TY - THES A1 - Andronic, Joseph T1 - Volumenregulatorische Transportwege von anorganischen und organischen Osmolyten in Säugetierzellen T1 - Volume ragulatory pathways of anorganic and organic osmolytes in mammalian cells N2 - Die Aufrechterhaltung des Zellvolumens unter variablen osmotischen Bedingungen stellt für nahezu alle tierischen Zellen eine essenzielle Aufgabe dar. Um regulatorische Volumenanpassungen vorzunehmen besitzen sie daher effektive Mechanismen, mit deren Hilfe der zelluläre Gehalt an organischen und anorganischen Osmolyten erhöht (= regulatorische Volumenzunahme; RVI) oder gesenkt (= regulatorische Volumenabnahme; RVD) werden kann. Trotz langjähriger Forschung auf diesem Gebiet konnten die hieran beteiligten Transportwege für Osmolyte bisher nur unvollständig aufgeklärt werden. Insbesondere bei T-Lymphozyten sind wichtige Zellfunktionen wie die Proliferation, Migration und die T-Zell-Aktivierung eng mit volumenregulatorischen Mechanismen verbunden. Bei all diesen Prozessen sind u. a. unterschiedliche Kaliumkanäle beteiligt, die insbesondere für die pharmakologische Manipulation von Immunsystemprozessen von wissenschaftlichem Interesse sind. Bisherige Modelle der hypotonen Volumenregulation von T-Lymphozyten berücksichtigen lediglich den spannungsabhängigen KV1.3 sowie den Ca2+-aktivierten IKCa1-Kanal, die zur Klasse der 6TM/P-K+-Kanäle gehören. Im ersten Teil der vorliegenden Arbeit wurde eine potentielle Rolle von kürzlich entdeckten Zwei-Poren Domänen Kaliumkanälen (K2P) am RVD von murinen und humanen primären CD4+-T-Lymphozyten untersucht. In einem kombinierten genetischen und pharmakologischen Ansatz mittels knockout-Tiermodellen und dem Einsatz kanalspezifischer Inhibitoren konnte mithilfe zellvolumetrischer Analysen gezeigt werden, dass die K2P-Vertreter TASK1, TASK2, TASK3 und TRESK maßgeblich am schwellungsaktivierten Efflux von K+ beteiligt sind. Beurteilt an den Ergebnissen dieser Untersuchung sind der spannungsabhängige TASK2- und der Ca2+-aktivierte TRESK-Kanal für die hypotone Volumenregulation in T-Zellen deutlich bedeutender als TASK1 und TASK3. Der Beitrag der Kanäle TASK2 und TRESK am RVD-Prozess war über dies vergleichbar mit dessen des bisher bekannten KV1.3-Kanals. In dieser Arbeit wurde damit erstmals eine Beteiligung der K2P-Kanäle am RVD muriner und humaner CD4+-Lymphozyten identifiziert. Aufgrund der engen Verbindung zwischen T-Zell-Funktion und der Volumenregulation können Zwei-Poren Domänen K+-Kanäle damit in den engeren Kreis potentieller immunmodulierende Angriffspunkte aufgefasst werden. Im zweiten und umfangreicheren Teil dieser Arbeit wurden darüber hinaus die schwellungsaktivierten Transportwege für organische Osmolyte (small organic osmolytes; SOOs) untersucht. SOOs stellen chemisch inerte Verbindungen dar, zu denen vor allem Polyole (Sorbitol, myo-Inositol), Methylamine (Betain, α-Glycerophosphocholin) sowie Aminosäuren (α- bzw. β-Alanin und Prolin) und deren Derivate (Taurin) zählen. Da SOOs weder die zelluläre Struktur noch die Funktion von Makromolekülen beeinträchtigen, sind sie wichtige Instrumente der Volumenregulation, die sich in hohen Konzentrationen im Zytosol nahezu aller Zellen wiederfinden. Werden tierische Zellen mit hypotonen Bedingungen konfrontiert, dann ist bei nahezu allen Zellen die Freisetzung organischer Osmolyte zu beobachten, wodurch die zelluläre Osmolarität unabhängig von Elektrolyten angepasst werden kann. Trotz der wichtigen Funktion der SOOs in der Osmoregulation tierischer Zellen konnte die molekulare Identität beteiligter Effluxwege (Kanäle bzw. Transporter) bisher nicht aufgeklärt werden. Ungeachtet der molekularen Identität der SOO-Effluxwege war es aus zahlreichen biotechnologischen Anwendungen zu Beginn dieser Arbeit bekannt, dass die schwellungsaktivierten Transportwege für organische Osmolyte eine größenselektive Permeabilität für eine Reihe monomerer Zucker und verwandter Verbindungen aufweisen. Um diese Größenselektivität näher zu charakterisieren, wurde im ersten Schritt die schwellungsaktivierte Membranpermeabilität für eine Reihe strukturell homogener Polyethylenglykole unterschiedlicher Polymerlänge (PEG200–1500; hydrodynamische Radien zwischen ~0,5-1,5 nm) unter iso- und hypotonen Bedingungen in Jurkat-Lymphozyten untersucht. Unter milden hypotonen Bedingungen (200 mOsm) war die Plasmamembran der untersuchten Lymphozyten für PEG300-1500 undurchlässig, was aus der Fähigkeit der Zellen zur hypotonen Volumenregulation geschlossen werden konnte. Darüber hinaus wurde RVD in stark hypotonen Lösungen (100 mOsm) mit PEG600-1500 beobachtet, während PEG300-400 unter vergleichbaren osmotischen Bedingungen die Volumenregulation der Zellen inhibierten. Dieses Ergebnis deutet darauf hin, dass starkes hypotones Zellschwellen der Lymphozyten zur Permeabilisierung der Plasmamembran für PEG300-400, nicht jedoch für PEG600-1500, führt. Anhand der hydrodynamischen Radien Rh der verwendeten PEGs konnte ein cutoff-Radius von ~0,74 nm für schwellungsaktivierte Transportwege organischer Osmolyte bestimmt werden. Da diese schwellungsaktivierten Transportwege vielfältig für Zellbeladungstechniken verwendet werden, könnte dieses Ergebnis für zahlreiche biotechnologische und biomedizinische Anwendungen von Interesse sein. Im zweiten Schritt wurde der Versuch unternommen, potentielle Transportwege für organische Osmolyte im RVD-Prozess molekular zu identifizieren. Da es grundlegend ungeklärt war, wie viele unterschiedliche Transporter bzw. Kanäle am Efflux der zahlreichen organischen Osmolyte beteiligt sind, erfolgte zunächst die vergleichende Analyse des schwellungsaktivierten Membrantransports strukturell verschiedener SOOs einschließlich der Aminosulfonsäure Taurin und des Polyols myo-Inositol. Hierbei wurde erstmals gezeigt, dass die schwellungsaktivierten Transportwege für Taurin und myo-Inositol deutlich unterschiedliche Aktivitätsprofile aufweisen. Während der Taurintransport bereits unter milden hypotonen Bedingungen, d.h. nach einer geringen Absenkung der Osmolalität von 300 auf ~230 mOsm, aktiviert wurde, erfolgte die Aktivierung der Membranpermeabilität für myo-Inositol bei einer viel niedrigeren Osmolalität von ~150 mOsm. Darüber hinaus wiesen die beiden Transportwege unter vergleichbarem hypotonen Stress von 100 mOsm deutlich unterschiedliche Aktivitätsdauern auf (Transport von Taurin ~95 min und myo-Inositol ~40 min). Somit deuteten diese Ergebnisse erstmals auf substrat-spezifische Transportwege für SOOs hin, die voneinander stark abweichende osmotische Aktivierungsprofile besitzen. Als aussichtsreiche Kandidaten für diese Transportwege wurden zwei Mitglieder der Gruppe der Solute Carrier (SLC) untersucht, die klare Übereinstimmungen mit den gesuchten Transportern für SOOs aufweisen. Daher wurde im Weiteren eine RVD-Beteiligung dieser Transportergruppe mit einer Kombination aus molekularbiologischer und konventioneller bzw. hochaufgelöster mikroskopischen Techniken überprüft. Die semiqantitativen RT-PCR-Ergebnisse dieser Arbeit zeigen dabei, dass die Gentranskription der potentiellen SOO-Transporter SLC5A3 und SLC6A6 in den untersuchten Zelllinien Jurkat, HEK wie auch HepG2-Zellen durch hypotone Bedingungen deutlich verstärkt wird. Hierbei nimmt der zelluläre mRNA-Gehalt der Gene SLC5A3 zwischen 20-60% und SLC6A6 um 30-100% innerhalb von 10-20 min zu, was auf eine potentielle RVD-Beteiligung von SLC-Transportern hindeutet. Ausgehend von diesem Ergebnis wurde daraufhin die zelluläre Lokalisation des SLC5A3-Transporters unter isotonen und hypotonen Bedingungen mikroskopisch untersucht. Wie anhand der konfokalen lasermikroskopischen Untersuchung zu erkennen ist, findet unter hypotoner Stimulation eine zelluläre Umverteilung des mit EGFP fluoreszenzmarkierten Proteins SLC5A3 statt. Innerhalb von 10 min wird der Transporter dabei von intrazellulären Regionen in Richtung Plasmamembran verlagert. Darüber hinaus konnte mit Hilfe der hochauflösenden Mikroskopie-Technik dSTORM gezeigt werden, dass der Transporter SLC5A3 unter hypotoner Stimulation verstärkt mit der Plasmamembran assoziiert vorliegt. Diese verstärkte Membranassoziation des SLC5A3-Proteins deutet damit auf einen schwellungsinduzierten exozytotischen Einbau des Transporters hin. Die Ergebnisse dieser Arbeit zeigen damit erstmals, dass SLC-Transporter wie SLC5A3, SLC6A6 und vermutlich andere Vertreter der SLC-Superfamilie potentiell am Mechanismus der hypotonen Volumenregulation beteiligt sind. Da SLC-Transporter als wichtige Transportsysteme für Therapeutika angesehen werden und die Mechanismen der Volumenregulation bereits in zahlreichen biotechnologischen Anwendungen implementiert sind, könnte der hier aufgedeckte Zusammenhang einen Erkenntnisgewinn für zahlreiche biomedizinische Forschungsgebiete darstellen. N2 - Cell volume homeostasis is critically important for the functional and structural integrity of mammalian cells. To counteract osmotically induced volume perturbations, cells possess efficient mechanisms that control the intracellular osmolyte composition. The volume regulatory mechanisms operating under hyper- and hypotonic conditions are known, respectively, as regulatory volume increase (RVI) and decrease (RVD). During both, RVI and RVD, cells adjust the cellular content of inorganic ions (most notably Na+, K+ and Cl-) and organic solutes in order to gain or lose osmotically obligated water. These mechanisms counteract osmotic cell damage and enable the adaptation of cells to a wide range of extracellular osmolarities. Despite decades of research in this field, many aspects of the mechanisms underlying RVD and RVI remain poorly understood. In case of T lymphocytes, various cellular functions, including proliferation, migration and T cell activation are closely associated with the cell volume regulatory machinery. Among other mechanisms, all these processes are tightly linked by a network of potassium channels. The identification of this network is of great biomedical interest as it provides a key to pharmacological manipulation of the immune system. Current models of hypotonic volume regulation (RVD) in T-lymphocytes consider primarily the voltage-gated KV1.3 and the calcium-activated IKCa1 channel. The first part of this thesis explores the potential role of two-pore domain (K2P) potassium channels in RVD in murine and human primary CD4+-T lymphocytes. Using a combined genetic and pharmacological approach, time-resolved cell volume analysis revealed an important role of the K2P channels TASK1, TASK2, TASK3 and TRESK in swelling activated K+ efflux from hypotonically swollen T cells. Based on the analysis carried out here, the voltage-gated TASK2 as well as the calcium-activated TRESK channel were found as the most important K2P channels involved in the RVD of both naïve and stimulated T cells. The importance of TASK2 and TRESK in the RVD process was comparable to that of KV1.3. In summary, the data provide first evidence that hypotonic volume regulation of murine and human CD4+-T lymphocytes relies on K2P channels. With respect to the close relationship of T-cell function and volume regulatory mechanisms K2P channels may thus be considered as potential targets for immunomodulation. In the second and major part of this thesis, the swelling-activated transport pathways for small organic osmolytes (SOOs) were investigated. Nearly all eukaryotic cells possess a considerable reservoir of SOOs, such as polyols (e.g. sorbitol, myo-inositol), methylamines (e.g. betaine, α-glycerophosphoryl choline) and small amino acids (e.g. α-/β- alanine, proline and the derivate taurine), which are synthesized within the cells or accumulated from the extracellular medium. Since SOOs do not interfere with the integrity of macromolecules and the membrane potential, cells tolerate great cytosolic fluctuations of these solutes without negative effects on cellular structure or function. Due to these properties, small organic osmolytes are important tools for cell volume regulatory mechanisms, by which the intracellular osmolarity can be adjusted independently of electrolytes. Although the importance of SOOs for hypotonic volume regulation has been known for long time, the molecular identity of participating membrane efflux pathways is far from being clear. Regardless of the involved transporters, swelling-activated pathways have been reported to exhibit a size selective permeability for a wide range of sugars and related compounds. To gain a deeper insight into this issue, in a first step the impact of the molecular size on the permeation of low-molecular-weight polyethylene glycols (PEG200–1500) through the plasma membrane of Jurkat cells under iso- and hypotonic conditions was analyzed. Upon moderate swelling in slightly hypotonic solutions (200 mOsm), the lymphocyte membrane was found to remain impermeable to PEG300–1500, which allowed the cells to accomplish regulatory volume decrease. RVD also occurred in strongly hypotonic solutions (100 mOsm) of PEG600–1500, whereas 100 mOsm solutions of PEG300–400 inhibited RVD. These findings suggest that extensive hypotonic swelling rendered the cell membrane highly permeable to PEG300–400, but not to PEG600–1500. Using the values of hydrodynamic radii Rh for PEGs, the observed size-selectivity of membrane permeation yielded an estimate of ∼0.74 nm for the cut-off radius of the swelling-activated pathway for organic osmolytes. This result may be of interest for many biotechnological and biomedical applications, where swelling-activated SOO-pathways are widely used for cell-loading techniques. As a second step, an attempt was made to elucidate the molecular identity of transporters for organic osmolytes potentially involved in RVD. Since it was not clear whether RVD-related efflux of SOOs is mediated by one common or several distinct transporter(s), at first, the plasma membrane permeability profiles for two structurally dissimilar SOOs, including the amino sulfonic acid taurine and the polyol myo-inositol were analyzed. The results of the time resolved volumetric measurements clearly showed that the membrane permeability to taurine was activated upon moderate cell swelling (by ~15%) in mildly hypotonic solutions (~230 mOsm). In sharp contrast, the membrane permeability to myo-inositol was activated after a much larger swelling (~50%) in strongly hypotonic media (<150 mOsm). Moreover, the swelling-activated permittivity to taurine during RVD in 100 mOsm medium persisted for about twice as long as that for myo-inositol (taurine ~95 min, myo-inositol ~40 min). These findings clearly showed that, taurine and myo-inositol utilized separate, apparently substrate-specific pathways, which were activated at different hypotonic thresholds. Since many members of SLC-family proteins (Solute Carrier) are known for their substrate selectivity and also for their contribution to osmoregulatory mechanisms a participation of SLCs was investigated in the context of RVD. To this end, a combination of molecular biological (semiquantitative RT-PCR) and fluorescence microscopy techniques (confocal and super-resolution microscopy) was used. The semiquantitative RT-PCR data showed a transcriptional upregulation for the SLC proteins SLC5A3 (myo-inositol transporter; SMIT) and SLC6A6 (taurine transporter TauT) in hypotonically stressed Jurkat lymphocytes, HEK293, and HepG2 cells. In all three human cell lines strongly hypotonic solutions (100 mOsm) increased the mRNA level of the genes SLC5A3 and SLC6A6 between 20-60% and 30-100%, respectively, suggesting a potential participation of SLC transporters in RVD. In addition, confocal microscopy images clearly showed the intracellular displacement of EGFP-tagged SLC5A3 expressed in HEK293 cells following strongly hypotonic stress (100 mOsm). Within 10 min the fluorescence of EGFP was shifted from intracellular regions towards the plasma membrane. Furthermore, super-resolution microscopy by means of dSTORM revealed a considerably increased membrane association of SLC5A3 in strongly hypotonic stressed (100 mOsm) HEK293 and Jurkat cells. This finding suggests that SLC5A3 is integrated into the plasma membrane by swelling-induced exocytosis. Taken together, the results of this investigation provided first evidence that transporters such as SLC5A3, SLC6A6 and probably other SLC-proteins participate in the mechanism of hypotonic volume regulation. Due to the relevance of SLC-proteins as potential drug delivery systems the possible role of these transporters might be of great interest for many biomedical research areas. KW - Säugetiere KW - Osmoregulation KW - Zelle KW - Regulatory Volume Decrease KW - Transporter SLC5A3 KW - Transporter SLC6A6 KW - small organic osmolytes KW - Zwei-Poren Domänen Kaliumkanäle KW - two-pore domain potassium channels KW - Zellvolumen KW - Volumenregulation Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-103255 ER - TY - JOUR A1 - Andreska, Thomas A1 - Lüningschrör, Patrick A1 - Wolf, Daniel A1 - McFleder, Rhonda L. A1 - Ayon-Olivas, Maurilyn A1 - Rattka, Marta A1 - Drechsler, Christine A1 - Perschin, Veronika A1 - Blum, Robert A1 - Aufmkolk, Sarah A1 - Granado, Noelia A1 - Moratalla, Rosario A1 - Sauer, Markus A1 - Monoranu, Camelia A1 - Volkmann, Jens A1 - Ip, Chi Wang A1 - Stigloher, Christian A1 - Sendtner, Michael T1 - DRD1 signaling modulates TrkB turnover and BDNF sensitivity in direct pathway striatal medium spiny neurons JF - Cell Reports N2 - Highlights • Dopamine receptor-1 activation induces TrkB cell-surface expression in striatal neurons • Dopaminergic deficits cause TrkB accumulation and clustering in the ER • TrkB clusters colocalize with cargo receptor SORCS-2 in direct pathway striatal neurons • Intracellular TrkB clusters fail to fuse with lysosomes after dopamine depletion Summary Disturbed motor control is a hallmark of Parkinson’s disease (PD). Cortico-striatal synapses play a central role in motor learning and adaption, and brain-derived neurotrophic factor (BDNF) from cortico-striatal afferents modulates their plasticity via TrkB in striatal medium spiny projection neurons (SPNs). We studied the role of dopamine in modulating the sensitivity of direct pathway SPNs (dSPNs) to BDNF in cultures of fluorescence-activated cell sorting (FACS)-enriched D1-expressing SPNs and 6-hydroxydopamine (6-OHDA)-treated rats. DRD1 activation causes enhanced TrkB translocation to the cell surface and increased sensitivity for BDNF. In contrast, dopamine depletion in cultured dSPN neurons, 6-OHDA-treated rats, and postmortem brain of patients with PD reduces BDNF responsiveness and causes formation of intracellular TrkB clusters. These clusters associate with sortilin related VPS10 domain containing receptor 2 (SORCS-2) in multivesicular-like structures, which apparently protects them from lysosomal degradation. Thus, impaired TrkB processing might contribute to disturbed motor function in PD. KW - motor learning KW - cortico-striatal synapse KW - basal ganglia KW - direct pathway KW - DRD1 KW - dSPN KW - BDNF KW - TrkB KW - synaptic plasticity KW - GPCR Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-349932 VL - 42 IS - 6 ER - TY - JOUR A1 - Andreska, Thomas A1 - Aufmkolk, Sarah A1 - Sauer, Markus A1 - Blum, Robert T1 - High abundance of BDNF within glutamatergic presynapses of cultured hippocampal neurons JF - Frontiers in Cellular Neuroscience N2 - In the mammalian brain, the neurotrophin brain-derived neurotrophic factor (BDNF) has emerged as a key factor for synaptic refinement, plasticity and learning. Although BDNF-induced signaling cascades are well known, the spatial aspects of the synaptic BDNF localization remained unclear. Recent data provide strong evidence for an exclusive presynaptic location and anterograde secretion of endogenous BDNF at synapses of the hippocampal circuit. In contrast, various studies using BDNF overexpression in cultured hippocampal neurons support the idea that postsynaptic elements and other dendritic structures are the preferential sites of BDNF localization and release. In this study we used rigorously tested anti-BDNF antibodies and achieved a dense labeling of endogenous BDNF close to synapses. Confocal microscopy showed natural BDNF close to many, but not all glutamatergic synapses, while neither GABAergic synapses nor postsynaptic structures carried a typical synaptic BDNF label. To visualize the BDNF distribution within the fine structure of synapses, we implemented super resolution fluorescence imaging by direct stochastic optical reconstruction microscopy (dSTORM). Two-color dSTORM images of neurites were acquired with a spatial resolution of ~20 nm. At this resolution, the synaptic scaffold proteins Bassoon and Homer exhibit hallmarks of mature synapses and form juxtaposed bars, separated by a synaptic cleft. BDNF imaging signals form granule-like clusters with a mean size of ~60 nm and are preferentially found within the fine structure of the glutamatergic presynapse. Individual glutamatergic presynapses carried up to 90% of the synaptic BDNF immunoreactivity, and only a minor fraction of BDNF molecules was found close to the postsynaptic bars. Our data proof that hippocampal neurons are able to enrich and store high amounts of BDNF in small granules within the mature glutamatergic presynapse, at a principle site of synaptic plasticity. KW - hippocampal neurons KW - synapse structure KW - presynapse KW - synaptic localization KW - BDNF Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-119793 SN - 1662-5102 VL - 8 IS - 107 ER - TY - THES A1 - Andreska, Thomas T1 - Effects of dopamine on BDNF / TrkB mediated signaling and plasticity on cortico-striatal synapses T1 - Effekte von Dopamin auf BDNF / TrkB vermittelte Signalwege und Plastizität an cortico-striatalen Synapsen N2 - Progressive loss of voluntary movement control is the central symptom of Parkinson's disease (PD). Even today, we are not yet able to cure PD. This is mainly due to a lack of understanding the mechanisms of movement control, network activity and plasticity in motor circuits, in particular between the cerebral cortex and the striatum. Brain-derived neurotrophic factor (BDNF) has emerged as one of the most important factors for the development and survival of neurons, as well as for synaptic plasticity. It is thus an important target for the development of new therapeutic strategies against neurodegenerative diseases. Together with its receptor, the Tropomyosin receptor kinase B (TrkB), it is critically involved in development and function of the striatum. Nevertheless, little is known about the localization of BDNF within presynaptic terminals in the striatum, as well as the types of neurons that produce BDNF in the cerebral cortex. Furthermore, the influence of midbrain derived dopamine on the control of BDNF / TrkB interaction in striatal medium spiny neurons (MSNs) remains elusive so far. Dopamine, however, appears to play an important role, as its absence leads to drastic changes in striatal synaptic plasticity. This suggests that dopamine could regulate synaptic activity in the striatum via modulation of BDNF / TrkB function. To answer these questions, we have developed a sensitive and reliable protocol for the immunohistochemical detection of endogenous BDNF. We find that the majority of striatal BDNF is provided by glutamatergic, cortex derived afferents and not dopaminergic inputs from the midbrain. In fact, we found BDNF in cell bodies of neurons in layers II-III and V of the primary and secondary motor cortex as well as layer V of the somatosensory cortex. These are the brain areas that send dense projections to the dorsolateral striatum for control of voluntary movement. Furthermore, we could show that these projection neurons significantly downregulate the expression of BDNF during the juvenile development of mice between 3 and 12 weeks. In parallel, we found a modulatory effect of dopamine on the translocation of TrkB to the cell surface in postsynaptic striatal Medium Spiny Neurons (MSNs). In MSNs of the direct pathway (dMSNs), which express dopamine receptor 1 (DRD1), we observed the formation of TrkB aggregates in the 6-hydroxydopamine (6-OHDA) model of PD. This suggests that DRD1 activity controls TrkB surface expression in these neurons. In contrast, we found that DRD2 activation has opposite effects in MSNs of the indirect pathway (iMSNs). Activation of DRD2 promotes a rapid decrease in TrkB surface expression which was reversible and depended on cAMP. In parallel, stimulation of DRD2 led to induction of phospho-TrkB (pTrkB). This effect was significantly slower than the effect on TrkB surface expression and indicates that TrkB is transactivated by DRD2. Together, our data provide evidence that dopamine triggers dual modes of plasticity on striatal MSNs by acting on TrkB surface expression in DRD1 and DRD2 expressing MSNs. This surface expression of the receptor is crucial for the binding of BDNF, which is released from corticostriatal afferents. This leads to the induction of TrkB-mediated downstream signal transduction cascades and long-term potentiation (LTP). Therefore, the dopamine-mediated translocation of TrkB could be a mediator that modulates the balance between dopaminergic and glutamatergic signaling to allow synaptic plasticity in a spatiotemporal manner. This information and the fact that TrkB is segregated to persistent aggregates in PD could help to improve our understanding of voluntary movement control and to develop new therapeutic strategies beyond those focusing on dopaminergic supply. N2 - Der fortschreitende Verlust der willkürlichen Bewegungskontrolle ist ein zentrales Symptom der Parkinson-Krankheit (PD). Auch heute sind wir noch nicht in der Lage, PD zu heilen. Dafür verantwortlich ist hauptsächlich ein mangelndes Verständnis von Mechanismen der Bewegungskontrolle, Netzwerkaktivität und Plastizität in motorischen Schaltkreisen, insbesondere zwischen Hirnrinde und Striatum. Der neurotrophe Faktor BDNF ist einer der wichtigsten Faktoren für die Entwicklung und das Überleben von Neuronen sowie für synaptische Plastizität im zentralen Nervensystem. BDNF ist daher ein Target für die Entwicklung neuer therapeutischer Strategien gegen neurodegenerative Erkrankungen. Zusammen mit seinem Rezeptor, der Tropomyosin-Rezeptorkinase B (TrkB), ist BDNF maßgeblich an der Entwicklung und Funktion des Striatums beteiligt. Dennoch ist nur wenig bekannt, wo BDNF an Synapsen im Striatum lokalisiert ist, und wo BDNF in Neuronen der Hirnrinde synthetisiert wird. Außerdem ist der Einfluss von Dopamin aus dem Mittelhirn auf die Kontrolle der BDNF / TrkB-Interaktion in striatalen Medium-Spiny-Neuronen (MSNs) bisher unklar. Dopamin scheint jedoch eine wichtige Rolle zu spielen, da dessen Abwesenheit zu drastischen Veränderungen der striatalen Plastizität führt. Dopamin könnte synaptische Plastizität im Striatum über eine Modulation der BDNF / TrkB-Interaktion regulieren. Um diese Fragen beantworten zu können, haben wir ein sensitives und zuverlässiges Protokoll für den immunhistochemischen Nachweis von endogenem BDNF entwickelt. Wir fanden heraus, dass BDNF im Striatum vor allem in glutamatergen Synapsen von Projektion aus dem Kortex lokalisiert ist und nicht in Terminalen dopaminerger Neurone aus dem Mittelhirn. Tatsächlich fanden wir BDNF in den Zellkörpern von Neuronen in den Schichten II-III und V des primären und sekundären motorischen Kortex sowie Schicht V des somatosensorischen Kortex. Es sind jene Hirnareale, welche dichte Projektionen zum dorsolateralen Striatum senden und entscheidend an der Steuerung von willkürlichen Bewegungen beteiligt sind. Weiterhin konnten wir zeigen, dass eben jene Projektionsneurone die Bildung von BDNF während der juvenilen Entwicklung von Mäusen zwischen 3 und 12 Wochen signifikant herunter regulieren. In striatalen MSN fanden wir zudem einen modulatorischen Effekt von Dopamin auf die Translokation von TrkB zur Zelloberfläche. In MSNs des direkten Signalweges (dMSNs), welche Dopaminrezeptor 1 (DRD1) exprimieren, konnten wir die Bildung von TrkB-Aggregaten im 6-Hydroxydopamin (6-OHDA) - Rattenmodell der Parkinson Erkankung beobachten. Dies deutet darauf hin, dass die DRD1-Aktivität die TrkB-Oberflächenexpression in diesen Neuronen steuert. Im Gegensatz dazu fanden wir heraus, dass die DRD2-Aktivierung in MSNs des indirekten Signalweges (iMSNs) eine gegensätzliche Wirkung hat. Die Aktivierung von DRD2 führt zu einer schnellen Reduktion der TrkB-Oberflächenexpression, die reversibel und von cAMP abhängig ist. Außerdem führte die Stimulation von DRD2 zu einer Induktion von Phospho-TrkB (pTrkB). Dieser Effekt war deutlich langsamer als die Wirkung auf die TrkB-Oberflächenexpression und deutet auf eine Transaktivierung von TrkB über DRD2 hin. Insgesamt scheint Dopamin entgegengesetzte Plastizitätsmodi in striatalen MSNs auszulösen, indem es auf die TrkB-Oberflächenexpression in DRD1- und DRD2-exprimierenden MSNs einwirkt. Diese Oberflächenexpression des Rezeptors ist entscheidend für die Bindung von BDNF, welches aus kortiko-striatalen Afferenzen freigesetzt wird. Dies führt zur Induktion von TrkB-vermittelten-Signaltransduktionskaskaden und Langzeitpotenzierung (LTP). Daher könnte die dopamin-vermittelte Translokalisation von TrkB das Gleichgewicht zwischen dopaminergen und glutamatergen Signalen modulieren, um die synaptische Plastizität in einer räumlich-zeitlich abgestimmten Weise zu ermöglichen. Diese Information und die Tatsache, dass TrkB bei PD stabile Aggregate bildet, könnte dazu beitragen, unser Verständnis der willkürlichen Bewegungskontrolle zu verbessern und neue therapeutische Strategien zu entwickeln, die über jene hinausgehen, welche sich auf die dopaminerge Versorgung konzentrieren. KW - Brain-derived neurotrophic factor KW - Parkinson Krankheit KW - Plastizität KW - Motorisches Lernen KW - Basalganglien KW - Brain-derived neurotrophic factor KW - TrkB KW - Basal Ganglia KW - Motor learning KW - Parkinson's disease KW - Synaptic plasticity KW - Striatum KW - Medium spiny neurons KW - Cortico-striatal projection neurons Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-174317 ER - TY - CHAP A1 - Anders, Fritz A1 - Schartl, Manfred A1 - Barnekow, Angelika T1 - Xiphophorus as an in vivo model for studies on oncogenes N2 - The capacity of Xiphophorus to develop neoplasia can be formally assigned to a "tumor gene" (Tu), which appears to be a normal part of the genome of all individuals. The wild fish have evolved population-specific and cell type-specific systems of regulatory genes (R) for Tu that protect the fish from neoplasia. Hybridization of members of different wild populations in the laborstory followed by treatment of the hybrids with carcinogens led to disintegration of the R systems permitting excessive expression of Tu and thus resulting in neoplasia. Certain hybrids developed neoplasia even spontaneously. Observations on the genuine phenotypic effect of the derepressed Tu in the early embryo indicated an essential normal function of this oncogene in cell differentiation, proliferation and cell-cell communication. Tu appeared to be indispensable in the genome but may also be present in accessory copics. Recently, c-src, the cellular homolog of the Rous sarcoma virus oncogene v-src, was detected in Xiphophorus. The protein product of c-src, pp60c-src, was identified and then examined by its associated kinase activity. This pp60c-src was found in all individuals tested, but, depending on the genotype, its kinase activity was different. The genetic characters of c-src, such as linkage relations, dosage relations, expression, etc., correspond to those of Tu. From a systematic study which showed that pp60c-src was present in all metazoa tested ranging from mammals down to sponges, we concluded that c-src has evolved with the multicellular organization of animals. Neoplasia of animals and humans is a characteristic closely related to this evolution. Our data showed that small aquariurn fish, besides being used successfully because they are time-, space-, and money-saving systems for carcinogenicity testing, are also highly suitable for basic studies on neoplasia at the populational, morphological, developmental, cell biological, and molecular levels. KW - Schwertkärpfling KW - In vivo KW - Onkogen Y1 - 1984 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-86398 ER - TY - CHAP A1 - Anders, F. A1 - Scholl, E. A1 - Schartl, Manfred T1 - Environmental and hereditary factors in the causation of neoplasia, based on studies of the Xiphophorus fish melanoma system N2 - Neoplasia in Xiphophorus can be classified into: a) a Jarge group triggered by carcinogens; b) a large group triggered by promoters; and c) a small group that develops "spontaneously" according to Mendelian Jaw. The process leading to susceptibility for neoplasia is represented by the disintegration of gene systems that normally protect the fish from neoplasia. Interpopulational arid interracial hybridization is the most effective process that Ieads to disintegration of the protective gene systems. Environmental factors may complete disintegration in somatic cells and thus may trigger neoplasia. The applications of the findings on Xiphophorus to humans are discussed. KW - Schwertkärpfling KW - Gen KW - Umweltfaktor KW - Tumor Y1 - 1981 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-86402 ER - TY - CHAP A1 - Anders, F. A1 - Scholl, E. A1 - Schartl, Manfred T1 - Xiphophorus als Modell in der Krebsforschung N2 - No abstract available. KW - Schwertkärpfling KW - Krebsforschung KW - Modell Y1 - 1979 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-72752 ER - TY - JOUR A1 - Anders, F. A1 - Schartl., Manfred A1 - Barnekow, A. A1 - Anders, A. T1 - Xiphophorus as an in vivo model for studies on normal and defective control of oncogenes N2 - The Xiphophorus tumor system has provided the opportunity to reduce the enormous complexity of cancer etiology to a few biological elements basically involved in neoplasia. The development of a tumor requires an oncogene which, after impairment, deletion, or elimination of its regulatory genes is permitted to mediate neoplastic transformation. Emphasis is being placed today in cancer research on the actual oncogenes themselves, but, in our opinion, the most important genes involved in neoplasia are these regulatory genes. However, although detected by c1assical genetics in the Xiphophorus system, th ese genes are not at present open to a more fin ely detailed molecular biological analysis. Their actual mode of action is therefore still far from being understood. KW - Xiphophorus Y1 - 1984 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-80721 ER - TY - CHAP A1 - Anders, F. A1 - Schartl, Manfred A1 - Scholl, E. T1 - Evaluation of environmental and hereditary factors in carcinogenesis, based on studies in Xiphophorus N2 - Neoplasia in Xiphophorus can be classified into a) a large group that is triggered by carcinogens; b) a large group triggered by promoters; c) a small group that develops "spontaneously" following interpopulational and interracial hybridizations; and d) a small group that develops "spontaneously" following germ line mutation. The process leading to susceptibility for neoplasia is represented by the disintegration of gene systems that normally protect the fish from neoplasia. Hybridization is the most effective process that leads to disintegration of the protection gene systems. Environmental factors may complete disintegration and thus may trigger neoplasia. It is discussed whether the findings on Xiphophorus may also apply to humans. KW - Schwertkärpfling KW - Gen KW - Umweltfaktor KW - Carcinogenese Y1 - 1981 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-72741 ER - TY - JOUR A1 - Anders, F. A1 - Schartl, Manfred A1 - Barnekow, A. A1 - Schmidt, C. R. A1 - Luke, W. A1 - Jaenel-Dess, G. A1 - Anders, A. T1 - The genes that carcinogens act upon N2 - No abstract available. KW - Onkogen Y1 - 1985 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-72704 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 - THES A1 - Amini, Emad T1 - How central and peripheral clocks and the neuroendocrine system interact to time eclosion behavior in \(Drosophila\) \(melanogaster\) T1 - Wie zentrale und periphere Uhren und das neuroendokrine System zusammenwirken, um das Schlupfverhalten von \(Drosophila\) \(melanogaster\) zeitlich festzulegen N2 - To grow larger, insects must shed their old rigid exoskeleton and replace it with a new one. This process is called molting and the motor behavior that sheds the old cuticle is called ecdysis. Holometabolic insects have pupal stages in between their larval and adult forms, during which they perform metamorphosis. The pupal stage ends with eclosion, i.e., the emergence of the adult from the pupal shell. Insects typically eclose at a specific time during the day, likely when abiotic conditions are at their optimum. A newly eclosed insect is fragile and needs time to harden its exoskeleton. Hence, eclosion is regulated by sophisticated developmental and circadian timing mechanisms. In Drosophila melanogaster, eclosion is limited to a daily time window in the morning, regarded as the “eclosion gate”. In a population of laboratory flies entrained by light/dark cycles, most of the flies eclose around lights on. This rhythmic eclosion pattern is controlled by the circadian clock and persists even under constant conditions. Developmental timing is under the control of complex hormonal signaling, including the steroid ecdysone, insulin-like peptides, and prothoracicotropic hormone (PTTH). The interactions of the central circadian clock in the brain and a peripheral clock in the prothoracic gland (PG) that produces ecdysone are important for the circadian timing of eclosion. These two clocks are connected by a bilateral pair of peptidergic PTTH neurons (PTTHn) that project to the PG. Before each molt, the ecdysone level rises and then falls shortly before ecdysis. The falling ecdysone level must fall below a certain threshold value for the eclosion gate to open. The activity of PTTHn is inhibited by short neuropeptide F (sNPF) from the small ventrolateral neurons (sLNvs) and inhibition is thought to lead to a decrease in ecdysone production. The general aim of this thesis is to further the understanding of how the circadian clock and neuroendocrinal pathways are coordinated to drive eclosion rhythmicity and to identify when these endocrinal signaling pathways are active. In Chapter I, a series of conditional PTTHn silencing-based behavioral assays, combined with neuronal activity imaging techniques such as non-invasive ARG-Luc show that PTTH signaling is active and required shortly before eclosion and may serve to phase-adjust the activity of the PG at the end of pupal development. Trans-synaptic anatomical stainings identified the sLNvs, dorsal neurons 1 (DN1), dorsal neurons 2 (DN2), and lateral posterior neurons (LPNs) clock neurons as directly upstream of the PTTHn. Eclosion motor behavior is initiated by Ecdysis triggering hormone (ETH) which activates a pair of ventromedial (Vm) neurons to release eclosion hormone (EH) which positively feeds back to the source of ETH, the endocrine Inka cells. In Chapter II trans-synaptic tracing showed that most clock neurons provide input to the Vm and non-canonical EH neurons. Hence, clock can potentially influence the ETH/EH feedback loop. The activity profile of the Inka cells and Vm neurons before eclosion is described. Vm and Inka cells are active around seven hours before eclosion. Interestingly, all EH neurons appear to be exclusively peptidergic. In Chapter III, using chemoconnectomics, PTTHns were found to express receptors for sNPF, allatostatin A (AstA), allatostatin C (AstC), and myosuppressin (Ms), while EH neurons expressed only Ms and AstA receptors. Eclosion assays of flies with impaired AstA, AstC, or Ms signaling do not show arrhythmicity under constant conditions. However, optogenetic activation of the AstA neurons strongly suppresses eclosion. Chapter IV focuses on peripheral ventral’ Tracheal dendrite (v’Td) and class IV dendritic arborization (C4da) neurons. The C4da neurons mediate larval light avoidance through endocrine PTTH signaling. The v’Td neurons mainly receive O2/CO2 input from the trachea and are upstream of Vm neurons but are not required for eclosion rhythmicity. Conditional ablation of the C4da neurons or torso (receptor of PTTH) knock-out in the C4da neurons impaired eclosion rhythmicity. Six to seven hours before eclosion, PTTHn, C4da, and Vm neurons are active based on ARG-Luc imaging. Thus, C4da neurons may indirectly connect the PTTHn to the Vm neurons. In summary, this thesis advances our knowledge of the temporal activity and role of PTTH signaling during pupal development and rhythmic eclosion. It further provides a comprehensive characterization of the synaptic and peptidergic inputs from clock neurons to PTTHn and EH neurons. AstA, AstC, and Ms are identified as potential modulators of eclosion circuits and suggest an indirect effect of PTTH signaling on EH signaling via the peripheral sensory C4da neurons. N2 - Um zu wachsen, müssen Insekten ihr altes, starres Exoskelett abwerfen und durch ein neues ersetzen. Dieser Vorgang wird als Häutung bezeichnet, und das motorische Verhalten, bei dem die alte Kutikula abgestoßen wird, heißt Ekdysis. Holometabole Insekten haben zwischen ihrer Larven- und Erwachsenenform ein Puppenstadium, in welchem sie eine Metamorphose durchlaufen. Das Puppenstadium endet mit dem Schlüpfen des erwachsenen Tieres aus der Puppenhülle. Die Insekten schlüpfen in der Regel zu einem bestimmten Zeitpunkt am Tag, wenn die abiotischen Bedingungen optimal sind, da das frisch geschlüpfte Insekt zerbrechlich ist und Zeit braucht, um sein Exoskelett auszuhärten. Daher wird der Schlupf durch ausgeklügelte Mechanismen der Entwicklung und der inneren Uhr gesteuert. Bei Drosophila melanogaster ist der Sclupf auf ein tägliches Zeitfenster am Morgen beschränkt, das als "Schlupffenster" bezeichnet wird. In einer Population von Laborfliegen, die durch Licht/Dunkel-Zyklen gesteuert wird, schlüpfen die meisten Fliegen in etwa um das Einschalten der Beleuchtung. Dieses rhythmische Schlupfmuster wird von der inneren Uhr gesteuert und bleibt auch unter konstanten Bedingungen bestehen. Das Timing der Entwicklung wird von komplexen hormonellen Signalen gesteuert, darunter das Steroid Ecdyson, insulinähnliche Peptide und das prothorakotrope Hormon (PTTH). Die Wechselwirkungen zwischen der zentralen zirkadianen Uhr im Gehirn und einer peripheren Uhr in der Prothorakaldrüse (PG), die Ecdyson produziert, sind wichtig für die zirkadiane Zeitsteuerung des Schlupfs. Diese beiden Uhren sind durch ein bilaterales Paar peptiderger PTTH-Neuronen (PTTHn) verbunden, die in die PG projizieren. Vor jeder Häutung steigt der Ecdysonspiegel an und fällt dann kurz vor danach wieder ab. Der fallende Ecdysonspiegel muss einen bestimmten Schwellenwert unterschreiten, damit sich das Schlupffenster öffnen kann. Die Aktivität der PTTHn wird durch das kurze Neuropeptid F (sNPF) aus den kleinen ventrolateralen Neuronen (sLNvs) gehemmt, und es wird angenommen, dass die Hemmung zu einer Abnahme der Ecdysonproduktion führt. Das allgemeine Ziel dieser Thesis besteht darin, die Koordination zwischen der zirkadianen Uhr und den neuroendokrinen Signalwegen zur Steuerung der Eklosionsrhythmik weiter zu charakterisieren und zu ermitteln, wann diese endokrinen Signalwege aktiv sind. In Kapitel I zeigen eine Reihe von Verhaltenstests, die auf der konditionalen Ausschaltung von PTTHn basieren, in Kombination mit Techniken zur Darstellung neuronaler Aktivität, wie z. B. nicht-invasives ARG-Luc imaging, dass PTTH-Signale kurz vor dem Schlupf aktiv und erforderlich sind und zur Phasenanpassung der Aktivität der PG am Ende der Puppenentwicklung dienen könnten. Trans-synaptische anatomische Färbungen identifizierten die sLNvs, die dorsalen Neuronen 1 (DN1), die dorsalen Neuronen 2 (DN2) und die lateralen posterioren Neuronen (LPNs) als Uhrneuronen, die dem PTTHn direkt vorgeschaltet sind. Das motorische Schlupfverhalten wird durch das Ecdysis-auslösende Hormon (ETH) ausgelöst, das ein Paar ventromedialer (Vm) Neuronen zur Freisetzung des Eklosionshormons (EH) anregt, welches positiv an die Quelle des ETH, die endokrinen Inka-Zellen, zurückkoppelt. In Kapitel II zeigte die trans-synaptische Nachverfolgung, dass die meisten Uhrneuronen Input für die Vm- und nicht-kanonischen EH-Neuronen liefern, sodass die Uhr möglicherweise die ETH/EH-Rückkopplungsschleife beeinflussen kann. Das Aktivitätsprofil der Inka-Zellen und Vm-Neuronen vor dem Schlupf wird beschrieben. Vm- und Inka-Zellen sind etwa sieben Stunden vor dem Schlupf aktiv. Interessanterweise scheinen alle EH-Neuronen ausschließlich peptiderg zu sein. In Kapitel III wurde mit Hilfe von Chemoconnectomics festgestellt, dass PTTH-Neuronen Rezeptoren für sNPF, Allatostatin A (AstA), Allatostatin C (AstC) und Myosuppressin (Ms) exprimieren, während EH nur Ms- und AstA-Rezeptoren exprimieren. Eklosionsversuche mit Fliegen, deren AstA-, AstC- oder Ms-Signalübertragung beeinträchtigt ist, zeigen unter konstanten Bedingungen keine Arrhythmie. Eine optogenetische Aktivierung der AstA-Neuronen führt jedoch zu einer starken Unterdrückung des Schlupfs. Kapitel IV konzentriert sich auf die peripheren ventralen Trachealdendritischen Neurone (v'Td) und dendritische Verzweigungsneurone der Klasse IV (C4da). Die C4da-Neuronen vermitteln die Lichtvermeidung der Larven durch endokrine PTTH-Signale. Die v'Td-Neuronen erhalten hauptsächlich O2/CO2-Input aus den Tracheen und sind den Vm-Neuronen vorgeschaltet, werden aber für die Schlupfrhythmik nicht benötigt. Die bedingte Ablation der C4da-Neuronen und das Knock-out von torso (Rezeptor für PTTH) in den C4da-Neuronen beeinträchtigten die Schlupfrhythmik. Sechs bis sieben Stunden vor dem Schlupf sind die PTTHn-, C4da- und Vm-Neuronen aktiv. Somit könnten C4da-Neuronen indirekt die PTTHn mit den Vm-Neuronen verbinden. Zusammenfassend lässt sich sagen, dass diese Arbeit unser Wissen über das zeitliche Aktivitätsmuster und der Rolle des PTTH signalling während der Puppenentwicklung und dem rhythmisches Schlupf erweitert. Sie liefert auch eine umfassende Charakterisierung der synaptischen und peptidergen Eingänge von Uhrneuronen zu PTTHn- und EH-Neuronen. AstA, AstC und Ms wurden als potenzielle Modulatoren der neuronalen Schlupfschaltkreise identifiziert und deuten auf einen indirekten Effekt der PTTH-Signalgebung auf das EH signalling über die peripheren sensorischen C4da-Neuronen hin. KW - Prothoracicotropic hormone KW - Prothoracic gland KW - Eclosion KW - Eclosion hormone KW - C4da KW - v’Td KW - Neuropeptide KW - Neuroendokrines System KW - Taufliege Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-361309 ER - TY - JOUR A1 - Ambrožová, Lucie A1 - Finnberg, Sven A1 - Feldmann, Benedikt A1 - Buse, Jörn A1 - Preuss, Henry A1 - Ewald, Jörg A1 - Thorn, Simon T1 - Coppicing and topsoil removal promote diversity of dung‐inhabiting beetles (Coleoptera: Scarabaeidae, Geotrupidae, Staphylinidae) in forests JF - Agricultural and Forest Entomology N2 - Central European forests experience a substantial loss of open-forest organisms due to forest management and increasing nitrogen deposition. However, management strategies, removing different levels of nitrogen, have been rarely evaluated simultaneously. We tested the additive effects of coppicing and topsoil removal on communities of dung-inhabiting beetles compared to closed forests. We sampled 57 021 beetles, using baited pitfall traps exposed on 27 plots. Experimental treatments resulted in significantly different communities by promoting open-habitat species. While alpha diversity did not differ among treatments, gamma diversity of Geotrupidae and Scarabaeidae and beta diversity of Staphylinidae were higher in coppice than in forest. Functional diversity of rove beetles was higher in both, coppice and topsoil-removed plots, compared to control plots. This was likely driven by higher habitat heterogeneity in established forest openings. Five dung beetle species and four rove beetle species benefitted from coppicing, one red-listed dung beetle and two rove beetle species benefitted from topsoil removal. Our results demonstrate that dung-inhabiting beetles related to open forest patches can be promoted by both, coppicing and additional topsoil removal. A mosaic of coppice and bare-soil-rich patches can hence promote landscape-level gamma diversity of dung and rove beetles within forests. KW - nitrogen uptake KW - dung beetle KW - forest management KW - functional diversity KW - insect decline KW - rove beetle Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-258296 VL - 24 IS - 1 ER - TY - JOUR A1 - Amatobi, Kelechi M. A1 - Ozbek-Unal, Ayten Gizem A1 - Schäbler, Stefan A1 - Deppisch, Peter A1 - Helfrich-Förster, Charlotte A1 - Mueller, Martin J. A1 - Wegener, Christian A1 - Fekete, Agnes T1 - The circadian clock is required for rhythmic lipid transport in Drosophila in interaction with diet and photic condition JF - Journal of Lipid Research N2 - Modern lifestyle is often at odds with endogenously driven rhythmicity, which can lead to circadian disruption and metabolic syndrome. One signature for circadian disruption is a reduced or altered metabolite cycling in the circulating tissue reflecting the current metabolic status. Drosophila is a well-established model in chronobiology, but day-time dependent variations of transport metabolites in the fly circulation are poorly characterized. Here, we sampled fly hemolymph throughout the day and analyzed diacylglycerols (DGs), phosphoethanolamines (PEs) and phosphocholines (PCs) using LC-MS. In wild-type flies kept on sugar-only medium under a light-dark cycle, all transport lipid species showed a synchronized bimodal oscillation pattern with maxima at the beginning and end of the light phase which were impaired in period01 clock mutants. In wild-type flies under constant dark conditions, the oscillation became monophasic with a maximum in the middle of the subjective day. In strong support of clock-driven oscillations, levels of the targeted lipids peaked once in the middle of the light phase under time-restricted feeding independent of the time of food intake. When wild-type flies were reared on full standard medium, the rhythmic alterations of hemolymph lipid levels were greatly attenuated. Our data suggest that the circadian clock aligns daily oscillations of DGs, PEs, and PCs in the hemolymph to the anabolic siesta phase, with a strong influence of light on phase and modality. KW - hemolymph lipids KW - lipidomics KW - circadian rhythm KW - feeding KW - locomotor activity KW - light-driven metabolism Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-349961 VL - 64 IS - 10 ER - TY - CHAP A1 - Altschmied, Joachim A1 - Schartl, Manfred T1 - Genetics and molecular biology of tumour formation in Xiphophorus N2 - No abstract available. KW - Schwertkärpfling KW - Tumor KW - Entstehung KW - Molekularbiologie KW - Genetik Y1 - 1994 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-69752 ER - TY - THES A1 - Altrock, Stefanie T1 - Genetische Organisation und Transkription eines Virulenz-assoziierten, instabilen Chromosomenabschnitts von Listeria ivanovii T1 - Genetic organisation and transcription of a virulence-associated, instable chromosomal region of Listeria ivanovii N2 - Unter den sechs Arten der Gattung Listeria finden sich nur zwei pathogene Spezies. L. monocytogenes ist pathogen für Mensch und Tier, L. ivanovii nur tierpathogen. Beide Arten besitzen ein Virulenzgencluster, das auch als Pathogenitätsinsel LIPI-1 bezeichnet wird. Pathogenitätsinseln (PAIs) sind bei gram-negativen Bakterien weit verbreitet, wurden bei gram-positiven Pathogenen bisher jedoch nur selten beschrieben. In L. ivanovii wurde nun ein weiterer Virulenz-assoziierter, instabiler Chromosomenabschnitt entdeckt, der in einem Teilbereich Eigenschaften einer Pathogenitätsinsel besitzt. Ausgehend von einem spontanen, aber reproduzierbaren Deletionsereignis eines großen Genomabschnitts, der einige schon bekannte Virulenz-assoziierte Gene umfasst (i-inlE, i-inlF, smcL), wurden in Zusammenarbeit mit den Kooperationspartnern an der "Universidad Complutense de Madrid", insbesondere mit G. Domínguez-Bernal die komplette deletierte Region sowie flankierende Genombereiche genauer analysiert. Im Rahmen dieser Arbeit konnten rechts von dem bereits charakterisierten Gen smcL 13 neue Open Reading Frames (ORFs) bzw. Gene (ydeI, rnaH, norA) von L. ivanovii identifiziert werden, die größtenteils in der Deletionsmutante L. ivanovii GD-3 deletiert waren. Für die meisten Open Reading Frames konnten Homologien zu ORFs in den Genomsequenzen von L. monocytogenes und der apathogenen Art L. innocua gefunden werden. Eigene experimentelle Analysen zeigten zudem, dass diese ORFs in ähnlicher Anordnung auch in den apathogenen Arten L. seeligeri und L. welshimeri vorhanden sind, was wahrscheinlich macht, dass sie nicht an der Virulenz von Listerien beteiligt sind. G. Domínguez-Bernal fand im links von smcL liegenden Bereich eine Reihe neuer Internalingene, die alle spezifisch für L. ivanovii sind. Für die Gene i-inlE, i-inlF und smcL ist bereits bekannt, dass diese Virulenz-assoziiert sind. Dies führte zur Definition einer neuen, LIPI-2 genannten Pathogenitätsinsel in L. ivanovii, die außer smcL und i-inlFE alle neu gefundenen Internalingene umfasst. In dieser Arbeit durchgeführte Untersuchungen der LIPI-2 flankierenden Bereiche zeigten, dass diese in L. monocytogenes und auch den apathogenen Arten L. innocua, L. seeligeri und L. welshimeri bemerkenswert konserviert sind. Durch Transkriptionsuntersuchungen mittels RT-PCR wurde die Expression der neu identifizierten Gene analysiert. Hierbei wurden verschiedene Kulturbedingungen untersucht sowie die Transkription nach Infektion mehrerer Zelllinien bestimmt. Bei der Sequenzanalyse wurde für fast alle Internalingene eine PrfA-Box identifiziert und es bestätigte sich in dieser Arbeit, dass die meisten der Internalingene PrfA-abhängig exprimiert werden. Allerdings wiesen die einzelnen Gene kein einheitliches Transkriptionsprofil unter verschiedenen in vitro-Bedingungen auf. Eine Analyse der Genexpression nach Infektion verschiedener Zelllinien zeigte schließlich, dass die Internalingene während einer Infektion differentiell transkribiert werden und möglicherweise am Infektionsgeschehen beteiligt sind. Das Expressionsmuster der zu LIPI-2 benachbarten Open Reading Frames bestätigte, dass diese Gene PrfA-unabhängig und unter verschiedenen Bedingungen konstitutiv exprimiert werden. Das Expressionsmuster dieser Gene läßt den Schluss zu, dass sie vermutlich nicht zur Virulenz von L. ivanovii beitragen. Die Untersuchung der Virulenzclustergene in LIPI-1 schließlich zeigte eine deutliche PrfA-Abhängigkeit der Genexpression. Es konnte bestätigt werden, dass deren Transkription unter PrfA-induzierenden Bedingungen verstärkt wird. Zudem fand sich auch nach Infektion eine deutliche Expression dieser Gene. N2 - Among the six species of Listeria only two are pathogenic. Whereas L. monocytogenes is pathogenic for men and animals, L. ivanovii only causes Listeriosis in animals. Both pathogenic species possess a virulence gene cluster, which is also designated as pathogenicity island LIPI-1. Pathogenicity islands (PAIs) are widespread among gram-negative bacteria, but so far have rarely been described for gram-positive pathogens. In L. ivanovii, an additional virulence-associated unstable part of the chromosome has recently been discovered, parts of which have some characteristics of a pathogenicity island. Starting from a spontaneous but reproducible deletion event of a big part of the genome which carries some known virulence associated genes (i-inlE, i-inlF, smcL), the complete deleted area plus flanking regions were analyzed in co-operation with G. Domínguez-Bernal from the "Universidad Complutense de Madrid". Within this work 13 new open reading frames (ORFs) resp. genes (ydeI, rnaH, norA) on the right side of the smcL gene could be identified in L. ivanovii. Most of them were deleted in the deletion mutant L ivanovii GD-3. Most of the open reading frames show homologies to ORFs also found in the genome sequences of L. monocytogenes and the apathogenic species L. innocua. Own experimental analyses showed, that the genes identified in this work are also present in the apathogenic species L. seeligeri and L. welshimeri. From this it can be concluded that they presumably are not involved in L. ivanovii virulence. G. Domínguez-Bernal discovered several new internalin genes on the left side of the smcL gene. All these genes are specific for L. ivanovii. For i-inlE, i-inlF and smcL it has already been shown that they are virulence associated. This lead to the definition of a new pathogenicity island (LIPI-2) in L. ivanovii, which, in addition to smcL and i-inlFE, comprises all newly found internalin genes. Study of the regions flanking LIPI-2 showed that these are considerably conserved in L. monocytogenes as well as in the apathogenic species L. innocua, L. seeligeri and L. welshimeri. By means of RT-PCR the expression of the new identified genes was analyzed. For this, different culture conditions and transcription after infection of several cell lines were examined. By sequence analysis, a PrfA-box has been identified in front of almost all internalin genes. This work confirmed, that the expression of most internalin genes is PrfA-dependent. However, the transcription pattern was not uniform under different in vitro conditions. Finally, the analysis of gene expression after infection of several cell lines showed, that the internalin genes are transcribed differentially during infection. From this it can be concluded that they may have a role in the infection process. The expression pattern of the open reading frames flanking LIPI-2 confirmed, that these genes are transcribed PrfA independently and constitutively in vitro. This suggests that they do not contribute to virulence of L. ivanovii. Examination of the virulence cluster genes finally showed, that there is a strong PrfA dependency in gene expression. It could be confirmed, that the transcription of these genes is increased under PrfA inducing conditions. In addition, after infection also a strong expression could be detected. KW - Listeria ivanovii KW - Virulenz KW - Molekulargenetik KW - Listeria KW - Listeria ivanovii KW - LIPI-2 KW - Pathogenitätsinsel KW - Internaline KW - ydeI KW - rnaH KW - norA KW - Genexpression KW - Listeria KW - Listeria ivanovii KW - LIPI-2 KW - pathogenicity island KW - internalins KW - ydeI KW - rnaH KW - norA KW - gene expression Y1 - 2002 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-3303 ER - TY - JOUR A1 - Alsheimer, Manfred A1 - Link, Jana A1 - Leubner, Monika A1 - Schmitt, Johannes A1 - Göb, Eva A1 - Benavente, Ricardo A1 - Jeang, Kuan-Teh A1 - Xu, Rener T1 - Analysis of Meiosis in SUN1 Deficient Mice Reveals a Distinct Role of SUN2 in Mammalian Meiotic LINC Complex Formation and Function N2 - LINC complexes are evolutionarily conserved nuclear envelope bridges, composed of SUN (Sad-1/UNC-84) and KASH (Klarsicht/ANC-1/Syne/homology) domain proteins. They are crucial for nuclear positioning and nuclear shape determination, and also mediate nuclear envelope (NE) attachment of meiotic telomeres, essential for driving homolog synapsis and recombination. In mice, SUN1 and SUN2 are the only SUN domain proteins expressed during meiosis, sharing their localization with meiosis-specific KASH5. Recent studies have shown that loss of SUN1 severely interferes with meiotic processes. Absence of SUN1 provokes defective telomere attachment and causes infertility. Here, we report that meiotic telomere attachment is not entirely lost in mice deficient for SUN1, but numerous telomeres are still attached to the NE through SUN2/KASH5-LINC complexes. In Sun12/2 meiocytes attached telomeres retained the capacity to form bouquetlike clusters. Furthermore, we could detect significant numbers of late meiotic recombination events in Sun12/2 mice. Together, this indicates that even in the absence of SUN1 telomere attachment and their movement within the nuclear envelope per se can be functional. Author summary: Correct genome haploidization during meiosis requires tightly regulated chromosome movements that follow a highly conserved choreography during prophase I. Errors in these movements cause subsequent meiotic defects, which typically lead to infertility. At the beginning of meiotic prophase, chromosome ends are tethered to the nuclear envelope (NE). This attachment of telomeres appears to be mediated by well-conserved membrane spanning protein complexes within the NE (LINC complexes). In mouse meiosis, the two main LINC components SUN1 and SUN2 were independently described to localize at the sites of telomere attachment. While SUN1 has been demonstrated to be critical for meiotic telomere attachment, the precise role of SUN2 in this context, however, has been discussed controversially in the field. Our current study was targeted to determine the factual capacity of SUN2 in telomere attachment and chromosome movements in SUN1 deficient mice. Remarkably, although telomere attachment is impaired in the absence of SUN1, we could find a yet undescribed SUN1-independent telomere attachment, which presumably is mediated by SUN2 and KASH5. This SUN2 mediated telomere attachment is stable throughout prophase I and functional in moving telomeres within the NE. Thus, our results clearly indicate that SUN1 and SUN2, at least partially, fulfill redundant meiotic functions. KW - telomeres KW - spermatocytes KW - Oocytes KW - meiosis KW - protein domains KW - cytoskeleton KW - synapsis KW - homologous chromosomes Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-111355 ER - TY - JOUR A1 - Alsheimer, Manfred A1 - Link, Jana A1 - Jahn, Daniel A1 - Schmitt, Johannes A1 - Göb, Eva A1 - Baar, Johannes A1 - Ortega, Sagrario A1 - Benavente, Ricardo T1 - The Meiotic Nuclear Lamina Regulates Chromosome Dynamics and Promotes Efficient Homologous Recombination in the Mouse JF - PLoS Genetics N2 - The nuclear lamina is the structural scaffold of the nuclear envelope and is well known for its central role in nuclear organization and maintaining nuclear stability and shape. In the past, a number of severe human disorders have been identified to be associated with mutations in lamins. Extensive research on this topic has provided novel important clues about nuclear lamina function. These studies have contributed to the knowledge that the lamina constitutes a complex multifunctional platform combining both structural and regulatory functions. Here, we report that, in addition to the previously demonstrated significance for somatic cell differentiation and maintenance, the nuclear lamina is also an essential determinant for germ cell development. Both male and female mice lacking the short meiosis-specific A-type lamin C2 have a severely defective meiosis, which at least in the male results in infertility. Detailed analysis revealed that lamin C2 is required for telomere-driven dynamic repositioning of meiotic chromosomes. Loss of lamin C2 affects precise synapsis of the homologs and interferes with meiotic double-strand break repair. Taken together, our data explain how the nuclear lamina contributes to meiotic chromosome behaviour and accurate genome haploidization on a mechanistic level. KW - homologous chromosomes KW - homologous recombination KW - lamins KW - Oocytes KW - spermatocytes KW - synapsis KW - telomeres KW - testes Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-96285 ER - TY - JOUR A1 - Alnusaire, Taghreed S. A1 - Sayed, Ahmed M. A1 - Elmaidomy, Abeer H. A1 - Al-Sanea, Mohammad M. A1 - Albogami, Sarah A1 - Albqmi, Mha A1 - Alowaiesh, Bassam F. A1 - Mostafa, Ehab M. A1 - Musa, Arafa A1 - Youssif, Khayrya A. A1 - Refaat, Hesham A1 - Othman, Eman M. A1 - Dandekar, Thomas A1 - Alaaeldin, Eman A1 - Ghoneim, Mohammed M. A1 - Abdelmohsen, Usama Ramadan T1 - An in vitro and in silico study of the enhanced antiproliferative and pro-oxidant potential of Olea europaea L. cv. Arbosana leaf extract via elastic nanovesicles (spanlastics) JF - Antioxidants N2 - The olive tree is a venerable Mediterranean plant and often used in traditional medicine. The main aim of the present study was to evaluate the effect of Olea europaea L. cv. Arbosana leaf extract (OLE) and its encapsulation within a spanlastic dosage form on the improvement of its pro-oxidant and antiproliferative activity against HepG-2, MCF-7, and Caco-2 human cancer cell lines. The LC-HRESIMS-assisted metabolomic profile of OLE putatively annotated 20 major metabolites and showed considerable in vitro antiproliferative activity against HepG-2, MCF-7, and Caco-2 cell lines with IC\(_{50}\) values of 9.2 ± 0.8, 7.1 ± 0.9, and 6.5 ± 0.7 µg/mL, respectively. The encapsulation of OLE within a (spanlastic) nanocarrier system, using a spraying method and Span 40 and Tween 80 (4:1 molar ratio), was successfully carried out (size 41 ± 2.4 nm, zeta potential 13.6 ± 2.5, and EE 61.43 ± 2.03%). OLE showed enhanced thermal stability, and an improved in vitro antiproliferative effect against HepG-2, MCF-7, and Caco-2 (IC\(_{50}\) 3.6 ± 0.2, 2.3 ± 0.1, and 1.8 ± 0.1 µg/mL, respectively) in comparison to the unprocessed extract. Both preparations were found to exhibit pro-oxidant potential inside the cancer cells, through the potential inhibitory activity of OLE against glutathione reductase and superoxide dismutase (IC\(_{50}\) 1.18 ± 0.12 and 2.33 ± 0.19 µg/mL, respectively). These inhibitory activities were proposed via a comprehensive in silico study to be linked to the presence of certain compounds in OLE. Consequently, we assume that formulating such a herbal extract within a suitable nanocarrier would be a promising improvement of its therapeutic potential. KW - olive KW - metabolomic profiling KW - antiproliferative KW - pro-oxidant KW - encapsulation KW - spanlastic KW - nanocarrier KW - docking KW - molecular dynamics simulation KW - Olea Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-250064 SN - 2076-3921 VL - 10 IS - 12 ER - TY - JOUR A1 - Alizadehrad, Davod A1 - Krüger, Timothy A1 - Engstler, Markus A1 - Stark, Holger T1 - Simulating the complex cell design of Trypanosoma brucei and its motility JF - PLOS Computational Biology N2 - The flagellate Trypanosoma brucei, which causes the sleeping sickness when infecting a mammalian host, goes through an intricate life cycle. It has a rather complex propulsion mechanism and swims in diverse microenvironments. These continuously exert selective pressure, to which the trypanosome adjusts with its architecture and behavior. As a result, the trypanosome assumes a diversity of complex morphotypes during its life cycle. However, although cell biology has detailed form and function of most of them, experimental data on the dynamic behavior and development of most morphotypes is lacking. Here we show that simulation science can predict intermediate cell designs by conducting specific and controlled modifications of an accurate, nature-inspired cell model, which we developed using information from live cell analyses. The cell models account for several important characteristics of the real trypanosomal morphotypes, such as the geometry and elastic properties of the cell body, and their swimming mechanism using an eukaryotic flagellum. We introduce an elastic network model for the cell body, including bending rigidity and simulate swimming in a fluid environment, using the mesoscale simulation technique called multi-particle collision dynamics. The in silico trypanosome of the bloodstream form displays the characteristic in vivo rotational and translational motility pattern that is crucial for survival and virulence in the vertebrate host. Moreover, our model accurately simulates the trypanosome's tumbling and backward motion. We show that the distinctive course of the attached flagellum around the cell body is one important aspect to produce the observed swimming behavior in a viscous fluid, and also required to reach the maximal swimming velocity. Changing details of the flagellar attachment generates less efficient swimmers. We also simulate different morphotypes that occur during the parasite's development in the tsetse fly, and predict a flagellar course we have not been able to measure in experiments so far. KW - multiparticle collision dynamics KW - human african trypanosomiasis KW - biology KW - cytoskeleton KW - flow KW - flagellar motility KW - tsetse fly KW - propulsion KW - cytokinesis KW - parasites Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-144610 VL - 11 IS - 1 ER -