@article{DoerkPeterlongoMannermaaetal.2019, author = {D{\"o}rk, Thilo and Peterlongo, Peter and Mannermaa, Arto and Bolla, Manjeet K. and Wang, Qin and Dennis, Joe and Ahearn, Thomas and Andrulis, Irene L. and Anton-Culver, Hoda and Arndt, Volker and Aronson, Kristan J. and Augustinsson, Annelie and Beane Freeman, Laura E. and Beckmann, Matthias W. and Beeghly-Fadiel, Alicia and Behrens, Sabine and Bermisheva, Marina and Blomqvist, Carl and Bogdanova, Natalia V. and Bojesen, Stig E. and Brauch, Hiltrud and Brenner, Hermann and Burwinkel, Barbara and Canzian, Federico and Chan, Tsun L. and Chang-Claude, Jenny and Chanock, Stephen J. and Choi, Ji-Yeob and Christiansen, Hans and Clarke, Christine L. and Couch, Fergus J. and Czene, Kamila and Daly, Mary B. and dos-Santos-Silva, Isabel and Dwek, Miriam and Eccles, Diana M. and Ekici, Arif B. and Eriksson, Mikael and Evans, D. Gareth and Fasching, Peter A. and Figueroa, Jonine and Flyger, Henrik and Fritschi, Lin and Gabrielson, Marike and Gago-Dominguez, Manuela and Gao, Chi and Gapstur, Susan M. and Garc{\´i}a-Closas, Montserrat and Garc{\´i}a-S{\´a}enz, Jos{\´e} A. and Gaudet, Mia M. and Giles, Graham G. and Goldberg, Mark S. and Goldgar, David E. and Guen{\´e}l, Pascal and Haeberle, Lothar and Haimann, Christopher A. and H{\aa}kansson, Niclas and Hall, Per and Hamann, Ute and Hartman, Mikael and Hauke, Jan and Hein, Alexander and Hillemanns, Peter and Hogervorst, Frans B. L. and Hooning, Maartje J. and Hopper, John L. and Howell, Tony and Huo, Dezheng and Ito, Hidemi and Iwasaki, Motoki and Jakubowska, Anna and Janni, Wolfgang and John, Esther M. and Jung, Audrey and Kaaks, Rudolf and Kang, Daehee and Kapoor, Pooja Middha and Khusnutdinova, Elza and Kim, Sung-Won and Kitahara, Cari M. and Koutros, Stella and Kraft, Peter and Kristensen, Vessela N. and Kwong, Ava and Lambrechts, Diether and Le Marchand, Loic and Li, Jingmei and Lindstr{\"o}m, Sara and Linet, Martha and Lo, Wing-Yee and Long, Jirong and Lophatananon, Artitaya and Lubiński, Jan and Manoochehri, Mehdi and Manoukian, Siranoush and Margolin, Sara and Martinez, Elena and Matsuo, Keitaro and Mavroudis, Dimitris and Meindl, Alfons and Menon, Usha and Milne, Roger L. and Mohd Taib, Nur Aishah and Muir, Kenneth and Mulligan, Anna Marie and Neuhausen, Susan L. and Nevanlinna, Heli and Neven, Patrick and Newman, William G. and Offit, Kenneth and Olopade, Olufunmilayo I. and Olshan, Andrew F. and Olson, Janet E. and Olsson, H{\aa}kan and Park, Sue K. and Park-Simon, Tjoung-Won and Peto, Julian and Plaseska-Karanfilska, Dijana and Pohl-Rescigno, Esther and Presneau, Nadege and Rack, Brigitte and Radice, Paolo and Rashid, Muhammad U. and Rennert, Gad and Rennert, Hedy S. and Romero, Atocha and Ruebner, Matthias and Saloustros, Emmanouil and Schmidt, Marjanka K. and Schmutzler, Rita K. and Schneider, Michael O. and Schoemaker, Minouk J. and Scott, Christopher and Shen, Chen-Yang and Shu, Xiao-Ou and Simard, Jaques and Slager, Susan and Smichkoska, Snezhana and Southey, Melissa C. and Spinelli, John J. and Stone, Jennifer and Surowy, Harald and Swerdlow, Anthony J. and Tamimi, Rulla M. and Tapper, William J. and Teo, Soo H. and Terry, Mary Beth and Toland, Amanda E. and Tollenaar, Rob A. E. M. and Torres, Diana and Torres-Mej{\´i}a, Gabriela and Troester, Melissa A. and Truong, Th{\´e}r{\`e}se and Tsugane, Shoichiro and Untch, Michael and Vachon, Celine M. and van den Ouweland, Ans M. W. and van Veen, Elke M. and Vijai, Joseph and Wendt, Camilla and Wolk, Alicja and Yu, Jyh-Cherng and Zheng, Wei and Ziogas, Argyrios and Ziv, Elad and Dunnig, Alison and Pharaoh, Paul D. P. and Schindler, Detlev and Devilee, Peter and Easton, Douglas F.}, title = {Two truncating variants in FANCC and breast cancer risk}, series = {Scientific Reports}, volume = {9}, journal = {Scientific Reports}, organization = {ABCTB Investigators, NBCS Collaborators}, doi = {10.1038/s41598-019-48804-y}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-222838}, year = {2019}, abstract = {Fanconi anemia (FA) is a genetically heterogeneous disorder with 22 disease-causing genes reported to date. In some FA genes, monoallelic mutations have been found to be associated with breast cancer risk, while the risk associations of others remain unknown. The gene for FA type C, FANCC, has been proposed as a breast cancer susceptibility gene based on epidemiological and sequencing studies. We used the Oncoarray project to genotype two truncating FANCC variants (p.R185X and p.R548X) in 64,760 breast cancer cases and 49,793 controls of European descent. FANCC mutations were observed in 25 cases (14 with p.R185X, 11 with p.R548X) and 26 controls (18 with p.R185X, 8 with p.R548X). There was no evidence of an association with the risk of breast cancer, neither overall (odds ratio 0.77, 95\%CI 0.44-1.33, p = 0.4) nor by histology, hormone receptor status, age or family history. We conclude that the breast cancer risk association of these two FANCC variants, if any, is much smaller than for BRCA1, BRCA2 or PALB2 mutations. If this applies to all truncating variants in FANCC it would suggest there are differences between FA genes in their roles on breast cancer risk and demonstrates the merit of large consortia for clarifying risk associations of rare variants.}, language = {en} } @article{ElMeseryRosenthalRauertWunderlichetal.2019, author = {El-Mesery, Mohamed and Rosenthal, Tina and Rauert-Wunderlich, Hilka and Schreder, Martin and St{\"u}hmer, Thorsten and Leich, Ellen and Schlosser, Andreas and Ehrenschwender, Martin and Wajant, Harald and Siegmund, Daniela}, title = {The NEDD8-activating enzyme inhibitor MLN4924 sensitizes a TNFR1+ subgroup of multiple myeloma cells for TNF-induced cell death}, series = {Cell Death \& Disease}, volume = {10}, journal = {Cell Death \& Disease}, doi = {10.1038/s41419-019-1860-2}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-226666}, year = {2019}, abstract = {The NEDD8-activating enzyme (NAE) inhibitor MLN4924 inhibits cullin-RING ubiquitin ligase complexes including the SKP1-cullin-F-box E3 ligase βTrCP. MLN4924 therefore inhibits also the βTrCP-dependent activation of the classical and the alternative NFĸB pathway. In this work, we found that a subgroup of multiple myeloma cell lines (e.g., RPMI-8226, MM.1S, KMS-12BM) and about half of the primary myeloma samples tested are sensitized to TNF-induced cell death by MLN4924. This correlated with MLN4924-mediated inhibition of TNF-induced activation of the classical NFκB pathway and reduced the efficacy of TNF-induced TNFR1 signaling complex formation. Interestingly, binding studies revealed a straightforward correlation between cell surface TNFR1 expression in multiple myeloma cell lines and their sensitivity for MLN4924/TNF-induced cell death. The cell surface expression levels of TNFR1 in the investigated MM cell lines largely correlated with TNFR1 mRNA expression. This suggests that the variable levels of cell surface expression of TNFR1 in myeloma cell lines are decisive for TNF/MLN4924 sensitivity. Indeed, introduction of TNFR1 into TNFR1-negative TNF/MLN4924-resistant KMS-11BM cells, was sufficient to sensitize this cell line for TNF/MLN4924-induced cell death. Thus, MLN4924 might be especially effective in myeloma patients with TNFR1+ myeloma cells and a TNFhigh tumor microenvironment.}, language = {en} } @article{KrebsBehrmannKalogirouetal.2019, author = {Krebs, Markus and Behrmann, Christoph and Kalogirou, Charis and Sokolakis, Ioannis and Kneitz, Susanne and Kruithof-de Julio, Marianna and Zoni, Eugenio and Rech, Anne and Schilling, Bastian and K{\"u}bler, Hubert and Spahn, Martin and Kneitz, Burkhard}, title = {miR-221 Augments TRAIL-mediated apoptosis in prostate cancer cells by inducing endogenous TRAIL expression and targeting the functional repressors SOCS3 and PIK3R1}, series = {BioMed Research International}, volume = {2019}, journal = {BioMed Research International}, doi = {10.1155/2019/6392748}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-202480}, pages = {6392748}, year = {2019}, abstract = {miR-221 is regarded as an oncogene in many malignancies, and miR-221-mediated resistance towards TRAIL was one of the first oncogenic roles shown for this small noncoding RNA. In contrast, miR-221 is downregulated in prostate cancer (PCa), thereby implying a tumour suppressive function. By using proliferation and apoptosis assays, we show a novel feature of miR-221 in PCa cells: instead of inducing TRAIL resistance, miR-221 sensitized cells towards TRAIL-induced proliferation inhibition and apoptosis induction. Partially responsible for this effect was the interferon-mediated gene signature, which among other things contained an endogenous overexpression of the TRAIL encoding gene TNFSF10. This TRAIL-friendly environment was provoked by downregulation of the established miR-221 target gene SOCS3. Moreover, we introduced PIK3R1 as a target gene of miR-221 in PCa cells. Proliferation assays showed that siRNA-mediated downregulation of SOCS3 and PIK3R1 mimicked the effect of miR-221 on TRAIL sensitivity. Finally, Western blotting experiments confirmed lower amounts of phospho-Akt after siRNA-mediated downregulation of PIK3R1 in PC3 cells. Our results further support the tumour suppressing role of miR-221 in PCa, since it sensitises PCa cells towards TRAIL by regulating the expression of the oncogenes SOCS3 and PIK3R1. Given the TRAIL-inhibiting effect of miR-221 in various cancer entities, our results suggest that the influence of miR-221 on TRAIL-mediated apoptosis is highly context- and entity-dependent.}, language = {en} } @article{MolinasGonzalezCastroGonzalezMegiasetal.2019, author = {Molinas-Gonz{\´a}lez, Carlos R. and Castro, Jorge and Gonz{\´a}lez-Meg{\´i}as, Adela and Leverkus, Alexandro B.}, title = {Effects of post-fire deadwood management on soil macroarthropod communities}, series = {Forests}, volume = {10}, journal = {Forests}, number = {11}, issn = {1999-4907}, doi = {10.3390/f10111046}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-193079}, year = {2019}, abstract = {Dead wood comprises a vast amount of biological legacies that set the scene for ecological regeneration after wildfires, yet its removal is the most frequent management strategy worldwide. Soil-dwelling organisms are conspicuous, and they provide essential ecosystem functions, but their possible affection by different post-fire management strategies has so far been neglected. We analyzed the abundance, richness, and composition of belowground macroarthropod communities under two contrasting dead-wood management regimes after a large wildfire in the Sierra Nevada Natural and National Park (Southeast Spain). Two plots at different elevation were established, each containing three replicates of two experimental treatments: partial cut, where trees were cut and their branches lopped off and left over the ground, and salvage logging, where all the trees were cut, logs were piled, branches were mechanically masticated, and slash was spread on the ground. Ten years after the application of the treatments, soil cores were extracted from two types of microhabitat created by these treatments: bare-soil (in both treatments) and under-logs (in the partial cut treatment only). Soil macroarthropod assemblages were dominated by Hemiptera and Hymenoptera (mostly ants) and were more abundant and richer in the lowest plot. The differences between dead-wood treatments were most evident at the scale of management interventions: abundance and richness were lowest after salvage logging, even under similar microhabitats (bare-soil). However, there were no significant differences between microhabitat types on abundance and richness within the partial cut treatment. Higher abundance and richness in the partial cut treatment likely resulted from higher resource availability and higher plant diversity after natural regeneration. Our results suggest that belowground macroarthropod communities are sensitive to the manipulation of dead-wood legacies and that management through salvage logging could reduce soil macroarthropod recuperation compared to other treatments with less intense management even a decade after application.}, language = {en} } @article{LatifiValbuena2019, author = {Latifi, Hooman and Valbuena, Ruben}, title = {Current trends in forest ecological applications of three-dimensional remote sensing: Transition from experimental to operational solutions?}, series = {Forests}, volume = {10}, journal = {Forests}, number = {10}, issn = {1999-4907}, doi = {10.3390/f10100891}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-193282}, year = {2019}, abstract = {The alarming increase in the magnitude and spatiotemporal patterns of changes in composition, structure and function of forest ecosystems during recent years calls for enhanced cross-border mitigation and adaption measures, which strongly entail intensified research to understand the underlying processes in the ecosystems as well as their dynamics. Remote sensing data and methods are nowadays the main complementary sources of synoptic, up-to-date and objective information to support field observations in forest ecology. In particular, analysis of three-dimensional (3D) remote sensing data is regarded as an appropriate complement, since they are hypothesized to resemble the 3D character of most forest attributes. Following their use in various small-scale forest structural analyses over the past two decades, these sources of data are now on their way to be integrated in novel applications in fields like citizen science, environmental impact assessment, forest fire analysis, and biodiversity assessment in remote areas. These and a number of other novel applications provide valuable material for the Forests special issue "3D Remote Sensing Applications in Forest Ecology: Composition, Structure and Function", which shows the promising future of these technologies and improves our understanding of the potentials and challenges of 3D remote sensing in practical forest ecology worldwide.}, language = {en} } @phdthesis{Derakhshani2019, author = {Derakhshani, Shaghayegh}, title = {Measles virus infection enhances dendritic cell migration in a 3D environment}, doi = {10.25972/OPUS-18918}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-189182}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {The respiratory system is amongst the most important compartments in the human body. Due to its connection to the external environment, it is one of the most common portals of pathogen entry. Airborne pathogens like measles virus (MV) carried in liquid droplets exhaled from the infected individuals via a cough or sneeze enter the body from the upper respiratory tract and travel down to the lower respiratory tract and reach the alveoli. There, pathogens are captured by the resident dendritic cells (DCs) or macrophages and brought to the lymph node where immune responses or, as in case of MV, dissemination via the hematopoietic cell compartment are initiated. Basic mechanisms governing MV exit from the respiratory tract, especially virus transmission from infected immune cells to the epithelial cells have not been fully addressed before. Considering the importance of these factors in the viral spread, a complex close-to-in-vivo 3D human respiratory tract model was generated. This model was established using de-cellularized porcine intestine tissue as a biological scaffold and H358 cells as targets for infection. The scaffold was embedded with fibroblast cells, and later on, an endothelial cell layer seeded at the basolateral side. This provided an environment resembling the respiratory tract where MV infected DCs had to transmigrate through the collagen scaffold and transmit the virus to epithelial cells in a Nectin-4 dependent manner. For viral transmission, the access of infected DCs to the recipient epithelial cells is an essential prerequisite and therefore, this important factor which is reflected by cell migration was analyzed in this 3D system. The enhanced motility of specifically MV-infected DCs in the 3D models was observed, which occurred independently of factors released from the other cell types in the models. Enhanced motility of infected DCs in 3D collagen matrices suggested infection-induced cytoskeletal remodeling, as also verified by detection of cytoskeletal polarization, uropod formation. This enforced migration was sensitive to ROCK inhibition revealing that MV infection induces an amoeboid migration mode in DCs. In support of this, the formation of podosome structures and filopodia, as well as their activity, were reduced in infected DCs and retained in their uninfected siblings. Differential migration modes of uninfected and infected DCs did not cause differential maturation, which was found to be identical for both populations. As an underlying mechanism driving this enforced migration, the role of sphingosine kinase (SphK) and sphingosine-1-phosphate (S1P) was studied in MV-exposed cultures. It was shown in this thesis that MV-infection increased S1P production, and this was identified as a contributing factor as inhibition sphingosine kinase activity abolished enforced migration of MV-infected DCs. These findings revealed that MV infection induces a fast push-and-squeeze amoeboid mode of migration, which is supported by SphK/S1P axis. However, this push-and-squeeze amoeboid migration mode did not prevent the transendothelial migration of MV-infected DCs. Altogether, this 3D system has been proven to be a suitable model to study specific parameters of mechanisms involved in infections in an in vivo-like conditions.}, subject = {Dendritische Zelle}, language = {en} } @article{HovestadtThomasMitesseretal.2019, author = {Hovestadt, Thomas and Thomas, Jeremy A. and Mitesser, Oliver and Sch{\"o}nrogge, Karsten}, title = {Multiple host use and the dynamics of host-switching in host-parasite systems}, series = {Insect Conservation and Diversity}, volume = {12}, journal = {Insect Conservation and Diversity}, number = {6}, doi = {10.1111/icad.12374}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-204747}, pages = {511-522}, year = {2019}, abstract = {The link between multi-host use and host switching in host-parasite interactions is a continuing area of debate. Lycaenid butterflies in the genus Maculinea, for example, exploit societies of different Myrmica ant species across their ranges, but there is only rare evidence that they simultaneously utilise multiple hosts at a local site, even where alternative hosts are present. We present a simple population-genetic model accounting for the proportion of two alternative hosts and the fitness of parasite genotypes on each host. In agreement with standard models, we conclude that simultaneous host use is possible whenever fitness of heterozygotes on alternative hosts is not too low. We specifically focus on host-shifting dynamics when the frequency of hosts changes. We find that (i) host shifting may proceed so rapidly that multiple host use is unlikely to be observed, (ii) back and forth transition in host use can exhibit a hysteresis loop, (iii) the parasites' host use may not be proportional to local host frequencies and be restricted to the rarer host under some conditions, and (iv) that a substantial decline in parasite abundance may typically precede a shift in host use. We conclude that focusing not just on possible equilibrium conditions but also considering the dynamics of host shifting in non-equilibrium situations may provide added insights into host-parasite systems.}, language = {en} } @phdthesis{Wilde2019, author = {Wilde, Sabrina}, title = {Einsatz von mechanistischen Biomarkern zur Charakterisierung und Bewertung von \(in\) \(vitro\) Genotoxinen}, doi = {10.25972/OPUS-18278}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-182782}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Die verf{\"u}gbaren in vitro Genotoxizit{\"a}tstests weisen hinsichtlich ihrer Spezifit{\"a}t und ihres Informationsgehalts zum vorliegenden Wirkmechanismus (Mode of Action, MoA) Einschr{\"a}nkungen auf. Um diese M{\"a}ngel zu {\"u}berwinden, wurden in dieser Arbeit zwei Ziele verfolgt, die zu der Entwicklung und Etablierung neuer in vitro Methoden zur Pr{\"u}fung auf Genotoxizit{\"a}t in der Arzneimittelentwicklung beitragen. 1. Etablierung und Bewertung einer neuen in vitro Genotoxizit{\"a}tsmethode (MultiFlow Methode) Die MultiFlow Methode basiert auf DNA-schadensassoziierten Proteinantworten von γH2AX (DNA-Doppelstrangbr{\"u}che), phosphorylierten H3 (S10) (mitotische Zellen), nukle{\"a}ren Protein p53 (Genotoxizit{\"a}t) und cleaved PARP1 (Apoptose) in TK6-Zellen. Insgesamt wurden 31 Modellsubstanzen mit dem MultiFlow Assay und erg{\"a}nzend mit dem etablierten Mikrokerntest (MicroFlow MNT), auf ihre F{\"a}higkeit verschiedene MoA-Gruppen (Aneugene/Klastogene/Nicht-Genotoxine) zu differenzieren, untersucht. Die Performance der „neuen" gegen{\"u}ber der „alten" Methode f{\"u}hrte zu einer verbesserten Sensitivit{\"a}t von 95\% gegen{\"u}ber 90\%, Spezifit{\"a}t von 90\% gegen{\"u}ber 72\% und einer MoA-Klassifizierungsrate von 85\% gegen{\"u}ber 45\% (Aneugen vs. Klastogen). 2. Identifizierung mechanistischer Biomarker zur Klassifizierung genotoxischer Substanzen Die Analyse 67 ausgew{\"a}hlter DNA-schadensassoziierter Gene in der QuantiGene Plex Methode zeigte, dass mehrere Gene gleichzeitig zur MoA-Klassifizierung beitragen k{\"o}nnen. Die Kombination der h{\"o}chstrangierten Marker BIK, KIF20A, TP53I3, DDB2 und OGG1 erm{\"o}glichte die beste Identifizierungsrate der Modellsubstanzen. Das synergetische Modell kategorisierte 16 von 16 Substanzen korrekt in Aneugene, Klastogene und Nicht-Genotoxine. Unter Verwendung der Leave-One-Out-Kreuzvalidierung wurde das Modell evaluiert und erreichte eine Sensitivit{\"a}t, Spezifit{\"a}t und Pr{\"a}diktivit{\"a}t von 86\%, 83\% und 85\%. Ergebnisse der traditionellen qPCR Methode zeigten, dass Genotoxizit{\"a}t mit TP53I3, Klastogenit{\"a}t mit ATR und RAD17 und oxidativer Stress mit NFE2L2 detektiert werden kann. Durch die Untersuchungen von posttranslationalen Modifikationen unter Verwendung der High-Content-Imaging-Technologie wurden mechanistische Assoziationen f{\"u}r BubR1 (S670) und pH3 (S28) mit Aneugenit{\"a}t, 53BP1 (S1778) und FANCD2 (S1404) mit Klastogenit{\"a}t, p53 (K373) mit Genotoxizit{\"a}t und Nrf2 (S40) mit oxidativem Stress identifiziert. Diese Arbeit zeigt, dass (Geno)toxine unterschiedliche Gen- und Proteinver{\"a}nderungen in TK6-Zellen induzieren, die zur Erfassung mechanistischer Aktivit{\"a}ten und Einteilung (geno)toxischer MoA-Gruppen (Aneugen/Klastogen/ Reaktive Sauerstoffspezies) eingesetzt werden k{\"o}nnen und daher eine bessere Risikobewertung von Wirkstoffkandidaten erm{\"o}glichen.}, subject = {Genotoxizit{\"a}t}, language = {de} } @article{VeyKapsnerFuchsetal.2019, author = {Vey, Johannes and Kapsner, Lorenz A. and Fuchs, Maximilian and Unberath, Philipp and Veronesi, Giulia and Kunz, Meik}, title = {A toolbox for functional analysis and the systematic identification of diagnostic and prognostic gene expression signatures combining meta-analysis and machine learning}, series = {Cancers}, volume = {11}, journal = {Cancers}, number = {10}, issn = {2072-6694}, doi = {10.3390/cancers11101606}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-193240}, year = {2019}, abstract = {The identification of biomarker signatures is important for cancer diagnosis and prognosis. However, the detection of clinical reliable signatures is influenced by limited data availability, which may restrict statistical power. Moreover, methods for integration of large sample cohorts and signature identification are limited. We present a step-by-step computational protocol for functional gene expression analysis and the identification of diagnostic and prognostic signatures by combining meta-analysis with machine learning and survival analysis. The novelty of the toolbox lies in its all-in-one functionality, generic design, and modularity. It is exemplified for lung cancer, including a comprehensive evaluation using different validation strategies. However, the protocol is not restricted to specific disease types and can therefore be used by a broad community. The accompanying R package vignette runs in ~1 h and describes the workflow in detail for use by researchers with limited bioinformatics training.}, language = {en} } @article{HicklHeintzBuschartTrautweinSchultetal.2019, author = {Hickl, Oskar and Heintz-Buschart, Anna and Trautwein-Schult, Anke and Hercog, Rajna and Bork, Peer and Wilmes, Paul and Becher, D{\"o}rte}, title = {Sample preservation and storage significantly impact taxonomic and functional profiles in metaproteomics studies of the human gut microbiome}, series = {Microorganisms}, volume = {7}, journal = {Microorganisms}, number = {9}, issn = {2076-2607}, doi = {10.3390/microorganisms7090367}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-195976}, year = {2019}, abstract = {With the technological advances of the last decade, it is now feasible to analyze microbiome samples, such as human stool specimens, using multi-omic techniques. Given the inherent sample complexity, there exists a need for sample methods which preserve as much information as possible about the biological system at the time of sampling. Here, we analyzed human stool samples preserved and stored using different methods, applying metagenomics as well as metaproteomics. Our results demonstrate that sample preservation and storage have a significant effect on the taxonomic composition of identified proteins. The overall identification rates, as well as the proportion of proteins from Actinobacteria were much higher when samples were flash frozen. Preservation in RNAlater overall led to fewer protein identifications and a considerable increase in the share of Bacteroidetes, as well as Proteobacteria. Additionally, a decrease in the share of metabolism-related proteins and an increase of the relative amount of proteins involved in the processing of genetic information was observed for RNAlater-stored samples. This suggests that great care should be taken in choosing methods for the preservation and storage of microbiome samples, as well as in comparing the results of analyses using different sampling and storage methods. Flash freezing and subsequent storage at -80 °C should be chosen wherever possible.}, language = {en} } @phdthesis{Beck2019, author = {Beck, Sebastian}, title = {Using optogenetics to influence the circadian clock of \(Drosophila\) \(melanogaster\)}, doi = {10.25972/OPUS-18495}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-184952}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Almost all life forms on earth have adapted to the most impactful and most predictable recurring change in environmental condition, the cycle of day and night, caused by the axial rotation of the planet. As a result many animals have evolved intricate endogenous clocks, which adapt and synchronize the organisms' physiology, metabolism and behaviour to the daily change in environmental conditions. The scientific field researching these endogenous clocks is called chronobiology and has steadily grown in size, scope and relevance since the works of the earliest pioneers in the 1960s. The number one model organism for the research of circadian clocks is the fruit fly, Drosophila melanogaster, whose clock serves as the entry point to understanding the basic inner workings of such an intricately constructed endogenous timekeeping system. In this thesis it was attempted to combine the research on the circadian clock with the techniques of optogenetics, a fairly new scientific field, launched by the discovery of Channelrhodopsin 2 just over 15 years ago. Channelrhodopsin 2 is a light-gated ion channel found in the green alga Chlamydomonas reinhardtii. In optogenetics, researches use these light-gated ion channels like Channelrhodopsin 2 by heterologously expressing them in cells and tissues of other organisms, which can then be stimulated by the application of light. This is most useful when studying neurons, as these channels provide an almost non-invasive tool to depolarize the neuronal plasma membranes at will. The goal of this thesis was to develop an optogenetic tool, which would be able to influence and phase shift the circadian clock of Drosophila melanogaster upon illumination. A phase shift is the adaptive response of the circadian clock to an outside stimulus that signals a change in the environmental light cycle. An optogenetic tool, able to influence and phase shift the circadian clock predictably and reliably, would open up many new ways and methods of researching the neuronal network of the clock and which neurons communicate to what extent, ultimately synchronizing the network. The first optogenetic tool to be tested in the circadian clock of Drosophila melanogaster was ChR2-XXL, a channelrhodopsin variant with dramatically increased expression levels and photocurrents combined with a prolonged open state. The specific expression of ChR2-XXL and of later constructs was facilitated by deploying the three different clock-specific GAL4-driver lines, clk856-gal4, pdf-gal4 and mai179-gal4. Although ChR2-XXL was shown to be highly effective at depolarizing neurons, these stimulations proved to be unable to significantly phase shift the circadian clock of Drosophila. The second series of experiments was conducted with the conceptually novel optogenetic tools Olf-bPAC and SthK-bPAC, which respectively combine a cyclic nucleotide-gated ion channel (Olf and SthK) with the light-activated adenylyl-cyclase bPAC. These tools proved to be quite useful when expressed in the motor neurons of instar-3 larvae of Drosophila, paralyzing the larvae upon illumination, as well as affecting body length. This way, these new tools could be precisely characterized, spawning a successfully published research paper, centered around their electrophysiological characterization and their applicability in model organisms like Drosophila. In the circadian clock however, these tools caused substantial damage, producing severe arrhythmicity and anomalies in neuronal development. Using a temperature-sensitive GAL80-line to delay the expression until after the flies had eclosed, yielded no positive results either. The last series of experiments saw the use of another new series of optogenetic tools, modelled after the Olf-bPAC, with bPAC swapped out for CyclOp, a membrane-bound guanylyl-cyclase, coupled with less potent versions of the Olf. This final attempt however also ended up being unsuccessful. While these tools could efficiently depolarize neuronal membranes upon illumination, they were ultimately unable to stimulate the circadian clock in way that would cause it to phase shift. Taken together, these mostly negative results indicate that an optogenetic manipulation of the circadian clock of Drosophila melanogaster is an extremely challenging subject. As light already constitutes the most impactful environmental factor on the circadian clock, the combination of chronobiology with optogenetics demands the parameters of the conducted experiments to be tuned with an extremely high degree of precision, if one hopes to receive positive results from these types of experiments at all.}, subject = {Chronobiologie}, language = {en} } @article{Puetz2019, author = {P{\"u}tz, Stephanie M.}, title = {Mbt/PAK4 together with SRC modulates N-Cadherin adherens junctions in the developing Drosophila eye}, series = {Biology Open}, volume = {8}, journal = {Biology Open}, doi = {10.1242/bio.038406}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-200898}, pages = {bio038406}, year = {2019}, abstract = {Tissue morphogenesis is accompanied by changes of adherens junctions (AJ). During Drosophila eye development, AJ reorganization includes the formation of isolated N-Cadherin AJ between photoreceptors R3/R4. Little is known about how these N-Cadherin AJ are established and maintained. This study focuses on the kinases Mbt/PAK4 and SRC, both known to alter E-Cadherin AJ across phyla. Drosophila p21-activated kinase Mbt and the non-receptor tyrosine kinases Src64 and Src42 regulate proper N-Cadherin AJ. N-Cadherin AJ elongation depends on SRC kinase activity. Cell culture experiments demonstrate binding of both Drosophila SRC isoforms to N-Cadherin and its subsequent tyrosine phosphorylation. In contrast, Mbt stabilizes but does not bind N-Cadherin in vitro. Mbt is required in R3/R4 for zipping the N-Cadherin AJ between these cells, independent of its kinase activity and Cdc42-binding. The mbt phenotype can be reverted by mutations in Src64 and Src42. Because Mbt neither directly binds to SRC proteins nor has a reproducible influence on their kinase activity, the conclusion is that Mbt and SRC signaling converge on N-Cadherin. N-Cadherin AJ formation during eye development requires a proper balance between the promoting effects of Mbt and the inhibiting influences of SRC kinases.}, language = {en} } @phdthesis{Breitenbach2019, author = {Breitenbach, Tim}, title = {A mathematical optimal control based approach to pharmacological modulation with regulatory networks and external stimuli}, doi = {10.25972/OPUS-17436}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-174368}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {In this work models for molecular networks consisting of ordinary differential equations are extended by terms that include the interaction of the corresponding molecular network with the environment that the molecular network is embedded in. These terms model the effects of the external stimuli on the molecular network. The usability of this extension is demonstrated with a model of a circadian clock that is extended with certain terms and reproduces data from several experiments at the same time. Once the model including external stimuli is set up, a framework is developed in order to calculate external stimuli that have a predefined desired effect on the molecular network. For this purpose the task of finding appropriate external stimuli is formulated as a mathematical optimal control problem for which in order to solve it a lot of mathematical methods are available. Several methods are discussed and worked out in order to calculate a solution for the corresponding optimal control problem. The application of the framework to find pharmacological intervention points or effective drug combinations is pointed out and discussed. Furthermore the framework is related to existing network analysis tools and their combination for network analysis in order to find dedicated external stimuli is discussed. The total framework is verified with biological examples by comparing the calculated results with data from literature. For this purpose platelet aggregation is investigated based on a corresponding gene regulatory network and associated receptors are detected. Furthermore a transition from one to another type of T-helper cell is analyzed in a tumor setting where missing agents are calculated to induce the corresponding switch in vitro. Next a gene regulatory network of a myocardiocyte is investigated where it is shown how the presented framework can be used to compare different treatment strategies with respect to their beneficial effects and side effects quantitatively. Moreover a constitutively activated signaling pathway, which thus causes maleficent effects, is modeled and intervention points with corresponding treatment strategies are determined that steer the gene regulatory network from a pathological expression pattern to physiological one again.}, subject = {Bioinformatik}, language = {en} } @article{MatosMachadoSchartletal.2019, author = {Matos, Isa and Machado, Miguel P. and Schartl, Manfred and Coelho, Maria Manuela}, title = {Allele-specific expression variation at different ploidy levels in Squalius alburnoides}, series = {Scientific Reports}, volume = {9}, journal = {Scientific Reports}, doi = {10.1038/s41598-019-40210-8}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-200910}, pages = {3688}, year = {2019}, abstract = {Allopolyploid plants are long known to be subject to a homoeolog expression bias of varying degree. The same phenomenon was only much later suspected to occur also in animals based on studies of single selected genes in an allopolyploid vertebrate, the Iberian fish Squalius alburnoides. Consequently, this species became a good model for understanding the evolution of gene expression regulation in polyploid vertebrates. Here, we analyzed for the first time genome-wide allele-specific expression data from diploid and triploid hybrids of S. alburnoides and compared homoeolog expression profiles of adult livers and of juveniles. Co-expression of alleles from both parental genomic types was observed for the majority of genes, but with marked homoeolog expression bias, suggesting homoeolog specific reshaping of expression level patterns in hybrids. Complete silencing of one allele was also observed irrespective of ploidy level, but not transcriptome wide as previously speculated. Instead, it was found only in a restricted number of genes, particularly ones with functions related to mitochondria and ribosomes. This leads us to hypothesize that allelic silencing may be a way to overcome intergenomic gene expression interaction conflicts, and that homoeolog expression bias may be an important mechanism in the achievement of sustainable genomic interactions, mandatory to the success of allopolyploid systems, as in S. alburnoides.}, language = {en} } @phdthesis{Jannasch2019, author = {Jannasch, Maren Annika}, title = {In vitro Fremdk{\"o}rpermodellsysteme zur Vorhersage von biomaterialinduzierten Immunreaktionen}, doi = {10.25972/OPUS-16289}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-162893}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Die Implantation eines Medizinprodukts in den menschlichen K{\"o}rper ruft eine Immunreaktion hervor, die zur fibr{\"o}sen Einkapselung f{\"u}hren kann. Makrophagen in direktem Kontakt mit der Oberfl{\"a}che des Implantats erfassen sensorisch den Fremdk{\"o}rper und {\"u}bersetzten das Signal in die Freisetzung zahlreicher l{\"o}slicher Mediatoren. Das generierte Entz{\"u}ndungsmilieu moduliert die Heilungsreaktion und kann zur Anreicherung von Fibroblasten sowie zur Erh{\"o}hung der Matrixsyntheserate in der Wundumgebung f{\"u}hren. Eine dichte fibr{\"o}se Kapsel um ein Medizinprodukt beeintr{\"a}chtigt den Ersatz von K{\"o}rperstrukturen, das Unterst{\"u}tzen physiologischer K{\"o}rperfunktionen sowie die Effizienz einer medizinischen Therapie. Zur Identifizierung potenzieller Biomaterialkandidaten mit optimalen Eigenschaften ist jedoch eine evidenzbasierte Entscheidungsfindung notwendig und diese wiederum muss durch geeignete Testmethoden unterst{\"u}tzt werden. Zur Erfassung lokaler Effekte nach Implantation eines Biomaterials begr{\"u}ndet die Komplexi-t{\"a}t der ablaufenden Fremdk{\"o}rperreaktion die Anwendung von Tiermodellen als Goldstandard. Die Eingliederung von in vitro Modellsystemen in standardisierte Testverfahren scheitert oft an der Verf{\"u}gbarkeit validierter, verl{\"a}sslicher und reproduzierbarer Methoden. Demnach ist kein standardisiertes in vitro Testverfahren beschrieben, das die komplexen dreidimensionalen Gewebsstrukturen w{\"a}hrend einer Fremdk{\"o}rperreaktion abbildet und sich zur Testung {\"u}ber l{\"a}ngere Kontaktphasen zwischen Blutkomponenten und Biomaterialien eignet. Jedoch k{\"o}nnen in vitro Testungen kosten- und zeiteffizienter sein und durch die Anwendung humaner Zellen eine h{\"o}here {\"U}bertragbarkeit auf den Menschen aufweisen. Zus{\"a}tzlich adressiert die Pr{\"a}ferenz zu in vitro Testmethoden den Aspekt „Reduzierung" der 3R-Prinzipien „Replacement, Reduction, Refinement" (Ersatz, Reduzierung, Verbesserung) von Russel und Burch (1959) zu einer bewussten und begr{\"u}ndeten Anwendung von Tiermodellen in der Wissenschaft. Ziel von diesem Forschungsvorhaben war die Entwicklung von humanen in vitro Modellsystemen, die den Kontakt zu Blutkomponenten sowie die Reaktion des umliegenden Bindegewebes bei lokaler Implantation eines Biomaterials abbilden. Referenzmaterialien, deren Gewebsantwort nach Implantation in Tiere oder den Menschen bekannt ist, dienten als Validierungskriterium f{\"u}r die entwickelten Modellsysteme. Die Anreicherung von Zellen sowie die Bildung extrazellul{\"a}rer Matrix in der Wundumgebung stellen wichtige Teilprozesse w{\"a}hrend einer Fremdk{\"o}rperreaktion dar. F{\"u}r beide Teilprozesse konnte in einem indirekten zellbasierten Modellsystem der Einfluss einer zellvermittelten Konditionierung wie die Freisetzung von l{\"o}slichen Mediatoren durch materialadh{\"a}rente Makrophagen auf die gerichtete Wanderung von Fibroblasten sowie den Umbau eines dreidimensionalen Bindegewebsmodells aufgezeigt werden. Des Weiteren ließ sich das Freisetzungsprofil von Zytokinen durch materialst{\"a}ndige Makrophagen unter verschiedenen Testbedingungen wie der Kontamination mit LPS, der Oberfl{\"a}chenbehandlung mit humanem Blutplasma und der Gegenwart von IL-4 bestimmen. Die anschließende vergleichende statistische Modellierung der generierten komplexen multifaktoriellen Datenmatrix erm{\"o}glichte die {\"U}bersetzung in eine Biomaterialbewertung. Dieses entwickelte Testverfahren eignete sich einerseits zur Validierung von in vitro Testbedingungen sowie andererseits zur Bewertung von Biomaterialien. Dar{\"u}ber hinaus konnte in einem dreidimensionalen Fremdk{\"o}rpermodell die komplexe dreidimensionale Struktur der extrazellul{\"a}ren Matrix in einer Wunde durch die Kombination unterschiedlicher Zell- und Matrixkomponenten biomimetisch nachgebaut werden. Diese neuartigen dreidimensionalen Fremdk{\"o}rpermodelle erm{\"o}glichten die Testung von Biomaterialien {\"u}ber l{\"a}ngere Testphasen und k{\"o}nnen in anschließenden Studien angewandt werden, um dynamische Prozesse zu untersuchen. Zusammenfassend konnten in dieser Arbeit drei unterschiedliche Teststrategien entwickelt werden, die (I) die Bewertung von Teilprozessen erm{\"o}glichen, (II) die Identifizierung verl{\"a}sslicher Testbedingungen unterst{\"u}tzen und (III) biomimetisch ein Wundgewebe abbilden. Wesentlich ist, dass biomimetisch ein dreidimensionales Gewebemodell entwickelt werden konnte, das eine verl{\"a}ssliche Unterscheidungskapazit{\"a}t zwischen Biomaterialien aufweist.}, subject = {Biomaterial}, language = {de} } @phdthesis{Yu2019, author = {Yu, Sung-Huan}, title = {Development and application of computational tools for RNA-Seq based transcriptome annotations}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-176468}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {In order to understand the regulation of gene expression in organisms, precise genome annotation is essential. In recent years, RNA-Seq has become a potent method for generating and improving genome annotations. However, this Approach is time consuming and often inconsistently performed when done manually. In particular, the discovery of non-coding RNAs benefits strongly from the application of RNA-Seq data but requires significant amounts of expert knowledge and is labor-intensive. As a part of my doctoral study, I developed a modular tool called ANNOgesic that can detect numerous transcribed genomic features, including non-coding RNAs, based on RNA-Seq data in a precise and automatic fashion with a focus on bacterial and achaeal species. The software performs numerous analyses and generates several visualizations. It can generate annotations of high-Resolution that are hard to produce using traditional annotation tools that are based only on genome sequences. ANNOgesic can detect numerous novel genomic Features like UTR-derived small non-coding RNAs for which no other tool has been developed before. ANNOgesic is available under an open source license (ISCL) at https://github.com/Sung-Huan/ANNOgesic. My doctoral work not only includes the development of ANNOgesic but also its application to annotate the transcriptome of Staphylococcus aureus HG003 - a strain which has been a insightful model in infection biology. Despite its potential as a model, a complete genome sequence and annotations have been lacking for HG003. In order to fill this gap, the annotations of this strain, including sRNAs and their functions, were generated using ANNOgesic by analyzing differential RNA-Seq data from 14 different samples (two media conditions with seven time points), as well as RNA-Seq data generated after transcript fragmentation. ANNOgesic was also applied to annotate several bacterial and archaeal genomes, and as part of this its high performance was demonstrated. In summary, ANNOgesic is a powerful computational tool for RNA-Seq based annotations and has been successfully applied to several species.}, subject = {Genom}, language = {en} } @phdthesis{Memmel2019, author = {Memmel, Simon}, title = {Automatisierte Algorithmen zur Analyse der Migration und der strahleninduzierten DNA-Sch{\"a}den humaner Glioblastomzellen nach kombinierter PI3K/mTOR/Hsp90-Inhibierung}, doi = {10.25972/OPUS-18571}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-185710}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Das hohe invasive Potential und die starke Resistenz gegen Radio-/Chemotherapie von Glioblastoma multiforme (GBM) Zellen machen sie zu dem t{\"o}dlichsten Tumor ihrer Art. Es ist deshalb von großem Interesse die Grundlagen, welche der Migrationsf{\"a}higkeit und DNA Reparatur zu Grunde liegen, besser zu verstehen. Im ersten Teil dieser Arbeit wurden zwei Algorithmen zur automatischen Analyse der Migration in der Einzelzellverfolgung und im Wundheilungsassay modifiziert. Die Auswertung der Daten konnte automatisch und somit schnell, effektiv und mit geringerem Arbeitsaufwand durchgef{\"u}hrt werden. Mit Hilfe dieser automatischen Algorithmen wurde die Migrationsf{\"a}higkeit von zwei GBM-Zelllinien (DK-MG und SNB19) untersucht. Zus{\"a}tzlich wurde die konfokale Laserscanning- sowie die hochaufl{\"o}sende dSTORM-Fluoreszenzmikroskopie verwendet um die, der Zellbewegung zu Grunde liegende, Struktur des F Aktin und der fokalen Adh{\"a}sionskinase (FAK) aufzul{\"o}sen und darzustellen. Unter Anwendung dieser genannten Methoden sind die Effekte des dualen PI3K/mTOR Inhibitors PI-103 alleine und in Kombination mit dem Hsp90 Inhibitor NVP AUY922 mit und ohne Bestrahlung auf die Bewegung untersucht worden. Es konnte festgestellt werden, dass sich beide Zelllinien deutlich in ihrem migratorischem Potential in vitro unterscheiden und zudem auch markante Unterschiede in ihrer Morphologie aufweisen. Die weniger invasiven DK MG-Zellen besitzen eine polarisierte Zellstruktur, wohingegen SNB19-Zellen sich durch multipolare ungerichtete Bewegung auszeichneten. Zudem wurde die Migration, durch PI3K/mTOR Inhibition mit PI-103 bei den DK-MG-Zellen (p53 wt, PTEN wt), sehr effektiv unterdr{\"u}ckt. Wohingegen sich die SNB19-Zellen (p53 mut, PTEN mut) resistent gegen diesen Inhibitor zeigten. Hsp90 Inhibition offenbarte in beiden Zelllinien einen starken inhibitorischen Effekt auf die Migration der Zellen sowie die Reorganisierung des F Aktinskelettes. In der zweiten H{\"a}lfte dieser Arbeit wurde ein Augenmerk auf die DNA-DSB-Reparatur der GBM Zellen nach ionisierender Strahlung gelegt. Zun{\"a}chst wurde eine automatische Analysesoftware „FocAn-3D" entwickelt, mit dessen Hilfe die DNA Doppelstrangbruchreparaturkinetik untersucht werden sollte. Diese Software erm{\"o}glicht es die gesamten Zellkerne mit ihren γH2AX-Foci in 3D-cLSM-Aufnahmen zu untersuchen. Es konnte somit eine Verbesserung der Genauigkeit in der Ausz{\"a}hlung der γH2AX-Foci erreicht werden, welche 2D beschr{\"a}nkter Software verwehrt bleibt. Mit FocAn-3D konnte der gesamte Verlauf der Induktions- und Abbauphase der γH2AX-Foci in DK MG- und SNB19-Zellen mit einem mathematischen Modell ausgewertet und dargestellt werden. Des Weiteren wurde die Nanometerstruktur von γH2AX- und pDNA-PKcs-Foci mittels hochaufl{\"o}sender dSTORM-Mikroskopie untersucht. Konventionelle Mikroskopiemethoden, begrenzt durch das Beugungslimit und einer Aufl{\"o}sung von ~200 nm, konnten die Nanometerstruktur (<100 nm) der Reparaturfoci bisher nicht darstellen. Mit Hilfe der beugungsunbegrenzten dSTORM-Mikroskopie war es m{\"o}glich in DK MG- und SNB19-Zellen die Nanometerstruktur genannten Reparaturproteine in den Foci mit einer Aufl{\"o}sung von bis zu ~20 nm darzustellen. γH2AX-Foci zeigten sich als eine Verteilung aus einzelnen Untereinheiten („Nanofoci") mit einem Durchmesser von ~45 nm. Dies l{\"a}sst die Vermutung zu, dass es sich hier um die elementare Substruktur der Foci und somit der γH2AX enthaltenen Nukleosome handelt. DNA-PK-Foci wiesen hingegen eine diffusere Verteilung auf. Die in dieser Arbeit ermittelten Unterschiede im Migrationsverhalten der Zellen rechtfertigen eine weitere pr{\"a}klinische Untersuchung der verwendeten Inhibitoren als potentielle Zelltherapeutika f{\"u}r die Behandlung von GBM. Zudem konnte sich dSTORM als machtvolles Hilfsmittel, sowohl zur Analyse der Migration zugrundeliegenden Zytoskelettstruktur und der Effekte der Hsp90 Inhibierung, als auch, der Nanostruktur der DNA-DSB-Reparaturfoci herausstellen. Es ist anzunehmen, dass beugungsunbegrenzte Mikroskopiemethoden sich als bedeutende Werkzeuge in der medizinischen und biologischen Erforschung der DNA-Reparaturmechanismen herausstellen werden. Das in dieser Arbeit entwickelte ImageJ Plugin „FocAn-3D" bewies sich ebenfalls als ein vielversprechendes Werkzeug f{\"u}r die Analyse der Reparaturkinetik. Mit Hilfe von „FocAn-3D" sollte es somit m{\"o}glich sein u.a. den Einfluss gezielter Inhibition auf den zeitlichen Verlauf der Induktion und des Abbaus der DNA-Reparaturmaschinerie genauer zu studieren.}, subject = {Glioblastom}, language = {de} } @phdthesis{Kaymak2019, author = {Kaymak, Irem}, title = {Identification of metabolic liabilities in 3D models of cancer}, doi = {10.25972/OPUS-18154}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-181544}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Inefficient vascularisation of solid tumours leads to the formation of oxygen and nutrient gradients. In order to mimic this specific feature of the tumour microenvironment, a multicellular tumour spheroid (SPH) culture system was used. These experiments were implemented in p53 isogenic colon cancer cell lines (HCT116 p53 +/+ and HCT116 p53-/-) since Tp53 has important regulatory functions in tumour metabolism. First, the characteristics of the cells cultured as monolayers and as spheroids were investigated by using RNA sequencing and metabolomics to compare gene expression and metabolic features of cells grown in different conditions. This analysis showed that certain features of gene expression found in tumours are also present in spheroids but not in monolayer cultures, including reduced proliferation and induction of hypoxia related genes. Moreover, comparison between the different genotypes revealed that the expression of genes involved in cholesterol homeostasis is induced in p53 deficient cells compared to p53 wild type cells and this difference was only detected in spheroids and tumour samples but not in monolayer cultures. In addition, it was established that loss of p53 leads to the induction of enzymes of the mevalonate pathway via activation of the transcription factor SREBP2, resulting in a metabolic rewiring that supports the generation of ubiquinone (coenzyme Q10). An adequate supply of ubiquinone was essential to support mitochondrial electron transport and pyrimidine biosynthesis in p53 deficient cancer cells under conditions of metabolic stress. Moreover, inhibition of the mevalonate pathway using statins selectively induced oxidative stress and apoptosis in p53 deficient colon cancer cells exposed to oxygen and nutrient deprivation. This was caused by ubiquinone being required for electron transfer by dihydroorotate dehydrogenase, an essential enzyme of the pyrimidine nucleotide biosynthesis pathway. Supplementation with exogenous nucleosides relieved the demand for electron transfer and restored viability of p53 deficient cancer cells under metabolic stress. Moreover, the mevalonate pathway was also essential for the synthesis of ubiquinone for nucleotide biosynthesis to support growth of intestinal tumour organoids. Together, these findings highlight the importance of the mevalonate pathway in cancer cells and provide molecular evidence for an enhanced sensitivity towards the inhibition of mitochondrial electron transfer in tumour-like metabolic environments.}, subject = {Tumor}, language = {en} } @phdthesis{Gorelashvili2019, author = {Gorelashvili, Maximilian Georg}, title = {Investigation of megakaryopoiesis and the acute phase of ischemic stroke by advanced fluorescence microscopy}, doi = {10.25972/OPUS-18600}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-186002}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {In mammals, anucleate platelets circulate in the blood flow and are primarily responsible for maintaining functional hemostasis. Platelets are generated in the bone marrow (BM) by megakaryocytes (MKs), which mainly reside directly next to the BM sinusoids to release proplatelets into the blood. MKs originate from hematopoietic stem cells and are thought to migrate from the endosteal to the vascular niche during their maturation, a process, which is, despite being intensively investigated, still not fully understood. Long-term intravital two photon microscopy (2PM) of MKs and vasculature in murine bone marrow was performed and mean squared displacement analysis of cell migration was performed. The MKs exhibited no migration, but wobbling-like movement on time scales of 3 h. Directed cell migration always results in non-random spatial distribution. Thus, a computational modelling algorithm simulating random MK distribution using real 3D light-sheet fluorescence microscopy data sets was developed. Direct comparison of real and simulated random MK distributions showed, that MKs exhibit a strong bias to vessel-contact. However, this bias is not caused by cell migration, as non-vessel-associated MKs were randomly distributed in the intervascular space. Furthermore, simulation studies revealed that MKs strongly impair migration of other cells in the bone marrow by acting as large-sized obstacles. MKs are thought to migrate from the regions close to the endosteum towards the vasculature during their maturation process. MK distribution as a function of their localization relative to the endosteal regions of the bones was investigated by light sheet fluorescence microscopy (LSFM). The results show no bone-region dependent distribution of MKs. Taken together, the newly established methods and obtained results refute the model of MK migration during their maturation. Ischemia reperfusion (I/R) injury is a frequent complication of cerebral ischemic stroke, where brain tissue damage occurs despite successful recanalization. Platelets, endothelial cells and immune cells have been demonstrated to affect the progression of I/R injury in experimental mouse models 24 h after recanalization. However, the underlying Pathomechanisms, especially in the first hours after recanalization, are poorly understood. Here, LSFM, 2PM and complemental advanced image analysis workflows were established for investigation of platelets, the vasculature and neutrophils in ischemic brains. Quantitative analysis of thrombus formation in the ipsilateral and contralateral hemispheres at different time points revealed that platelet aggregate formation is minimal during the first 8 h after recanalization and occurs in both hemispheres. Considering that maximal tissue damage already is present at this time point, it can be concluded that infarct progression and neurological damage do not result from platelet aggregated formation. Furthermore, LSFM allowed to confirm neutrophil infiltration into the infarcted hemisphere and, here, the levels of endothelial cell marker PECAM1 were strongly reduced. However, further investigations must be carried out to clearly identify the role of neutrophils and the endothelial cells in I/R injury.}, subject = {Fluoreszenzmikroskopie}, language = {en} } @phdthesis{Fuellsack2019, author = {F{\"u}llsack, Simone Alexandra}, title = {Die Bedeutung von Todesdom{\"a}ne Adapterproteinen f{\"u}r die Signaltransduktion des TNFR1 und der TRAIL Todesrezeptoren}, doi = {10.25972/OPUS-18451}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-184518}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Die NFκB-Signalwege, Apoptose und Nekroptose sind essentielle Prozesse in der Immunantwort. Außerdem sind diese Signalwege Teil der Regulation von Zelldifferenzierung, -proliferation, -tod und Entz{\"u}ndungsreaktionen. Dabei wird zuerst der Rezeptor (TNFR1 oder TRAILR 1/2) aktiviert, die rekrutierten DD-Adapterproteine TRADD, FADD und RIPK1 leiten dann die entsprechende Signalkaskade weiter und bestimmen durch ihre Zusammenwirkung, ob der NFκB-Signalweg, Apoptose oder Nekroptose induziert wird. TNFR1 und TRAILR 1/2 ben{\"o}tigen die DD-Adapterproteine TRADD, FADD und RIPK1 f{\"u}r die Zelltodinduktion, deren konkrete Bedeutung in Bezug auf Rezeptor-Spezifit{\"a}t, Zusammenwirken und Relevanz allerdings noch unklar ist. Um das Zusammenspiel dieser Proteine besser zu verstehen, wurden in dieser Arbeit Nekroptose-kompetente RIPK3-exprimierende HeLa-Zellen verwendet, bei denen die DD-Adapterproteine FADD, TRADD und RIPK1 einzeln oder in Kombination von zweien ausgeknockt wurden. Es stellte sich heraus, dass RIPK1 essentiell f{\"u}r die TNFR1- und TRAILR 1/2-vermittelte Nekroptose-Induktion ist, doch RIPK1 alleine, d.h. ohne FADD- oder TRADD-Mitbeteiligung, nur bei der TNFR1-Nekroptose-Induktion ausreicht. Wiederum inhibiert TRADD die TNFR1- und TRAILR 1/2-induzierte Nekroptose. RIPK1 und TRADD sind aber unverzichtbar f{\"u}r die NFκB-Aktivierung durch TNFR1 oder TRAILR 1/2 und spielen eine wichtige Rolle bei TNFR1-induzierter Apoptose. Andererseits ist FADD alleine ausreichend f{\"u}r die TRAILR 1/2-bezogene Caspase-8 Aktivierung. Zudem ist FADD notwendig f{\"u}r die TRAIL-induzierte NFκB-Signalaktivierung. In Abwesenheit von FADD und TRADD vermittelt RIPK1 die TNF-induzierte Caspase-8 Aktivierung. FADD wird f{\"u}r die TRAIL-induzierte Nekroptose ben{\"o}tigt, aber gegenl{\"a}ufig wirkt die TNF-induzierte Nektroptose in einer Caspase-8 abh{\"a}ngigen und unabh{\"a}ngigen Weise. Zudem sensitiviert TWEAK die TNF- und TRAIL-induzierte Nekroptose. Zusammenfassend wurde in dieser Arbeit die Auswirkung von TNFR1 und TRAILR 1/2 auf die Aktivierung der unterschiedlichen Signalkaskaden untersucht. Des Weiteren wurde gezeigt, in welcher Weise sich das Zusammenspiel von TRADD, FADD und RIPK1 auf die Induktion von NFκB, Apoptose und Nekroptose auswirkt.}, subject = {Signaltransduktion}, language = {de} } @phdthesis{Koenig2019, author = {K{\"o}nig, Eva-Maria}, title = {Pathogenese von Kraniosynostosen}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-175181}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Das humane Sch{\"a}deldach besteht aus f{\"u}nf Sch{\"a}delplatten, die durch intramembran{\"o}se Ossifikation entstehen. Wenn diese in der Embryonalentwicklung aufeinandertreffen, bilden sich Sch{\"a}deln{\"a}hte aus, die eine Fusion der Sch{\"a}delplatten verhindern und damit ein Sch{\"a}delwachstum parallel zu Gehirnentwicklung erm{\"o}glichen. F{\"u}r diesen Prozess ist eine Balance aus Zellproliferation und Differenzierung n{\"o}tig, deren Aufrechterhaltung wiederum durch eine komplexe Regulation von verschiedenen Signalwegen gew{\"a}hrleistet wird. St{\"o}rungen in diesem regulatorischen System k{\"o}nnen zu einer vorzeitigen Fusion der Sch{\"a}delplatten, Kraniosynostose genannt, f{\"u}hren. Die Kraniosynostose ist eine der h{\"a}ufigsten kraniofazialen Fehlbildungen beim Menschen. Durch kompensatorisches Wachstum an den nicht fusionierten Suturen entstehen charakteristische Sch{\"a}deldeformationen, die sekund{\"a}r einen erh{\"o}hten intrakranialen Druck zur Folge haben k{\"o}nnen. Eine vorzeitige Fusion der Suturen kann sowohl isoliert als auch syndromal zusammen mit weiteren klinischen Auff{\"a}lligkeiten vorliegen. Bisher sind {\"u}ber 150 verschiedene Kraniosynostose Syndrome beschrieben und insgesamt 25-30\% aller Kraniosynostose Patienten sind von einer syndromalen Form betroffen. Da die klinischen Merkmale der Kraniosynostose Syndrome variabel sind und zum Teil {\"u}berlappen, ist eine klare klinische Diagnose h{\"a}ufig erschwert. Sowohl Umwelteinfl{\"u}sse als auch genetische Ver{\"a}nderungen k{\"o}nnen die Ursache f{\"u}r Kraniosynostosen sein. Vor allem bei syndromalen Kraniosynostosen wurden genetische Ver{\"a}nderungen, wie beispielsweise Mutationen in den Genen FGFR2, FGFR3, TWIST1 und EFNB1, identifiziert. Dar{\"u}ber hinaus wurden chromosomale Ver{\"a}nderungen wie partielle Monosomien von 7p, 9p oder 11p sowie partielle Trisomien von 5q, 13q oder 15q mit Kraniosynostose assoziiert. Trotzdem ist in {\"u}ber 50\% der F{\"a}lle die genetische Ursache unbekannt und die Pathogenese von Kraniosynostosen noch nicht vollst{\"a}ndig gekl{\"a}rt. Ziel dieser Arbeit war es neue genetische Ursachen bei Kraniosynostose Patienten zu identifizieren und so zur Aufkl{\"a}rung der Pathogenese beizutragen. Es wurde die genomische DNA von 83 Patienten molekulargenetisch durch Mikroarray basierte vergleichende Genomhybridisierung (Array-CGH) oder durch ein speziell entworfenes Next Generation Sequencing (NGS) Genpanel untersucht. Bei 30\% der Patienten konnte eine potentiell pathogene Ver{\"a}nderung identifiziert werden. Davon waren 23\% chromosomale Aberrationen wie unbalancierte Translokationen, isolierte interstitielle Verluste und ein Zugewinn an genomischen Material. Bei zwei Patienten wurden unbalancierte Translokationen mit partieller 5q Trisomie nachgewiesen. Das Gen MSX2 liegt innerhalb des duplizierten Bereichs, sodass m{\"o}glicherweise eine MSX2 {\"U}berexpression vorliegt. F{\"u}r ein normales Sch{\"a}delwachstum ist jedoch die richtige Menge an MSX2 kritisch. Des Weiteren wurde eine partielle Deletion von TCF12 detektiert, die in einer Haploinsuffizienz von TCF12 resultiert. TCF12 Mutationen sind mit Koronarnahtsynosten assoziiert. In einem anderen Fall lag das Gen FGF10 innerhalb der duplizierten 5p15.1-p12 Region. Das Gen kodiert f{\"u}r einen Liganden des FGF Signalwegs und wurde bisher noch nicht mit Kraniosynostose assoziiert. Aufgrund dessen wurden Analysen im Tiermodell Danio rerio durchgef{\"u}hrt. Eine simulierte {\"U}berexpression durch Injektion der fgf10a mRNA in das 1-Zell Stadium f{\"u}hrte zu schweren Gehirn-, Herz- und Augendefekten. Mittels NGS wurden 77\% der potentiell pathogenen genetischen Ver{\"a}nderungen identifiziert. Hierf{\"u}r wurde in dieser Arbeit ein Genpanel erstellt, das 68 Gene umfasst. Es wurden sowohl bekannte Kraniosynostose- als auch Kandidaten-Gene sowie Gene, die mit der Ossifikation assoziiert sind, in die Analyse eingeschlossen. Das Genpanel wurde durch die Sequenzierung von f{\"u}nf Kontrollproben mit bekannten Mutationen erfolgreich validiert. Anschließend wurde die genomische DNA von 66 Patienten analysiert. Es konnten 20 (potentiell) pathogene Varianten identifiziert werden. Neben bereits bekannten Mutationen in den Genen FGFR1, FGFR2, FGFR3 und TWIST1, konnten zus{\"a}tzlich 8 neue, potentiell pathogene Varianten in den Genen ERF, MEGF8, MSX2, PTCH1 und TCF12 identifiziert werden. Die Ergebnisse dieser Arbeit tragen dazu bei das Mutationsspektrum dieser Gene zu erweitern. Bei zwei der Varianten handelte es sich um potentielle Spleißvarianten. F{\"u}r diese konnte in einem in vitro Spleißsystem gezeigt werden, dass sie eine {\"A}nderung des Spleißmusters bewirken. Der Nachweis von zwei seltenen Varianten in den Genen FGFR2 und HUWE1 hat außerdem dazu beigetragen die Pathogenit{\"a}t dieser spezifischen Varianten zu bekr{\"a}ftigen. Eine Variante in POR, die aufgrund bioinformatischer Analysen als potentiell pathogen bewertet wurde, wurde nach der Segregationsanalyse als wahrscheinlich benigne eingestuft. Zusammenfassend konnten bei etwa einem Drittel der Patienten, die mit dem NGS Genpanel analysiert wurden, eine genetische Ursache identifiziert werden. Dieses Genpanel stellt somit ein effizientes diagnostisches Tool dar, das zuk{\"u}nftig in der genetischen Routine-Diagnostik von Kraniosynostose-Patienten eingesetzt werden kann. Die Ergebnisse dieser Arbeit zeigen, dass sowohl eine Untersuchung auf CNVs als auch auf Sequenz{\"a}nderungen bei Kraniosynostose Patienten sinnvoll ist.}, subject = {Kraniosynostose}, language = {de} } @phdthesis{Tiwarekar2019, author = {Tiwarekar, Vishakha Rakesh}, title = {The APOBEC3G-regulated host factors REDD1 and KDELR2 restrict measles virus replication}, doi = {10.25972/OPUS-17952}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-179526}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Measles is an extremely contagious vaccine-preventable disease responsible for more than 90000 deaths worldwide annually. The number of deaths has declined from 8 million in the pre-vaccination era to few thousands every year due to the highly efficacious vaccine. However, this effective vaccine is still unreachable in many developing countries due to lack of infrastructure, while in developed countries too many people refuse vaccination. Specific antiviral compounds are not yet available. In the current situation, only an extensive vaccination approach along with effective antivirals could help to have a measles-free future. To develop an effective antiviral, detailed knowledge of viral-host interaction is required. This study was undertaken to understand the interaction between MV and the innate host restriction factor APOBEC3G (A3G), which is well-known for its activity against human immunodeficiency virus (HIV). Restriction of MV replication was not attributed to the cytidine deaminase function of A3G, instead, we identified a novel role of A3G in regulating cellular gene functions. Among two of the A3G regulated host factors, we found that REDD1 reduced MV replication, whereas, KDELR2 hampered MV haemagglutinin (H) surface transport thereby affecting viral release. REDD1, a negative regulator of mTORC1 signalling impaired MV replication by inhibiting mTORC1. A3G regulated REDD1 expression was demonstrated to inversely correlate with MV replication. siRNA mediated silencing of A3G in primary human blood lymphocytes (PBL) reduced REDD1 levels and simultaneously increased MV titres. Also, direct depletion of REDD1 improved MV replication in PBL, indicating its role in A3G mediated restriction of MV. Based on these finding, a new role of rapamycin, a pharmacological inhibitor of mTORC1, was uncovered in successfully diminishing MV replication in Vero as well as in human PBL. The ER and Golgi resident receptor KDELR2 indirectly affected MV by competing with MV-H for cellular chaperones. Due to the sequestering of chaperones by KDELR2, they can no longer assist in MV-H folding and subsequent surface expression. Taken together, the two A3G-regulated host factors REDD1 and KDELR2 are mainly responsible for mediating its antiviral activity against MV.}, language = {en} } @phdthesis{Anany2019, author = {Anany, Mohamed Ahmed Mohamed Mohamed}, title = {Enhancement of Toll-like receptor3 (TLR3)-induced death signaling by TNF-like weak inducer of apoptosis (TWEAK)}, doi = {10.25972/OPUS-18975}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-189757}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a member of the TNF superfamily (TNFSF) and is as such initially expressed as type II class transmembrane glycoprotein from which a soluble ligand form can be released by proteolytic processing. While the expression of TWEAK has been detected at the mRNA level in various cell lines and cell types, its cell surface expression has so far only been documented for dendritic cells, monocytes and interferon-γ stimulated NK cells. The fibroblast growth factor-inducible-14 (Fn14) is a TRAF2-interacting receptor of the TNF receptor superfamily (TNFRSF) and is the only receptor for TWEAK. The expression of Fn14 is strongly induced in a variety of non-hematopoietic cell types after tissue injury. The TWEAK/Fn14 system induces pleiotropic cellular activities such as induction of proinflammatory genes, stimulation of cellular angiogenesis, proliferation, differentiation, migration and in rare cases induction of apoptosis. On the other side, Toll-like receptor3 (TLR3) is one of DNA- and RNA-sensing pattern recognition receptors (PRRs), plays a crucial role in the first line of defense against virus and invading foreign pathogens and cancer cells. Polyinosinic-polycytidylic acid poly(I:C) is a synthetic analog of dsRNA, binds to TLR3 which acts through the adapter TRIF/TICAM1, leading to cytokine secretion, NF-B activation, IRF3 nuclear translocation, inflammatory response and may also elicit the cell death. TWEAK sensitizes cells for TNFR1-induced apoptosis and necroptosis by limiting the availability of protective TRAF2-cIAP1 and TRAF2-cIAP2 complexes, which interact with the TNFR1-binding proteins TRADD and RIPK1. In accordance with the fact that poly(I:C)-induced signaling also involves these proteins, we found enhanced necroptosis-induction in HaCaT and HeLa-RIPK3 by poly(I:C) in the presence of TWEAK (Figure 24). Analysis of a panel of TRADD, FADD, RIPK1 and caspase-8 knockout cells revealed furthermore similarities and differences in the way how these molecules act in cell death signaling by poly(I:C)/TWEAK and TNF and TRAIL. RIPK1 turned out to be essential for poly(I:C)/TWEAK-induced caspase-8-mediated apoptosis but was dispensable for these responses in TNF and TRAIL signaling. Lack of FADD protein abrogated TRAIL- but not TNF- and poly(I:C)-induced necroptosis. Moreover, we observed that both long and short FLIP rescued HaCaT and HeLa-RIPK3 cells from poly(I:C)-induced apoptosis or necroptosis. To sum up, our results demonstrate that TWEAK, which is produced by interferon stimulated myeloid cells, controls the induction of apoptosis and necroptosis by the TLR3 ligand poly(I:C) and may thus contribute to cancer or anti-viral immunity treatment.}, subject = {Immunologe}, language = {en} } @phdthesis{Turakhiya2019, author = {Turakhiya, Ankit}, title = {Functional characterization of the role of ZFAND1 in stress granule turnover}, doi = {10.25972/OPUS-16375}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-163751}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Protein quality control systems are critical for cellular proteostasis and survival under stress conditions. The ubiquitin proteasome system (UPS) plays a pivotal role in proteostasis by eliminating misfolded and damaged proteins. However, exposure to the environmental toxin arsenite results in the accumulation of polyubiquitylated proteins, indicating an overload of the UPS. Arsenite stress induces the rapid formation of stress granules (SGs), which are cytoplasmic assemblies of mRNPs stalled in translation initiation. The mammalian proteins ZFAND2A/B (also known as AIRAP and AIRAPL, respectively) bind to the 26S proteasome, and ZFAND2A has been shown to adapt proteasome activity to arsenite stress. They belong to a small subfamily of AN1 type zinc finger containing proteins that also comprises the unexplored mammalian member ZFAND1 and its yeast homolog Cuz1. In this thesis, the cellular function of Cuz1 and ZFAND1 was investigated. Cuz1/ZFAND1 was found to interact with the ubiquitin-selective, chaperone-like ATPase Cdc48/p97 and with the 26S proteasome. The interaction between Cuz1/ZFAND1 and Cdc48/p97 requires a predicted ubiquitin-like domain of Cuz1/ZFAND1. In vivo, this interaction was strongly dependent on acute arsenite stress, suggesting that it is a part of the cellular arsenite stress response. Lack of Cuz1/ZFAND1 caused a defect in the clearance of arsenite induced SG clearance. ZFAND1 recruits both, the 26S proteasome and p97, to arsenite-induced SGs for their normal clearance. In the absence of ZFAND1, SGs lack the 26S proteasome and p97, accumulate defective ribosomal products and become aberrant. These aberrant SGs persist after arsenite removal and undergo degradation via autophagy. ZFAND1 depletion is epistatic to the expression of pathogenic mutant p97 with respect to SG clearance, suggesting that ZFAND1 function is relevant to the multisystem degenerative disorder, inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia and amyotrophic lateral sclerosis (IBMPFD/ALS).}, subject = {ubiquitin}, language = {en} } @phdthesis{KaltdorfgebSchuch2019, author = {Kaltdorf [geb. Schuch], Kristin Verena}, title = {Mikroskopie, Bildverarbeitung und Automatisierung der Analyse von Vesikeln in \(C.\) \(elegans\) und anderen biologischen Strukturen}, doi = {10.25972/OPUS-16062}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-160621}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Thema dieser Thesis ist die Analyse sekretorischer Vesikelpools auf Ultrastrukturebene in unterschiedlichen biologischen Systemen. Der erste und zweite Teil dieser Arbeit fokussiert sich auf die Analyse synaptischer Vesikelpools in neuromuskul{\"a}ren Endplatten (NME) im Modellorganismus Caenorhabditis elegans. Dazu wurde Hochdruckgefrierung und Gefriersubstitution angewandt, um eine unverz{\"u}gliche Immobilisation der Nematoden und somit eine Fixierung im nahezu nativen Zustand zu gew{\"a}hrleisten. Anschließend wurden dreidimensionale Aufnahmen der NME mittels Elektronentomographie erstellt. Im ersten Teil dieser Arbeit wurden junge adulte, wildtypische C. elegans Hermaphroditen mit Septin-Mutanten verglichen. Um eine umfassende Analyse mit hoher Stichprobenzahl zu erm{\"o}glichen und eine automatisierte L{\"o}sung f{\"u}r {\"a}hnliche Untersuchungen von Vesikelpools bereit zu stellen wurde eine Software namens 3D ART VeSElecT zur automatisierten Vesikelpoolanalyse entwickelt. Die Software besteht aus zwei Makros f{\"u}r ImageJ, eines f{\"u}r die Registrierung der Vesikel und eines zur Charakterisierung. Diese Trennung in zwei separate Schritte erm{\"o}glicht einen manuellen Verbesserungsschritt zum Entfernen falsch positiver Vesikel. Durch einen Vergleich mit manuell ausgewerteten Daten neuromuskul{\"a}rer Endplatten von larvalen Stadien des Modellorganismus Zebrafisch (Danio rerio) konnte erfolgreich die Funktionalit{\"a}t der Software bewiesen werden. Die Analyse der neuromuskul{\"a}ren Endplatten in C. elegans ergab kleinere synaptische Vesikel und dichtere Vesikelpools in den Septin-Mutanten verglichen mit Wildtypen. Im zweiten Teil der Arbeit wurden neuromuskul{\"a}rer Endplatten junger adulter C. elegans Hermaphroditen mit Dauerlarven verglichen. Das Dauerlarvenstadium ist ein spezielles Stadium, welches durch widrige Umweltbedingungen induziert wird und in dem C. elegans {\"u}ber mehrere Monate ohne Nahrungsaufnahme {\"u}berleben kann. Da hier der Vergleich der Abundanz zweier Vesikelarten, der „clear-core"-Vesikel (CCV) und der „dense-core"-Vesikel (DCV), im Fokus stand wurde eine Erweiterung von 3D ART VeSElecT entwickelt, die einen „Machine-Learning"-Algorithmus zur automatisierten Klassifikation der Vesikel integriert. Durch die Analyse konnten kleinere Vesikel, eine erh{\"o}hte Anzahl von „dense-core"-Vesikeln, sowie eine ver{\"a}nderte Lokalisation der DCV in Dauerlarven festgestellt werden. Im dritten Teil dieser Arbeit wurde untersucht ob die f{\"u}r synaptische Vesikelpools konzipierte Software auch zur Analyse sekretorischer Vesikel in Thrombozyten geeignet ist. Dazu wurden zweidimensionale und dreidimensionale Aufnahmen am Transmissionselektronenmikroskop erstellt und verglichen. Die Untersuchung ergab, dass hierf{\"u}r eine neue Methodik entwickelt werden muss, die zwar auf den vorherigen Arbeiten prinzipiell aufbauen kann, aber den besonderen Herausforderungen der Bilderkennung sekretorischer Vesikel aus Thrombozyten gerecht werden muss.}, subject = {Mikroskopie}, language = {de} } @phdthesis{Bach2019, author = {Bach, Matthias}, title = {Massenspektrometrische Analyse der Interaktionen von Protein mit Proteinen und Proteinen mit niedermolekularen Verbindungen}, doi = {10.25972/OPUS-16046}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-160462}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Proteine k{\"o}nnen aufgrund ihrer biochemischen Vielfalt eine Vielzahl von Interaktionen mit anderen Proteinen oder chemischen Verbindungen eingehen. Im ersten Teil dieser Arbeit wurden Protein-Protein Interaktionen mittels chemischen Quervernetzens untersucht. Das Ziel war, neue und verbesserte Methoden zu entwickeln, um Interaktionsnetzwerke zu erstellen. Im zweiten Teil wurden die Interaktionen von Proteinen mit niedermolekularen Verbindungen untersucht, um Drug Targets zu identifizieren und zu validieren. Die Untersuchung von Protein-Protein Interaktionen mittels Massenspektrometrie (MS) ist eine leistungsf{\"a}hige Methode, um alle potentiellen Interaktionen eines Proteins nach einer Anreicherung (Co-IP) aus einem Zelllysat zu detektieren. Durch das zus{\"a}tzliche Quervernetzen dieser Proteine und anschließender MS kann ein Interaktionsnetzwerk erstellt werden, um direkte von indirekten Interaktionen unterscheiden zu k{\"o}nnen (Topology Mapping). Zur Methodenetablierung wurden kommerzielle Crosslinker und rekombinante Proteine von bekannten Interaktionspartnern mit niedriger Komplexit{\"a}t verwendet. Die beiden Interaktionspartner NPL4 und UFD1 konnten mit dem Crosslinker BS3 erfolgreich quervernetzt und anhand der vernetzten Peptide identifiziert werden. Im n{\"a}chsten Schritt wurde dieser Arbeitsablauf auf eine Co-IP des Mediatorkomplexes aus Hefe angewendet. Die Probenkomplexit{\"a}t ist hierbei 500 - 1000-fach h{\"o}her als bei der Verwendung von rekombinanten Proteinen. Nach der erfolgreichen Quervernetzung konnte innerhalb des Komplexes ein Interaktionsnetzwerk erstellt werden. Diese Daten passen zu dem bereits bekannten Modell des Mediatorkomplexes. Interaktionen zu bekannten Interaktionspartnern, wie der RNA-Pol II, konnten aufgrund deren subst{\"o}chiometrischen Anreicherung nicht identifiziert werden. Aufgrund der genannten Limitationen beim Quervernetzen von Proteinen wurden folgende neue und verbesserte Methoden entwickelt: 1. Verwendung des spaltbaren Crosslinkers (DSSO), der w{\"a}hrend der Messung selektiv durch niedrige Kollisionsenergie gespalten werden kann, um die Datenbanksuche zu vereinfachen. Die Funktionalit{\"a}t der DSSO-Strategie konnte erfolgreich am Protein Cytochrom C getestet werden. Bei der ersten Fragmentierung wird der Linker gespalten, anschließend k{\"o}nnen die getrennten Peptide separat fragmentiert werden. Die erzeugten Daten sind mit einer Standarddatenbanksuche kompatibel, was bei gemischten Spektren von zwei Peptiden nicht der Fall w{\"a}re. Beim Quervernetzen der rekombinanten Interaktionspartner UBX und p97N mit DSSO konnte der zu best{\"a}tigende Crosslink zwischen zwei Lysinen nicht identifziert werden. Grund hierf{\"u}r k{\"o}nnte eine zu kurze Linkerl{\"a}nge von DSSO sein. Diese Versuche brachten jedoch einige Limitationen des Ansatzes zum Vorschein, wie die Beschr{\"a}nkung auf die Protease Trypsin, aufgrund der positiven Ladung am C-Terminus und die Notwendigkeit von großen Proteinmengen, da das Spalten des Linkers einen zus{\"a}tzlichen Intensit{\"a}tsverlust f{\"u}r die folgende Identifizierung der Peptide mit sich bringt. 2. Da die niedrige Abundanz von quervernetzten Peptiden das Hauptproblem bei deren Identifizierung ist, wurde eine Methode entwickelt, um w{\"a}hrend der Messung direkt nach diesen niedrig abundanten Spezies zu suchen. Entscheidendes Kriterium hierf{\"u}r war, dass quervernetzte Peptide zwei C-Termini haben. Diese wurden zur H{\"a}lfte enzymatisch mit 18O bzw. 16O markiert und wieder vereinigt. Der resultierende Massenunterschied von 8 Da (4 x 18O) kommt ausschließlich bei zwei quervernetzten Peptiden vor und kann w{\"a}hrend der Messung direkt gesucht werden. Die vollst{\"a}ndige Markierung von Peptiden mit 18O wurde zun{\"a}chst am Protein Beta-Galaktosidase getestet. Bereits hier stellte sich heraus, dass der enzymatische R{\"u}cktausch von 18O zu 16O ein Problem darstellt und die Markierungseffizienz von Aminos{\"a}uren beeinflusst wird, die sich C-terminal nach der Spaltstelle befinden. Mit dieser Strategie ließ sich somit keine vollst{\"a}ndige Markierung f{\"u}r alle Peptide erreichen, was f{\"u}r diese Strategie essentiell gewesen w{\"a}re. 3. Um alle Probleme zu umgehen, die bei der Identifizierung von quervernetzten Peptiden auftreten, wurde eine Methode entwickelt, um quervernetzte Proteine anhand von Profilen nach einer Auftrennung im Polyacrylamidgel (SDS-PAGE) zu identifizieren. Durch das Quervernetzen von Proteinen entstehen zus{\"a}tzliche Proteinbanden nach einer SDSPAGE, die im Gel nach oben verschoben sind. Alle Proteine in diesen neu erzeugten Bereichen stellen somit potentielle Interaktionspartner dar. Als Modellsystem wurde der Mediatorkomplex verwendet. Er wurde aus einem Zelllysat mittels Co-IP angereichert und anschließend quervernetzt. Aus den mittels LC-MS/MS gemessenen Gelfraktionen wurden Proteinprofile erstellt und miteinander verglichen. Die Intensit{\"a}tsmaxima der Proteine des Mediatorkomplexes konnten in bestimmten zus{\"a}tzlichen Fraktionen gefunden werden, was den indirekten Nachweis f{\"u}r eine Interaktion darstellt. Die Funktionalit{\"a}t der Strategie konnte somit best{\"a}tigt werden. Ein verbleibender Nachteil ist jedoch die zu geringe Trennleistung von Polyacrylamidgelen. Befinden sich mehr als 50 Proteine in einer Fraktion, k{\"o}nnen potentielle Interaktionspartner nicht eindeutig zu einer Untereinheit eines Komplexes zugeordnet werden. Im zweiten Teil der Arbeit wurde im Rahmen der Klinischen Forschergruppe 216 (CRU216) Interaktionen von Proteinen mit verschiedenen niedermolekularen Verbindungen massenspektrometrisch untersucht, um potentielle Drug Targets zu identifizieren. Diese Versuche sind vergleichbar mit Co-IP Experimenten, da sich der Arbeitsablauf nur durch die Anreicherung mittels chemischer Verbindung unterscheidet. Hierzu wurden biotinylierte Verbindungen immobilisiert und potentielle Drug Targets aus einem komplexen Zelllysat angereichert. Die Identifzierung der echten Bindungspartner wurde {\"u}ber quantitive Massenspektrometrie erreicht. Dabei wurden die angereicherten Proteine, die an die niedermolekularen Substanzen binden mit einer geeigneten Kontrollanreicherung verglichen. Mit den getesteten α-acyl Aminocarboxamiden konnten verschiedene Proteinkomplexe und interagierende Proteine spezifisch angereichert werden. Hierbei waren die vier Kinasen DNA-PK, ATM, ATR und mTOR besonders interessant, da sie mit onkogenem Signalling und {\"U}berlebensmechanismen wie der Hitzeschockantwort in Zellen des Multiplen Myeloms (MM) in Verbidnung stehen. Die Inhibition der DNA-PK, ATM, ATR und mTOR mit α- acyl Aminocarboxamiden stellt somit einen m{\"o}glichen Therapieansatz dar, wenn er zusammen mit hitzestressausl{\"o}senden Inhibitoren verwendet wird. Weiterhin konnte gezeigt werden, dass die Armadillodom{\"a}ne innerhalb der potentiellen Drug targets signifkant angereichert wurde. Sie stellt damit eine potentielle Bindestelle der α-acyl Aminocarboxamide dar. Abschließend wurden Proteine mit biotinylierten Naphtylisochinolinen aus einem MMZelllysat angereichert, deren Vorl{\"a}ufersubstanzen eine Wirkung auf Tumorzellen und den Malariaparasit Plasmodium falciparum gezeigt hatten. Hierbei konnten vor allem RNAbindende- und mRNA-Splicing Proteine identifiziert werden, die zum Teil essentiell f{\"u}r das Spleißen in-vivo sind. Hierzu geh{\"o}ren mehrere Untereinheiten der Splicing Factoren 3A und 3B. Die Ver{\"a}nderung der transkriptionellen Regulation und der resultierende Effekt auf Krebszellen konnte bereits in anderen Studien mit dem Inhibitor Spliceostatin A gezeigt werden, der das Spleißen beeinflusst.}, subject = {Massenspektrometrie}, language = {de} } @phdthesis{Gulve2019, author = {Gulve, Nitish}, title = {Subversion of Host Genome Integrity by Human Herpesvirus 6 and \(Chlamydia\) \(trachomatis\)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-162026}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Ovarian cancer is one of the most common gynecological malignancies in the world. The prevalence of a microbial signature in ovarian cancer has been reported by several studies till date. In these microorganisms, Human herpesvirus 6 (HHV-6) and Chlamydia trachomatis (C.tr) are especially important as they have significantly high prevalence rate. Moreover, these pathogens are directly involved in causing DNA damage and thereby disrupting the integrity of host genome which is the underlying cause of any cancer. This study focuses on how the two pathogens, HHV-6 and C. trachomatis can affect the genome integrity in their individual capacities and thereby may drive ovarian epithelial cells towards transformation. HHV-6 has unique tendency to integrate its genome into the host genome at subtelomeric regions and achieve a state of latency. This latent virus may get reactivated during the course of life by stress, drugs such as steroids, during transplantation, pregnancy etc. The study presented here began with an interesting observation wherein the direct repeat (DR) sequences flanking the ends of double stranded viral genome were found in unusually high numbers in human blood samples as opposed to normal ratio of two DR copies per viral genome. This study was corroborated with in vitro data where cell lines were generated to mimic the HHV-6 status in human samples. The same observation of unusually high DR copies was found in these cell lines as well. Interestingly, fluorescence in situ hybridization (FISH) and inverse polymerase chain reaction followed by southern blotting showed that DR sequences were found to be integrated in nontelomeric regions as opposed to the usual sub-telomeric integration sites in both human samples and in cell lines. Sanger sequencing confirmed the non-telomeric integration of viral DR sequences in the host genome. Several studies have shown that C. trachomatis causes DNA damage and inhibits the signaling cascade of DNA damage response. However, the effect of C. trachomatis infection on process of DNA repair itself was not addressed. In this study, the effect of C. trachomatis infection on host base excision repair (BER) has been addressed. Base excision repair is a pathway which is responsible for replacing the oxidized bases with new undamaged ones. Interestingly, it was found that C. trachomatis infection downregulated polymerase β expression and attenuated polymerase β- mediated BER in vitro. The mechanism of the polymerase β downregulation was found to be associated with the changes in the host microRNAs and downregulation of tumor suppressor, p53. MicroRNA-499 which has a binding site in the polymerase β 3'UTR was shown to be upregulated during C. trachomatis infection. Inhibition of miR-499 using synthetic miR-499 inhibitor indeed improved the repair efficiency during C. trachomatis infection in the in vitro repair assay. Moreover, p53 transcriptionally regulates polymerase β and stabilizing p53 during C. trachomatis infection enhanced the repair efficiency. Previous studies have shown that C. trachomatis can reactivate latent HHV-6. Therefore, genomic instability due to insertions of unstable 'transposon-like' HHV-6 DR followed by compromised BER during C. trachomatis infection cumulatively support the hypothesis of pathogenic infections as a probable cause of ovarian cancer}, subject = {Chlamydia trachomatis}, language = {en} } @article{PaulsHamaratTrufasuetal.2019, author = {Pauls, Dennis and Hamarat, Yasmin and Trufasu, Luisa and Schendzielorz, Tim M. and Gramlich, Gertrud and Kahnt, J{\"o}rg and Vanselow, Jens and Schlosser, Andreas and Wegener, Christian}, title = {Drosophila carboxypeptidase D (SILVER) is a key enzyme in neuropeptide processing required to maintain locomotor activity levels and survival rate}, series = {European Journal of Neuroscience}, volume = {50}, journal = {European Journal of Neuroscience}, number = {9}, doi = {10.1111/ejn.14516}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-204863}, pages = {3502-3519}, year = {2019}, abstract = {Neuropeptides are processed from larger preproproteins by a dedicated set of enzymes. The molecular and biochemical mechanisms underlying preproprotein processing and the functional importance of processing enzymes are well-characterised in mammals, but little studied outside this group. In contrast to mammals, Drosophila melanogaster lacks a gene for carboxypeptidase E (CPE ), a key enzyme for mammalian peptide processing. By combining peptidomics and neurogenetics, we addressed the role of carboxypeptidase D (dCPD ) in global neuropeptide processing and selected peptide-regulated behaviours in Drosophila . We found that a deficiency in dCPD results in C-terminally extended peptides across the peptidome, suggesting that dCPD took over CPE function in the fruit fly. dCPD is widely expressed throughout the nervous system, including peptidergic neurons in the mushroom body and neuroendocrine cells expressing adipokinetic hormone. Conditional hypomorphic mutation in the dCPD -encoding gene silver in the larva causes lethality, and leads to deficits in starvation-induced hyperactivity and appetitive gustatory preference, as well as to reduced viability and activity levels in adults. A phylogenomic analysis suggests that loss of CPE is not common to insects, but only occurred in Hymenoptera and Diptera. Our results show that dCPD is a key enzyme for neuropeptide processing and peptide-regulated behaviour in Drosophila . dCPD thus appears as a suitable target to genetically shut down total neuropeptide production in peptidergic neurons. The persistent occurrence of CPD in insect genomes may point to important further CPD functions beyond neuropeptide processing which cannot be fulfilled by CPE.}, language = {en} } @article{DechaudVolffSchartletal.2019, author = {Dechaud, Corentin and Volff, Jean-Nicolas and Schartl, Manfred and Naville, Magali}, title = {Sex and the TEs: transposable elements in sexual development and function in animals}, series = {Mobile DNA}, volume = {10}, journal = {Mobile DNA}, doi = {10.1186/s13100-019-0185-0}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-202510}, pages = {42}, year = {2019}, abstract = {Transposable elements are endogenous DNA sequences able to integrate into and multiply within genomes. They constitute a major source of genetic innovations, as they can not only rearrange genomes but also spread ready-to-use regulatory sequences able to modify host gene expression, and even can give birth to new host genes. As their evolutionary success depends on their vertical transmission, transposable elements are intrinsically linked to reproduction. In organisms with sexual reproduction, this implies that transposable elements have to manifest their transpositional activity in germ cells or their progenitors. The control of sexual development and function can be very versatile, and several studies have demonstrated the implication of transposable elements in the evolution of sex. In this review, we report the functional and evolutionary relationships between transposable elements and sexual reproduction in animals. In particular, we highlight how transposable elements can influence expression of sexual development genes, and how, reciprocally, they are tightly controlled in gonads. We also review how transposable elements contribute to the organization, expression and evolution of sexual development genes and sex chromosomes. This underscores the intricate co-evolution between host functions and transposable elements, which regularly shift from a parasitic to a domesticated status useful to the host.}, language = {en} } @phdthesis{Sauer2019, author = {Sauer, Markus}, title = {DHX36 function in RNA G-quadruplex-mediated posttranscriptional gene regulation}, doi = {10.25972/OPUS-18395}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-183954}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {The expression of genetic information into proteins is a key aspect of life. The efficient and exact regulation of this process is essential for the cell to produce the correct amounts of these effector molecules to a given situation. For this purpose, eukaryotic cells have developed many different levels of transcriptional and posttranscriptional gene regulation. These mechanisms themselves heavily rely on interactions of proteins with associated nucleic acids. In the case of posttranscriptional gene regulation an orchestrated interplay between RNA-binding proteins, messenger RNAs (mRNA), and non-coding RNAs is compulsory to achieve this important function. A pivotal factor hereby are RNA secondary structures. One of the most stable and diverse representatives is the G-quadruplex structure (G4) implicated in many cellular mechanisms, such as mRNA processing and translation. In protein biosynthesis, G4s often act as obstacles but can also assist in this process. However, their presence has to be tightly regulated, a task which is often fulfilled by helicases. One of the best characterized G4-resolving factors is the DEAH-box protein DHX36. The in vitro function of this helicase is extensively described and individual reports aimed to address diverse cellular functions as well. Nevertheless, a comprehensive and systems-wide study on the function of this specific helicase was missing, so far. The here-presented doctoral thesis provides a detailed view on the global cellular function of DHX36. The binding sites of this helicase were defined in a transcriptome-wide manner, a consensus binding motif was deviated, and RNA targets as well as the effect this helicase exerts on them were examined. In human embryonic kidney cells, DHX36 is a mainly cytoplasmic protein preferentially binding to G-rich and G4-forming sequence motifs on more than 4,500 mRNAs. Loss of DHX36 leads to increased target mRNA levels whereas ribosome occupancy on and protein output of these transcripts are reduced. Furthermore, DHX36 knockout leads to higher RNA G4 levels and concomitant stress reactions in the cell. I hypothesize that, upon loss of this helicase, translationally-incompetent structured DHX36 target mRNAs, prone to localize in stress granules, accumulate in the cell. The cell reacts with basal stress to avoid cytotoxic effects produced by these mis-regulated and structured transcripts.}, subject = {RNS}, language = {en} } @phdthesis{RoesergebAssmus2019, author = {R{\"o}ser [geb. Aßmus], Benjamin}, title = {SPRED2 (Sprouty-related EVH1 domain containing 2) reguliert die Autophagie in Kardiomyozyten}, doi = {10.25972/OPUS-18270}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-182700}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Das Sprouty-related, EVH1 domain containing protein 2 (SPRED2) ist ein inhibitorisches, downstream von Ras wirkendes Protein des MAP-Kinase Signalwegs, welches entscheidenden Einfluss auf die Regulation von Proliferation, Expression von Proteinen und der zellul{\"a}ren Hom{\"o}ostase hat. Der kardiale Ph{\"a}notyp von SPRED2- defizienten M{\"a}usen zeigt nicht nur eine deutliche linksventrikul{\"a}re Hypertrophie, sondern auch eine erh{\"o}hte Fibrosierung des Herzgewebes. Zellul{\"a}r wird die SPRED2- Defizienz durch die Akkumulation von vesikul{\"a}ren Strukturen innerhalb der Zelle, sowie eine markant erh{\"o}hte Anzahl von Vesikeln entlang der longitudinalen Reihen der Mitochondrien gekennzeichnet. Ziel dieser Arbeit war es, den Charakter dieser vesikul{\"a}ren Strukturen n{\"a}her zu beleuchten und festzustellen, in welchem Zusammenhang die subzellul{\"a}r ver{\"a}nderte Architektur mit der Hypertrophie der SPRED2-defizienten Tiere steht. Um diese Fragestellung zu beantworten, wurde zun{\"a}chst nach einem vesikul{\"a}ren Degradationsmechanismus gesucht, der in SPRED2-/--Cardiomyocyten betroffen sein k{\"o}nnte. Die Macroautophagie, im folgenden Autophagie bezeichnet, ist ein solcher Degradationsmechanismus, bei dem selektiv langlebige Proteine und Zellorganellen abgebaut werden. Es konnten signifikante Ver{\"a}nderung der Protein-Level an Schl{\"u}sselpositionen der Autophagie identifiziert werden. Das Ubiquitin-aktivierende (E1) Enzym Homolog Atg7 sowie die Cystein-Protease Atg4B zeigen sich im SPRED2- KO deutlich reduziert. Ebenso Atg16L, das als essentieller Bestandteil des Atg5- Atg12-Atg16-Konjugationssystems bei der Konjugation von MAPLC3-II an das Phospholipid Phosphatidylethanolamin beteiligt ist. Die Autophagie-Rate als Verh{\"a}ltnis von konjugiertem zu unkonjugiertem MAPLC3 ist ebenfalls reduziert. Die Akkumulation der autophagischen Vesikel zeigt sich kongruent zu dem erh{\"o}hten Protein-Level der autophagischen Cargo-Rezeptoren SQSTM1 und NBR1, sowie des lysosomalen Markers CathepsinD. Außer der verringerten Autophagie-Rate zeigt sich in Einklang mit der Fibrosierung des Herzgewebes eine erh{\"o}ht aktive Caspase-3 als Marker f{\"u}r Apoptose. Um die mitochondriale Integrit{\"a}t n{\"a}her zu beleuchten, wurde die Menge an reaktiven Sauerstoffspezies (ROS) in Wildtyp und SPRED2-KO untersucht. Hierbei zeigte sich eine erh{\"o}hte Menge an ROS im KO, was ein Hinweis auf eine Beeintr{\"a}chtigung der Mitochondrien darstellt. Letztlich wurde die Hypothese {\"u}berpr{\"u}ft, ob ein gest{\"o}rter Transport der Vesikel durch eine Beeintr{\"a}chtigung der Motorproteine Dynein und Kinesin vorliegt. In der Tat zeigte sich die Aktivit{\"a}t der Dynein-ATPase verringert in der Abwesenheit von SPRED2. Diese Beobachtung wird durch die erh{\"o}hten Mengen des vSNARE-Proteins VTI1b unterst{\"u}tzt, was letztlich die Akkumulation der autophagischen Vesikel mit einer verringerten F{\"a}higkeit zur Membranfusion und dem ineffizienteren Transport der Vesikel in Einklang bringt. Da die gesamten Experimente in einem globalen SPRED2-KO System durchgef{\"u}hrt wurden, k{\"o}nnen eventuelle Auswirkungen der beeinflussten hormonellen Situation der SPRED2-KO Tiere auf den Herzph{\"a}notyp nicht final ausgeschlossen werden. Um die genaue Wirkung einer SPRED2-Defizienz auf das Herzgewebe und das Herz als Organ zu untersuchen, wurde im Rahmen dieser Arbeit eine SPRED2- defiziente knockout Mauslinie mit konditionalem Potential generiert, die eine gesteuerte Deletion von SPRED2 im Herzgewebe erlaubt.}, subject = {Spred-Proteine}, language = {de} } @phdthesis{MendesPereira2019, author = {Mendes Pereira, Lenon}, title = {Morphological and Functional Ultrashort Echo Time (UTE) Magnetic Resonance Imaging of the Human Lung}, doi = {10.25972/OPUS-18317}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-183176}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {In this thesis, a 3D Ultrashort echo time (3D-UTE) sequence was introduced in the Self-gated Non-Contrast-Enhanced Functional Lung Imaging (SENCEFUL) framework. The sequence was developed and implemented on a 3 Tesla MR scanner. The 3D-UTE technique consisted of a nonselective RF pulse followed by a koosh ball quasi-random sampling order of the k-space. Measurements in free-breathing and without contrast agent were performed in healthy subjects and a patient with lung cancer. A gating technique, using a combination of different coils with high signal correlation, was evaluated in-vivo and compared with a manual approach of coil selection. The gating signal offered an estimation of the breathing motion during measurement and was used as a reference to segment the acquired data into different breathing phases. Gradient delays and trajectory errors were corrected during post-processing using the Gradient Impulse Response Function. Iterative SENSE was then applied to determine the fully sampled data. In order to eliminate signal changes caused by motion, a 3D image registration was employed, and the results were compared to a 2D image registration method. Ventilation was assessed in 3D and regionally quantified by monitoring the signal changes in the lung parenchyma. Finally, image quality and quantitative ventilation values were compared to the standard 2D-SENCEFUL technique. 3D-UTE, combined with an automatic gating technique and SENCEFUL MRI, offered ventilation maps with high spatial resolution and SNR. Compared to the 2D method, UTE-SENCEFUL greatly improved the clinical quality of the structural images and the visualization of the lung parenchyma. Through-plane motion, partial volume effects and ventilation artifacts were also reduced with a three-dimensional method for image registration. UTE-SENCEFUL was also able to quantify regional ventilation and presented similar results to previous studies.}, subject = {Kernspintomografie}, language = {en} } @phdthesis{delOlmoToledo2019, author = {del Olmo Toledo, Valentina}, title = {Evolution of DNA binding preferences in a family of eukaryotic transcription regulators}, doi = {10.25972/OPUS-18789}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-187890}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Regulation of gene expression by the control of transcription is essential for any cell to adapt to the environment and survive. Transcription regulators, i.e. sequence-specific DNA binding proteins that regulate gene expression, are central elements within the gene networks of most organisms. Transcription regulators are grouped into distinct families based on structural features that determine, to a large extent, the DNA sequence(s) that they can recognise and bind. Less is known, however, about how the DNA binding preferences can diversify within transcription regulator families during evolutionary timescales, and how such diversification can affect the biology of the organism. In this dissertation I study the SREBP (sterol regulatory element binding protein) family of transcriptional regulators in yeasts, and in Candida albicans in particular, as an experimental system to address these questions. The SREBPs are conserved from fungi to humans and represent a subgroup of basic helix-loop-helix DNA binding proteins. Early chromatin immunoprecipitation experiments with SREBPs from humans and yeasts showed that these proteins bound in vivo to the canonical DNA sequence, termed E-box, most basic helix-loop-helix proteins bind to. By contrast, most recent analysis carried out with less-studied fungal SREBPs revealed a non-canonical DNA motif to be the most overrepresented sequence in the bound regions. This study aims to establish the intrinsic DNA binding preferences of key branches of this family and to determine how the divergence in DNA binding affinities originated. To this end, I combined phylogenetic and ancestral reconstruction with extensive biochemical characterisation of key SREBP proteins. The results indicated that while the most-studied SREBPs (in mammals) indeed show preference for the E-box, a second branch of the family preferentially binds the non-E-box, and a third one is able to bind both sequences with similar affinity. The preference for one or the other DNA sequence is an intrinsic property of each protein because their purified DNA binding domain was sufficient to recapitulate their in vivo binding preference. The ancestor that gave rise to these two different types of SREBPs (the branch that binds E-box and the one that binds non-E-box DNA) appears to be a protein with a broader DNA binding capability that had a slight preference for the non-canonical motif. Thus, the results imply these two branches originated by either enhancing the original ancestral preference for non-E-box or tilting it towards the E-box DNA and flipping the preference for this sequence. The main function associated with members of the SREBP family in most eukaryotes is the control of lipid biosynthesis. I have further studied the function of these proteins in the lineage that encompasses the human associated yeast C. albicans. Strikingly, the three SREBPs present in the fungus' genome contribute to the colonisation of the mammalian gut by regulating cellular processes unrelated to lipid metabolism. Here I describe that two of the three C. albicans SREBPs form a regulatory cascade that regulates morphology and cell wall modifications under anaerobic conditions, whereas the third SREBP has been shown to be involved in the regulation of glycolysis genes. Therefore, I posit that the described diversification in DNA binding specificity in these proteins and the concomitant expansion of targets of regulation were key in enabling this fungal lineage to associate with animals.}, subject = {Candida albicans}, language = {en} } @article{LiuMaierhoferRybaketal.2019, author = {Liu, Yi and Maierhofer, Tobias and Rybak, Katarzyna and Sklenar, Jan and Breakspear, Andy and Johnston, Matthew G. and Fliegmann, Judith and Huang, Shouguang and Roelfsema, M. Rob G. and Felix, Georg and Faulkner, Christine and Menke, Frank L.H. and Geiger, Dietmar and Hedrich, Rainer and Robatzek, Silke}, title = {Anion channel SLAH3 is a regulatory target of chitin receptor-associated kinase PBL27 in microbial stomatal closure}, series = {eLife}, volume = {8}, journal = {eLife}, doi = {10.7554/eLife.44474}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-202631}, pages = {e44474}, year = {2019}, abstract = {In plants, antimicrobial immune responses involve the cellular release of anions and are responsible for the closure of stomatal pores. Detection of microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) induces currents mediated via slow-type (S-type) anion channels by a yet not understood mechanism. Here, we show that stomatal closure to fungal chitin is conferred by the major PRRs for chitin recognition, LYK5 and CERK1, the receptor-like cytoplasmic kinase PBL27, and the SLAH3 anion channel. PBL27 has the capacity to phosphorylate SLAH3, of which S127 and S189 are required to activate SLAH3. Full activation of the channel entails CERK1, depending on PBL27. Importantly, both S127 and S189 residues of SLAH3 are required for chitin-induced stomatal closure and anti-fungal immunity at the whole leaf level. Our results demonstrate a short signal transduction module from MAMP recognition to anion channel activation, and independent of ABA-induced SLAH3 activation.}, language = {en} } @article{MammadovaBachBraun2019, author = {Mammadova-Bach, Elmina and Braun, Attila}, title = {Zinc homeostasis in platelet-related diseases}, series = {International Journal of Molecular Sciences}, volume = {20}, journal = {International Journal of Molecular Sciences}, number = {21}, issn = {1422-0067}, doi = {10.3390/ijms20215258}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-285554}, year = {2019}, abstract = {Zn\(^{2+}\) deficiency in the human population is frequent in underdeveloped countries. Worldwide, approximatively 2 billion people consume Zn\(^{2+}\)-deficient diets, accounting for 1-4\% of deaths each year, mainly in infants with a compromised immune system. Depending on the severity of Zn\(^{2+}\) deficiency, clinical symptoms are associated with impaired wound healing, alopecia, diarrhea, poor growth, dysfunction of the immune and nervous system with congenital abnormalities and bleeding disorders. Poor nutritional Zn\(^{2+}\) status in patients with metastatic squamous cell carcinoma or with advanced non-Hodgkin lymphoma, was accompanied by cutaneous bleeding and platelet dysfunction. Forcing Zn\(^{2+}\) uptake in the gut using different nutritional supplementation of Zn\(^{2+}\) could ameliorate many of these pathological symptoms in humans. Feeding adult rodents with a low Zn\(^{2+}\) diet caused poor platelet aggregation and increased bleeding tendency, thereby attracting great scientific interest in investigating the role of Zn\(^{2+}\) in hemostasis. Storage protein metallothionein maintains or releases Zn\(^{2+}\) in the cytoplasm, and the dynamic change of this cytoplasmic Zn\(^{2+}\) pool is regulated by the redox status of the cell. An increase of labile Zn\(^{2+}\) pool can be toxic for the cells, and therefore cytoplasmic Zn\(^{2+}\) levels are tightly regulated by several Zn\(^{2+}\) transporters located on the cell surface and also on the intracellular membrane of Zn\(^{2+}\) storage organelles, such as secretory vesicles, endoplasmic reticulum or Golgi apparatus. Although Zn\(^{2+}\) is a critical cofactor for more than 2000 transcription factors and 300 enzymes, regulating cell differentiation, proliferation, and basic metabolic functions of the cells, the molecular mechanisms of Zn\(^{2+}\) transport and the physiological role of Zn\(^{2+}\) store in megakaryocyte and platelet function remain elusive. In this review, we summarize the contribution of extracellular or intracellular Zn\(^{2+}\) to megakaryocyte and platelet function and discuss the consequences of dysregulated Zn\(^{2+}\) homeostasis in platelet-related diseases by focusing on thrombosis, ischemic stroke and storage pool diseases.}, language = {en} } @article{HerzBrehm2019, author = {Herz, Michaela and Brehm, Klaus}, title = {Evidence for densovirus integrations into tapeworm genomes}, series = {Parasites \& Vectors}, volume = {12}, journal = {Parasites \& Vectors}, doi = {10.1186/s13071-019-3820-1}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-202478}, pages = {560}, year = {2019}, abstract = {Background Tapeworms lack a canonical piRNA-pathway, raising the question of how they can silence existing mobile genetic elements (MGE). Investigation towards the underlying mechanisms requires information on tapeworm transposons which is, however, presently scarce. Methods The presence of densovirus-related sequences in tapeworm genomes was studied by bioinformatic approaches. Available RNA-Seq datasets were mapped against the Echinococcus multilocularis genome to calculate expression levels of densovirus-related genes. Transcription of densovirus loci was further analyzed by sequencing and RT-qPCR. Results We herein provide evidence for the presence of densovirus-related elements in a variety of tapeworm genomes. In the high-quality genome of E. multilocularis we identified more than 20 individual densovirus integration loci which contain the information for non-structural and structural virus proteins. The majority of densovirus loci are present as head-to-tail concatemers in isolated repeat containing regions of the genome. In some cases, unique densovirus loci have integrated close to histone gene clusters. We show that some of the densovirus loci of E. multilocularis are actively transcribed, whereas the majority are transcriptionally silent. RT-qPCR data further indicate that densovirus expression mainly occurs in the E. multilocularis stem cell population, which probably forms the germline of this organism. Sequences similar to the non-structural densovirus genes present in E. multilocularis were also identified in the genomes of E. canadensis, E. granulosus, Hydatigera taeniaeformis, Hymenolepis diminuta, Hymenolepis microstoma, Hymenolepis nana, Taenia asiatica, Taenia multiceps, Taenia saginata and Taenia solium. Conclusions Our data indicate that densovirus integration has occurred in many tapeworm species. This is the first report on widespread integration of DNA viruses into cestode genomes. Since only few densovirus integration sites were transcriptionally active in E. multilocularis, our data are relevant for future studies into gene silencing mechanisms in tapeworms. Furthermore, they indicate that densovirus-based vectors might be suitable tools for genetic manipulation of cestodes.}, language = {en} } @article{PaponovDindas Krol etal.2019, author = {Paponov, Ivan A. and Dindas , Julian and Kr{\´o}l , Elżbieta and Friz, Tatyana and Budnyk, Vadym and Teale, William and Paponov, Martina and Hedrich , Rainer and Palme, Klaus}, title = {Auxin-Induced plasma membrane depolarization is regulated by Auxin transport and not by AUXIN BINDING PROTEIN1}, series = {Frontiers in Plant Science}, volume = {9}, journal = {Frontiers in Plant Science}, issn = {1664-462X}, doi = {10.3389/fpls.2018.01953}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-195914}, year = {2019}, abstract = {Auxin is a molecule, which controls many aspects of plant development through both transcriptional and non-transcriptional signaling responses. AUXIN BINDING PROTEIN1 (ABP1) is a putative receptor for rapid non-transcriptional auxin-induced changes in plasma membrane depolarization and endocytosis rates. However, the mechanism of ABP1-mediated signaling is poorly understood. Here we show that membrane depolarization and endocytosis inhibition are ABP1-independent responses and that auxin-induced plasma membrane depolarization is instead dependent on the auxin influx carrier AUX1. AUX1 was itself not involved in the regulation of endocytosis. Auxin-dependent depolarization of the plasma membrane was also modulated by the auxin efflux carrier PIN2. These data establish a new connection between auxin transport and non-transcriptional auxin signaling.}, language = {en} } @article{RothDoerflerBaessleretal.2019, author = {Roth, Nicolas and Doerfler, Inken and B{\"a}ssler, Claus and Blaschke, Markus and Bussler, Heinz and Gossner, Martin M. and Heideroth, Antje and Thorn, Simon and Weisser, Wolfgang W. and M{\"u}ller, J{\"o}rg}, title = {Decadal effects of landscape-wide enrichment of dead wood on saproxylic organisms in beech forests of different historic management intensity}, series = {Diversity and Distributions}, volume = {25}, journal = {Diversity and Distributions}, number = {3}, doi = {10.1111/ddi.12870}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-227061}, pages = {430-441}, year = {2019}, abstract = {Aim: European temperate forests have lost dead wood and the associated biodiversity owing to intensive management over centuries. Nowadays, some of these forests are being restored by enrichment with dead wood, but mostly only at stand scales. Here, we investigated effects of a seminal dead-wood enrichment strategy on saproxylic organisms at the landscape scale. Location: Temperate European beech forest in southern Germany. Methods: In a before-after control-impact design, we compared assemblages and gamma diversities of saproxylic organisms in strictly protected old-growth forest areas (reserves) and historically moderately and intensively managed forest areas before and a decade after starting a landscape-wide strategy of dead-wood enrichment. Results: Before enrichment with dead wood, the gamma diversity of saproxylic organisms in historically intensively managed forest stands was significantly lower than in reserves and historically moderately managed forest stands; this difference disappeared after 10 years of dead-wood enrichment. The species composition of beetles in forest stands of the three historical management intensities differed before the enrichment strategy, but a decade thereafter, the species compositions of previously intensively logged and forest reserve plots were similar. However, the differences in fungal species composition between historical management categories before and after 10 years of enrichment persisted. Main conclusions: Our results demonstrate that intentional enrichment of dead wood at the landscape scale is a powerful tool for rapidly restoring saproxylic beetle communities and for restoring wood-inhabiting fungal communities, which need longer than a decade for complete restoration. We propose that a strategy of area-wide active restoration combined with some permanent strict refuges is a promising means of promoting the biodiversity of age-long intensively managed Central European beech forests.}, language = {en} } @article{BluemelZinkKlopockietal.2019, author = {Bl{\"u}mel, Rabea and Zink, Miriam and Klopocki, Eva and Liedtke, Daniel}, title = {On the traces of tcf12: Investigation of the gene expression pattern during development and cranial suture patterning in zebrafish (Danio rerio)}, series = {PLoS ONE}, volume = {14}, journal = {PLoS ONE}, number = {6}, doi = {10.1371/journal.pone.0218286}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201428}, pages = {e0218286}, year = {2019}, abstract = {The transcription factor 12 (tcf12) is a basic Helix-Loop-Helix protein (bHLH) of the E-protein family, proven to play an important role in developmental processes like neurogenesis, mesoderm formation, and cranial vault development. In humans, mutations in TCF12 lead to craniosynostosis, a congenital birth disorder characterized by the premature fusion of one or several of the cranial sutures. Current research has been primarily focused on functional studies of TCF12, hence the cellular expression profile of this gene during embryonic development and early stages of ossification remains poorly understood. Here we present the establishment and detailed analysis of two transgenic tcf12:EGFP fluorescent zebrafish (Danio rerio) reporter lines. Using these transgenic lines, we analyzed the general spatiotemporal expression pattern of tcf12 during different developmental stages and put emphasis on skeletal development and cranial suture patterning. We identified robust tcf12 promoter-driven EGFP expression in the central nervous system (CNS), the heart, the pronephros, and the somites of zebrafish embryos. Additionally, expression was observed inside the muscles and bones of the viscerocranium in juvenile and adult fish. During cranial vault development, the transgenic fish show a high amount of tcf12 expressing cells at the growth fronts of the ossifying frontal and parietal bones and inside the emerging cranial sutures. Subsequently, we tested the transcriptional activity of three evolutionary conserved non-coding elements (CNEs) located in the tcf12 locus by transient transgenic assays and compared their in vivo activity to the expression pattern determined in the transgenic tcf12:EGFP lines. We could validate two of them as tcf12 enhancer elements driving specific gene expression in the CNS during embryogenesis. Our newly established transgenic lines enhance the understanding of tcf12 gene regulation and open up the possibilities for further functional investigation of these novel tcf12 enhancer elements in zebrafish.}, language = {en} } @article{LiedtkeOrthMeissleretal.2019, author = {Liedtke, Daniel and Orth, Melanie and Meissler, Michelle and Geuer, Sinje and Knaup, Sabine and K{\"o}blitz, Isabell and Klopocki, Eva}, title = {ECM alterations in fndc3a (fibronectin domain containing protein 3A) deficient zebrafish cause temporal fin development and regeneration defects}, series = {Scientific Reports}, volume = {9}, journal = {Scientific Reports}, doi = {10.1038/s41598-019-50055-w}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-202141}, pages = {13383}, year = {2019}, abstract = {Fin development and regeneration are complex biological processes that are highly relevant in teleost fish. They share genetic factors, signaling pathways and cellular properties to coordinate formation of regularly shaped extremities. Especially correct tissue structure defined by extracellular matrix (ECM) formation is essential. Gene expression and protein localization studies demonstrated expression of fndc3a (fibronectin domain containing protein 3a) in both developing and regenerating caudal fins of zebrafish (Danio rerio). We established a hypomorphic fndc3a mutant line (fndc3a\(^{wue1/wue1}\)) via CRISPR/Cas9, exhibiting phenotypic malformations and changed gene expression patterns during early stages of median fin fold development. These developmental effects are mostly temporary, but result in a fraction of adults with permanent tail fin deformations. In addition, caudal fin regeneration in adult fndc3a\(^{wue1/wue1}\) mutants is hampered by interference with actinotrichia formation and epidermal cell organization. Investigation of the ECM implies that loss of epidermal tissue structure is a common cause for both of the observed defects. Our results thereby provide a molecular link between these developmental processes and foreshadow Fndc3a as a novel temporal regulator of epidermal cell properties during extremity development and regeneration in zebrafish.}, language = {en} } @article{KimShustaDoran2019, author = {Kim, Brandon J. and Shusta, Eric V. and Doran, Kelly S.}, title = {Past and current perspectives in modeling bacteria and blood-brain barrier interactions}, series = {Frontiers in Microbiology}, volume = {10}, journal = {Frontiers in Microbiology}, number = {1336}, doi = {10.3389/fmicb.2019.01336}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201766}, year = {2019}, abstract = {The central nervous system (CNS) barriers are highly specialized cellular barriers that promote brain homeostasis while restricting pathogen and toxin entry. The primary cellular constituent regulating pathogen entry in most of these brain barriers is the brain endothelial cell (BEC) that exhibits properties that allow for tight regulation of CNS entry. Bacterial meningoencephalitis is a serious infection of the CNS and occurs when bacteria can cross specialized brain barriers and cause inflammation. Models have been developed to understand the bacterial - BEC interaction that lead to pathogen crossing into the CNS, however, these have been met with challenges due to these highly specialized BEC phenotypes. This perspective provides a brief overview and outlook of the in vivo and in vitro models currently being used to study bacterial brain penetration, and opinion on improved models for the future.}, language = {en} } @article{StreinzerChakravortyNeumayeretal.2019, author = {Streinzer, Martin and Chakravorty, Jharna and Neumayer, Johann and Megu, Karsing and Narah, Jaya and Schmitt, Thomas and Bharti, Himender and Spaethe, Johannes and Brockmann, Axel}, title = {Species composition and elevational distribution of bumble bees (Hymenoptera, Apidae, Bombus Latreille) in the East Himalaya, Arunachal Pradesh, India}, series = {ZooKeys}, volume = {851}, journal = {ZooKeys}, doi = {10.3897/zookeys.851.32956}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201937}, pages = {71-89}, year = {2019}, abstract = {The East Himalaya is one of the world's most biodiverse ecosystems. However, very little is known about the abundance and distribution of many plant and animal taxa in this region. Bumble bees are a group of cold-adapted and high elevation insects that fulfil an important ecological and economical function as pollinators of wild and agricultural flowering plants and crops. The Himalayan mountain range provides ample suitable habitats for bumble bees. Systematic study of Himalayan bumble bees began a few decades ago and the main focus has centred on the western region, while the eastern part of the mountain range has received little attention and only a few species have been verified. During a three-year survey, more than 700 bumble bee specimens of 21 species were collected in Arunachal Pradesh, the largest of the north-eastern states of India. The material included a range of species that were previously known from a limited number of collected specimens, which highlights the unique character of the East Himalayan ecosystem. Our results are an important first step towards a future assessment of species distribution, threat, and conservation. Clear elevation patterns of species diversity were observed, which raise important questions about the functional adaptations that allow bumble bees to thrive in this particularly moist region in the East Himalaya.}, language = {en} } @article{HarnošCanizalJuraseketal.2019, author = {Harnoš, Jakub and Ca{\~n}izal, Maria Consuelo Alonso and Jur{\´a}sek, Miroslav and Kumar, Jitender and Holler, Cornelia and Schambony, Alexandra and Han{\´a}kov{\´a}, Kateřina and Bernat{\´i}k, Ondřej and Zdr{\´a}hal, Zbyn{\^e}k and G{\"o}m{\"o}ryov{\´a}, Krist{\´i}na and Gybeľ, Tom{\´a}š and Radaszkiewicz, Tomasz Witold and Kravec, Marek and Trant{\´i}rek, Luk{\´a}š and Ryneš, Jan and Dave, Zankruti and Fern{\´a}ndez-Llamazares, Ana Iris and V{\´a}cha, Robert and Tripsianes, Konstantinos and Hoffmann, Carsten and Bryja, V{\´i}tězslav}, title = {Dishevelled-3 conformation dynamics analyzed by FRET-based biosensors reveals a key role of casein kinase 1}, series = {Nature Communications}, volume = {10}, journal = {Nature Communications}, doi = {10.1038/s41467-019-09651-7}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-227837}, year = {2019}, abstract = {Dishevelled (DVL) is the key component of the Wnt signaling pathway. Currently, DVL conformational dynamics under native conditions is unknown. To overcome this limitation, we develop the Fluorescein Arsenical Hairpin Binder- (FlAsH-) based FRET in vivo approach to study DVL conformation in living cells. Using this single-cell FRET approach, we demonstrate that (i) Wnt ligands induce open DVL conformation, (ii) DVL variants that are predominantly open, show more even subcellular localization and more efficient membrane recruitment by Frizzled (FZD) and (iii) Casein kinase 1 ɛ (CK1ɛ) has a key regulatory function in DVL conformational dynamics. In silico modeling and in vitro biophysical methods explain how CK1ɛ-specific phosphorylation events control DVL conformations via modulation of the PDZ domain and its interaction with DVL C-terminus. In summary, our study describes an experimental tool for DVL conformational sampling in living cells and elucidates the essential regulatory role of CK1ɛ in DVL conformational dynamics.}, language = {en} } @article{KrausBrinkSiegel2019, author = {Kraus, Amelie J. and Brink, Benedikt G. and Siegel, T. Nicolai}, title = {Efficient and specific oligo-based depletion of rRNA}, series = {Scientific Reports}, volume = {9}, journal = {Scientific Reports}, doi = {10.1038/s41598-019-48692-2}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-224829}, year = {2019}, abstract = {In most organisms, ribosomal RNA (rRNA) contributes to >85\% of total RNA. Thus, to obtain useful information from RNA-sequencing (RNA-seq) analyses at reasonable sequencing depth, typically, mature polyadenylated transcripts are enriched or rRNA molecules are depleted. Targeted depletion of rRNA is particularly useful when studying transcripts lacking a poly(A) tail, such as some non-coding RNAs (ncRNAs), most bacterial RNAs and partially degraded or immature transcripts. While several commercially available kits allow effective rRNA depletion, their efficiency relies on a high degree of sequence homology between oligonucleotide probes and the target RNA. This restricts the use of such kits to a limited number of organisms with conserved rRNA sequences. In this study we describe the use of biotinylated oligos and streptavidin-coated paramagnetic beads for the efficient and specific depletion of trypanosomal rRNA. Our approach reduces the levels of the most abundant rRNA transcripts to less than 5\% with minimal off-target effects. By adjusting the sequence of the oligonucleotide probes, our approach can be used to deplete rRNAs or other abundant transcripts independent of species. Thus, our protocol provides a useful alternative for rRNA removal where enrichment of polyadenylated transcripts is not an option and commercial kits for rRNA are not available.}, language = {en} } @article{KimAmoresKangetal.2019, author = {Kim, Bo-Mi and Amores, Angel and Kang, Seunghyun and Ahn, Do-Hwan and Kim, Jin-Hyoung and Kim, Il-Chan and Lee, Jun Hyuck and Lee, Sung Gu and Lee, Hyoungseok and Lee, Jungeun and Kim, Han-Woo and Desvignes, Thomas and Batzel, Peter and Sydes, Jason and Titus, Tom and Wilson, Catherine A. and Catchen, Julian M. and Warren, Wesley C. and Schartl, Manfred and Detrich, H. William III and Postlethwait, John H. and Park, Hyun}, title = {Antarctic blackfin icefish genome reveals adaptations to extreme environments}, series = {Nature Ecology \& Evolution}, volume = {3}, journal = {Nature Ecology \& Evolution}, doi = {10.1038/s41559-019-0812-7}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-325811}, pages = {469-478}, year = {2019}, abstract = {Icefishes (suborder Notothenioidei; family Channichthyidae) are the only vertebrates that lack functional haemoglobin genes and red blood cells. Here, we report a high-quality genome assembly and linkage map for the Antarctic blackfin icefish Chaenocephalus aceratus, highlighting evolved genomic features for its unique physiology. Phylogenomic analysis revealed that Antarctic fish of the teleost suborder Notothenioidei, including icefishes, diverged from the stickleback lineage about 77 million years ago and subsequently evolved cold-adapted phenotypes as the Southern Ocean cooled to sub-zero temperatures. Our results show that genes involved in protection from ice damage, including genes encoding antifreeze glycoprotein and zona pellucida proteins, are highly expanded in the icefish genome. Furthermore, genes that encode enzymes that help to control cellular redox state, including members of the sod3 and nqo1 gene families, are expanded, probably as evolutionary adaptations to the relatively high concentration of oxygen dissolved in cold Antarctic waters. In contrast, some crucial regulators of circadian homeostasis (cry and per genes) are absent from the icefish genome, suggesting compromised control of biological rhythms in the polar light environment. The availability of the icefish genome sequence will accelerate our understanding of adaptation to extreme Antarctic environments.}, language = {en} } @article{GotruvanGeffenNagyetal.2019, author = {Gotru, Sanjeev Kiran and van Geffen, Johanna P. and Nagy, Magdolna and Mammadova-Bach, Elmina and Eilenberger, Julia and Volz, Julia and Manukjan, Georgi and Schulze, Harald and Wagner, Leonard and Eber, Stefan and Schambeck, Christian and Deppermann, Carsten and Brouns, Sanne and Nurden, Paquita and Greinacher, Andreas and Sachs, Ulrich and Nieswandt, Bernhard and Hermanns, Heike M. and Heemskerk, Johan W. M. and Braun, Attila}, title = {Defective Zn2+ homeostasis in mouse and human platelets with α- and δ-storage pool diseases}, series = {Scientific Reports}, volume = {9}, journal = {Scientific Reports}, doi = {10.1038/s41598-019-44751-w}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-227455}, year = {2019}, abstract = {Zinc (Zn2+) can modulate platelet and coagulation activation pathways, including fibrin formation. Here, we studied the (patho)physiological consequences of abnormal platelet Zn2+ storage and release. To visualize Zn2+ storage in human and mouse platelets, the Zn2+ specific fluorescent dye FluoZin3 was used. In resting platelets, the dye transiently accumulated into distinct cytosolic puncta, which were lost upon platelet activation. Platelets isolated from Unc13d-/- mice, characterized by combined defects of α/δ granular release, showed a markedly impaired Zn2+ release upon activation. Platelets from Nbeal2-/- mice mimicking Gray platelet syndrome (GPS), characterized by primarily loss of the α-granule content, had strongly reduced Zn2+ levels, which was also confirmed in primary megakaryocytes. In human platelets isolated from patients with GPS, Hermansky-Pudlak Syndrome (HPS) and Storage Pool Disease (SPD) altered Zn2+ homeostasis was detected. In turbidity and flow based assays, platelet-dependent fibrin formation was impaired in both Nbeal2-/- and Unc13d-/- mice, and the impairment could be partially restored by extracellular Zn2+. Altogether, we conclude that the release of ionic Zn2+ store from secretory granules upon platelet activation contributes to the procoagulant role of Zn2+ in platelet-dependent fibrin formation.}, language = {en} } @article{NerreterLetschertGoetzetal.2019, author = {Nerreter, Thomas and Letschert, Sebastian and G{\"o}tz, Ralph and Doose, S{\"o}ren and Danhof, Sophia and Einsele, Hermann and Sauer, Markus and Hudecek, Michael}, title = {Super-resolution microscopy reveals ultra-low CD19 expression on myeloma cells that triggers elimination by CD19 CAR-T}, series = {Nature Communications}, volume = {10}, journal = {Nature Communications}, doi = {10.1038/s41467-019-10948-w}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-232258}, year = {2019}, abstract = {Immunotherapy with chimeric antigen receptor-engineered T-cells (CAR-T) is under investigation in multiple myeloma. There are reports of myeloma remission after CD19 CAR-T therapy, although CD19 is hardly detectable on myeloma cells by flow cytometry (FC). We apply single molecule-sensitive direct stochastic optical reconstruction microscopy (dSTORM), and demonstrate CD19 expression on a fraction of myeloma cells (10.3-80\%) in 10 out of 14 patients (density: 13-5,000 molecules per cell). In contrast, FC detects CD19 in only 2 of these 10 patients, on a smaller fraction of cells. Treatment with CD19 CAR-T in vitro results in elimination of CD19-positive myeloma cells, including those with <100 CD19 molecules per cell. Similar data are obtained by dSTORM analyses of CD20 expression on myeloma cells and CD20 CAR-T. These data establish a sensitivity threshold for CAR-T and illustrate how super-resolution microscopy can guide patient selection in immunotherapy to exploit ultra-low density antigens.}, language = {en} } @article{SchurrSpindlerKurzetal.2019, author = {Schurr, Yvonne and Spindler, Markus and Kurz, Hendrikje and Bender, Markus}, title = {The cytoskeletal crosslinking protein MACF1 is dispensable for thrombus formation and hemostasis}, series = {Scientific Reports}, volume = {9}, journal = {Scientific Reports}, doi = {10.1038/s41598-019-44183-6}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-234966}, year = {2019}, abstract = {Coordinated reorganization of cytoskeletal structures is critical for key aspects of platelet physiology. While several studies have addressed the role of microtubules and filamentous actin in platelet production and function, the significance of their crosstalk in these processes has been poorly investigated. The microtubule-actin cross-linking factor 1 (MACF1; synonym: Actin cross-linking factor 7, ACF7) is a member of the spectraplakin family, and one of the few proteins expressed in platelets, which possess actin and microtubule binding domains thereby facilitating actin-microtubule interaction and regulation. We used megakaryocyte- and platelet-specific Macf1 knockout (Macf1fl/fl, Pf4-Cre) mice to study the role of MACF1 in platelet production and function. MACF1 deficient mice displayed comparable platelet counts to control mice. Analysis of the platelet cytoskeletal ultrastructure revealed a normal marginal band and actin network. Platelet spreading on fibrinogen was slightly delayed but platelet activation and clot traction was unaffected. Ex vivo thrombus formation and mouse tail bleeding responses were similar between control and mutant mice. These results suggest that MACF1 is dispensable for thrombopoiesis, platelet activation, thrombus formation and the hemostatic function in mice.}, language = {en} } @article{SteuerCostaVanderAuweraGlocketal.2019, author = {Steuer Costa, Wagner and Van der Auwera, Petrus and Glock, Caspar and Liewald, Jana F. and Bach, Maximilian and Sch{\"u}ler, Christina and Wabnig, Sebastian and Oranth, Alexandra and Masurat, Florentin and Bringmann, Henrik and Schoofs, Liliane and Stelzer, Ernst H. K. and Fischer, Sabine C. and Gottschalk, Alexander}, title = {A GABAergic and peptidergic sleep neuron as a locomotion stop neuron with compartmentalized Ca2+ dynamics}, series = {Nature Communications}, volume = {10}, journal = {Nature Communications}, doi = {10.1038/s41467-019-12098-5}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-223273}, year = {2019}, abstract = {Animals must slow or halt locomotion to integrate sensory inputs or to change direction. In Caenorhabditis elegans, the GABAergic and peptidergic neuron RIS mediates developmentally timed quiescence. Here, we show RIS functions additionally as a locomotion stop neuron. RIS optogenetic stimulation caused acute and persistent inhibition of locomotion and pharyngeal pumping, phenotypes requiring FLP-11 neuropeptides and GABA. RIS photoactivation allows the animal to maintain its body posture by sustaining muscle tone, yet inactivating motor neuron oscillatory activity. During locomotion, RIS axonal Ca2+ signals revealed functional compartmentalization: Activity in the nerve ring process correlated with locomotion stop, while activity in a branch correlated with induced reversals. GABA was required to induce, and FLP-11 neuropeptides were required to sustain locomotion stop. RIS attenuates neuronal activity and inhibits movement, possibly enabling sensory integration and decision making, and exemplifies dual use of one cell across development in a compact nervous system.}, language = {en} } @article{DammertBraegelmannOlsenetal.2019, author = {Dammert, Marcel A. and Br{\"a}gelmann, Johannes and Olsen, Rachelle R. and B{\"o}hm, Stefanie and Monhasery, Niloufar and Whitney, Christopher P. and Chalishazar, Milind D. and Tumbrink, Hannah L. and Guthrie, Matthew R. and Klein, Sebastian and Ireland, Abbie S. and Ryan, Jeremy and Schmitt, Anna and Marx, Annika and Ozretić, Luka and Castiglione, Roberta and Lorenz, Carina and Jachimowicz, Ron D. and Wolf, Elmar and Thomas, Roman K. and Poirier, John T. and B{\"u}ttner, Reinhard and Sen, Triparna and Byers, Lauren A. and Reinhardt, H. Christian and Letai, Anthony and Oliver, Trudy G. and Sos, Martin L.}, title = {MYC paralog-dependent apoptotic priming orchestrates a spectrum of vulnerabilities in small cell lung cancer}, series = {Nature Communications}, volume = {10}, journal = {Nature Communications}, doi = {10.1038/s41467-019-11371-x}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-223569}, year = {2019}, abstract = {MYC paralogs are frequently activated in small cell lung cancer (SCLC) but represent poor drug targets. Thus, a detailed mapping of MYC-paralog-specific vulnerabilities may help to develop effective therapies for SCLC patients. Using a unique cellular CRISPR activation model, we uncover that, in contrast to MYCN and MYCL, MYC represses BCL2 transcription via interaction with MIZ1 and DNMT3a. The resulting lack of BCL2 expression promotes sensitivity to cell cycle control inhibition and dependency on MCL1. Furthermore, MYC activation leads to heightened apoptotic priming, intrinsic genotoxic stress and susceptibility to DNA damage checkpoint inhibitors. Finally, combined AURK and CHK1 inhibition substantially prolongs the survival of mice bearing MYC-driven SCLC beyond that of combination chemotherapy. These analyses uncover MYC-paralog-specific regulation of the apoptotic machinery with implications for genotype-based selection of targeted therapeutics in SCLC patients.}, language = {en} } @article{KrahBuentgenSchaeferetal.2019, author = {Krah, Franz-Sebastian and B{\"u}ntgen, Ulf and Schaefer, Hanno and M{\"u}ller, J{\"o}rg and Andrew, Carrie and Boddy, Lynne and Diez, Jeffrey and Egli, Simon and Freckleton, Robert and Gange, Alan C. and Halvorsen, Rune and Heegaard, Einar and Heideroth, Antje and Heibl, Christoph and Heilmann-Clausen, Jacob and H{\o}iland, Klaus and Kar, Ritwika and Kauserud, H{\aa}vard and Kirk, Paul M. and Kuyper, Thomas W. and Krisai-Greilhuber, Irmgard and Norden, Jenni and Papastefanou, Phillip and Senn-Irlet, Beatrice and B{\"a}ssler, Claus}, title = {European mushroom assemblages are darker in cold climates}, series = {Nature Communications}, volume = {10}, journal = {Nature Communications}, doi = {10.1038/s41467-019-10767-z}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-224815}, year = {2019}, abstract = {Thermal melanism theory states that dark-colored ectotherm organisms are at an advantage at low temperature due to increased warming. This theory is generally supported for ectotherm animals, however, the function of colors in the fungal kingdom is largely unknown. Here, we test whether the color lightness of mushroom assemblages is related to climate using a dataset of 3.2 million observations of 3,054 species across Europe. Consistent with the thermal melanism theory, mushroom assemblages are significantly darker in areas with cold climates. We further show differences in color phenotype between fungal lifestyles and a lifestyle differentiated response to seasonality. These results indicate a more complex ecological role of mushroom colors and suggest functions beyond thermal adaption. Because fungi play a crucial role in terrestrial carbon and nutrient cycles, understanding the links between the thermal environment, functional coloration and species' geographical distributions will be critical in predicting ecosystem responses to global warming.}, language = {en} } @article{MilaneseMendePaolietal.2019, author = {Milanese, Alessio and Mende, Daniel R and Paoli, Lucas and Salazar, Guillem and Ruscheweyh, Hans-Joachim and Cuenca, Miguelangel and Hingamp, Pascal and Alves, Renato and Costea, Paul I and Coelho, Luis Pedro and Schmidt, Thomas S. B. and Almeida, Alexandre and Mitchell, Alex L and Finn, Robert D. and Huerta-Cepas, Jaime and Bork, Peer and Zeller, Georg and Sunagawa, Shinichi}, title = {Microbial abundance, activity and population genomic profiling with mOTUs2}, series = {Nature Communications}, volume = {10}, journal = {Nature Communications}, doi = {10.1038/s41467-019-08844-4}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-224089}, year = {2019}, abstract = {Metagenomic sequencing has greatly improved our ability to profile the composition of environmental and host-associated microbial communities. However, the dependency of most methods on reference genomes, which are currently unavailable for a substantial fraction of microbial species, introduces estimation biases. We present an updated and functionally extended tool based on universal (i.e., reference-independent), phylogenetic marker gene (MG)-based operational taxonomic units (mOTUs) enabling the profiling of >7700 microbial species. As more than 30\% of them could not previously be quantified at this taxonomic resolution, relative abundance estimates based on mOTUs are more accurate compared to other methods. As a new feature, we show that mOTUs, which are based on essential housekeeping genes, are demonstrably well-suited for quantification of basal transcriptional activity of community members. Furthermore, single nucleotide variation profiles estimated using mOTUs reflect those from whole genomes, which allows for comparing microbial strain populations (e.g., across different human body sites).}, language = {en} } @article{LeeLiRuanetal.2019, author = {Lee, Hong-Jen and Li, Chien-Feng and Ruan, Diane and He, Jiabei and Montal, Emily D. and Lorenz, Sonja and Girnun, Geoffrey D. and Chan, Chia-Hsin}, title = {Non-proteolytic ubiquitination of Hexokinase 2 by HectH9 controls tumor metabolism and cancer stem cell expansion}, series = {Nature Communications}, volume = {10}, journal = {Nature Communications}, doi = {10.1038/s41467-019-10374-y}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-236445}, year = {2019}, abstract = {Enormous efforts have been made to target metabolic dependencies of cancer cells for developing new therapies. However, the therapeutic efficacy of glycolysis inhibitors is limited due to their inability to elicit cell death. Hexokinase 2 (HK2), via its mitochondrial localization, functions as a central nexus integrating glycolysis activation and apoptosis resilience. Here we identify that K63-linked ubiquitination by HectH9 regulates the mitochondrial localization and function of HK2. Through stable isotope tracer approach and functional metabolic analyses, we show that HectH9 deficiency impedes tumor glucose metabolism and growth by HK2 inhibition. The HectH9/HK2 pathway regulates cancer stem cell (CSC) expansion and CSC-associated chemoresistance. Histological analyses show that HectH9 expression is upregulated and correlated with disease progression in prostate cancer. This work uncovers that HectH9 is a novel regulator of HK2 and cancer metabolism. Targeting HectH9 represents an effective strategy to achieve long-term tumor remission by concomitantly disrupting glycolysis and inducing apoptosis.}, language = {en} } @article{MercierWolmaransSchubertetal.2019, author = {Mercier, Rebecca and Wolmarans, Annemarie and Schubert, Jonathan and Neuweiler, Hannes and Johnson, Jill L. and LaPointe, Paul}, title = {The conserved NxNNWHW motif in Aha-type co-chaperones modulates the kinetics of Hsp90 ATPase stimulation}, series = {Nature Communications}, volume = {10}, journal = {Nature Communications}, doi = {10.1038/s41467-019-09299-3}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-224007}, year = {2019}, abstract = {Hsp90 is a dimeric molecular chaperone that is essential for the folding and activation of hundreds of client proteins. Co-chaperone proteins regulate the ATP-driven Hsp90 client activation cycle. Aha-type co-chaperones are the most potent stimulators of the Hsp90 ATPase activity but the relationship between ATPase regulation and in vivo activity is poorly understood. We report here that the most strongly conserved region of Aha-type co-chaperones, the N terminal NxNNWHW motif, modulates the apparent affinity of Hsp90 for nucleotide substrates. The ability of yeast Aha-type co-chaperones to act in vivo is ablated when the N terminal NxNNWHW motif is removed. This work suggests that nucleotide exchange during the Hsp90 functional cycle may be more important than rate of catalysis.}, language = {en} } @article{LuebckeEbbersVolzkeetal.2019, author = {L{\"u}bcke, Paul M. and Ebbers, Meinolf N. B. and Volzke, Johann and Bull, Jana and Kneitz, Susanne and Engelmann, Robby and Lang, Hermann and Kreikemeyer, Bernd and M{\"u}ller-Hilke, Brigitte}, title = {Periodontal treatment prevents arthritis in mice and methotrexate ameliorates periodontal bone loss}, series = {Scientific Reports}, volume = {9}, journal = {Scientific Reports}, doi = {10.1038/s41598-019-44512-9}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-237355}, year = {2019}, abstract = {Recent studies indicate a causal relationship between the periodontal pathogen P. gingivalis and rheumatoid arthritis involving the production of autoantibodies against citrullinated peptides. We therefore postulated that therapeutic eradication P. gingivalis may ameliorate rheumatoid arthritis development and here turned to a mouse model in order to challenge our hypothesis. F1 (DBA/1 x B10.Q) mice were orally inoculated with P. gingivalis before collagen-induced arthritis was provoked. Chlorhexidine or metronidazole were orally administered either before or during the induction phase of arthritis and their effects on arthritis progression and alveolar bone loss were compared to intraperitoneally injected methotrexate. Arthritis incidence and severity were macroscopically scored and alveolar bone loss was evaluated via microcomputed tomography. Serum antibody titres against P. gingivalis were quantified by ELISA and microbial dysbiosis following oral inoculation was monitored in stool samples via microbiome analyses. Both, oral chlorhexidine and metronidazole reduced the incidence and ameliorated the severity of collagen-induced arthritis comparable to methotrexate. Likewise, all three therapies attenuated alveolar bone loss. Relative abundance of Porphyromonadaceae was increased after oral inoculation with P. gingivalis and decreased after treatment. This is the first study to describe beneficial effects of non-surgical periodontal treatment on collagen-induced arthritis in mice and suggests that mouthwash with chlorhexidine or metronidazole may also be beneficial for patients with rheumatoid arthritis and a coexisting periodontitis. Methotrexate ameliorated periodontitis in mice, further raising the possibility that methotrexate may also positively impact on the tooth supporting tissues of patients with rheumatoid arthritis.}, language = {en} } @article{WoodcockGarrattPowneyetal.2019, author = {Woodcock, B. A. and Garratt, M. P. D. and Powney, G. D. and Shaw, R. F. and Osborne, J. L. and Soroka, J. and Lindstr{\"o}m, S. A. M. and Stanley, D. and Ouvrard, P. and Edwards, M. E. and Jauker, F. and McCracken, M. E. and Zou, Y. and Potts, S. G. and Rundl{\"o}f, M. and Noriega, J. A. and Greenop, A. and Smith, H. G. and Bommarco, R. and van der Werf, W. and Stout, J. C. and Steffan-Dewenter, I. and Morandin, L. and Bullock, J. M. and Pywell, R. F.}, title = {Meta-analysis reveals that pollinator functional diversity and abundance enhance crop pollination and yield}, series = {Nature Communications}, volume = {10}, journal = {Nature Communications}, doi = {10.1038/s41467-019-09393-6}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-233787}, year = {2019}, abstract = {How insects promote crop pollination remains poorly understood in terms of the contribution of functional trait differences between species. We used meta-analyses to test for correlations between community abundance, species richness and functional trait metrics with oilseed rape yield, a globally important crop. While overall abundance is consistently important in predicting yield, functional divergence between species traits also showed a positive correlation. This result supports the complementarity hypothesis that pollination function is maintained by non-overlapping trait distributions. In artificially constructed communities (mesocosms), species richness is positively correlated with yield, although this effect is not seen under field conditions. As traits of the dominant species do not predict yield above that attributed to the effect of abundance alone, we find no evidence in support of the mass ratio hypothesis. Management practices increasing not just pollinator abundance, but also functional divergence, could benefit oilseed rape agriculture.}, language = {en} } @article{AnnunziatavandeVlekkertWolfetal.2019, author = {Annunziata, Ida and van de Vlekkert, Diantha and Wolf, Elmar and Finkelstein, David and Neale, Geoffrey and Machado, Eda and Mosca, Rosario and Campos, Yvan and Tillman, Heather and Roussel, Martine F. and Weesner, Jason Andrew and Fremuth, Leigh Ellen and Qiu, Xiaohui and Han, Min-Joon and Grosveld, Gerard C. and d'Azzo, Alessandra}, title = {MYC competes with MiT/TFE in regulating lysosomal biogenesis and autophagy through an epigenetic rheostat}, series = {Nature Communications}, volume = {10}, journal = {Nature Communications}, doi = {10.1038/s41467-019-11568-0}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-221189}, year = {2019}, abstract = {Coordinated regulation of the lysosomal and autophagic systems ensures basal catabolism and normal cell physiology, and failure of either system causes disease. Here we describe an epigenetic rheostat orchestrated by c-MYC and histone deacetylases that inhibits lysosomal and autophagic biogenesis by concomitantly repressing the expression of the transcription factors MiT/TFE and FOXH1, and that of lysosomal and autophagy genes. Inhibition of histone deacetylases abates c-MYC binding to the promoters of lysosomal and autophagy genes, granting promoter occupancy to the MiT/TFE members, TFEB and TFE3, and/or the autophagy regulator FOXH1. In pluripotent stem cells and cancer, suppression of lysosomal and autophagic function is directly downstream of c-MYC overexpression and may represent a hallmark of malignant transformation. We propose that, by determining the fate of these catabolic systems, this hierarchical switch regulates the adaptive response of cells to pathological and physiological cues that could be exploited therapeutically.}, language = {en} } @article{HersterBittnerCodreaetal.2019, author = {Herster, Franziska and Bittner, Zsofia and Codrea, Marius Cosmin and Archer, Nathan K. and Heister, Martin and L{\"o}ffler, Markus W. and Heumos, Simon and Wegner, Joanna and Businger, Ramona and Schindler, Michael and Stegner, David and Sch{\"a}kel, Knut and Grabbe, Stephan and Ghoreschi, Kamran and Miller, Lloyd S. and Weber, Alexander N. R.}, title = {Platelets Aggregate With Neutrophils and Promote Skin Pathology in Psoriasis}, series = {Frontiers in Immunology}, volume = {10}, journal = {Frontiers in Immunology}, doi = {10.3389/fimmu.2019.01867}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-320175}, year = {2019}, abstract = {Psoriasis is a frequent systemic inflammatory autoimmune disease characterized primarily by skin lesions with massive infiltration of leukocytes, but frequently also presents with cardiovascular comorbidities. Especially polymorphonuclear neutrophils (PMNs) abundantly infiltrate psoriatic skin but the cues that prompt PMNs to home to the skin are not well-defined. To identify PMN surface receptors that may explain PMN skin homing in psoriasis patients, we screened 332 surface antigens on primary human blood PMNs from healthy donors and psoriasis patients. We identified platelet surface antigens as a defining feature of psoriasis PMNs, due to a significantly increased aggregation of neutrophils and platelets in the blood of psoriasis patients. Similarly, in the imiquimod-induced experimental in vivo mouse model of psoriasis, disease induction promoted PMN-platelet aggregate formation. In psoriasis patients, disease incidence directly correlated with blood platelet counts and platelets were detected in direct contact with PMNs in psoriatic but not healthy skin. Importantly, depletion of circulating platelets in mice in vivo ameliorated disease severity significantly, indicating that both PMNs and platelets may be relevant for psoriasis pathology and disease severity.}, language = {en} }