = {100},
  doi       = {10.1186/s12967-019-1829-z},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-226312},
  year      = {2019},
  abstract  = {Background Previous studies have identified IFNγ as an important early barrier to oncolytic viruses including vaccinia. The existing innate and adaptive immune barriers restricting oncolytic virotherapy, however, can be overcome using autologous or allogeneic mesenchymal stem cells as carrier cells with unique immunosuppressive properties. Methods To test the ability of mesenchymal stem cells to overcome innate and adaptive immune barriers and to successfully deliver oncolytic vaccinia virus to tumor cells, we performed flow cytometry and virus plaque assay analysis of ex vivo co-cultures of stem cells infected with vaccinia virus in the presence of peripheral blood mononuclear cells from healthy donors. Comparative analysis was performed to establish statistically significant correlations and to evaluate the effect of stem cells on the activity of key immune cell populations. Results Here, we demonstrate that adipose-derived stem cells (ADSCs) have the potential to eradicate resistant tumor cells through a combination of potent virus amplification and sensitization of the tumor cells to virus infection. Moreover, the ADSCs demonstrate ability to function as a virus-amplifying Trojan horse in the presence of both autologous and allogeneic human PBMCs, which can be linked to the intrinsic immunosuppressive properties of stem cells and their unique potential to overcome innate and adaptive immune barriers. The clinical application of ready-to-use ex vivo expanded allogeneic stem cell lines, however, appears significantly restricted by patient-specific allogeneic differences associated with the induction of potent anti-stem cell cytotoxic and IFNγ responses. These allogeneic responses originate from both innate (NK)- and adaptive (T)- immune cells and might compromise therapeutic efficacy through direct elimination of the stem cells or the induction of an anti-viral state, which can block the potential of the Trojan horse to amplify and deliver vaccinia virus to the tumor. Conclusions Overall, our findings and data indicate the feasibility to establish simple and informative assays that capture critically important patient-specific differences in the immune responses to the virus and stem cells, which allows for proper patient-stem cell matching and enables the effective use of off-the-shelf allogeneic cell-based delivery platforms, thus providing a more practical and commercially viable alternative to the autologous stem cell approach.},
  language  = {en}
}
@article{DuggalGeissingerZhangetal.2013,
  author    = {Duggal, Rohit and Geissinger, Ulrike and Zhang, Qian and Aguilar, Jason and Chen, Nanhai G. and Binda, Elena and Vescovi, Angelo L. and Szalay, Aladar A.},
  title     = {Vaccinia virus expressing bone morphogenetic protein-4 in novel glioblastoma orthotopic models facilitates enhanced tumor regression and long-term survival},
  series = {Journal of Translational Medicine},
  volume    = {11},
  journal   = {Journal of Translational Medicine},
  number    = {155},
  doi       = {10.1186/1479-5876-11-155},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-129626},
  year      = {2013},
  abstract  = {No abstract availableBackground: Glioblastoma multiforme (GBM) is one of the most aggressive forms of cancer with a high rate of recurrence. We propose a novel oncolytic vaccinia virus (VACV)-based therapy using expression of the bone morphogenetic protein (BMP)-4 for treating GBM and preventing recurrence. Methods: We have utilized clinically relevant, orthotopic xenograft models of GBM based on tumor-biopsy derived, primary cancer stem cell (CSC) lines. One of the cell lines, after being transduced with a cDNA encoding firefly luciferase, could be used for real time tumor imaging. A VACV that expresses BMP-4 was constructed and utilized for infecting several primary glioma cultures besides conventional serum-grown glioma cell lines. This virus was also delivered intracranially upon implantation of the GBM CSCs in mice to determine effects on tumor growth. Results: We found that the VACV that overexpresses BMP-4 demonstrated heightened replication and cytotoxic activity in GBM CSC cultures with a broad spectrum of activity across several different patient-biopsy cultures. Intracranial inoculation of mice with this virus resulted in a tumor size equal to or below that at the time of injection. This resulted in survival of 100\% of the treated mice up to 84 days post inoculation, significantly superior to that of a VACV lacking BMP-4 expression. When mice with a higher tumor burden were injected with the VACV lacking BMP-4, 80\% of the mice showed tumor recurrence. In contrast, no recurrence was seen when mice were injected with the VACV expressing BMP-4, possibly due to induction of differentiation in the CSC population and subsequently serving as a better host for VACV infection and oncolysis. This lack of recurrence resulted in superior survival in the BMP-4 VACV treated group. Conclusions: Based on these findings we propose a novel VACV therapy for treating GBM, which would allow tumor specific production of drugs in the future in combination with BMPs which would simultaneously control tumor maintenance and facilitate CSC differentiation, respectively, thereby causing sustained tumor regression without recurrence.},
  language  = {en}
}
@phdthesis{Eggert2005,
  author    = {Eggert, Christian},
  title     = {Untersuchungen zur Biogenese spleißosomaler UsnRNPs und ihrer Bedeutung f{\"u}r die Pathogenese der SMA},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-15334},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2005},
  abstract  = {Die neurodegenerative Krankheit Spinale Muskelatrophie (SMA) wird durch den Mangel an funktionellem Survival Motor Neuron Protein (SMN) verursacht. Eine Funktion von SMN liegt in der Biogenese spleißosomaler UsnRNPs (U-rich small nuclear ribonucleoprotein particles). Diese Arbeit zeigt in einem SMA-Modell in Hela-Zellkultur, dass der SMN-Mangel zu einer reduzierten de novo-Produktion der spleißosomalen UsnRNPs f{\"u}hrt. In einem Zebrafisch-Modell f{\"u}r SMA wurde nachgewiesen, dass die reduzierte UsnRNP-Produktion die Degenerationen von Axonen der Motoneuronen verursacht, einen Ph{\"a}notyp wie er bei SMA auftritt. Damit konnte erstmals eine direkte Verbindung zwischen einer zellul{\"a}ren Funktion von SMN und der Entstehung von SMA hergestellt werden.},
  subject      = {Spinale Muskelatrophie},
  language  = {de}
}
@phdthesis{Ehrig2012,
  author    = {Ehrig, Klaas},
  title     = {Effects of stem cell transcription factor-expressing vaccinia viruses in oncolytic virotherapy},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85139},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2012},
  abstract  = {Krebserkrankungen bleiben auch im Jahr 2012 die zweith{\"a}uftigste Todesursache in der industralisierten Welt. Zus{\"a}tzlich hat die Etablierung der Krebsstammzell-Hypothese grunds{\"a}tzliche Auswirkungen auf die Erfolgsaussichten konventioneller Krebstherapie, wie Chemotherapie oder Strahlentherapie. Deswegen ist es von gr{\"o}βter Notwendigkeit, dass neue Ans{\"a}tze zur Krebstherapie entwickelt werden, die den Ausgang der Behandlung verbessern und zu weniger Nebenwirkungen f{\"u}hren. Diverse vorklinische Studien haben gezeigt, dass die onkolytische Virotherapie mit Vaccinia-Viren ein potentes und gut tolerierbares neues Werkzeug in der Krebstherapie darstellt. Die Effizienz des Vaccinia-Virus als Therapeutikum allein oder in Kombination mit Strahlen- oder Chemotherapie wird aktuell in mehreren klinischen Studien der Phasen I \& II getestet. Krebsstammzellen und Stammzellen teilen eine Vielzahl von Eigenschaften, wie die F{\"a}higkeit zur Selbst-Erneuerung und Pluripotenz, Stilllegung der Zellproliferation, Resistenz gegen Medikamente oder Bestrahlung, die Expression von diversen Zelloberfl{\"a}chen-molek{\"u}len, die Aktivierung und Hemmung spezifischer Signaltransduktionswege oder die Expression von Stammzell-spezifischen Genen. In dieser Arbeit wurden zwei neue rekombinante Vaccinia-Viren entwickelt, welche die Stammzell-Transkriptionsfaktoren Nanog (GLV-1h205) und Oct4 (GLV-1h208) exprimieren, um tiefere Einblicke in die Rolle dieser Masterregulatoren in der Entstehung von Krebs und ihrem Einfluss auf die onkolytische Virotherapie zu gewinnen. Das Replikationspotential beider Virusst{\"a}mme in menschlichen A549-Zellen und PC-3-Zellen wurde anhand von Replikations-Assays bestimmt. Die Expression der Virus-spezifischen Markergene Ruc-GFP und beta-Galaktosidase, wie auch die Expression der Transkriptionsfaktoren Nanog und Oct4 wurde mit Hilfe von RT-PCR, SDS-PAGE und Western blotting, sowie immunozytochemischen Experimenten nachgewiesen. Des Weiteren wurde der Einfluss einer GLV-1h205-Infektion von A549-Zellen auf den Zellzyklus untersucht. Zudem wurde die Bedeutung der Virus-vermittelten Transkriptionsfaktor-Expression auf die Behandlung von subkutanen A549-Tumoren in einem Xenograft-Modell untersucht. Zur Untersuchung, ob die beobachteten Vorteile in der Behandlung von Lungenadenokarzinomen in M{\"a}usen mit GLV-1h205 Promoter- oder Transkriptionsfaktor-abh{\"a}ngig sind, wurde ein Kontroll-Virus (GLV-1h321) hergestellt, dass f{\"u}r eine unfunktionale Nanog-Mutante codiert. Mittels SDS-PAGE und Western blotting sowie Immunozytochemie wurde die Transgen-Expression analysiert. Ein weitere Aspekt dieser Arbeit war die Fragestellung, ob sich das onkolyische Vaccinia-Virus GLV-1h68 eignet, als neues und weniger invasives Therapeutikum effizient Darmkrebszellen zu infizieren um sich in ihnen zu replizieren und diese anschlieβend zu lysieren. Ein derartiger Therapieansatz w{\"u}rde besonders im Hinblick auf sp{\"a}t diagnostizierten, metastasierenden Darmkrebs eine interessante Behandlungsalternative darstellen. Virale Markergen-expression wurde anhand von Fluoreszenzmikroskopie und FACS-Analyse untersucht. Desweiteren wurde gezeigt, dass die einmalige Administration von GLV-1h68 in mindestens zwei verschiedenen Darmkrebszelllinien zu einer signifikanten Inhibierung des Tumorwachstums in vivo und zu signifikant verbessertem {\"U}berleben f{\"u}hrt. Der Transkriptionsfaktor Klf4 wird zwar stark in ruhenden, ausdifferenzierten Zellen des Darmepithels exprimiert, ist hingegen bei Darmkrebs generell dramatisch herabreguliert. Die Expression von Klf4 f{\"u}hrt zu einem Stop der Zellproliferation und inhibiert die Aktivit{\"a}t des Wnt-Signalweges, indem es im Zellkern an die Transaktivierungsdom{\"a}ne von beta-Catenin bindet. Um die Behandlung von Darmkrebs mit Hilfe onkolytischer Virotherapie weiter zu verbessern, wurden verschiedene Vaccinia-Viren (GLV-1h290-292) erzeugt, die durch verschiedene Promoterst{\"a}rken die Expression unterschiedlicher Mengen an Tumorsuppressor Klf4 vermitteln. Die anf{\"a}ngliche Charakterisierung der drei Virusst{\"a}mme mittels Replikations-Assay, Zytotoxizit{\"a}tstudien, SDS-PAGE und Western blotting, Immunozytochemie sowie die Analyse der Proteinfunktion mit Hilfe von qPCR- und ELISA-Analysen zur Bestimmung von zellul{\"a}rem beta-Catenin, zeigten eine Promoter-abh{\"a}ngige Expression und Wirkung von Klf4. F{\"u}r weitere Analysen wurde das Virus GLV-1h291 gew{\"a}hlt, welches nach Infektion die gr{\"o}βte Menge an Klf4 produziert und zus{\"a}tzlich durch die C-terminale Fusion einer TAT Transduktionsdom{\"a}ne Membran-g{\"a}ngig gemacht (GLV-1h391). Die erhaltenen Befunde machen das Klf4-TAT-kodierende Vaccinia-Virus GLV-1h391 zu einem vielversprechenden Kandidaten f{\"u}r eine Behandlung von Darmkrebs beim Menschen.},
  subject      = {Lungenkrebs},
  language  = {en}
}
@article{EhrigKilincChenetal.2013,
  author    = {Ehrig, Klaas and Kilinc, Mehmet O. and Chen, Nanhai G. and Stritzker, Jochen and Buckel, Lisa and Zhang, Qian and Szalay, Aladar A.},
  title     = {Growth inhibition of different human colorectal cancer xenografts after a single intravenous injection of oncolytic vaccinia virus GLV-1h68},
  series = {Journal of Translational Medicine},
  volume    = {11},
  journal   = {Journal of Translational Medicine},
  number    = {79},
  doi       = {10.1186/1479-5876-11-79},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-129619},
  year      = {2013},
  abstract  = {Background: Despite availability of efficient treatment regimens for early stage colorectal cancer, treatment regimens for late stage colorectal cancer are generally not effective and thus need improvement. Oncolytic virotherapy using replication-competent vaccinia virus (VACV) strains is a promising new strategy for therapy of a variety of human cancers. Methods: Oncolytic efficacy of replication-competent vaccinia virus GLV-1h68 was analyzed in both, cell cultures and subcutaneous xenograft tumor models. Results: In this study we demonstrated for the first time that the replication-competent recombinant VACV GLV-1h68 efficiently infected, replicated in, and subsequently lysed various human colorectal cancer lines (Colo 205, HCT-15, HCT-116, HT-29, and SW-620) derived from patients at all four stages of disease. Additionally, in tumor xenograft models in athymic nude mice, a single injection of intravenously administered GLV-1h68 significantly inhibited tumor growth of two different human colorectal cell line tumors (Duke's type A-stage HCT-116 and Duke's type C-stage SW-620), significantly improving survival compared to untreated mice. Expression of the viral marker gene ruc-gfp allowed for real-time analysis of the virus infection in cell cultures and in mice. GLV-1h68 treatment was well-tolerated in all animals and viral replication was confined to the tumor. GLV-1h68 treatment elicited a significant up-regulation of murine immune-related antigens like IFN-γ, IP-10, MCP-1, MCP-3, MCP-5, RANTES and TNF-γ and a greater infiltration of macrophages and NK cells in tumors as compared to untreated controls. Conclusion: The anti-tumor activity observed against colorectal cancer cells in these studies was a result of direct viral oncolysis by GLV-1h68 and inflammation-mediated innate immune responses. The therapeutic effects occurred in tumors regardless of the stage of disease from which the cells were derived. Thus, the recombinant vaccinia virus GLV-1h68 has the potential to treat colorectal cancers independently of the stage of progression.},
  language  = {en}
}
@phdthesis{Englert2005,
  author    = {Englert, Markus},
  title     = {Mechanismus des pre-tRNA-Spleißens : Struktur und Funktion pflanzlicher und animaler RNA-Ligasen},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-14420},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2005},
  abstract  = {Transfer Ribonukleins{\"a}uren werden von der RNA Polymerase III als Vorl{\"a}ufer tRNA transkribiert und durchlaufen eine Vielzahl von Reifungsschritten hin zur maturen tRNA. Neben der Hydrolyse der 5´- und 3´-Flanke durch die RNase P und die tRNase Z, sowie einer Vielzahl von Basenmodifizierungen, wird bei einigen pre-tRNAs das Intron herausgespleißt. Die ersten intronhaltigen tRNA Gene wurden in der Hefe Saccharomyces cerevisiae nachgewiesen und folglich wurde der Spleißmechanismus in diesem Organismus als erstes untersucht. Eine tetramere tRNA Spleißendonuklease spaltet das Intron an den Exongrenzen heraus und eine tRNA Ligase ligiert die entstandenen tRNA H{\"a}lften zur gespleißten tRNA. Einzig in der Hefe und anderen Pilzen konnten bisher die Gene f{\"u}r die tRNA Ligase identifiziert werden. Weder molekularbiologische Ans{\"a}tze - wie z.B. DNA Hybridisierung, Expressions-"Screening" und funktionelle Komplementationsstudien mit einem tRNA Ligase-defizienten Hefestamm - noch Datenbanksuchen mit der bekannten Hefe tRNA Ligasesequenz haben in den vergangenen Jahren zur Identifizierung eines pflanzlichen oder animalen tRNA Ligase Gens gef{\"u}hrt. In dieser Arbeit ist es erstmals gelungen, das tRNA Ligase Protein aus Weizenkeimen bis zur Homogenit{\"a}t zu isolieren und mit Hilfe erhaltener Peptidsequenzen die entsprechenden Kern-codierten Gene in h{\"o}heren und niederer Pflanzen zu identifizieren. Die Ligaseaktivit{\"a}t wurde f{\"u}r das klonierte, rekombinant {\"u}berexprimierte tRNA Ligaseprotein best{\"a}tigt. Weiterhin wurde zum ersten Mal das Ligaseprotein aus Schweineleber aufgereinigt und das zugeh{\"o}rige Gen im humanen Genom identifiziert.},
  subject      = {Pflanzen},
  language  = {de}
}
@phdthesis{Faul2004,
  author    = {Faul, Thomas},
  title     = {Lokalisation und Dynamik der Replikationsproteine des murinen pr{\"a}-replikativen Komplexes},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-10473},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2004},
  abstract  = {Ein Ziel der vorliegenden Arbeit war die Untersuchung der Lokalisation und der Dynamik der Replikationsproteine des murinen pr{\"a}-replikativen Komplexes in vivo. Dazu wurden die zu untersuchenden Replikationsproteine als EGFP-Fusionsproteine in LTK--Zellen exprimiert und am konfokalen Laserscanning-Mikroskop untersucht. CDC6-EGFP war in der G1-Phase diffus in Zellkern und Cytoplasma verteilt, am G1/S-{\"U}bergang ausschließlich im Zellkern lokalisiert und w{\"a}hrend der S-Phase in zahlreichen Foci im Kern akkumuliert. CDC6-EGFP war mit Replikationsfoci colokalisiert. Endogenes Cdc6p wies dieselbe subzellul{\"a}re Verteilung wie CDC6-EGFP auf. Auch Fusionsproteine des humanen Proteins Cdc6p waren in HEK-293T-Zellen in Replikationsfoci lokalisiert. FRAP-Studien ergaben, dass 80-90 \% von CDC6-EGFP w{\"a}hrend der gesamten S-Phase stabil mit der Replikationsmaschinerie assoziiert sind. Durch Mutation der Phosphoryliersstellen f{\"u}r Cyclin-abh{\"a}ngige Proteinkinasen wurde der Einfluss des Phosphorylierungsstatus der konservierten Serinreste der Cdk-Phosphorylierungsstellen auf die Lokalisation von CDC6-EGFP in vivo untersucht. Alle Mutanten bei denen die Cdk-Serinreste zu nicht-phosphorylierbaren Alaninresten mutiert wurden waren in Replikationsfoci lokalisiert. Dies zeigt, dass die Phosphorylierung dieser Serinreste f{\"u}r die Lokalisation von CDC6-EGFP an Stellen aktiver DNA-Replikation nicht essentiell ist. Durch Mutation der Serinreste zu Phosphatreste-simulierenden Aspartatresten konnte gezeigt werden, dass die Phosphorylierung des Serinrests S102 zum Export von CDC6-EGFP aus dem Zellkern f{\"u}hrt. FRAP-Studien ergaben, dass CDC6-EGFP in Replikationsfoci an Serinrest 82 phosphoryliert und an Serinrest 102 dephosphoryliert vorliegt. Mit Immunfluoreszenz-Analysen konnte gezeigt werden, dass Chromatin in Replikationsfoci nicht acetyliert ist. Dies deutet darauf hin, dass die Elongation der DNA-Replikation an nicht-acetyliertem Chromatin erfolgt. Trichostatin A-induzierte Hyperacetylierung des Chromatins hatte keinen Einfluss auf Lokalisation und Mobilit{\"a}t von CDC6-EGFP in Replikationsfoci. Die Mobilit{\"a}t des nucleoplasmatischen CDC6-EGFP-Pools wurde dadurch erh{\"o}ht. In der G1-Phase wurde die Mobilit{\"a}t von CDC6-EGFP durch TSA verringert, woraus gefolgert werden kann, dass der Acetylierungsstatus des Chromatins in der G1-Phase die Mobilit{\"a}t von CDC6-EGFP beeinflusst. ORC1-EGFP war im Zellkern in großen kugelf{\"o}rmigen Strukturen lokalisiert, ORC2-EGFP war diffus in Cytoplasma und Zellkern verteilt. ORC3-EGFP akkumulierte in PML nuclear bodies. W{\"a}hrend ORC4-EGFP und ORC5-EGFP am Centrosom lokalisiert waren konnte ORC6-EGFP in Nucleoli nachgewiesen werden. Die EGFP-Fusionsproteine von Cdc45p, PCNA und DNA-Ligase-I waren im Zellkern lokalisiert, die Nucleoli waren ausgespart. Ein weiterer Aspekt dieser Arbeit war die Untersuchung der Substratspezifit{\"a}t der murinen Cdc7p/Dbf4p-Proteinkinase. Die in Sf9-Zellen exprimierte und aufgereinigte Kinase phosphorylierte Orc2p, Orc6p, Cdc45p und Mcm6p. Mit Phosphopeptidkartierungen konnte gezeigt werden, dass Cdc7p von CylinE/Cdk2 an zwei Stellen und von CyclinA/Cdk2 an einer Stelle in vitro phosphoryliert wird. CDC7-EGFP war in der G1-Phase, am G1/S-{\"U}bergang und in der S-Phase im Kern lokalisiert. Durch FISH-Experimente konnte der genomische Locus des murinen Cdc7-Gens der Bande E von Chromosom 5 zugeordnet werden. Mit Kinase-Assays wurde untersucht, ob die murine Plk1p-Kinase Initiationsfaktoren der DNA-Replikation in vitro phosphoryliert. Die in Sf9-Zellen exprimierte Plk1p phosphorylierte Cdc7p, Orc2p und Orc6p. Cdc7p und Orc6p sind mit Plk1p am Midbody w{\"a}hrend der Telophase in vivo colokalisiert. Ein weiteres Ziel dieser Arbeit war die Messung der Mobilit{\"a}t des murinen Transkriptions-Terminationsfaktors TTF-I mittels FRAP. EGFP-TTF-I und EGFP-NRD waren diffus in den Nucleoli verteilt, einzelne Areale waren ausgespart. EGFP-TTFdeltaN185 war hingegen in distinkten nucleol{\"a}ren Stellen akkumuliert. Mit FRAP-Studien konnte gezeigt werden, dass EGFP-TTFdeltaN185 in einer 10 \%igen immobilen Fraktion vorlag w{\"a}hrend das Gesamtprotein EGFP-TTF-I zu 100\% mobil war. Das Protein TIP5 interagiert mit TTF-I. EGFP-TIP5 war diffus im Nucleoplasma verteilt, die Ncleoli waren ausgespart. Durch Cotransfektionen verschiedener EYFP-TTF-I-Konstrukte mit EGFP-TIP5 konnte gezeigt werden, dass EGFP-TIP5 von EYFP-TTFdeltaN185 nicht in Nucleoli cotransportiert wird. Mit BRET-Studien ergaben, dass Orc6p mit TTF-I in vivo interagiert. Eine Interaktion mit TTFdeltaN185 war nicht nachweisbar.},
  subject      = {Maus},
  language  = {de}
}
@article{FetivaLissGertzmannetal.2023,
  author    = {Fetiva, Maria Camila and Liss, Franziska and Gertzmann, D{\"o}rthe and Thomas, Julius and Gantert, Benedikt and Vogl, Magdalena and Sira, Nataliia and Weinstock, Grit and Kneitz, Susanne and Ade, Carsten P. and Gaubatz, Stefan},
  title     = {Oncogenic YAP mediates changes in chromatin accessibility and activity that drive cell cycle gene expression and cell migration},
  series = {Nucleic Acids Research},
  volume    = {51},
  journal   = {Nucleic Acids Research},
  number    = {9},
  doi       = {10.1093/nar/gkad107},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-350218},
  pages     = {4266-4283},
  year      = {2023},
  abstract  = {YAP, the key protein effector of the Hippo pathway, is a transcriptional co-activator that controls the expression of cell cycle genes, promotes cell growth and proliferation and regulates organ size. YAP modulates gene transcription by binding to distal enhancers, but the mechanisms of gene regulation by YAP-bound enhancers remain poorly understood. Here we show that constitutive active YAP5SA leads to widespread changes in chromatin accessibility in untransformed MCF10A cells. Newly accessible regions include YAP-bound enhancers that mediate activation of cycle genes regulated by the Myb-MuvB (MMB) complex. By CRISPR-interference we identify a role for YAP-bound enhancers in phosphorylation of Pol II at Ser5 at MMB-regulated promoters, extending previously published studies that suggested YAP primarily regulates the pause-release step and transcriptional elongation. YAP5SA also leads to less accessible 'closed' chromatin regions, which are not directly YAP-bound but which contain binding motifs for the p53 family of transcription factors. Diminished accessibility at these regions is, at least in part, a consequence of reduced expression and chromatin-binding of the p53 family member ΔNp63 resulting in downregulation of ΔNp63-target genes and promoting YAP-mediated cell migration. In summary, our studies uncover changes in chromatin accessibility and activity that contribute to the oncogenic activities of YAP.},
  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{Geissinger2010,
  author    = {Geissinger, Ulrike},
  title     = {Vaccinia Virus-mediated MR Imaging of Tumors in Mice: Overexpression of Iron-binding Proteins in Colonized Xenografts},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-48099},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2010},
  abstract  = {Vaccinia virus plays an important role in human medicine and molecular biology ever since the 18th century after E. Jenner discovered its value as a vaccination virus against smallpox. After the successful eradication of smallpox, vaccinia virus, apart from its use as a vaccine carrier, is today mainly used as a viral vector in molecular biology and increasingly in cancer therapy. The capability to specifically target and destroy cancer cells makes it a perfect agent for oncolytic virotherapy. Furthermore, the virus can easily be modified by inserting genes encoding therapeutic or diagnostic proteins to be expressed within the tumor. The emphasis in this study was the diagnosis of tumors using different vaccinia virus strains. Viruses with metal-accumulating capabilities for tumor detection via MRI technology were generated and tested for their usefulness in cell culture and in vivo. The virus strains GLV-1h131, GLV-1h132, and GLV-1h133 carry the gene encoding the two subunits of the iron storage protein ferritin under the control of three different promoters. GLV-1h110, GLV-1h111, and GLV-1h112 encode the bacterial iron storage protein bacterioferritin, whereas GLV-1h113 encodes the codon-optimized version of bacterioferritin for more efficient expression in human cells. GLV-1h22 contains the transferrin receptor gene, which plays an important role in iron uptake, and GLV-1h114 and GLV-1h115 contain the murine transferrin receptor gene. For possibly better iron uptake the virus strains GLV-1h154, GLV-1h155, GLV-1h156, and GLV-1h157 were generated, each with a version of a ferritin gene and a transferrin receptor gene. GLV-1h154 carries the genes that encode bacterioferritin and human transferrin receptor, GLV-1h155 the human ferritin H-chain gene and the human transferrin receptor gene. GLV-1h156 and GLV-1h157 infected cells both express the mouse transferrin receptor and bacterioferritin or human ferritin H-chain, respectively. The virus strains GLV-1h186 and GLV-1h187 were generated to contain a mutated form of the ferritin light chain, which was shown to result in iron overload and the wildtype light chain gene, respectively. The gene encoding the Divalent Metal Transporter 1, which is a major protein in the uptake of iron, was inserted in the virus strain GLV-1h102. The virus strain GLV-1h184 contains the magA gene of the magnetotactic bacterium Magnetospirillum magnetotacticum, which produces magnetic nanoparticles for orientation in the earth's magnetic field. Initially the infection and replication capability of all the virus strains were analyzed and compared to that of the parental virus strain GLV-1h68, revealing that all the viruses were able to infect cells of the human cancer cell lines A549 and GI-101A. All constructs exhibited a course of infection comparable to that of GLV-1h68. Next, to investigate the expression of the foreign proteins in GI-101A and A549 cells with protein analytical methods, SDS-gelelectrophoresis, Western blots and ELISAs were performed. The proteins, which were expressed under the control of the strong promoters, could be detected using these methods. To be able to successfully detect the protein expression of MagA and DMT1, which were expressed under the control of the weak promoter, the more sensitive method RT-PCR was used to at least confirm the transcription of the inserted genes. The determination of the iron content in infected GI-101A and A549 cells showed that infection with all used virus strains led to iron accumulation in comparison to uninfected cells, even infection with the parental virus strain GLV-1h68. The synthetic phytochelatin EC20 was also shown to enhance the accumulation of different heavy metals in bacterial cultures. In vivo experiments with A549 tumor-bearing athymic nude mice revealed that 24 days post infection virus particles were found mainly in the tumor. The virus-mediated expression of recombinant proteins in the tumors was detected successfully by Western blot. Iron accumulation in tumor lysates was investigated by using the ferrozine assay and led to the result that GLV-1h68-infected tumors had the highest iron content. Histological stainings confirmed the finding that iron accumulation was not a direct result of the insertion of genes encoding iron-accumulating proteins in the virus genome. Furthermore virus-injected tumorous mice were analyzed using MRI technology. Two different measurements were performed, the first scan being done with a seven Tesla small animal scanner seven days post infection whereas the second scan was performed using a three Tesla human scanner 21 days after virus injection. Tumors of mice injected with the virus strains GLV-1h113 and GLV-1h184 were shown to exhibit shortened T2 and T2* relaxation times, which indicates enhanced iron accumulation. In conclusion, the experiments in this study suggest that the bacterioferritin-encoding virus strain GLV-1h113 and the magA-encoding virus strain GLV-1h184 are promising candidates to be used for cancer imaging after further analyzation and optimization.},
  subject      = {Vaccinia-Virus},
  language  = {en}
}
@article{GentschevAdelfingerJosupeitetal.2012,
  author    = {Gentschev, Ivaylo and Adelfinger, Marion and Josupeit, Rafael and Rudolph, Stephan and Ehrig, Klaas and Donat, Ulrike and Weibel, Stephanie and Chen, Nanhai G. and Yu, Yong A. and Zhang, Qian and Heisig, Martin and Thamm, Douglas and Stritzker, Jochen and MacNeill, Amy and Szalay, Aladar A.},
  title     = {Preclinical Evaluation of Oncolytic Vaccinia Virus for Therapy of Canine Soft Tissue Sarcoma},
  series = {PLoS One},
  volume    = {7},
  journal   = {PLoS One},
  number    = {5},
  doi       = {10.1371/journal.pone.0037239},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-129998},
  year      = {2012},
  abstract  = {Virotherapy using oncolytic vaccinia virus (VACV) strains is one promising new strategy for canine cancer therapy. In this study we describe the establishment of an in vivo model of canine soft tissue sarcoma (CSTS) using the new isolated cell line STSA-1 and the analysis of the virus-mediated oncolytic and immunological effects of two different Lister VACV LIVP1.1.1 and GLV-1h68 strains against CSTS. Cell culture data demonstrated that both tested VACV strains efficiently infected and destroyed cells of the canine soft tissue sarcoma line STSA-1. In addition, in our new canine sarcoma tumor xenograft mouse model, systemic administration of LIVP1.1.1 or GLV-1h68 viruses led to significant inhibition of tumor growth compared to control mice. Furthermore, LIVP1.1.1 mediated therapy resulted in almost complete tumor regression and resulted in long-term survival of sarcoma-bearing mice. The replication of the tested VACV strains in tumor tissues led to strong oncolytic effects accompanied by an intense intratumoral infiltration of host immune cells, mainly neutrophils. These findings suggest that the direct viral oncolysis of tumor cells and the virus-dependent activation of tumor-associated host immune cells could be crucial parts of anti-tumor mechanism in STSA-1 xenografts. In summary, the data showed that both tested vaccinia virus strains and especially LIVP1.1.1 have great potential for effective treatment of CSTS.},
  language  = {en}
}
@article{GentschevMuellerAdelfingeretal.2011,
  author    = {Gentschev, Ivaylo and M{\"u}ller, Meike and Adelfinger, Marion and Weibel, Stephanie and Grummt, Friedrich and Zimmermann, Martina and Bitzer, Michael and Heisig, Martin and Zhang, Qian and Yu, Yong A. and Chen, Nanhai G. and Stritzker, Jochen and Lauer, Ulrich M. and Szalay, Aladar A.},
  title     = {Efficient Colonization and Therapy of Human Hepatocellular Carcinoma (HCC) Using the Oncolytic Vaccinia Virus Strain GLV-1h68},
  series = {PLOS ONE},
  volume    = {6},
  journal   = {PLOS ONE},
  number    = {7},
  doi       = {10.1371/journal.pone.0022069},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-135319},
  pages     = {e22069},
  year      = {2011},
  abstract  = {Virotherapy using oncolytic vaccinia virus strains is one of the most promising new strategies for cancer therapy. In this study, we analyzed for the first time the therapeutic efficacy of the oncolytic vaccinia virus GLV-1h68 in two human hepatocellular carcinoma cell lines HuH7 and PLC/PRF/5 (PLC) in cell culture and in tumor xenograft models. By viral proliferation assays and cell survival tests, we demonstrated that GLV-1h68 efficiently colonized, replicated in, and did lyse these cancer cells in culture. Experiments with HuH7 and PLC xenografts have revealed that a single intravenous injection (i.v.) of mice with GLV-1h68 resulted in a significant reduction of primary tumor sizes compared to uninjected controls. In addition, replication of GLV-1h68 in tumor cells led to strong inflammatory and oncolytic effects resulting in intense infiltration of MHC class II-positive cells like neutrophils, macrophages, B cells and dendritic cells and in up-regulation of 13 pro-inflammatory cytokines. Furthermore, GLV-1h68 infection of PLC tumors inhibited the formation of hemorrhagic structures which occur naturally in PLC tumors. Interestingly, we found a strongly reduced vascular density in infected PLC tumors only, but not in the non-hemorrhagic HuH7 tumor model. These data demonstrate that the GLV-1h68 vaccinia virus may have an enormous potential for treatment of human hepatocellular carcinoma in man.},
  language  = {en}
}
@article{GentschevPatilPetrovetal.2014,
  author    = {Gentschev, Ivaylo and Patil, Sadeep S. and Petrov, Ivan and Cappello, Joseph and Adelfinger, Marion and Szalay, Aladar A.},
  title     = {Oncolytic Virotherapy of Canine and Feline Cancer},
  series = {Viruses},
  volume    = {6},
  journal   = {Viruses},
  number    = {5},
  doi       = {10.3390/v6052122},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-119753},
  pages     = {2122-2137},
  year      = {2014},
  abstract  = {Cancer is the leading cause of disease-related death in companion animals such as dogs and cats. Despite recent progress in the diagnosis and treatment of advanced canine and feline cancer, overall patient treatment outcome has not been substantially improved. Virotherapy using oncolytic viruses is one promising new strategy for cancer therapy. Oncolytic viruses (OVs) preferentially infect and lyse cancer cells, without causing excessive damage to surrounding healthy tissue, and initiate tumor-specific immunity. The current review describes the use of different oncolytic viruses for cancer therapy and their application to canine and feline cancer.},
  language  = {en}
}
@article{GerovaWickeChiharaetal.2021,
  author    = {Gerova, Milan and Wicke, Laura and Chihara, Kotaro and Schneider, Cornelius and Lavigne, Rob and Vogel, J{\"o}rg},
  title     = {A grad-seq view of RNA and protein complexes in Pseudomonas aeruginosa under standard and bacteriophage predation conditions},
  series = {mbio},
  volume    = {12},
  journal   = {mbio},
  number    = {1},
  doi       = {10.1128/mBio.03454-20},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259054},
  pages     = {e03454-20},
  year      = {2021},
  abstract  = {The Gram-negative rod-shaped bacterium Pseudomonas aeruginosa is not only a major cause of nosocomial infections but also serves as a model species of bacterial RNA biology. While its transcriptome architecture and posttranscriptional regulation through the RNA-binding proteins Hfq, RsmA, and RsmN have been studied in detail, global information about stable RNA-protein complexes in this human pathogen is currently lacking. Here, we implement gradient profiling by sequencing (Grad-seq) in exponentially growing P. aeruginosa cells to comprehensively predict RNA and protein complexes, based on glycerol gradient sedimentation profiles of >73\% of all transcripts and ∼40\% of all proteins. As to benchmarking, our global profiles readily reported complexes of stable RNAs of P. aeruginosa, including 6S RNA with RNA polymerase and associated product RNAs (pRNAs). We observe specific clusters of noncoding RNAs, which correlate with Hfq and RsmA/N, and provide a first hint that P. aeruginosa expresses a ProQ-like FinO domain-containing RNA-binding protein. To understand how biological stress may perturb cellular RNA/protein complexes, we performed Grad-seq after infection by the bacteriophage ΦKZ. This model phage, which has a well-defined transcription profile during host takeover, displayed efficient translational utilization of phage mRNAs and tRNAs, as evident from their increased cosedimentation with ribosomal subunits. Additionally, Grad-seq experimentally determines previously overlooked phage-encoded noncoding RNAs. Taken together, the Pseudomonas protein and RNA complex data provided here will pave the way to a better understanding of RNA-protein interactions during viral predation of the bacterial cell. IMPORTANCE Stable complexes by cellular proteins and RNA molecules lie at the heart of gene regulation and physiology in any bacterium of interest. It is therefore crucial to globally determine these complexes in order to identify and characterize new molecular players and regulation mechanisms. Pseudomonads harbor some of the largest genomes known in bacteria, encoding ∼5,500 different proteins. Here, we provide a first glimpse on which proteins and cellular transcripts form stable complexes in the human pathogen Pseudomonas aeruginosa. We additionally performed this analysis with bacteria subjected to the important and frequently encountered biological stress of a bacteriophage infection. We identified several molecules with established roles in a variety of cellular pathways, which were affected by the phage and can now be explored for their role during phage infection. Most importantly, we observed strong colocalization of phage transcripts and host ribosomes, indicating the existence of specialized translation mechanisms during phage infection. All data are publicly available in an interactive and easy to use browser.},
  language  = {en}
}
@phdthesis{Gimple2004,
  author    = {Gimple, Olaf},
  title     = {Substratbindung und Katalyse in RNase P RNA vom cyanobakteriellen Typ},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-11283},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2004},
  abstract  = {Ribonuklease P (RNase P) ist eine essentielle Endonuklease, welche die 5'-Flanke von pre-tRNAs entfernt. Die RNase P RNA des Cyanobakteriums Prochlorococcus marinus ist in vitro katalytisch aktiv und bevorzugt in heterologen Prozessierungssystemen Substrate mit vollst{\"a}ndigem 3'-CCA-Ende. Diese Substratspezifit{\"a}t widerspricht den Erwartungen, da tRNAs in P. marinus nicht mit dem CCA-Ende codiert sind und die RNase P RNA auch nicht das GGU-Bindungsmotiv f{\"u}r diese CCA-Enden aufweist. Um die Substratspezifit{\"a}t und Aufbau des Ribozym-Substrat-Komplex von P. marinus RNase P RNA im homologen System untersuchen zu k{\"o}nnen, wurden Transkriptionsklone f{\"u}r P. marinus pre- und mat-tRNAArgCCU konstruiert, mit denen nach entsprechender Restriktionshydrolyse Transkripte mit stufenweise verk{\"u}rzten 3'-CCA-Ende synthetisiert werden k{\"o}nnen. Durch enzymkinetische Untersuchungen der Prozessierung durch P. marinus RNase P RNA wurde unter steady-state-Bedingungen f{\"u}r pre-tRNACCA eine Michaelis-Menten Konstante von 6,92 µM ermittelt. Die Entfernung von A76 und C75 des 3'-CCA-Endes f{\"u}hrt zu einer Erh{\"o}hung der KM (7,13 µM bzw. 19,68µM). Diese Substrate werden folglich weniger stark gebunden, was sich auch in der freien Bindungsenthalpie von 0,02 und 0,65 kcal/mol ausdr{\"u}ckt. Die Entfernung des vollst{\"a}ndigen 3'-CCA-Endes f{\"u}hrt zu einer erheblichen Erniedrigung der KM (0,83µM) und zu einer energetisch beg{\"u}nstigten, st{\"a}rkeren Substratbindung (-1,31 kcal/mol). P. marinus RNase RNase P RNA zeigt folglich bei der in vitro Prozessierung im homologen System unter steady-state-Bedingungen eine Substratspezifit{\"a}t f{\"u}r das Substrat mit deletiertem 3'-CCA-Ende. Durch die Methode des Crosslinking, die in dieser Arbeit etabliert und optimiert wurde, k{\"o}nnen RNA-Protein und RNA-RNA Interaktionen nachgewiesen werden. Mit ihr wurde die Bindung von Substrat und Produkt im Komplex mit der RNase P RNA untersucht. Durch interne Modifizierung der P. marinus RNase P RNA-Komponente mit dem photosensiblen Nukleotidanalogon s4U wurden Kontaktstellen in 5'-Flanke, Acceptor-Stamm, D-Stamm, D-Schleife, Anticodon-Schleife und in der variablen Schleife der P. marinus pre-tRNAArg identifiziert. Diese lokalisierten Kontaktstellen stehen denen in der 5'-Flanke, dem Acceptor-Stamm und der 3'-Flanke, wie sie f{\"u}r den Ribozym-Substrat-Komplex mit E. coli RNase P RNA identifiziert wurden, gegen{\"u}ber. In P. marinus RNase P RNA werden folglich alternative Kontaktstellen zur Substratbindung benutzt. Mit Hilfe der hier {\"u}berexprimierten E. coli Nukleotidyltransferase, konnte pre- und mat-tRNAArg durch eine neue Synthesestrategie am 3'-CCA-Ende mit dem Crosslink-Reagenz Azidophenacyl (APA) modifiziert werden. Durch die Positionierung von APA am 5'-Terminus von pre- und mat-tRNAArg wurden weitere modifizierte tRNAs synthetisiert. Durch Crosslink-Experimente im homologen P. marinus System mit diesen modifizierten pre- und mat-tRNAArg-Varianten wurden die selben Regionen des katalytischen Zentrums (J18/2, Region P15/P16, J5/15) der RNase P RNA identifiziert, wie sie von E. coli und B. subtilis RNase P RNA bekannt sind. Dies bedeutet, dass die 5'-Flanke, die Prozessierungsstelle und das 3'-CCA-Ende der tRNAs auf einer vergleichbaren Oberfl{\"a}che positioniert werden wie in anderen Ribozymen. Durch die fehlende Fixierung des 3'-CCA-Endes {\"u}ber Basenpaarungen mit dem GGU-Bindungsmotiv werden die tRNAs in P. marinus RNase P RNA weniger starr an das Ribozym gebunden und das 3'-CCA-Ende besitzt eine flexiblere Positionierung im Komplex mit dem Ribozym. Die Existenz unterschiedlicher Crosslink-Muster in P6, P18, J5/15 und J3/4 zeigt, dass pre-tRNAs und reife tRNAs durch verschiedene Modi an das P. marinus Ribozym gebunden werden. Die Identifizierung von vernetzten Nukleotiden in P15, J15/16, P16 und J16/15, die mit vergleichbaren modifizierten tRNAs in E. coli RNase P RNA nicht gefunden wurden, belegen, dass in P. marinus RNase P RNA ein anderer Produkt-Bindungs-Modus existiert als in E. coli. Erstmals konnten in dieser Arbeit auch zu erwartende Interaktionen mit dem katalytischen Zentrum identifiziert werden, die in bisherigen Crosslink-Experimenten in E. coli und B. subtilis RNase P RNA nicht oder nur geringf{\"u}gig auftraten. Um die erhaltenen Ergebnisse besser veranschaulichen zu k{\"o}nnen, wurde mit dem Programm ERNA 3D ein Raumstrukturmodell f{\"u}r P. marinus RNase P RNA und tRNAArg erstellt. Die RNase P RNA der Cyanellen von Cyanophora paradoxa, ist in vitro katalytisch inaktiv. Um zu kl{\"a}ren, ob die fehlende Ribozym-Aktivit{\"a}t dieser RNase P RNA auf eine fehlerhafte Substratbindung zur{\"u}ckzuf{\"u}hren ist, sollten Crosslink-Experimente mit den modifizierten P. marinus tRNAArg durchgef{\"u}hrt werden. Es konnte gezeigt werden, dass 5'- und 3'-modifizierte pre-tRNAs in C. paradoxa in einem anderen Modus gebunden werden, als durch die katalytisch aktive P. marinus RNase P RNA.},
  subject      = {Prochlorococcus marinus},
  language  = {de}
}
@article{GoncharovaRuzhenkovaPetrovetal.2016,
  author    = {Goncharova, Elena P. and Ruzhenkova, Julia S. and Petrov, Ivan S. and Shchelkunov, Sergey N. and Zenkova, Marina A.},
  title     = {Oncolytic virus efficiency inhibited growth of tumour cells with multiple drug resistant phenotype in vivo and in vitro},
  series = {Journal of Translational Medicine},
  volume    = {14},
  journal   = {Journal of Translational Medicine},
  number    = {241},
  doi       = {10.1186/s12967-016-1002-x},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-165714},
  pages     = {1-14},
  year      = {2016},
  abstract  = {Background Tumour resistance to a wide range of drugs (multiple drug resistant, MDR) acquired after intensive chemotherapy is considered to be the main obstacle of the curative treatment of cancer patients. Recent work has shown that oncolytic viruses demonstrated prominent potential for effective treatment of diverse cancers. Here, we evaluated whether genetically modified vaccinia virus (LIVP-GFP) may be effective in treatment of cancers displaying MDR phenotype. Methods LIVP-GFP replication, transgene expression and cytopathic effects were analysed in human cervical carcinomas KB-3-1 (MDR-), KB-8-5 (MDR+) and in murine melanoma B-16 (MDR-), murine lymphosarcomas RLS and RLS-40 (MDR+). To investigate the efficacy of this therapy in vivo, we treated immunocompetent mice bearing murine lymphosarcoma RLS-40 (MDR+) (6- to 8-week-old female CBA mice; n = 10/group) or melanoma B-16 (MDR-) (6- to 8-week-old female C57Bl mice; n = 6/group) with LIVP-GFP (5 × 107 PFU of virus in 0.1 mL of IMDM immediately and 4 days after tumour implantation). Results We demonstrated that LIVP-GFP replication was effective in human cervical carcinomas KB-3-1 (MDR-) and KB-8-5 (MDR+) and in murine melanoma B-16 (MDR-), whereas active viral production was not detected in murine lymphosarcomas RLS and RLS-40 (MDR+). Additionally, it was found that in tumour models in immunocompetent mice under the optimized regimen intratumoural injections of LIVP-GFP significantly inhibited melanoma B16 (33 \% of mice were with complete response after 90 days) and RLS-40 tumour growth (fourfold increase in tumour doubling time) as well as metastasis. Conclusion The anti-tumour activity of LIVP-GFP is a result of direct oncolysis of tumour cells in case of melanoma B-16 because the virus effectively replicates and destroys these cells, and virus-mediated activation of the host immune system followed by immunologically mediated destruction of of tumour cells in case of lymphosarcoma RLS-40. Thus, the recombinant vaccinia virus LIVP-GFP is able to inhibit the growth of malignant cells with the MDR phenotype and tumour metastasis when administered in the early stages of tumour development.},
  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{GrimmPelzSchneideretal.2020,
  author    = {Grimm, Clemens and Pelz, Jann-Patrick and Schneider, Cornelius and Sch{\"a}ffler, Katrin and Fischer, Utz},
  title     = {Crystal Structure of a Variant PAM2 Motif of LARP4B Bound to the MLLE Domain of PABPC1},
  series = {Biomolecules},
  volume    = {10},
  journal   = {Biomolecules},
  number    = {6},
  issn      = {2218-273X},
  doi       = {10.3390/biom10060872},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-207800},
  year      = {2020},
  abstract  = {Eukaryotic cells determine the protein output of their genetic program by regulating mRNA transcription, localization, translation and turnover rates. This regulation is accomplished by an ensemble of RNA-binding proteins (RBPs) that bind to any given mRNA, thus forming mRNPs. Poly(A) binding proteins (PABPs) are prominent members of virtually all mRNPs that possess poly(A) tails. They serve as multifunctional scaffolds, allowing the recruitment of diverse factors containing a poly(A)-interacting motif (PAM) into mRNPs. We present the crystal structure of the variant PAM motif (termed PAM2w) in the N-terminal part of the positive translation factor LARP4B, which binds to the MLLE domain of the poly(A) binding protein C1 cytoplasmic 1 (PABPC1). The structural analysis, along with mutational studies in vitro and in vivo, uncovered a new mode of interaction between PAM2 motifs and MLLE domains.},
  language  = {en}
}