TY  - JOUR
A1  - Ruf, Franziska
A1  - Fraunholz, Martin
A1  - Öchsner, Konrad
A1  - Kaderschabeck, Johann
A1  - Wegener, Christian
T1  - WEclMon - A simple and robust camera-based system to monitor Drosophila eclosion under optogenetic manipulation and natural conditions
JF  - PLoS ONE
N2  - Eclosion in flies and other insects is a circadian-gated behaviour under control of a central and a peripheral clock. It is not influenced by the motivational state of an animal, and thus presents an ideal paradigm to study the relation and signalling pathways between central and peripheral clocks, and downstream peptidergic regulatory systems. Little is known, however, about eclosion rhythmicity under natural conditions, and research into this direction is hampered by the physically closed design of current eclosion monitoring systems.
We describe a novel open eclosion monitoring system (WEclMon) that allows the puparia to come into direct contact with light, temperature and humidity. We demonstrate that the system can be used both in the laboratory and outdoors, and shows a performance similar to commercial closed funnel-type monitors. Data analysis is semi-automated based on a macro toolset for the open imaging software Fiji. Due to its open design, the WEclMon is also well suited for optogenetic experiments. A small screen to identify putative neuroendocrine signals mediating time from the central clock to initiate eclosion showed that optogenetic activation of ETH-, EH and myosuppressin neurons can induce precocious eclosion. Genetic ablation of myosuppressin-expressing neurons did, however, not affect eclosion rhythmicity.
KW  - chronobiology
KW  - infrared radiation
KW  - light pulses
KW  - molting
KW  - Drosophila melanogaster
KW  - optogenetics
KW  - eclosion
Y1  - 2017
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170755
VL  - 12
IS  - 6
ER  - 
TY  - JOUR
A1  - Seher, Axel
A1  - Lagler, Charlotte
A1  - Stühmer, Thorsten
A1  - Müller-Richter, Urs Dietmar Achim
A1  - Kübler, Alexander Christian
A1  - Sebald, Walter
A1  - Müller, Thomas Dieter
A1  - Nickel, Joachim
T1  - Utilizing BMP-2 muteins for treatment of multiple myeloma
JF  - PLoS ONE
N2  - Multiple myeloma (MM) represents a haematological cancer characterized by the pathological hyper proliferation of antibody-producing B-lymphocytes. Patients typically suffer from kidney malfunction and skeletal disorders. In the context of MM, the transforming growth factor β (TGFβ) member Activin A was recently identified as a promoter of both accompanying symptoms. Because studies have shown that bone morphogenetic protein (BMP)-2-mediated activities are counteracted by Activin A, we analysed whether BMP2, which also binds to the Activin A receptors ActRII and ActRIIB but activates the alternative SMAD-1/5/8 pathway, can be used to antagonize Activin A activities, such as in the context of MM. Therefore three BMP2 derivatives were generated with modified binding activities for the type II (ActRIIB) and/or type I receptor (BMPRIA) showing either increased or decreased BMP2 activity. In the context of MM these BMP2 muteins show two functionalities since they act as a) an anti-proliferative/apoptotic agent against neoplastic B-cells, b) as a bone-formation promoting growth factor. The molecular basis of both activities was shown in two different cellular models to clearly rely on the properties of the investigated BMP2 muteins to compete for the binding of Activin A to the Activin type II receptors. The experimental outcome suggests new therapeutic strategies using BMP2 variants in the treatment of MM-related pathologies.
KW  - multiple myeloma
KW  - signaling
KW  - cell proliferation
KW  - cell binding
KW  - membrane receptor signaling
KW  - BMP
KW  - gene expression
KW  - B cell receptors
KW  - B cells
Y1  - 2017
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-158144
VL  - 12
IS  - 5
ER  - 
TY  - JOUR
A1  - Ampattu, Biju Joseph
A1  - Hagmann, Laura
A1  - Liang, Chunguang
A1  - Dittrich, Marcus
A1  - Schlüter, Andreas
A1  - Blom, Jochen
A1  - Krol, Elizaveta
A1  - Goesmann, Alexander
A1  - Becker, Anke
A1  - Dandekar, Thomas
A1  - Müller, Tobias
A1  - Schoen, Christoph
T1  - Transcriptomic buffering of cryptic genetic variation contributes to meningococcal virulence
JF  - BMC Genomics
N2  - Background:
Commensal bacteria like Neisseria meningitidis sometimes cause serious disease. However, genomic comparison of hyperinvasive and apathogenic lineages did not reveal unambiguous hints towards indispensable virulence factors. Here, in a systems biological approach we compared gene expression of the invasive strain MC58 and the carriage strain α522 under different ex vivo conditions mimicking commensal and virulence compartments to assess the strain-specific impact of gene regulation on meningococcal virulence.
Results:
Despite indistinguishable ex vivo phenotypes, both strains differed in the expression of over 500 genes under infection mimicking conditions. These differences comprised in particular metabolic and information processing genes as well as genes known to be involved in host-damage such as the nitrite reductase and numerous LOS biosynthesis genes. A model based analysis of the transcriptomic differences in human blood suggested ensuing metabolic flux differences in energy, glutamine and cysteine metabolic pathways along with differences in the activation of the stringent response in both strains. In support of the computational findings, experimental analyses revealed differences in cysteine and glutamine auxotrophy in both strains as well as a strain and condition dependent essentiality of the (p)ppGpp synthetase gene relA and of a short non-coding AT-rich repeat element in its promoter region.
Conclusions:
Our data suggest that meningococcal virulence is linked to transcriptional buffering of cryptic genetic variation in metabolic genes including global stress responses. They further highlight the role of regulatory elements for bacterial virulence and the limitations of model strain approaches when studying such genetically diverse species as N. meningitidis.
KW  - neisseria meningitidis
KW  - MITE
KW  - virulenceregulatory evolution
KW  - systems biology
KW  - metabolism
KW  - cryptic
KW  - genetic variation
KW  - stringent response
KW  - relA
Y1  - 2017
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-157534
VL  - 18
IS  - 282
ER  - 
TY  - JOUR
A1  - Wegert, Jenny
A1  - Vokuh, Christian
A1  - Ziegler, Barbara
A1  - Ernestus, Karen
A1  - Leuschner, Ivo
A1  - Furtwängler, Rhoikos
A1  - Graf, Norbert
A1  - Gessler, Manfred
T1  - TP53 alterations in Wilms tumour represent progression events with strong intratumour heterogeneity that are closely linked but not limited to anaplasia
JF  - The Journal of Pathology: Clinical Research
N2  - TP53 mutations have been associated with anaplasia in Wilms tumour, which conveys a high risk for relapse and fatal outcome. Nevertheless, TP53 alterations have been reported in no more than 60% of anaplastic tumours, and recent data have suggested their presence in tumours that do not fulfil the criteria for anaplasia, questioning the clinical utility of TP53 analysis. Therefore, we characterized the TP53 status in 84 fatal cases of Wilms tumour, irrespective of histological subtype. We identified TP53 alterations in at least 90% of fatal cases of anaplastic Wilms tumour, and even more when diffuse anaplasia was present, indicating a very strong if not absolute coupling between anaplasia and deregulation of p53 function. Unfortunately, TP53 mutations do not provide additional predictive value in anaplastic tumours since the same mutation rate was found in a cohort of non-fatal anaplastic tumours. When classified according to tumour stage, patients with stage I diffuse anaplastic tumours still had a high chance of survival (87%), but this rate dropped to 26% for stages II–IV. Thus, volume of anaplasia or possible spread may turn out to be critical parameters. Importantly, among non-anaplastic fatal tumours, 26% had TP53 alterations, indicating that TP53 screening may identify additional cases at risk. Several of these non-anaplastic tumours fulfilled some criteria for anaplasia, for example nuclear unrest, suggesting that such partial phenotypes should be under special scrutiny to enhance detection of high-risk tumours via TP53 screening. A major drawback is that these alterations are secondary changes that occur only later in tumour development, leading to striking intratumour heterogeneity that requires multiple biopsies and analysis guided by histological criteria. In conclusion, we found a very close correlation between histological signs of anaplasia and TP53 alterations. The latter may precede development of anaplasia and thereby provide diagnostic value pointing towards aggressive disease.
KW  - tumour heterogeneity
KW  - Wilms tumour
KW  - nephroblastoma
KW  - anaplasia
KW  - TP53
Y1  - 2017
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-158302
VL  - 3
ER  - 
TY  - JOUR
A1  - Kollmannsberger, Philip
A1  - Kerschnitzki, Michael
A1  - Repp, Felix
A1  - Wagermaier, Wolfgang
A1  - Weinkamer, Richard
A1  - Fratzl, Peter
T1  - The small world of osteocytes: connectomics of the lacuno-canalicular network in bone
JF  - New Journal of Physics
N2  - Osteocytes and their cell processes reside in a large, interconnected network of voids pervading the mineralized bone matrix of most vertebrates. This osteocyte lacuno-canalicular network (OLCN) is believed to play important roles in mechanosensing, mineral homeostasis, and for the mechanical properties of bone. While the extracellular matrix structure of bone is extensively studied on ultrastructural and macroscopic scales, there is a lack of quantitative knowledge on how the cellular network is organized. Using a recently introduced imaging and quantification approach, we analyze the OLCN in different bone types from mouse and sheep that exhibit different degrees of structural organization not only of the cell network but also of the fibrous matrix deposited by the cells. We define a number of robust, quantitative measures that are derived from the theory of complex networks. These measures enable us to gain insights into how efficient the network is organized with regard to intercellular transport and communication. Our analysis shows that the cell network in regularly organized, slow-growing bone tissue from sheep is less connected, but more efficiently organized compared to irregular and fast-growing bone tissue from mice. On the level of statistical topological properties (edges per node, edge length and degree distribution), both network types are indistinguishable, highlighting that despite pronounced differences at the tissue level, the topological architecture of the osteocyte canalicular network at the subcellular level may be independent of species and bone type. Our results suggest a universal mechanism underlying the self-organization of individual cells into a large, interconnected network during bone formation and mineralization.
KW  - bone
KW  - osteocytes
KW  - networks
KW  - biomaterials
KW  - mechanobiology
KW  - image analysis
Y1  - 2017
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170662
VL  - 19
IS  - 073019
ER  - 
TY  - THES
A1  - Iltzsche, Fabian
T1  - The Role of DREAM/MMB-mediated mitotic gene expression downstream of mutated K-Ras in lung cancer
T1  - Die Rolle DREAM/MMB-vermittelter mitotischer Genexpression unterhalb von mutiertem K-Ras in Lungenkrebs
N2  - The evolutionary conserved Myb-MuvB (MMB) multiprotein complex has an essential role in transcriptional activation of mitotic genes. MMB target genes as well as the MMB associated transcription factor B-Myb and FoxM1 are highly expressed in a range of different cancer types. The elevated expression of these genes correlates with an advanced tumor state and a poor prognosis. This suggests that MMB could contribute to tumorigenesis by mediating overexpression of mitotic genes. Although MMB has been extensively characterized biochemically, the requirement for MMB to tumorigenesis in vivo remains largely unknown and has not been tested directly so far.
In this study, conditional knockout of the MMB core member Lin9 inhibits tumor formation in vivo in a mouse model of lung cancer driven by oncogenic K-Ras and loss of p53. The incomplete recombination observed within tumors points towards an enormous selection pressure against the complete loss of Lin9. RNA interference (RNAi)-mediated depletion of Lin9 or the MMB associated subunit B-Myb provides evidence that MMB is required for the expression of mitotic genes in lung cancer cells. Moreover, it was demonstrated that proliferation of lung cancer cells strongly depends on MMB. Furthermore, in this study, the relationship of MMB to the p53 tumor suppressor was investigated in a primary lung cancer cell line with restorable p53 function. Expression analysis revealed that mitotic genes are downregulated after p53 re-expression. Moreover, activation of p53 induces formation of the repressive DREAM complex and results in enrichment of DREAM at mitotic gene promoters. Conversely, MMB is displaced at these promoters.
Based on these findings the following model is proposed: In p53-negative cells, mitogenic stimuli foster the switch from DREAM to MMB. Thus, mitotic genes are overexpressed and may promote chromosomal instability and tumorigenesis.
This study provides evidence that MMB contributes to the upregulation of G2/M phase-specific genes in p53-negative cells and suggests that inhibition of MMB (or its target genes) might be a strategy for treatment of lung cancer.
N2  - Der evolutionär konservierte Myb-MuvB (MMB) Multiproteinkomplex hat eine wesentliche Rolle in der transkriptionellen Aktivierung mitotischer Gene. Zielgene des MMB sowie die MMB assoziierten Transkriptionsfaktoren B-Myb und FoxM1 sind hoch exprimiert in einer Bandbreite verschiedener Krebsarten. Die erhöhte Expression dieser Gene korreliert mit einem fortgeschrittenen Tumorstadium und einer geringen Prognose. Das weißt auf darauf hin, dass MMB an der Tumorentstehung beteiligt sein könnte indem es die Überexpression mitotischer Gene fördert. Obwohl MMB biochemisch eingehend untersucht wurde, ist die Erfordernis von MMB zur Tumorentstehung in vivo weitestgehend unbekannt und wurde bisher nicht direkt getestet.
In dieser Studie hemmt der konditionale Knockout der MMB Kerneinheit Lin9 die Tumorbildung in vivo in einem Lungenkrebs-Mausmodell angetrieben durch onkogenes K-Ras und den Verlust von p53. Die unvollständige Rekombination welche in Tumoren beobachtet wurde deutet auf einen starken Selektionsdruck gegen den kompletten Verlust von Lin9 hin. Die Verminderung von Lin9 und der MMB- assoziierten Untereinheit B-Myb durch RNAi-Interferenz (RNAi) liefert Beweise dafür, dass MMB für die Expression mitotischer Gene in Lungenkrebszellen notwendig ist. Zudem wurde gezeigt, dass das Zellwachstum von Lungenkrebszellen stark von MMB abhängig ist. Weiterhin wurde der Zusammenhang zwischen MMB und dem p53-Tumorsuppressor in einer primären Lungenkrebszelllinie mit wiederherstellbarer p53-Funktion untersucht. Expressionsanalysen zeigen, dass mitotische Gene nach Re-expression von p53 runterreguliert werden. Außerdem induziert die Aktivierung von p53 die Bildung des repressiven DREAM-Komplexes und führt zu einer Anreicherung von DREAM an Promotoren mitotischer Gene. Im Gegenzug wird MMB an den Promotoren verdrängt.
Basierend auf den Ergebnissen wird das folgende Model vorgeschlagen: In p53- negativen Zellen begünstigen mitogene Reize den Wechsel von DREAM zu MMB. Dadurch werden mitotische Gene überexprimiert und können so chromosomale Instabilität und Tumorentstehung fördern
Diese Studie liefert Hinweise, dass MMB an der Hochregulation G2/M- Phasenspezifischer Gene in p53-negativen Zellen beteiligt ist und dass die Hemmung von MMB (oder seiner Zielgene) eine Strategie zur Behandlung von Lungenkrebs sein könnte.
KW  - Nicht-kleinzelliges Bronchialkarzinom (NSCLC)
KW  - Lungenkrebs
KW  - lung cancer
KW  - DREAM complex
KW  - MMB
KW  - K-Ras
KW  - mitotic gene expression
KW  - Mitose
Y1  - 2017
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-154108
ER  - 
TY  - JOUR
A1  - Grob, Robin
A1  - Fleischmann, Pauline N.
A1  - Grübel, Kornelia
A1  - Wehner, Rüdiger
A1  - Rössler, Wolfgang
T1  - The role of celestial compass information in Cataglyphis ants during learning walks and for neuroplasticity in the central complex and mushroom bodies
JF  - Frontiers in Behavioral Neuroscience
N2  - Central place foragers are faced with the challenge to learn the position of their nest entrance in its surroundings, in order to find their way back home every time they go out to search for food. To acquire navigational information at the beginning of their foraging career, Cataglyphis noda performs learning walks during the transition from interior worker to forager. These small loops around the nest entrance are repeatedly interrupted by strikingly accurate back turns during which the ants stop and precisely gaze back to the nest entrance—presumably to learn the landmark panorama of the nest surroundings. However, as at this point the complete navigational toolkit is not yet available, the ants are in need of a reference system for the compass component of the path integrator to align their nest entrance-directed gazes. In order to find this directional reference system, we systematically manipulated the skylight information received by ants during learning walks in their natural habitat, as it has been previously suggested that the celestial compass, as part of the path integrator, might provide such a reference system. High-speed video analyses of distinct learning walk elements revealed that even exclusion from the skylight polarization pattern, UV-light spectrum and the position of the sun did not alter the accuracy of the look back to the nest behavior. We therefore conclude that C. noda uses a different reference system to initially align their gaze directions. However, a comparison of neuroanatomical changes in the central complex and the mushroom bodies before and after learning walks revealed that exposure to UV light together with a naturally changing polarization pattern was essential to induce neuroplasticity in these high-order sensory integration centers of the ant brain. This suggests a crucial role of celestial information, in particular a changing polarization pattern, in initially calibrating the celestial compass system.
KW  - sky-compass pathway
KW  - visual orientation
KW  - look-back behavior
KW  - desert ants
KW  - vector navigation
KW  - memory
KW  - central complex
KW  - mushroom body
Y1  - 2017
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-159235
VL  - 11
IS  - 226
ER  - 
TY  - JOUR
A1  - Goos, Carina
A1  - Dejung, Mario
A1  - Janzen, Christian J.
A1  - Butter, Falk
A1  - Kramer, Susanne
T1  - The nuclear proteome of Trypanosoma brucei
JF  - PLoS ONE
N2  - Trypanosoma brucei is a protozoan flagellate that is transmitted by tsetse flies into the mammalian bloodstream. The parasite has a huge impact on human health both directly by causing African sleeping sickness and indirectly, by infecting domestic cattle. The biology of trypanosomes involves some highly unusual, nuclear-localised processes. These include polycistronic transcription without classical promoters initiated from regions defined by histone variants, trans-splicing of all transcripts to the exon of a spliced leader RNA, transcription of some very abundant proteins by RNA polymerase I and antigenic variation, a switch in expression of the cell surface protein variants that allows the parasite to resist the immune system of its mammalian host. Here, we provide the nuclear proteome of procyclic Trypanosoma brucei, the stage that resides within the tsetse fly midgut. We have performed quantitative label-free mass spectrometry to score 764 significantly nuclear enriched proteins in comparison to whole cell lysates. A comparison with proteomes of several experimentally characterised nuclear and non-nuclear structures and pathways confirmed the high quality of the dataset: the proteome contains about 80% of all nuclear proteins and less than 2% false positives. Using motif enrichment, we found the amino acid sequence KRxR present in a large number of nuclear proteins. KRxR is a sub-motif of a classical eukaryotic monopartite nuclear localisation signal and could be responsible for nuclear localization of proteins in Kinetoplastida species. As a proof of principle, we have confirmed the nuclear localisation of six proteins with previously unknown localisation by expressing eYFP fusion proteins. While proteome data of several T. brucei organelles have been published, our nuclear proteome closes an important gap in knowledge to study trypanosome biology, in particular nuclear-related processes.
KW  - Trypanosoma
KW  - gambiense
KW  - Trypanosoma brucei
KW  - proteomes
KW  - yellow fluorescent protein
KW  - mitochondria
KW  - protein structure
KW  - histones
Y1  - 2017
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-158572
VL  - 12
IS  - 7
ER  - 
TY  - JOUR
A1  - Scheiner, Ricarda
A1  - Entler, Brian V.
A1  - Barron, Andrew B.
A1  - Scholl, Christina
A1  - Thamm, Markus
T1  - The Effects of Fat Body Tyramine Level on Gustatory Responsiveness of Honeybees (Apis mellifera) Differ between Behavioral Castes
JF  - Frontiers in Systems Neuroscience
N2  - Division of labor is a hallmark of social insects. In the honeybee (Apis mellifera) each sterile female worker performs a series of social tasks. The most drastic changes in behavior occur when a nurse bee, who takes care of the brood and the queen in the hive, transitions to foraging behavior. Foragers provision the colony with pollen, nectar or water. Nurse bees and foragers differ in numerous behaviors, including responsiveness to gustatory stimuli. Differences in gustatory responsiveness, in turn, might be involved in regulating division of labor through differential sensory response thresholds. Biogenic amines are important modulators of behavior. Tyramine and octopamine have been shown to increase gustatory responsiveness in honeybees when injected into the thorax, thereby possibly triggering social organization. So far, most of the experiments investigating the role of amines on gustatory responsiveness have focused on the brain. The potential role of the fat body in regulating sensory responsiveness and division of labor has large been neglected. We here investigated the role of the fat body in modulating gustatory responsiveness through tyramine signaling in different social roles of honeybees. We quantified levels of tyramine, tyramine receptor gene expression and the effect of elevating fat body tyramine titers on gustatory responsiveness in both nurse bees and foragers. Our data suggest that elevating the tyramine titer in the fat body pharmacologically increases gustatory responsiveness in foragers, but not in nurse bees. This differential effect of tyramine on gustatory responsiveness correlates with a higher natural gustatory responsiveness of foragers, with a higher tyramine receptor (Amtar1) mRNA expression in fat bodies of foragers and with lower baseline tyramine titers in fat bodies of foragers compared to those of nurse bees. We suggest that differential tyramine signaling in the fat body has an important role in the plasticity of division of labor through changing gustatory responsiveness.
KW  - behavior
KW  - biogenic amines
KW  - division of labor
KW  - nurse bee
KW  - forager
KW  - PER
KW  - octopamine
KW  - insect
Y1  - 2017
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-157874
VL  - 11
IS  - 55
ER  - 
TY  - JOUR
A1  - Liu, Han
A1  - Chen, Chunhai
A1  - Gao, Zexia
A1  - Min, Jiumeng
A1  - Gu, Yongming
A1  - Jian, Jianbo
A1  - Jiang, Xiewu
A1  - Cai, Huimin
A1  - Ebersberger, Ingo
A1  - Xu, Meng
A1  - Zhang, Xinhui
A1  - Chen, Jianwei
A1  - Luo, Wei
A1  - Chen, Boxiang
A1  - Chen, Junhui
A1  - Liu, Hong
A1  - Li, Jiang
A1  - Lai, Ruifang
A1  - Bai, Mingzhou
A1  - Wei, Jin
A1  - Yi, Shaokui
A1  - Wang, Huanling
A1  - Cao, Xiaojuan
A1  - Zhou, Xiaoyun
A1  - Zhao, Yuhua
A1  - Wei, Kaijian
A1  - Yang, Ruibin
A1  - Liu, Bingnan
A1  - Zhao, Shancen
A1  - Fang, Xiaodong
A1  - Schartl, Manfred
A1  - Qian, Xueqiao
A1  - Wang, Weimin
T1  - The draft genome of blunt snout bream (Megalobrama amblycephala) reveals the development of intermuscular bone and adaptation to herbivorous diet
JF  - GigaScience
N2  - The blunt snout bream Megalobrama amblycephala is the economically most important cyprinid fish species. As an herbivore, it can be grown by eco-friendly and resource-conserving aquaculture. However, the large number of intermuscular bones in the trunk musculature is adverse to fish meat processing and consumption. As a first towards optimizing this aquatic livestock, we present a 1.116-Gb draft genome of M. amblycephala, with 779.54 Mb anchored on 24 linkage groups. Integrating spatiotemporal transcriptome analyses, we show that intermuscular bone is formed in the more basal teleosts by intramembranous ossification and may be involved in muscle contractibility and coordinating cellular events. Comparative analysis revealed that olfactory receptor genes, especially of the beta type, underwent an extensive expansion in herbivorous cyprinids, whereas the gene for the umami receptor T1R1 was specifically lost in M. amblycephala. The composition of gut microflora, which contributes to the herbivorous adaptation of M. amblycephala, was found to be similar to that of other herbivores. As a valuable resource for the improvement of M. amblycephala livestock, the draft genome sequence offers new insights into the development of intermuscular bone and herbivorous adaptation.
KW  - Megalobrama amblycephala
KW  - whole genome
KW  - herbivorous diet
KW  - intermuscular bone
KW  - transcriptome
KW  - gut microflora
Y1  - 2017
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170844
VL  - 6
IS  - 7
ER  - 
TY  - JOUR
A1  - Halboth, Florian
A1  - Roces, Flavio
T1  - The construction of ventilation turrets in Atta vollenweideri leaf-cutting ants: Carbon dioxide levels in the nest tunnels, but not airflow or air humidity, influence turret structure
JF  - PLoS ONE
N2  - Nest ventilation in the leaf-cutting ant Atta vollenweideri is driven via a wind-induced mechanism. On their nests, workers construct small turrets that are expected to facilitate nest ventilation. We hypothesized that the construction and structural features of the turrets would depend on the colony’s current demands for ventilation and thus might be influenced by the prevailing environmental conditions inside the nest. Therefore, we tested whether climate-related parameters, namely airflow, air humidity and CO\(_{2}\) levels in the outflowing nest air influenced turret construction in Atta vollenweideri. In the laboratory, we simulated a semi-natural nest arrangement with fungus chambers, a central ventilation tunnel providing outflow of air and an aboveground building arena for turret construction. In independent series, different climatic conditions inside the ventilation tunnel were experimentally generated, and after 24 hours, several features of the built turret were quantified, i.e., mass, height, number and surface area (aperture) of turret openings. Turret mass and height were similar in all experiments even when no airflow was provided in the ventilation tunnel. However, elevated CO\(_{2}\) levels led to the construction of a turret with several minor openings and a larger total aperture. This effect was statistically significant at higher CO\(_{2}\) levels of 5% and 10% but not at 1% CO\(_{2}\). The construction of a turret with several minor openings did not depend on the strong differences in CO\(_{2}\) levels between the outflowing and the outside air, since workers also built permeated turrets even when the CO\(_{2}\) levels inside and outside were both similarly high. We propose that the construction of turrets with several openings and larger opening surface area might facilitate the removal of CO\(_{2}\) from the underground nest structure and could therefore be involved in the control of nest climate in leaf-cutting ants.
KW  - carbon dioxide
KW  - animal sociality
KW  - ants
KW  - fungi
KW  - humidity
KW  - social systems
KW  - nesting habits
KW  - fungal structure
Y1  - 2017
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-159133
VL  - 12
IS  - 11
ER  - 
TY  - JOUR
A1  - Kramer, Susanne
T1  - The ApaH-like phosphatase TbALPH1 is the major mRNA decapping enzyme of trypanosomes
JF  - PLoS Pathogens
N2  - 5’-3’ decay is the major mRNA decay pathway in many eukaryotes, including trypanosomes. After deadenylation, mRNAs are decapped by the nudix hydrolase DCP2 of the decapping complex and finally degraded by the 5’-3’ exoribonuclease. Uniquely, trypanosomes lack homologues to all subunits of the decapping complex, while deadenylation and 5’-3’ degradation are conserved. Here, I show that the parasites use an ApaH-like phosphatase (ALPH1) as their major mRNA decapping enzyme. The protein was recently identified as a novel trypanosome stress granule protein and as involved in mRNA binding. A fraction of ALPH1 co-localises exclusively with the trypanosome 5’-3’ exoribonuclease XRNA to a special granule at the posterior pole of the cell, indicating a connection between the two enzymes. RNAi depletion of ALPH1 is lethal and causes a massive increase in total mRNAs that are deadenylated, but have not yet started 5’-3’ decay. These data suggest that ALPH1 acts downstream of deadenylation and upstream of mRNA degradation, consistent with a function in mRNA decapping. In vitro experiments show that recombinant, N-terminally truncated ALHP1 protein, but not a catalytically inactive mutant, sensitises the capped trypanosome spliced leader RNA to yeast Xrn1, but only if an RNA 5’ polyphosphatase is included. This indicates that the decapping mechanism of ALPH1 differs from the decapping mechanism of Dcp2 by leaving more than one phosphate group at the mRNA’s 5’ end. This is the first reported function of a eukaryotic ApaH-like phosphatase, a bacterial-derived class of enzymes present in all phylogenetic super-groups of the eukaryotic kingdom. The substrates of eukaryotic ApaH-like phosphatases are unknown. However, the substrate of the related bacterial enzyme ApaH, diadenosine tetraphosphate, is highly reminiscent of a eukaryotic mRNA cap.
KW  - eukaryota
KW  - Trypanosoma
KW  - RNA interference
KW  - messenger RNA
Y1  - 2017
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-158482
VL  - 13
IS  - 6
ER  - 
TY  - THES
A1  - Brühlmann, David
T1  - Tailoring Recombinant Protein Quality by Rational Media Design
T1  - Der Einfluss von Zellkulturmedien auf Qualitätsattribute von rekombinanten Proteinen
N2  - Nowadays, more than half of the biotherapeutics are produced in mammalian cell lines as a result of correct protein folding and assembly as well as their faculty to bring about a variety of post-translational modifications. The widespread progression of biosimilars has moved the focus in mammalian cell-culture process development. Thereby, the modulation of quality attributes of recombinant therapeutic proteins has increasingly gained importance from early process development stages. Protein quality directly shapes the clinical efficacy and safety in vivo, and therefore, the control of the complex post-translational modifications, such as glycosylation (e.g. high mannose, fucosylation, galactosylation and sialylation), charge variants, aggregates and low-molecular-weight species formation, is pivotal for efficient receptor binding and for triggering the desired immune responses in patients. In the frame of biosimilar development, product quality modulation methods using the potential of the host cell line are particularly sought after to match the quality profile of the targeted reference medicinal product (RMP) as closely as possible. The environment the cell is dwelling in directly influences its metabolism and the resulting quality profile of the expressed protein. Thereby the cell culture medium plays a central role in upstream manufacturing. In this work, concentration adjustment of selected media components and supplementation with a variety of compounds was performed to alter various metabolic pathways, enzyme activities and in some cases the gene expression levels of Chinese Hamster Ovary (CHO) cells in culture. The supplementation of cell culture medium with the trisaccharide raffinose in fed-batch cultures entailed an increase of the abundance of high mannose glycans in two different CHO cell lines. Raffinose especially favored mannose 5 glycans. At the same time, it impaired cell culture performance, induced changes on the intracellular nucleotide levels and even varied the expression levels of glycosylation-related genes. Supplementation with a number of galactosyltransferase inhibiting compounds, in particular fluorinated galactose analogs (alpha- and beta-2F-peracetyl-galactose), consistently decreased the production of galactosylated monoclonal antibodies (mAb). By means of targeted addition during the culture rather than at the beginning, the inhibition was further increased, while limiting detrimental effects on both growth and productivity. High-throughput screening in 96-deepwell plates showed that spermine and L-ornithine also reduced the level of galactosylation. On the other hand, exploratory screening of a variety of potentially disulfide-bridge-reducing agents highlighted that the inherent low-molecular-species level of the proprietary platform cell culture process was likely due to favored reduction. This hypothesis was reinforced by the observation that supplementation of cysteine and N-acetylcysteine promoted fragmentation. Additionally, fragmentation decreased with higher protein expression. 
At that point, aiming to improve the efficiency in process development, a rational experimental design method was developed to identify and to define the optimal concentration range of quality modulating compounds by calling on a combination of high throughput fed-batch testing and multivariate data analysis. Seventeen medium supplements were tested in five parallel 96-deepwell plate experiments. The selection process of promising modulators for the follow-up experiment in shake tubes consisted in a three-step procedure, including principal component analysis, quantitative evaluation of their performance with respect to the specifications for biosimilarity and selection following a hierarchical order of decisions using a decision tree. The method resulted in a substantial improvement of the targeted glycosylation profile in only two experimental rounds. Subsequent development stages, namely validation and transfer to industrial-scale facilities require tight control of product quality. Accordingly, further mechanistic understanding of the underlying processes was acquired by non-targeted metabolomic profiling of a CHO cell line expressing a mAb cultured in four distinct process formats. Univariate analysis of intra- and extracellular metabolite and temporal glycosylation profiles provided insights in various pathways. The numerous of parameters were the main driver to carry out principal component analysis, and then, using the methodology of partial-least-square (PLS) projection on latent structures, a multivariate model was built to correlate the extracellular data with the distinct glycosylation profiles. The PLS observation model proved to be reliable and showed its great benefit for glycan pattern control in routine manufacturing, especially at large scale. Rather than relying on post-production interpretation of glycosylation results, glycosylation can be predicted in real-time based on the extracellular metabolite levels in the bioreactor. 
Finally, for the bioactivity assessment of the glycan differences between the biosimilar and the reference medicinal product (RMP), the health agencies may ask for in the drug registration process, extended ranges of glycan variants need to be generated so that the in vitro assays pick up the changes. The developed glycosylation modulator library enabled the generation of extreme glycosylation variants, including high mannose, afucosylated, galactosylated as well as sialic acid species of both a mAb and an antibody fusion molecule with three N-glycosylation sites. Moreover, to create increased variety, enzymatic glycoengineering was explored for galactosylation and sialylation. The glyco variants induced significant responses in the respective in vitro biological activity assays. The data of this work highlight the immense potential of cell culture medium optimization to adjust product quality. Medium and feed supplementation of a variety of compounds resulted in reproducible and important changes of the product quality profile of both mAbs and a fusion antibody. In addition to the intermediate modulation ranges that largely met the requirements for new-biological-entity and biosimilar development, medium supplementation even enabled quick and straightforward generation of extreme glycan variants suitable for biological activity testing.
N2  - Mehr als die Hälfte der Biotherapeutika werden heutzutage aufgrund korrekter Proteinfaltung und korrektem Zusammenbau in tierischen Zelllinien hergestellt, welche zudem die Fähigkeit besitzen, verschiedene posttranslationale Modifikationen zu bewerkstelligen, hergestellt. Der ausgeprägte Aufschwung von Biosimilars hat den Entwicklungsschwerpunkt von Zellkulturverfahren verlagert. Dabei hat die Modulierung der Qualitätsattribute von rekombinanten Proteinen bereits in frühen Entwicklungsstadien eine wichtige Bedeutung erlangt. Die Qualitätsattribute beeinflussen die klinische Wirksamkeit und die In-Vivo-Sicherheit direkt. Somit ist die Regulierung der posttranslationalen Modifikationen, einschließlich der Glykosylierung (mannosereiche, fukosylierte, galaktosylierte und sialylierte Glykane), der Ladungsvarianten, sowie die Bildung von Aggregaten und niedermolekularen Spezien, für effiziente Rezeptorbindung und das Auslösen der gewünschten Immunantwort in Patienten entscheidend. Im Rahmen der Biosimilarentwicklung werden Methoden zur Anpassung der Produktqualität innerhalb des Potentials der Wirtszelle gesucht, um sie möglichst genau dem Referenzarzneimittel anzugleichen. Die Umgebung, in der die Zelle verweilt, beeinflusst ihren Metabolismus und das resultierende Produktqualitätsprofil. Dabei spielen Medien eine zentrale Rolle in der Zellkultur. Im Rahmen dieser Doktorarbeit wurden durch Adjustierung von ausgewählten Medienbestandteilen und Ergänzung mit einer Vielfalt von Stoffen diverse Stoffwechselwege, Enzymaktivitäten und in einigen Fällen das Genexpressionsniveau von kultivierten Chinesischen Hamster-Ovarialzellen (CHO) verändert. Die Ergänzung von Zellkulturmedium mit Raffinose, ein Trisaccharid, führte zu einer Erhöhung des mannosereichen Glykosylierungsmusters in zwei unterschiedlichen CHO-Zelllinien. Raffinose begünstigte hauptsächlich Mannose-5-Spezien. Gleichzeitig wurde die Zellkulturleistung beeinträchtigt und zudem intrazelluläre Nukleotidkonzentrationen sowie das Expressionsniveau von Glykosylierungsgenen verändert. Ergänzung mit mehreren Inhibitoren der Galaktosyltransferase, insbesondere fluorierte Galaktosenachbildungen (Alpha- und Beta-2F-Peracetyl-Galaktose), verringerte stetig die Produktion von galaktosylierten monoklonalen Antikörpern (mAb). Durch gezielte Zugabe im Verlauf der Kultur, statt bereits am Anfang, wurde die Inhibition weiter erhöht, und dabei die Einwirkung auf das Zellwachstum und die Produktivität beschränkt. Ein Hochdurchsatz-Screening in 96-Deep-Well-Platten zeigte, dass Spermin und L-Ornithin auch das Ausmaß der Galaktosylierung reduzierte. Andererseits zeigten erste Nachforschungen anhand eines Screenings einer Auswahl von potenziellen Disulfidbrücken-Reduktionsmittel, dass wahrscheinlich begünstigte Reduktion das inhärente Niedermolekular-Speziesniveau des firmeneigenen Zellkulturplattformverfahrens verursacht. Die Hypothese wurde durch die Beigabe von Cystein und N-Acetylcystein bekräftigt. Diese Stoffe begünstigten die Fragmentierung, wohingegen sie bei höherer Proteinexpression abnahm. 
Mit dem Ziel die Entwicklungseffizienz zu steigern, wurde daraufhin zur Identifikation von qualitätsverändernden Stoffen und Bestimmung der optimalen Konzentrationsbereichen eine rationale Versuchsanordnungsmethode entwickelt. Dazu wurde eine Kombination von Hochdurchsatz-Fed-Batch-Tests und multivariater Datenanalyse herbeigezogen. Siebzehn Mediumergänzungsstoffe wurden in fünf parallelen 96-Deep-Well-Platten-Experimenten getestet. Das Auswahlverfahren von erfolgsversprechenden Modulatoren fürs Nachfolgeexperiment in Schüttelröhrchen umfasste drei Schritte: Hauptkomponentenanalyse, quantitative Evaluierung der Leistung der Modulatoren hinsichtlich der Biosimilaritätsspezifikationen und die Auswahl in Anlehnung an eine hierarchische Entscheidungsreihenfolge mit Hilfe eines Entscheidungsbaums. Die Methode führte in nur zwei Versuchsreihen zu einer erheblichen Annäherung an das gewünschte Glykosylierungsprofil. Anschließende Entwicklungsschritte (Validierung und Transfer in die großtechnische Anlage) erforden eine rigorose Kontrolle der Produktqualität. Demzufolge konnte dank der Non-Targeted Metabolomics Analyse von vier verschiedenen Herstellungsverfahren einer mAb exprimierenden CHO-Zelllinie weitere mechanistische Kenntnisse der zugrunde liegenden Vorgängen gewonnen werden. Univariate Analysen der intra- und extrazellulären Stoffwechselprodukte und die zeitliche Glykosylierungsprofile lieferten einen Einblick in verschiedene Stoffwechselwege. Die Vielzahl von Parametern führte dazu, nach dem Prinzip der Hauptkomponentenanalyse vorzugehen, und dann anhand der Partial Least Squares (PLS)-Projektion auf latente Strukturen ein multivariates Modell zu erstellen, das die extrazellulären Daten mit den individuellen Glykosylierungsprofilen korreliert. Das PLS Beobachtungsmodell stellte sich als verlässlich heraus und zeigte seinen außerordentlichen Nutzen zur Regulierung der Glykanen in der Routineherstellung, insbesondere in der Großanlage. Anstatt sich auf Glykosylierungsresultate nach dem Ende der Produktion zu verlassen, kann die Glykosylierung, basierend auf den Niveaus der extrazellulären Stoffwechselprodukte im Bioreaktor, in Echtzeit vorausgesagt werden. 
Schließlich können im Rahmen des Arzneigenehmigungsverfahrens Gesundheitsbehörden verlangen, die Glykanunterschiede zwischen dem Biosimilar und dem Referenzarzneimittel zu untersuchen. Damit der biologische Test die Unterschiede nachweisen kann, muss eine erweiterte Palette von Glykanvarianten hergestellt werden. Die entwickelte Glykosylierungsmodulierungsbibliothek ermöglichte, extreme Varianten für mannosereiche, afukosylierte, galaktosylierte und sialylierte Glykane von mAb und einem Antikörperfusionsmolekül mit drei N-Glykosylierungsstellen zu generieren. Für erhöhte Variantenvielfalt wurde die enzymatische Glykoengineering Technologie für die Galaktosylierung und Sialylierung untersucht. Die Glykanvarianten erzeugten signifikante Antworten in der jeweiligen In-Vitro-Bestimmung der biologischen Aktivität. Die Ergebnisse unterstreichen das immense Potential von Zellkulturmediumoptimierung zur Anpassung der Produktqualität. Ergänzung des Mediums und der Nährstofflösung brachte reproduzierbare und beträchtliche Veränderungen der Produktqualität von mAb und eines Fusionsantikörpers hervor. Zusätzlich zu den intermediären Modulierungsbereichen, die mehr als ausreichend den Anforderungen für die Entwicklung von neuen biologischen Wirkstoffen und Biosimilars genügen, ermöglichte die Mediumergänzung auf schnelle und einfache Art und Weise selbst extreme Glykanvarianten zu bilden, die für die Bestimmung der biologischen Aktivität geeignet waren.
KW  - CHO cell culture
KW  - product qualitymodulation
KW  - media design
KW  - metabolism
KW  - glycosylation
KW  - high throughput
KW  - Zellkultur
KW  - CHO-Zelle
KW  - Produktivität
KW  - Nährboden
KW  - Stoffwechsel
Y1  - 2017
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-147345
ER  - 
TY  - JOUR
A1  - Kadochová, Štěpánka
A1  - Frouz, Jan
A1  - Roces, Flavio
T1  - Sun basking in red wood ants Formica polyctena (Hymenoptera, Formicidae): Individual behaviour and temperature-dependent respiration rates
JF  - PLoS ONE
N2  - In early spring, red wood ants Formica polyctena are often observed clustering on the nest surface in large numbers basking in the sun. It has been hypothesized that sun-basking behaviour may contribute to nest heating because of both heat carriage into the nest by sunbasking workers, and catabolic heat production from the mobilization of the workers’ lipid reserves. We investigated sun-basking behaviour in laboratory colonies of F. polyctena exposed to an artificial heat source. Observations on identified individuals revealed that not all ants bask in the sun. Sun-basking and non-sun-basking workers did not differ in body size nor in respiration rates. The number of sun-basking ants and the number of their visits to the hot spot depended on the temperature of both the air and the hot spot. To investigate whether sun basking leads to a physiological activation linked with increased lipolysis, we measured respiration rates of individual workers as a function of temperature, and compared respiration rates of sun-basking workers before and two days after they were allowed to expose themselves to a heat source over 10 days, at self-determined intervals. As expected for ectothermic animals, respiration rates increased with increasing temperatures in the range 5 to 35˚C. However, the respiration rates of sun-basking workers measured two days after a long-term exposure to the heat source were similar to those before sun basking, providing no evidence for a sustained increase of the basal metabolic rates after prolonged sun basking. Based on our measurements, we argue that self-heating of the nest mound in early spring has therefore to rely on alternative heat sources, and speculate that physical transport of heat in the ant bodies may have a significant effect.
KW  - biology
KW  - ants
KW  - social systems
KW  - animal sociality
KW  - lipids
KW  - nesting habit
KW  - video recording
KW  - catabolism
Y1  - 2017
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-171936
VL  - 12
IS  - 1
ER  - 
TY  - JOUR
A1  - Costea, Paul I.
A1  - Coelho, Louis Pedro
A1  - Sunagawa, Shinichi
A1  - Munch, Robin
A1  - Huerta-Cepas, Jaime
A1  - Forslund, Kristoffer
A1  - Hildebrand, Falk
A1  - Kushugulova, Almagul
A1  - Zeller, Georg
A1  - Bork, Peer
T1  - Subspecies in the global human gut microbiome
JF  - Molecular Systems Biology
N2  - Population genomics of prokaryotes has been studied in depth in only a small number of primarily pathogenic bacteria, as genome sequences of isolates of diverse origin are lacking for most species. Here, we conducted a large‐scale survey of population structure in prevalent human gut microbial species, sampled from their natural environment, with a culture‐independent metagenomic approach. We examined the variation landscape of 71 species in 2,144 human fecal metagenomes and found that in 44 of these, accounting for 72% of the total assigned microbial abundance, single‐nucleotide variation clearly indicates the existence of sub‐populations (here termed subspecies). A single subspecies (per species) usually dominates within each host, as expected from ecological theory. At the global scale, geographic distributions of subspecies differ between phyla, with Firmicutes subspecies being significantly more geographically restricted. To investigate the functional significance of the delineated subspecies, we identified genes that consistently distinguish them in a manner that is independent of reference genomes. We further associated these subspecies‐specific genes with properties of the microbial community and the host. For example, two of the three Eubacterium rectale subspecies consistently harbor an accessory pro‐inflammatory flagellum operon that is associated with lower gut community diversity, higher host BMI, and higher blood fasting insulin levels. Using an additional 676 human oral samples, we further demonstrate the existence of niche specialized subspecies in the different parts of the oral cavity. Taken together, we provide evidence for subspecies in the majority of abundant gut prokaryotes, leading to a better functional and ecological understanding of the human gut microbiome in conjunction with its host.
KW  - biology
KW  - genetic variation
KW  - metagenomics
KW  - microbiome
KW  - population structure
KW  - prokaryotic subspecies
Y1  - 2017
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-172674
VL  - 13
IS  - 12
ER  - 
TY  - JOUR
A1  - Kasaragod, Prasad
A1  - Midekessa, Getnet B.
A1  - Sridhar, Shruthi
A1  - Schmitz, Werner
A1  - Kiema, Tiila-Riikka
A1  - Hiltunen, Jukka K.
A1  - Wierenga, Rik K.
T1  - Structural enzymology comparisons of multifunctional enzyme, type-1 (MFE1): the flexibility of its dehydrogenase part
JF  - FEBS Open Bio
N2  - Multifunctional enzyme, type-1 (MFE1) is a monomeric enzyme with a 2E-enoyl-CoA hydratase and a 3S-hydroxyacyl-CoA dehydrogenase (HAD) active site. Enzyme kinetic data of rat peroxisomal MFE1 show that the catalytic efficiencies for converting the short-chain substrate 2E-butenoyl-CoA into acetoacetyl-CoA are much lower when compared with those of the homologous monofunctional enzymes. The mode of binding of acetoacetyl-CoA (to the hydratase active site) and the very similar mode of binding of NAD\(^+\) and NADH (to the HAD part) are described and compared with those of their monofunctional counterparts. Structural comparisons suggest that the conformational flexibility of the HAD and hydratase parts of MFE1 are correlated. The possible importance of the conformational flexibility of MFE1 for its biocatalytic properties is discussed.
KW  - biology
KW  - CoA
KW  - crotonase
KW  - dehydrogenase
KW  - NAD
KW  - substrate channeling
Y1  - 2017
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-172732
VL  - 7
IS  - 12
ER  - 
TY  - THES
A1  - Hagen, Franziska
T1  - Sphingolipids in gonococcal infection
T1  - Sphingolipide in der Gonokokken Infektion
N2  - Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea, has the potential to spread in the human host and cause a severe complication called disseminated gonococcal infection (DGI). The expression of the major outer membrane porin PorBIA is a characteristic of most gonococci associated with DGI. PorBIA binds to the scavenger receptor expressed on endothelial cells (SREC-I), which mediates the so-called low phosphate-dependent invasion (LPDI). This uptake mechanism enables N. gonorrhoeae to rapidly invade epithelial and endothelial cells in a phosphate-sensitive manner. 
We recently demonstrated that the neutral sphingomyelinase, which catalyses the hydrolysis of sphingomyelin to ceramide and phosphorylcholine, is required for the LPDI of gonococci in non-phagocytic cells. Neutral sphingomyelinase 2 (NSM2) plays a key role in the early PorBIA signaling by recruiting the PI3 kinase to caveolin. The following activation of the PI3 kinase-dependent downstream signaling leads to the engulfment of the bacteria. As a part of this work, I could confirm the involvement of the NSM2. The role of the enzyme was further elucidated by the generation of antibodies directed against NSM2 and the construction of an epithelium-based NSM2 knockout cell line using CRISPR/Cas9. The knockout of the NSM2 strongly inhibits the LPDI. The invasion could be, however, restored by the complementation of the knockout using an NSM2-GFP construct. However, the results could not be reproduced. 
In this work, I could show the involvement of further members of the sphingolipid pathway in the PorBIA-mediated invasion. Lipidome analysis revealed an increase of the bioactive molecules ceramide and sphingosine due to gonococcal infection. Both molecules do not only affect the host cell, but seem to influence the bacteria as well: while ceramide seems to be incorporated by the gonococci, sphingosine is toxic for the bacteria. Furthermore, the sphingosine kinase 2 (SPHK2) plays an important role in invasion, since the inhibition and knockdown of the enzyme revealed a negative effect on gonococcal invasion. To elucidate the role of the sphingosine kinases in invasion in more detail, an activity assay was established in this study. Additionally, the impact of the sphingosine-1-phosphate lyase (S1PL) on invasion was investigated. Inhibitor studies and infection experiments conducted with a CRISPR/Cas9 HeLa S1PL knockout cell line revealed a role of the enzyme not only in the PorBIA-mediated invasion, but also in the Opa50/HSPG-mediated gonococcal invasion. The signaling experiments allowed the categorization of the SPHK and S1PL activation in the context of infection. Like the NSM2, both enzymes play a role in the early PorBIA signaling events leading to the uptake of the bacteria. All those findings indicate an important role of sphingolipids in the invasion and survival of N. gonorrhoeae. 
In the last part of this work, the role of the NSM2 in the inhibition of apoptosis in neutrophils due to gonococcal infection was investigated. It could be demonstrated that the delayed onset of apoptosis is independent of neisserial porin and Opa proteins. Furthermore, the influence of neisserial peptidoglycan on PMN apoptosis was analysed using mutant strains, but no connection could be determined. Since the NSM2 is the most prominent sphingomyelinase in PMNs, fulfils manifold cell physiological functions and has already been connected to apoptosis, the impact of the enzyme on apoptosis inhibition due to gonococcal infection was investigated using inhibitors, with no positive results.
N2  - Neisseria gonorrhoeae, der Auslöser der sexuell übertragbaren Krankheit Gonorrhö, hat das Potenzial sich im menschlichen Wirt auszubreiten und eine schwere Komplikation, die disseminierende Gonokokkeninfektion (DGI), hervorzurufen. Die Expression des Porins PorBIA, das eines der häufigsten Proteine der äußeren Membran ist, stellt ein Charakteristikum der mit DGI assoziierten Gonokokken dar. PorBIA bindet an SREC-I (scavenger receptor expressed on endothelial cells), der die phosphatabhängige Invasion (low phosphate-dependent invasion LPDI) vermittelt. Dieser Aufnahmemechanismus erlaubt es N. gonorrhoeae Epithel- sowie Endothelzellen, schnell zu invadieren.
Wir haben kürzlich gezeigt, dass die neutrale Sphingomyelinase 2 (NSM2), welche die Hydrolyse von Sphingomyelin zu Ceramid und Phosphorylcholin katalysiert, für die LPDI der Gonokokken in nicht-phagozytische Zellen benötigt wird. Dabei spielt die neutrale Sphingomyelinase 2 eine Schlüsselrolle in der frühen PorBIA Signalübertragung, indem sie die PI3 Kinase zu Caveolin rekrutiert. Die darauffolgende Aktivierung von nachgeschalteten Signalwegen, die von der PI3 Kinase abhängig sind, führt zur Aufnahme der Bakterien. Als Teil dieser Arbeit konnte ich die Beteiligung der NSM2 bestätigen. Die Rolle des Enzyms sollte durch die Herstellung von NSM2-spezifischen Antikörpern und einer auf Epithelzellen basierenden NSM2 knockout Zelllinie, die mit Hilfe des CRISPR/Cas9 Systems hergestellt wurde, aufgeklärt werden. Der knockout der NSM2 führte zu einer starken Inhibition der LPDI. Die Invasion konnte jedoch durch die Komplementation mit Hilfe eines NSM2-GFP Konstruktes wiederhergestellt werden. Wobei die Ergebnisse jedoch nicht reproduziert werden konnten.
In dieser Arbeit konnte ich die Beteiligung weiterer Mitglieder des Sphingolipid Signalwegs an der PorBIA-vermittelten Invasion zeigen. Die Lipidomanalysen zeigten einen Anstieg der bioaktiven Moleküle Ceramide und Sphingosin aufgrund der Gonokokkeninfektion. Beide Moleküle beeinflussen nicht nur die Wirtszelle, sondern schienen auch Auswirkungen auf die Bakterien selbst zu haben: während Ceramid anscheinend von den Gonokokken aufgenommen wird, ist Sphingosin für die Bakterien toxisch. Weiterhin spielt die Sphingosinkinase 2 (SPHK2) eine wichtige Rolle in der Invasion, da die Inhibierung und der Knockdown des Enzyms die Gonokokkeninfektion negativ beeinflussen. Um die Rolle der Sphingosinkinasen in der Invasion im Detail zu erforschen, wurde in dieser Arbeit ein Aktivitätsassay etabliert. Außerdem wurde der Einfluss der Sphingosin-1-phosphat Lyase (S1PL) auf die Invasion erforscht. Inhibitorstudien und Infektionsexperimente, die mit einer CRISPR/Cas9 HeLa S1PL knockout Zelllinie durchgeführt wurden, zeigten, dass das Enzym nicht nur eine Rolle in der PorBIA-vermittelten, sondern auch in der Opa50/HSPG-vermittelten Gonokokkeninfektion spielt. Die Experimente, die bezüglich der zugrundeliegenden Signalwege durchgeführt wurden, erlaubten die Einordnung der Aktivierung der SPHK und der S1PL im Kontext der Invasion. Wie auch die NSM2, spielen beide Enzyme in der frühen PorBIA Signalübertragung eine Rolle, die schließlich zur Aufnahme der Bakterien führt. Alle diese Ergebnisse weisen auf eine wichtige Rolle der Sphingolipide für die Invasion und das Überleben von N. gonorrhoeae hin.
Im letzten Teil dieser Arbeit, wurde die Inhibierung der Apoptose von Neutrophilen aufgrund der Gonokokkeninfektion untersucht. Es konnte gezeigt werden, dass das verspätete Einsetzen der Apoptose von neisseriellen Porinen und Opa Proteinen unabhängig ist. Weiterhin wurde der Einfluss von neisseriellem Peptidoglycan auf die Apoptose der Neutrophilen mit Hilfe von Mutanten untersucht, wobei eine Verbindung nicht bestätigt werden konnte. Da die NSM2 die bedeutendste Sphingomyelinase in Neutrophilen darstellt, sowie vielfältige zellphysiologische Funktionen erfüllt und im Vorfeld schon mit der Apoptose in Verbindung gebracht wurde, wurde der Einfluss des Enzymes auf die Inhibierung der Apoptose durch die Gonokokkeninfektion mit Hilfe von Inhibitoren überprüft.
KW  - gonococcal
KW  - sphingolipids
KW  - gonococcal infection
Y1  - 2017
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-153852
ER  - 
TY  - JOUR
A1  - Mitjans, M.
A1  - Begemann, M.
A1  - Ju, A.
A1  - Dere, E.
A1  - Wüstefeld, L.
A1  - Hofer, S.
A1  - Hassouna, I.
A1  - Balkenhol, J.
A1  - Oliveira, B.
A1  - Van der Auwera, S.
A1  - Tammer, R.
A1  - Hammerschmidt, K.
A1  - Völzke, H.
A1  - Homuth, G.
A1  - Cecconi, F.
A1  - Chowdhury, K.
A1  - Grabe, H.
A1  - Frahm, J.
A1  - Boretius, S.
A1  - Dandekar, T.
A1  - Ehrenreich, H.
T1  - Sexual dimorphism of \(AMBRA1\)-related autistic features in human and mouse
JF  - Translational Psychiatry
N2  - \(Ambra1\) is linked to autophagy and neurodevelopment. Heterozygous \(Ambra1\) deficiency induces autism-like behavior in a sexually dimorphic manner. Extraordinarily, autistic features are seen in female mice only, combined with stronger Ambra1 protein reduction in brain compared to males. However, significance of \(AMBRA1\) for autistic phenotypes in humans and, apart from behavior, for other autism-typical features, namely early brain enlargement or increased seizure propensity, has remained unexplored. Here we show in two independent human samples that a single normal \(AMBRA1\) genotype, the intronic SNP rs3802890-AA, is associated with autistic features in women, who also display lower \(AMBRA1\) mRNA expression in peripheral blood mononuclear cells relative to female GG carriers. Located within a non-coding RNA, likely relevant for mRNA and protein interaction, rs3802890 (A versus G allele) may affect its stability through modification of folding, as predicted by \(in\) \(silico\) analysis. Searching for further autism-relevant characteristics in \(Ambra1^{+/−}\) mice, we observe reduced interest of female but not male mutants regarding pheromone signals of the respective other gender in the social intellicage set-up. Moreover, altered pentylentetrazol-induced seizure propensity, an \(in\) \(vivo\) readout of neuronal excitation–inhibition dysbalance, becomes obvious exclusively in female mutants. Magnetic resonance imaging reveals mild prepubertal brain enlargement in both genders, uncoupling enhanced brain dimensions from the primarily female expression of all other autistic phenotypes investigated here. These data support a role of \(AMBRA1/Ambra1\) partial loss-of-function genotypes for female autistic traits. Moreover, they suggest \(Ambra1\) heterozygous mice as a novel multifaceted and construct-valid genetic mouse model for female autism.
KW  - biology
KW  - clinical genetics
KW  - molecular neuroscience
Y1  - 2017
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-173782
VL  - 2017
IS  - 7
ER  - 
TY  - THES
A1  - Contar Adolfi, Mateus
T1  - Sex determination and meiosis in medaka: The role of retinoic acid
T1  - Geschlechtsbestimmung und Meiose in Medaka: Die Rolle der Retinsäure
N2  - Sex determination (SD) is a complex and diverse developmental process that leads to the decision whether the bipotential gonad anlage will become a testis or an ovary. This mechanism is regulated by gene cascades, networks and/or chromosomal systems, and can be influenced by fluctuations of extrinsic factors like temperature, exposure to hormones and pollution. Within vertebrates, the group of fish show the widest variety of sex determination mechanism. This whole diversity of processes and mechanisms converges to the formation of two different gametes, the eggs and the sperm, the first bigger and static, and the second smaller and motile. Meiosis is crucial for the formation of both types of gametes, and the timing of meiosis entry is one of the first recognizable differences between male and female in vertebrates. The germ cells go into meiosis first in female than in male, and in mammals, this event has been shown to be regulated by retinoic acid (RA). This small polar molecule induces in the germ cells the expression of the pre-meiotic marker Stra8 (stimulated by retinoic acid gene 8), which is necessary for meiosis initiation. Interestingly, genome analyzes have shown that the majority of fish (including medaka) lack the stra8 gene, adding a question mark to the role of RA in meiosis induction in this group. Since a role of RA in entry of meiosis and sexual development of fish is still far from being understood, I investigated in medaka (Oryzias latipes) a possible signaling function of RA during the SD period in embryos and in reproductively active gonads of adults. I generated a transgenic medaka line that reports responsiveness to RA in vivo. With this tool, I compared RA responsiveness with the expression of the main gene involved in the synthesis of RA. My results show that there is a de-correlation between the action of RA with its source. In adults, expression of the RA metabolizing enzymes show sexually dimorphic RA levels, with aldh1a2 levels being higher in testis, and cyp26a1 stronger in female gonad. In ovary, the responsiveness is restricted to the early meiotic oocytes. In testis, RA is acting directly in the pre-meiotic cells, but also in Sertoli and Leydig cells. Treatment experiments on testis organ culture showed that RA pathway activation leads to a decrease in meiosis markers expression levels. During the development, RA responsiveness in the germ cells was observed in both sexes much earlier than the first female meiosis entry. Treatments with RA-synthesis inhibitor show a decrease in meiosis markers expression levels only after the sex differentiation period in female. Expression analyzes of embryos treated with exogenous RA showed induction of dmrt1a at the gonad levels and an increase of amh levels. Both genes are not only involved in male formation, but also in the regulation of germ cell proliferation and differentiation. RA is important in meiosis induction and gametogenesis in adult medaka. However, there is no evidence for a similar role of RA in initiating the first meiosis in female germ cells at the SD stage. Moreover, contrary to common expectation, RA seems to induce sex related genes that are involved indirectly in meiosis inhibition. In this thesis, I showed for the first time that RA can be involved in both induction and inhibition of meiosis entry, depending on the sex and the developmental stage in a stra8-independent model organism.
N2  - Geschlechtsbestimmung ist ein komplexer und vielfältiger Entwicklungsprozess, der zu der Entscheidung führt, ob sich aus der bipotenten Gonadenanlage Hoden oder Ovarien entwickeln. Dieser Mechanismus ist durch Genkaskaden, Netzwerke und/oder chromosomale Systeme reguliert, kann aber auch durch Fluktuation äußerer Faktoren wie beispielsweise Temperatur, durch Hormonexposition oder durch Umweltverschmutzung beeinflusst werden. Innerhalb der Wirbeltiere zeigen Fische die größte Vielfalt in Bezug auf die Mechanismen der Geschlechtsbestimmung. Die unterschiedlichen Mechanismen der Geschlechtsbestimmung konvergieren in der Entstehung von der beiden unterschiedlichen Geschlechtszellen, der Eizelle und des Spermiums. Die Eizelle ist groß und statisch, das Spermium hingegen ist kleiner und beweglich. Die entscheidende Rolle für die Entstehung der Geschlechtzellen spielt die Meiose. Der Zeitpunkt, an dem zum ersten Mal in der Entwicklung die Meiose einsetzt, ist der erste erkennbare Unterschied in der Gonadenentwicklung zwischen männlichen und weiblichen Wirbeltieren. Die Meiose der Keimzellen beginnt bei Weibchen früher als bei Männchen. Bei Säugetieren reguliert Retinsäure (RA) diesen Prozess. Dieses kleine polare Molekül induziert die Expression des Prä-Meiose-Markers Stra8 (stimulated by retinoic acid gene 8) in den Keimzellen, welcher für den Eintritt in die Meiose essentiell ist. Interessanterweise haben Genomanalyzen gezeigt, dass das stra8 Gen in Medaka sowie in den meisten anderen Fischarten nicht vorhanden ist. Dies stellt eine vergleichbare Rolle von RA für die Induktion der Meiose wie bei Säugetieren in diesen Fischen in Frage. Da die Rolle von RA für den Eintritt in die Meiose sowie für die Geschlechtsentwicklung in Fischen bisher nur unzureichend untersucht und verstanden ist, habe ich bei Medaka (Oryzias latipes) eine mögliche Funktion von RA für die Geschlechtsdetermination in Embryonen sowie in Gonaden geschlechtsreifer Tiere untersucht. Ich habe im Rahmen dieser Arbet eine transgene Medakalinie generiert, die in vivo eine RA induzierte Genexpression durch ein GFP Reportergen anzeigt. Mit Hilfe dieser Linie wurde die transkriptionsreulierende Aktivität von RAmit der Expression der wichtigsten Gene, die in die RA Synthese involviert sind, verglichen. Meine Ergebnisse zeigen eine Diskrepanz zwischen dem Wirkungs- und Syntheseort von RA. Die RA metabolisierenden Enzyme zeigten eine geschlechtsdimorphe Expression in adulten Medakas, mit einer höheren aldh1a2 Expression im Hoden sowie einer stärkeren cyp26a1 Expression in weiblichen Gonaden. Im Ovar sind lediglich frühe meiotische Eizellen RA-sensitiv. Im Hoden wirkt RA direkt in prä-meiotische Zellen, aber auch in Sertoli und Leydig Zellen. Stimulations-Experimente an Hoden Organkulturen ergaben, dass eine Aktivierung des RA Signalwegs zu einer Abnahme des Expressionslevels von Meiose-Markern führt. Während der Embryonalentwicklung konnte in den Keimzellen beider Geschlechter eine transkriptions-induziernende Aktivität von RA bereits zu einem Zeitpunkt beobachtet werden, der deutlich vor dem ersten Meiose Eintritt in Weibchen liegt. Behandlungen mit einem RA-Synthese Inhibitor zeigten lediglich nach der Geschlechtsdifferenzierung in Weibchen eine verminderte Expression der Meiose-Marker. Expressionsanalysen von Embryonen, die mit exogener RA behandelt wurden, ergaben eine Induktion von dmrt1a in den Gonaden und ein Anstieg von amh. Beide Gene sind sowohl in die männliche Geschlechtsentwicklung involviert, als auch in die Regulation der Keimzellproliferation und –differenzierung. Zusammen ergaben meine Untersuchungen, dass RA für die Induktion der Meiose und der Gametogenese in adulten Medakas wichtig ist. Allerdings gibt es keinen Hinweis für eine ähnliche Rolle
11
von RA bei der Initiierung der ersten Meiose in weiblichen Keimzellen während der Geschlechtsdetermination. Im Gegensatz zur bisher beschriebenen Situation, scheint darüber hinaus RA die Expression geschlechtsspezifischer Gene zu induzieren, die indirekt in die Inhibition der Meiose involviert sind. In der vorliegenden Arbeit konnte in einem stra8-unabhängigen Modelorganismus das erste Mal gezeigt werden, dass RA – abhängig vom Geschlecht und vom Stadium der Entwicklung - sowohl in die Induktion als auch in die Inhibition des Meiose-Eintritts involviert ist.
KW  - Japankärpfling
KW  - Meiose
KW  - Sex determination
KW  - Meiosis
KW  - Retinoic acid
KW  - Medaka
KW  - Geschlechtsdifferenzierung
KW  - Retinoesäure
KW  - Geschlechtsbestimmung
Y1  - 2017
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-136335
ER  - 
TY  - JOUR
A1  - Sbirkov, Yordan
A1  - Kwok, Colin
A1  - Bhamra, Amandeep
A1  - Thompson, Andrew J.
A1  - Gil, Veronica
A1  - Zelent, Arthur
A1  - Petrie, Kevin
T1  - Semi-quantitative mass spectrometry in AML cells identifies new non-genomic targets of the EZH2 methyltransferase
JF  - International Journal of Molecular Sciences
N2  - Alterations to the gene encoding the EZH2 (KMT6A) methyltransferase, including both gain-of-function and loss-of-function, have been linked to a variety of haematological malignancies and solid tumours, suggesting a complex, context-dependent role of this methyltransferase. The successful implementation of molecularly targeted therapies against EZH2 requires a greater understanding of the potential mechanisms by which EZH2 contributes to cancer. One aspect of this effort is the mapping of EZH2 partner proteins and cellular targets. To this end we performed affinity-purification mass spectrometry in the FAB-M2 HL-60 acute myeloid leukaemia (AML) cell line before and after all-trans retinoic acid-induced differentiation. These studies identified new EZH2 interaction partners and potential non-histone substrates for EZH2-mediated methylation. Our results suggest that EZH2 is involved in the regulation of translation through interactions with a number of RNA binding proteins and by methylating key components of protein synthesis such as eEF1A1. Given that deregulated mRNA translation is a frequent feature of cancer and that eEF1A1 is highly expressed in many human tumours, these findings present new possibilities for the therapeutic targeting of EZH2 in AML.
KW  - acute myeloid leukaemia
KW  - EZH2
KW  - mass spectrometry
KW  - methylation
KW  - eEF1A1
Y1  - 2017
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-285541
SN  - 1422-0067
VL  - 18
IS  - 7
ER  -