TY - THES A1 - Stief, Anna T1 - Genetics and ecology of plant heat stress memory Y1 - 2016 ER - TY - THES A1 - Rolke, Daniel T1 - Räumliche und zeitliche Expressionsmuster sowie Funktionen der Serotonin-Rezeptor-Subtypen der Honigbiene, Apis mellifera L., 1758 T1 - Spatial and temporal expression patterns as well as functions of the serotonin-receptor-subtypes in the honey bee, Apis mellifera L., 1758 N2 - Das biogene Amin Serotonin (5-Hydroxytryptamin, 5-HT) agiert als wichtiger chemischer Botenstoff bei einer Vielzahl von Organismen. Das durch 5 HT vermittelte Signal wird dabei durch spezifische Rezeptoren wahrgenommen und in eine zelluläre Reaktion umgesetzt. Diese 5 HT Rezeptoren gehören überwiegend zur Familie der G Protein gekoppelten Rezeptoren (GPCRs). Die Honigbiene Apis mellifera bietet unter anderem aufgrund ihrer eusozialen Lebensweise vielfältige Ansatzpunkte zur Erforschung der Funktionen des serotonergen Systems in Insekten. Bei A. mellifera wurden bereits vier 5-HT-Rezeptor-Subtypen beschrieben und molekular sowie pharmakologisch charakterisiert: Am5 HT1A, Am5 HT2α, Am5 HT2β und Am5 HT7. Ziel dieser Arbeit war es, gewebespezifische sowie alters- und tageszeitabhängige Expressionsmuster der 5 HT Rezeptor-Subtypen zu untersuchen, um zu einem umfassenden Verständnis des serotonergen Systems der Honigbiene beizutragen und eine Basis zur Hypothesenentwicklung für mögliche physiologische Funktionen zu schaffen. Es wurde die Expression der 5 HT Rezeptorgene sowohl im zentralen Nervensystem, als auch in Teilen des Verdauungs-, Exkretions- und Speicheldrüsensystems gemessen. Dabei konnte gezeigt werden, dass die untersuchten 5-HT-Rezeptor-Subtypen generell weit im Organismus der Honigbiene verbreitet sind. Interessanterweise unterschieden sich die untersuchten Gewebe hinsichtlich der mRNA-Expressionsmuster der untersuchten Rezeptoren. Während beispielsweise im Gehirn Am5 ht1A und Am5 ht7 stärker als Am5 ht2α und Am5 ht2β exprimiert wurden, zeigte sich in Darmgewebe ein umgekehrtes Muster. Es war bereits bekannt, dass es bei der Expression der Am5-ht2-Gene zu alternativem Spleißen kommt. Dies führt zur Entstehung der verkürzten mRNA-Varianten Am5 ht2αΔIII und Am5 ht2βΔII. Die daraus resultierenden Proteine können nicht als funktionelle GPCRs agieren. Es konnte gezeigt werden, dass diese verkürzten Spleißvarianten dennoch ubiquitär in der Honigbiene exprimiert werden. Bemerkenswerterweise wurden gewebeübergreifende Ähnlichkeiten der Expressionsmuster der Spleißvarianten gegenüber deren zugehörigen Volllängenvarianten festgestellt, welche auf Funktionen der verkürzten Varianten in vivo hindeuten. Im Hinblick auf die bei A. mellifera hauptsächlich altersbedingte Arbeitsteilung wurde die Expression der 5 HT Rezeptor-Subtypen in Gehirnen von unterschiedlich alten Arbeiterinnen mit unterschiedlichen sozialen Rollen verglichen. Während auf mRNA-Ebene keines der vier 5 HT Rezeptor-Subtypen eine altersabhängig unterschiedliche Expression zeigte, konnte für das Am5-HT1A-Protein eine höhere Konzentration in den Gehirnen älterer Tiere gefunden werden. Dies deutet auf eine posttranskriptionale Regulation der 5 HT1A Rezeptorexpression hin, welche im Zusammenhang mit der Arbeitsteilung stehen könnte. Es erfolgte die Untersuchung tageszeitlicher Änderungen sowohl der Expression der 5 HT Rezeptor-Subtypen, als auch des biogenen Amins 5 HT selbst. Während es in den Gehirnen von Arbeiterinnen, welche unter natürlichen Bedingungen gehalten wurden, zu keiner tageszeitabhängigen Veränderung des 5 HT-Titers kam, zeigte die mRNA-Expression von Am5-ht2α und Am5-ht2β eine periodische Oszillation mit Zunahme während des Tages und Abnahme während der Nacht. Diese Regulation wird durch externe Faktoren hervorgerufen und ist nicht auf einen endogenen circadianen Rhythmus zurückzuführen. Dies ging aus der Wiederholung der Expressionsmessungen an Gehirnen von Bienen, welche unter konstanten Laborbedingungen gehalten wurden, hervor. Weiterhin wurde die Beteiligung des serotonergen Systems an der Steuerung von Aspekten des circadianen lokomotorischen Aktivitätsrhythmus anhand von Verhaltensexperimenten untersucht. Mit 5 HT gefütterte Arbeiterinnen zeigten dabei unter konstanten Bedingungen eine längere Periode des Aktivitätsrhythmus als Kontrolltiere. Dies deutet auf einen Einfluss von 5 HT auf die Modulation der Synchronisation der inneren Uhr hin. Die vorliegenden Ergebnisse tragen wesentlich zum tieferen Verständnis des serotonergen Systems der Honigbiene bei und bieten Ansatzpunkte für weitergehende Studien zur Funktion von 5 HT im Zusammenhang mit der Modulation von physiologischen Prozessen, Arbeitsteilung und circadianen Rhythmen. N2 - The biogenic amine serotonin (5-hydroxytryptamine, 5-HT) acts as an important chemical messenger in a variety of organisms. The 5 HT-mediated signal is perceived by specific receptors and converted to a cellular response. This 5 HT receptors mainly belong to the family of G protein-coupled receptors (GPCRs). The honeybee offers various starting points to explore the functions of the serotonergic system in insects, among other things because of their eusocial lifestyle. In A. mellifera four 5-HT receptor subtypes have been described and molecularly and pharmacologically characterized: Am5 HT1A, Am5 HT2α, Am5 HT2β and Am5 HT7. The aim of this study was to investigate the tissue-specific and age- and daytime-dependent expression patterns of the 5 HT receptor subtypes in order to contribute to a comprehensive understanding of the serotonergic system in A. mellifera. The expression of the 5 HT receptor genes was measured in the central nervous system, as well as in parts of the digestive, excretory and salivary system. It was shown that the investigated 5-HT receptor subtypes are widely distributed in the honeybee. Interestingly, the tissues examined differed with regard to the mRNA expression pattern of the studied receptors. While in the brain the expression of Am5 ht1A and Am5 ht7 was higher than that of Am5 ht2α and Am5 ht2β, the opposite held true for intestinal tissue. It was already known that alternative splicing occurs in the expression of both Am5-ht2 genes. This leads to the formation of the truncated mRNA variants Am5 ht2αΔIII and Am5 ht2βΔII. The resulting proteins cannot act as functional GPCRs. However, it was demonstrated that these truncated splice variants are still ubiquitously expressed in the honeybee. Remarkably, similarities in the expression patterns of the shortened splice variants towards their corresponding full length variants were found throughout different tissues that indicate in vivo functions of the shortened variants. In view of the mainly age related division of labor in A. mellifera, the expression of the 5 HT receptor subtypes was compared in brains of different aged workers with different social roles. While none of the four 5 HT receptor subtypes showed an age-dependent differential expression at the mRNA level, a higher concentration in the brains of older animals could be found for the Am5-HT1A protein. This points to a post-transcriptional regulation of 5 HT1A receptor expression, which could be associated with the division of labor. It was carried out the investigation of diurnal changes in both the expression of the 5 HT receptor subtypes, and of the biogenic amine 5 HT itself. While in the brains of workers, which were kept under natural conditions, no daytime-dependent changes of the 5 HT titer could be found, the mRNA expression of Am5 ht2α and Am5-ht2β showed a periodic oscillation with an increase during the day and a decrease at night. This regulation is caused by external factors and not due to an endogenous circadian rhythm. That was shown by the repetition of the expression measurements on brains of bees, which were kept under constant laboratory conditions. Furthermore, the involvement of the serotonergic system in controlling aspects of circadian locomotor activity rhythm was investigated in behavioral experiments. Under constant conditions, worker bees which were fed with 5 HT showed a longer period of locomotor rhythm than control animals. This suggests an influence of 5 HT in the modulation of the synchronization of the internal clock. In conclusion, the present results contribute to a more detailed understanding of the serotonergic system of the honeybee and provide a basis for further studies on the function of 5 HT in connection with the modulation of physiological processes, division of labor and circadian rhythms. KW - Serotonin KW - Rezeptor KW - Honigbiene KW - serotonin KW - receptors KW - honey bee Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-96667 ER - TY - THES A1 - Beltran, Juan Camilo Moreno T1 - Characterization of the Clp protease complex and identification of putative substrates in N. tabacum Y1 - 2016 ER - TY - GEN A1 - Zhu, Fangjun A1 - Schlupp, Ingo A1 - Tiedemann, Ralph T1 - Sequence Evolution and Expression of the Androgen Receptor and Other Pathway-Related Genes in a Unisexual Fish, the Amazon Molly, Poecilia formosa, and Its Bisexual Ancestors N2 - The all-female Amazon molly (Poecilia formosa) originated from a single hybridization of two bisexual ancestors, Atlantic molly (Poecilia mexicana) and sailfin molly (Poecilia latipinna). As a gynogenetic species, the Amazon molly needs to copulate with a heterospecific male, but the genetic information of the sperm-donor does not contribute to the next generation, as the sperm only acts as the trigger for the diploid eggs’ embryogenesis. Here, we study the sequence evolution and gene expression of the duplicated genes coding for androgen receptors (ars) and other pathway-related genes, i.e., the estrogen receptors (ers) and cytochrome P450, family19, subfamily A, aromatase genes (cyp19as), in the Amazon molly, in comparison to its bisexual ancestors. Mollies possess–as most other teleost fish—two copies of the ar, er, and cyp19a genes, i.e., arα/arβ, erα/erβ1, and cyp19a1 (also referred as cyp19a1a)/cyp19a2 (also referred to as cyp19a1b), respectively. Non-synonymous single nucleotide polymorphisms (SNPs) among the ancestral bisexual species were generally predicted not to alter protein function. Some derived substitutions in the P. mexicana and one in P. formosa are predicted to impact protein function. We also describe the gene expression pattern of the ars and pathway-related genes in various tissues (i.e., brain, gill, and ovary) and provide SNP markers for allele specific expression research. As a general tendency, the levels of gene expression were lowest in gill and highest in ovarian tissues, while expression levels in the brain were intermediate in most cases. Expression levels in P. formosa were conserved where expression did not differ between the two bisexual ancestors. In those cases where gene expression levels significantly differed between the bisexual species, P. formosa expression was always comparable to the higher expression level among the two ancestors. Interestingly, erβ1 was expressed neither in brain nor in gill in the analyzed three molly species, which implies a more important role of erα in the estradiol synthesis pathway in these tissues. Furthermore, our data suggest that interactions of steroid-signaling pathway genes differ across tissues, in particular the interactions of ars and cyp19as. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 265 Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-97119 ER - TY - JOUR A1 - Zhu, Fangjun A1 - Schlupp, Ingo A1 - Tiedemann, Ralph T1 - Sequence Evolution and Expression of the Androgen Receptor and Other Pathway-Related Genes in a Unisexual Fish, the Amazon Molly, Poecilia formosa, and Its Bisexual Ancestors JF - PLoS one N2 - The all-female Amazon molly (Poecilia formosa) originated from a single hybridization of two bisexual ancestors, Atlantic molly (Poecilia mexicana) and sailfin molly (Poecilia latipinna). As a gynogenetic species, the Amazon molly needs to copulate with a heterospecific male, but the genetic information of the sperm-donor does not contribute to the next generation, as the sperm only acts as the trigger for the diploid eggs’ embryogenesis. Here, we study the sequence evolution and gene expression of the duplicated genes coding for androgen receptors (ars) and other pathway-related genes, i.e., the estrogen receptors (ers) and cytochrome P450, family19, subfamily A, aromatase genes (cyp19as), in the Amazon molly, in comparison to its bisexual ancestors. Mollies possess–as most other teleost fish—two copies of the ar, er, and cyp19a genes, i.e., arα/arβ, erα/erβ1, and cyp19a1 (also referred as cyp19a1a)/cyp19a2 (also referred to as cyp19a1b), respectively. Non-synonymous single nucleotide polymorphisms (SNPs) among the ancestral bisexual species were generally predicted not to alter protein function. Some derived substitutions in the P. mexicana and one in P. formosa are predicted to impact protein function. We also describe the gene expression pattern of the ars and pathway-related genes in various tissues (i.e., brain, gill, and ovary) and provide SNP markers for allele specific expression research. As a general tendency, the levels of gene expression were lowest in gill and highest in ovarian tissues, while expression levels in the brain were intermediate in most cases. Expression levels in P. formosa were conserved where expression did not differ between the two bisexual ancestors. In those cases where gene expression levels significantly differed between the bisexual species, P. formosa expression was always comparable to the higher expression level among the two ancestors. Interestingly, erβ1 was expressed neither in brain nor in gill in the analyzed three molly species, which implies a more important role of erα in the estradiol synthesis pathway in these tissues. Furthermore, our data suggest that interactions of steroid-signaling pathway genes differ across tissues, in particular the interactions of ars and cyp19as. Y1 - 2016 U6 - https://doi.org/10.1371/JOURNAL.PONE.0156209 SN - 1932-6203 VL - 11 IS - 6 PB - PLoS CY - Lawrence, Kan. ER - TY - GEN A1 - Batsios, Petros A1 - Ren, Xiang A1 - Baumann, Otto A1 - Larochelle, Denis A. A1 - Gräf, Ralph T1 - Src1 is a Protein of the Inner Nuclear Membrane Interacting with the Dictyostelium Lamin NE81 N2 - The nuclear envelope (NE) consists of the outer and inner nuclear membrane (INM), whereby the latter is bound to the nuclear lamina. Src1 is a Dictyostelium homologue of the helix-extension-helix family of proteins, which also includes the human lamin-binding protein MAN1. Both endogenous Src1 and GFP-Src1 are localized to the NE during the entire cell cycle. Immuno-electron microscopy and light microscopy after differential detergent treatment indicated that Src1 resides in the INM. FRAP experiments with GFP-Src1 cells suggested that at least a fraction of the protein could be stably engaged in forming the nuclear lamina together with the Dictyostelium lamin NE81. Both a BioID proximity assay and mis-localization of soluble, truncated mRFP-Src1 at cytosolic clusters consisting of an intentionally mis-localized mutant of GFP-NE81 confirmed an interaction of Src1 and NE81. Expression GFP-Src11–646, a fragment C-terminally truncated after the first transmembrane domain, disrupted interaction of nuclear membranes with the nuclear lamina, as cells formed protrusions of the NE that were dependent on cytoskeletal pulling forces. Protrusions were dependent on intact microtubules but not actin filaments. Our results indicate that Src1 is required for integrity of the NE and highlight Dictyostelium as a promising model for the evolution of nuclear architecture. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 263 KW - Dictyostelium KW - HeH-protein KW - LEM-domain protein KW - lamin KW - nuclear lamina KW - nucleolus KW - nucleus Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-97033 ER - TY - JOUR A1 - Batsios, Petros A1 - Ren, Xiang A1 - Baumann, Otto A1 - Larochelle, Denis A. A1 - Gräf, Ralph T1 - Src1 is a Protein of the Inner Nuclear Membrane Interacting with the Dictyostelium Lamin NE81 JF - Cells N2 - The nuclear envelope (NE) consists of the outer and inner nuclear membrane (INM), whereby the latter is bound to the nuclear lamina. Src1 is a Dictyostelium homologue of the helix-extension-helix family of proteins, which also includes the human lamin-binding protein MAN1. Both endogenous Src1 and GFP-Src1 are localized to the NE during the entire cell cycle. Immuno-electron microscopy and light microscopy after differential detergent treatment indicated that Src1 resides in the INM. FRAP experiments with GFP-Src1 cells suggested that at least a fraction of the protein could be stably engaged in forming the nuclear lamina together with the Dictyostelium lamin NE81. Both a BioID proximity assay and mis-localization of soluble, truncated mRFP-Src1 at cytosolic clusters consisting of an intentionally mis-localized mutant of GFP-NE81 confirmed an interaction of Src1 and NE81. Expression GFP-Src11–646, a fragment C-terminally truncated after the first transmembrane domain, disrupted interaction of nuclear membranes with the nuclear lamina, as cells formed protrusions of the NE that were dependent on cytoskeletal pulling forces. Protrusions were dependent on intact microtubules but not actin filaments. Our results indicate that Src1 is required for integrity of the NE and highlight Dictyostelium as a promising model for the evolution of nuclear architecture. KW - Dictyostelium KW - lamin KW - nuclear lamina KW - nucleus KW - nucleolus KW - HeH-protein KW - LEM-domain protein Y1 - 2016 U6 - https://doi.org/10.3390/cells5010013 SN - 2073-4409 VL - 5 IS - 1 PB - MDPI CY - Basel ER - TY - THES A1 - Orf, Isabel T1 - Photorespiratory metabolism in the cyanobacterial model Synechocystis sp. strain PCC 6803 BT - a systems biology approach Y1 - 2016 ER - TY - THES A1 - Peter, Tatjana T1 - Molekulare Charakterisierung von CP75, einem neuen centrosomalen Protein in Dictyostelium discoideum T1 - Molecular characterization of CP75, a novel Dictyostelium centrosome protein N2 - Das Centrosom ist ein Zellkern-assoziiertes Organell, das nicht von einer Membran umschlossen ist. Es spielt eine wichtige Rolle in vielen Mikrotubuli- abhängigen Prozessen wie Organellenpositionierung, Zellpolarität oder die Organisation der mitotischen Spindel. Das Centrosom von Dictyostelium besteht aus einer dreischichtigen Core-Struktur umgeben von einer Corona, die Mikrotubuli-nukleierende Komplexe enthält. Die Verdoppelung des Centrosoms in Dictyostelium findet zu Beginn der Mitose statt. In der Prophase vergrößert sich die geschichtete Core-Struktur und die Corona löst sich auf. Anschließend trennen sich die beiden äußeren Lagen der Core-Struktur und bilden in der Metaphase die beiden Spindelpole, die in der Telophase zu zwei vollständigen Centrosomen heranreifen. Das durch eine Proteom-Analyse identifizierte Protein CP75 lokalisiert am Centrosom abhängig von den Mitosephasen. Es dissoziiert von der Core-Struktur in der Prometaphase und erscheint an den Spindelpolen in der Telophase wieder. Dieses Verhalten korreliert mit dem Verhalten der mittleren Lage der Core-Struktur in der Mitose, was darauf hinweist, dass CP75 eine Komponente dieser Schicht sein könnte. Die FRAP-Experimente am Interphase- Centrosom zeigen, dass GFP-CP75 dort nicht mobil ist. Das deutet darauf hin, dass das Protein wichtige Funktionen im Strukturerhalt der centrosomalen Core- Struktur übernehmen könnte. Sowohl die C- als auch die N-terminale Domäne von CP75 enthalten centrosomale Targeting-Domäne. Als GFP-Fusionsproteine (GFP-CP75-N und -C) lokalisieren die beiden Fragmente am Centrosom in der Interphase. Während GFP-CP75-C in der Mitose am Centrosom verbleibt, verschwindet GFP-CP75-N in der Metaphase und kehrt erst in der späten Telophase zurück. GFP-CP75-C und GFP-CP75O/E kolokalisieren mit F-Aktin am Zellcortex, zeigen aber keine Interaktion mit Aktin mit der BioID-Methode. Die N-terminale Domäne von CP75 enthält eine potentielle Plk1- Phosphorylierungssequenz. Die Überexpression der nichtphosphorylierbaren Punktmutante (GFP-CP75-Plk-S143A) ruft verschiedene Phänotypen wie verlängerte oder überzählige Centrosomen, vergrößerte Zellkerne und Anreicherung von detyrosinierten Mikrotubuli hervor. Die ähnlichen Phänotypen konnten auch bei GFP-CP75-N und CP75-RNAi beobachtet werden. Der Phänotyp der detyrosinierten Mikrotubuli bringt erstmals den Beweis dafür, dass I in Dictyostelium posttranslationale Modifikation an Tubulinen stattfindet. Außerdem zeigten CP75-RNAi-Zellen Defekte in der Organisation der mitotischen Spindel. Mittels BioID-Methode konnten drei potentielle Interaktionspartner von CP75 identifiziert werden. Diese drei Proteine CP39, CP91 und Cep192 sind ebenfalls Bestandteile des Centrosoms. N2 - The centrosome is a nonmembranous, nucleus-associated organelle. It plays crucial roles in a variety of mucrotubule-dependent processes, such as organelle positioning, cell polarization and mitotic spindle organization. The Dictyostelium centrosome consists of a core structure with three major layers, surrounded by a corona containing micrutubule-nucleation complexes. Dictyostelium centrosome replication starts at the onset of mitosis. In prophase, the core structure enlarges and the corona disappears. Afterwards, the core structure splits and the outer layers form two spindle poles maturating to two new, complete centrosomes in telophase. CP75 is one of nine novel proteins identified in a centrosomal proteome analysis. Endogenous CP75 localizes to the centrosome in a cell cycle- dependent manner. It dissociates from the core structure in early prometaphase and reappears at spindle poles in telophase. Since this pattern fits to the disappearance and reappearance of the central layer of the core structure, this indicates that CP75 is a constituent of this layer. During interphase FRAP experiments reveal that GFP-CP75 exhibits no mobility at the centrosome. This indicates a role in structural maintenance of the centrosomal core. Both the C- and N-terminal domains of CP75 contain a centrosomal targeting domain. Fused to GFP (GFP-CP75-N and -C) both fragments localized to the centrosome in interphase, but only GFP-CP75-N disappears from the centrosome during mitosis. GFP-CP75-C and GFP-CP75O/E also colocalized with F-actin at the cell cortex. But it shows no direct interaction with actin in BioID. The N-terminal domain of CP75 contains Plk1 phosphorylation sites. Overexpression of phosphorylation site-defficient mutant of GFP-CP75 (GFP-CP75-Plk-S143A) causes different phenotypes like enlarged and disrupted nuclei, enlarged and supernumerary centrosomes and detyrosinated microtubules. A very similar phenotype was observed upon overexpression of GFP-CP75-N and upon knockdown of CP75 expression by RNAi. The phenotype of detyrosinated microtubules for the first time shows that posttranslational modification of Tubulin takes place in Dictyostelium. Live cell imaging showed that lack of CP75 caused defect in spindle formation. CP75 interacts in BioID with CP39, CP91 and Cep192, three other components of the centrosome. KW - Dictyostelium KW - Centrosom KW - CP75 KW - dictyostelium KW - centrosome KW - CP75 Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-96472 ER - TY - THES A1 - Reinecke, Antje Adriana T1 - Impact of protein structure on the mechanics and assembly of mytilus byssal threads Y1 - 2016 ER -