TY - JOUR A1 - Albrecht, Marco A1 - Sharma, Cynthia M. A1 - Reinhardt, Richard A1 - Vogel, Joerg A1 - Rudel, Thomas T1 - Deep sequencing-based discovery of the Chlamydia trachomatis transcriptome N2 - Chlamydia trachomatis is an obligate intracellular pathogenic bacterium that has been refractory to genetic manipulations. Although the genomes of several strains have been sequenced, very little information is available on the gene structure of these bacteria. We used deep sequencing to define the transcriptome of purified elementary bodies (EB) and reticulate bodies (RB) of C. trachomatis L2b, respectively. Using an RNAseq approach, we have mapped 363 transcriptional start sites (TSS) of annotated genes. Semiquantitative analysis of mapped cDNA reads revealed differences in the RNA levels of 84 genes isolated from EB and RB, respectively. We have identified and in part confirmed 42 genome- and 1 plasmid-derived novel non-coding RNAs. The genome encoded non-coding RNA, ctrR0332 was one of the most abundantly and differentially expressed RNA in EB and RB, implying an important role in the developmental cycle of C. trachomatis. The detailed map of TSS in a thus far unprecedented resolution as a complement to the genome sequence will help to understand the organization, control and function of genes of this important pathogen. KW - Biologie Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68389 ER - TY - JOUR A1 - Koetschan, Christian A1 - Foerster, Frank A1 - Keller, Alexander A1 - Schleicher, Tina A1 - Ruderisch, Benjamin A1 - Schwarz, Roland A1 - Mueller, Tobias A1 - Wolf, Matthias A1 - Schultz, Joerg T1 - The ITS2 Database III-sequences and structures for phylogeny N2 - The internal transcribed spacer 2 (ITS2) is a widely used phylogenetic marker. In the past, it has mainly been used for species level classifications. Nowadays, a wider applicability becomes apparent. Here, the conserved structure of the RNA molecule plays a vital role. We have developed the ITS2 Database (http://its2.bioapps .biozentrum.uni-wuerzburg.de) which holds information about sequence, structure and taxonomic classification of all ITS2 in GenBank. In the new version, we use Hidden Markov models (HMMs) for the identification and delineation of the ITS2 resulting in a major redesign of the annotation pipeline. This allowed the identification of more than 160 000 correct full ength and more than 50 000 partial structures. In the web interface, these can now be searched with a modified BLAST considering both sequence and structure, enabling rapid taxon sampling. Novel sequences can be annotated using the HMM based approach and modelled according to multiple template structures. Sequences can be searched for known and newly identified motifs. Together, the database and the web server build an exhaustive resource for ITS2 based phylogenetic analyses. KW - Biologie Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68390 ER - TY - JOUR A1 - Meierjohann, Svenja A1 - Hufnagel, Anita A1 - Wende, Elisabeth A1 - Kleinschmidt, Markus A. A1 - Wolf, Katarina A1 - Friedl, Peter A1 - Gaubatz, Stefan A1 - Schartl, Manfred T1 - MMP13 mediates cell cycle progression in melanocytes and melanoma cells: in vitro studies of migration and proliferation N2 - Background: Melanoma cells are usually characterized by a strong proliferative potential and efficient invasive migration. Among the multiple molecular changes that are recorded during progression of this disease, aberrant activation of receptor tyrosine kinases (RTK) is often observed. Activation of matrix metalloproteases goes along with RTK activation and usually enhances RTK-driven migration. The purpose of this study was to examine RTKdriven three-dimensional migration of melanocytes and the pro-tumorigenic role of matrix metalloproteases for melanocytes and melanoma cells. Results: Using experimental melanocyte dedifferentiation as a model for early melanomagenesis we show that an activated EGF receptor variant potentiates migration through three-dimensional fibrillar collagen. EGFR stimulation also resulted in a strong induction of matrix metalloproteases in a MAPK-dependent manner. However, neither MAPK nor MMP activity were required for migration, as the cells migrated in an entirely amoeboid mode. Instead, MMPs fulfilled a function in cell cycle regulation, as their inhibition resulted in strong growth inhibition of melanocytes. The same effect was observed in the human melanoma cell line A375 after stimulation with FCS. Using sh- and siRNA techniques, we could show that MMP13 is the protease responsible for this effect. Along with decreased proliferation, knockdown of MMP13 strongly enhanced pigmentation of melanocytes. Conclusions: Our data show for the first time that growth stimuli are mediated via MMP13 in melanocytes and melanoma, suggesting an autocrine MMP13-driven loop. Given that MMP13-specific inhibitors are already developed, these results support the evaluation of these inhibitors in the treatment of melanoma. KW - Medizin Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68335 ER - TY - JOUR A1 - Zeeshan, Ahmed T1 - Towards Performance Measurement and Metrics Based Analysis of PLA Applications N2 - This article is about a measurement analysis based approach to help software practitioners in managing the additional level complexities and variabilities in software product line applications. The architecture of the proposed approach i.e. ZAC is designed and implemented to perform preprocessesed source code analysis, calculate traditional and product line metrics and visualize results in two and three dimensional diagrams. Experiments using real time data sets are performed which concluded with the results that the ZAC can be very helpful for the software practitioners in understanding the overall structure and complexity of product line applications. Moreover the obtained results prove strong positive correlation between calculated traditional and product line measures. KW - Programmierbare logische Anordnung KW - Analysis KW - Measurement KW - Software product lines KW - Variability Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68188 ER - TY - JOUR A1 - Bollazzi, Martin A1 - Roces, Flavio T1 - The thermoregulatory function of thatched nests in the South American grass-cutting ant, Acromyrmex heyeri N2 - The construction of mound-shaped nests by ants is considered as a behavioral adaptation to low environmental temperatures, i.e., colonies achieve higher and more stables temperatures than those of the environment. Besides the well-known nests of boreal Formica wood-ants, several species of South American leaf-cutting ants of the genus Acromyrmex construct thatched nests. Acromyrmex workers import plant fragments as building material, and arrange them so as to form a thatch covering a central chamber, where the fungus garden is located. Thus, the degree of thermoregulation attained by the fungus garden inside the thatched nest largely depends on how the thatch affects the thermal relations between the fungus and the environment. This work was aimed at studying the thermoregulatory function of the thatched nests built by the grass-cutting ant Acromyrmex heyeri Forel (Hymenoptera: Formicidae: Myrmicinae). Nest and environmental temperatures were measured as a function of solar radiation on the long-term. The thermal diffusivity of the nest thatch was measured and compared to that of the surrounding soil, in order to assess the influence of the building material on the nest’s thermoregulatory ability. The results showed that the average core temperature of thatched nests was higher than that of the environment, but remained below values harmful for the fungus. This thermoregulation was brought about by the low thermal diffusivity of the nest thatch built by workers with plant fragments, instead of the readily-available soil particles that have a higher thermal diffusivity. The thatch prevented diurnal nest overheating by the incoming solar radiation, and avoided losses of the accumulated daily heat into the cold air during the night. The adaptive value of thatching behavior in Acromyrmex leaf-cutting ants occurring in the southernmost distribution range is discussed. KW - Acromyrmex heyeri KW - building behaviour KW - thermal biology KW - nest material KW - heat transfer KW - leaf-cutting ants Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68225 ER - TY - THES A1 - Förster, Frank T1 - Making the most of phylogeny: Unique adaptations in tardigrades and 216374 internal transcribed spacer 2 structures T1 - Einzigartige Anpassungen in Tardigraden und 216374 "internal transcribed spacer 2" Strukturen N2 - The phylum Tardigrada consists of about 1000 described species to date. The animals live in habitats within marine, freshwater and terrestrial ecosystems allover the world. Tardigrades are polyextremophiles. They are capable to resist extreme temperature, pressure or radiation. In the event of desiccation, tardigrades enter a so-called tun stage. The reason for their great tolerance capabilities against extreme environmental conditions is not discovered yet. Our Funcrypta project aims at finding answers to the question what mechanisms underlie these adaption capabilities particularly with regard to the species Milnesium tardigradum. The first part of this thesis describes the establishment of expressed sequence tags (ESTs) libraries for different stages of M. tardigradum. From proteomics data we bioinformatically identified 144 proteins with a known function and additionally 36 proteins which seemed to be specific for M. tardigradum. The generation of a comprehensive web-based database allows us to merge the proteome and transcriptome data. Therefore we created an annotation pipeline for the functional annotation of the protein and nucleotide sequences. Additionally, we clustered the obtained proteome dataset and identified some tardigrade-specific proteins (TSPs) which did not show homology to known proteins. Moreover, we examined the heat shock proteins of M. tardigradum and their different expression levels depending on the actual state of the animals. In further bioinformatical analyses of the whole data set, we discovered promising proteins and pathways which are described to be correlated with the stress tolerance, e.g. late embryogenesis abundant (LEA) proteins. Besides, we compared the tardigrades with nematodes, rotifers, yeast and man to identify shared and tardigrade specific stress pathways. An analysis of the 50 and 30 untranslated regions (UTRs) demonstrates a strong usage of stabilising motifs like the 15-lipoxygenase differentiation control element (15-LOX-DICE) but also reveals a lack of other common UTR motifs normally used, e.g. AU rich elements. The second part of this thesis focuses on the relatedness between several cryptic species within the tardigrade genus Paramacrobiotus. Therefore for the first time, we used the sequence-structure information of the internal transcribed spacer 2 (ITS2) as a phylogenetic marker in tardigrades. This allowed the description of three new species which were indistinguishable using morphological characters or common molecular markers like the 18S ribosomal ribonucleic acid (rRNA) or the Cytochrome c oxidase subunit I (COI). In a large in silico simulation study we also succeeded to show the benefit for the phylogenetic tree reconstruction by adding structure information to the ITS2 sequence. Next to the genus Paramacrobiotus we used the ITS2 to corroborate a monophyletic DO-group (Sphaeropleales) within the Chlorophyceae. Additionally we redesigned another comprehensive database—the ITS2 database resulting in a doubled number of sequence-structure pairs of the ITS2. In conclusion, this thesis shows the first insights (6 first author publications and 4 coauthor publications) into the reasons for the enormous adaption capabilities of tardigrades and offers a solution to the debate on the phylogenetic relatedness within the tardigrade genus Paramacrobiotus. N2 - Der Tierstamm Tardigrada besteht aus derzeitig etwa 1000 beschriebenen Arten. Die Tiere leben in Habitaten in marinen, limnischen und terrestrischen Ökosystemen auf der ganzen Welt. Tardigraden sind polyextremophil. Sie können extremer Temperatur, Druck oder Strahlung widerstehen. Beim Austrocknen bilden sie ein so genanntes Tönnchenstadium. Der Grund für die hohe Toleranz gegenüber extremen Umweltbedingungen ist bis jetzt nicht aufgeklärt worden. Unser Funcrypta Projekt versucht Antworten darauf zu finden, was die hinter dieser Anpassungsfähigkeit liegenden Mechanismen sind. Dabei steht die Art Milnesium tardigradum im Mittelpunkt. Der erste Teil dieser Arbeit beschreibt die Etablierung einer expressed sequence tags (ESTs) Bibliothek für verschiedene Stadien von M. tardigradum. Aus unseren Proteomansatz konnten wir bislang 144 Proteine bioinformatisch identifizieren, denen eine Funktion zugeordnet werden konnte. Darüber hinaus wurden 36 Proteine gefunden, welche spezifisch für M. tardigradum zu sein scheinen. Die Erstellung einer umfassenden internetbasierenden Datenbank erlaubt uns die Verknüpfung der Proteom und Transkriptomdaten. Dafür wurde eine Annotations-Pipeline erstellt um den Sequenzen Funktionen zuordnen zu können. Außerdem wurden die erhaltenen Proteindaten von uns geclustert. Dabei konnten wir einige Tardigraden-spezifische Proteine (tardigrade-specific protein, TSP) identifizieren die keinerlei Homologie zu bekannten Proteinen zeigen. Außerdem untersuchten wir die Hitze-Schock-Proteine von M. tardigradum und deren differenzielle Expression in Abhängigkeit vom Stadium der Tiere. In weiteren bioinformatischen Analysen konnten wir viel versprechende Proteine und Stoffwechselwege entdecken für die beschrieben ist, dass sie mit Stressreaktionen in Verbindung stehen, beispielsweise late embryogenesis abundant (LEA) Proteine. Des Weiteren verglichen wir Tardigraden mit Nematoden, Rotatorien, Hefe und dem Menschen, um gemeinsame und Tardigraden-spezifische Stoffwechselwege identifizieren zu können. Analysen der 50 und 30 untranslatierten Bereiche zeigen eine verstärkte Nutzung von stabilisierenden Motiven, wie dem 15-lipoxygenase differentiation control element (LEA). Im Gegensatz dazu werden häufig benutzte Motive, wie beispielsweise AU-reiche Bereiche, gar nicht gefunden. Der zweite Teil der Doktorarbeit beschäftigt sich mit den Verwandtschaftsverhältnissen einiger kryptischer Arten in der Tardigradengattung Paramacrobiotus. Hierfür haben wir, zum ersten Mal in Tardigraden, die Sequenz-Struktur-Informationen der internal transcribed spacer 2 Region als phylogenetischen Marker verwendet. Dies erlaubte uns die Beschreibung von drei neuen Arten, welche mit klassischen morphologischen Merkmalen oder anderen molekularen Markern wie 18S ribosomaler RNA oder Cytochrome c oxidase subunit I (COI) nicht unterschieden werden konnten. In einer umfangreichen in silico Simulationsstudie zeigten wir den Vorteil der bei der Rekonstruktion phylogenetischer Bäume unter der Hinzunahme der Strukturinformationen zur Sequenz der ITS2 entsteht. ITS2 Sequenz-Struktur-Informationen wurden außerdem auch dazu benutzt, eine monophyletische DO-Gruppe (Sphaeropleales) in den Chlorophyceae zu bestätigen. Zusätzlich haben wir eine umfassende Datenbank, die ITS2-Datenbank, überarbeitet. Dadurch konnten die Sequenz-Struktur-Informationen verdoppelt werden, die in dieser Datenbank verfügbar sind. Die vorliegende Doktorarbeit zeigt erste Einblicke (6 Erstautor- und 4 Koautor-Publikationen) in die Ursachen für die hervorragende Anpassungsfähigkeit der Tardigraden und beschreibt die erfolgreiche Aufklärung der Verwandtschaftsverhältnisse in der Tardigradengattung Paramacrobiotus. KW - Phylogenie KW - Bioinformatik KW - Würzburg / Universität / Lehrstuhl für Bioinformatik KW - Anpassung KW - Datenbank KW - ITS2 KW - Marker KW - Tardigraden KW - Bärtierchen KW - ITS2 KW - Marker KW - Tardigrades KW - Waterbear Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-51466 ER - TY - JOUR A1 - Shityakov, Sergey A1 - Dandekar, Thomas T1 - Lead expansion and virtual screening of Indinavir derivate HIV-1 protease inhibitors using pharmacophoric - shape similarity scoring function N2 - Indinavir (Crivaxan®) is a potent inhibitor of the HIV (human immunodeficiency virus) protease. This enzyme has an important role in viral replication and is considered to be very attractive target for new antiretroviral drugs. However, it becomes less effective due to highly resistant new viral strains of HIV, which have multiple mutations in their proteases. For this reason, we used a lead expansion method to create a new set of compounds with a new mode of action to protease binding site. 1300 compounds chemically diverse from the initial hit were generated and screened to determine their ability to interact with protease and establish their QSAR properties. Further computational analyses revealed one unique compound with different protease binding ability from the initial hit and its role for possible new class of protease inhibitors is discussed in this report. KW - Proteasen KW - protease; Indinavir; lead expansion; docking; pharmacophore Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-67824 ER - TY - JOUR A1 - Keller, Alexander A1 - Foerster, Frank A1 - Mueller, Tobias A1 - Dandekar, Thomas A1 - Schultz, Joerg A1 - Wolf, Matthias T1 - Including RNA secondary structures improves accuracy and robustness in reconstruction of phylogenetic trees N2 - Background: In several studies, secondary structures of ribosomal genes have been used to improve the quality of phylogenetic reconstructions. An extensive evaluation of the benefits of secondary structure, however, is lacking. Results: This is the first study to counter this deficiency. We inspected the accuracy and robustness of phylogenetics with individual secondary structures by simulation experiments for artificial tree topologies with up to 18 taxa and for divergency levels in the range of typical phylogenetic studies. We chose the internal transcribed spacer 2 of the ribosomal cistron as an exemplary marker region. Simulation integrated the coevolution process of sequences with secondary structures. Additionally, the phylogenetic power of marker size duplication was investigated and compared with sequence and sequence-structure reconstruction methods. The results clearly show that accuracy and robustness of Neighbor Joining trees are largely improved by structural information in contrast to sequence only data, whereas a doubled marker size only accounts for robustness. Conclusions: Individual secondary structures of ribosomal RNA sequences provide a valuable gain of information content that is useful for phylogenetics. Thus, the usage of ITS2 sequence together with secondary structure for taxonomic inferences is recommended. Other reconstruction methods as maximum likelihood, bayesian inference or maximum parsimony may equally profit from secondary structure inclusion. Reviewers: This article was reviewed by Shamil Sunyaev, Andrea Tanzer (nominated by Frank Eisenhaber) and Eugene V. Koonin. Open peer review: Reviewed by Shamil Sunyaev, Andrea Tanzer (nominated by Frank Eisenhaber) and Eugene V. Koonin. For the full reviews, please go to the Reviewers’ comments section. KW - Phylogenie KW - phylogenetics Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-67832 ER - TY - THES A1 - Heinecke, Kai T1 - Die Dynamik der primären Erkennungsschritte von BMP-Rezeptoren T1 - Dynamics of the primary recognition steps of BMP receptors N2 - Bone Morphogenetic Proteins (BMPs) bilden zusammen mit den Activinen, Growth and Differentiation Factors (GDFs) und Transforming Growth Factor β (TGF-β) die Transforming Growth Factor β-Superfamilie von sekretierten Signalproteinen. Sie spielen eine wichtige Rolle in der Entwicklung, Erhaltung und Regeneration von Geweben und Organen. Die Signalvermittlung dieser Proteine erfolgt durch die Bindung von zwei verschiedenen Typen von Serin-/Threonin-Kinaserezeptoren, die als Typ-I- und Typ-II-Rezeptoren bezeichnet werden. Im ersten Schritt erfolgt die Bindung an den hochaffinen Rezeptor (im Fall von BMP-2 der Typ-I-Rezeptor), im nächsten Schritt wird der niederaffine Rezeptor in den Komplex rekrutiert. Bis heute sind lediglich sieben Typ-I- und fünf Typ-II-Rezeptoren bekannt, was auf eine Promiskuität in der Liganden-Rezeptor-Interaktion schließen lässt. Die Architektur beider Rezeptorsubtypen ist dabei relativ ähnlich. Beide bestehen aus einer ligandenbindenden extrazellulären Domäne, einer Transmembrandomäne sowie einer intrazellulären Kinasedomäne. Eine nacheinander ablaufende Transphosphorylierung der intrazellulären Domänen führt zu einer Phosphorylierung von SMAD-Proteinen, die dann als nachgeschaltete Vermittler fungieren und die Transkription regulierter Gene auslösen. Im Hauptteil dieser Arbeit wurden die initialen Schritte der Rezeptorkomplexformierung sowie die Mobilität der Rezeptoren mit Hilfe von fluoreszenzmikroskopischen Methoden untersucht. Dabei konnte festgestellt werden, dass für die Bildung eines Signalkomplexes eine bestimmte Schwellenkonzentration des Liganden nötig ist und dass der Mechanismus nach einem Alles-oder-Nichts-Prinzip wie ein Schalter funktioniert. Außerdem konnten Unterschiede in der Nutzung der gleichen Rezeptoren durch verschiedene Liganden festgestellt werden. Die anderen Teile der Arbeit befassen sich mit der Funktionalität der verschiedenen Rezeptordomänen in der Signalübermittlung, der Analyse von hoch- und niederaffinen Ligandenbindestellen auf ganzen Zellen sowie dem Einfluss des SMAD- und des MAPK-Signalwegs auf die Induktion der Alkalischen Phosphatase. Dabei konnte gezeigt werden, dass die Art der SMAD-Phosphorylierung allein vom Typ der Kinasedomäne abhängig ist, dass auf einer Zelle verschiedene Rezeptorpopulationen existieren, welche von unterschiedlichen Ligandenkonzentrationen angesprochen werden, und dass die Induktion der Alkalischen Phosphatase stark vom zeitlichen Verlauf der SMAD- und MAPK-Aktivierung abhängig ist. N2 - Bone Morphogenetic Proteins (BMPs), together with Activins, Growth and Differentiation Factors (GDFs) and Transforming Growth Factor β (TGFβ), are secreted signalling proteins that belong to the Transforming Growth Factor β superfamily. They play an important role in regulating the development, maintenance and regeneration of tissues and organs. Signalling of TGFβ superfamily members occurs by binding to two types of serine-/threonine kinase receptors termed type I and type II. First, the high affinity receptor (in case of BMP2 the type I receptor) is bound, and then the low affinity receptor is recruited into the signalling complex. The fact that there are only seven type-I and five type-II receptors are known implies a limited promiscuity in ligand-receptor interaction. The architecture of both receptor subtypes is quite similar, with a small extracellular ligand-binding domain, a single transmembrane domain and an intracellular kinase domain. Subsequent transphosphorylation of the intracellular receptor domains leads to phosphorylation of SMAD proteins, which then act as downstream mediators and activate gene transcription. The main part of this work was to analyze the initial steps in receptor complex formation and the mobility of TGFβ-superfamily receptors with fluorescence microscopy techniques. It could be shown that complex formation requires a certain ligand threshold concentration and shows an all-or-nothing switch-like behaviour. Furthermore, differences between different ligands using the same receptors could be visualized. The other parts of this work deal with the functionality of the different receptor domains in signal transduction, the analysis of high- and low-affinity binding sites on whole cells and the influence of the SMAD- and MAPK-pathways on alkaline phosphatase induction. It could be shown that the type of SMAD phosphorylation ist solely dependent on the type of the kinase domain, that there exist different receptor populations on a cell that are addressed by different ligand concentrations and that alkaline phosphatase induction is highly dependent on the time course of SMAD- and MAPK-pathway activation. KW - Knochen-Morphogenese-Proteine KW - Wirkstoff-Rezeptor-Bindung KW - Signaltransduktion KW - Signalkomplex KW - Rezeptormobilität KW - Rezeptor-Liganden-Interaktion KW - Physiologische Chemie KW - Molekulare Biophysik KW - Molekularbiologie KW - Signaling complex KW - receptor mobility KW - receptor-ligand-interaction Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-49257 ER - TY - THES A1 - Kirchmaier, Bettina Carmen T1 - Characterization of the Popeye domain containing gene family in zebrafish T1 - Charakterisierung der Popeye domain containing Genfamilie im Zebrafisch N2 - The Popeye domain containing (Popdc) gene family of membrane proteins is predominantly expressed in striated and smooth muscle tissues and has been shown to act as novel cAMP-binding proteins. In mice, loss of Popdc1 and Popdc2, respectively, affects sinus node function in the postnatal heart in an age and stress-dependent manner. In this thesis, I examined gene expression pattern and function of the Popdc gene family during zebrafish development with an emphasis on popdc2. Expression of the zebrafish popdc2 was exclusively present in cardiac and skeletal muscle during cardiac development, whereas popdc3 was expressed in striated muscle tissue and in distinct regions of the brain. In order to study the function of these genes, an antisense morpholino-based knockdown approach was used. Knockdown of popdc2 resulted in aberrant development of facial and tail musculature. In the heart, popdc2 morphants displayed irregular ventricular contractions with 2:1 and 3:1 ventricular pauses. Recordings of calcium transients using a transgenic indicator line Tg(cmlc2:gCaMP)s878 and selective plane illumination microscopy (SPIM) revealed the presence of an atrioventricular (AV) block in popdc2 morphants as well as a complete heart block. Interestingly, preliminary data revealed that popdc3 morphants developed a similar phenotype. In order to find a morphological correlate for the observed AV conduction defect, I studied the structure of the AV canal in popdc2 morphants using confocal analysis of hearts of the transgenic line Tg(cmlc2:eGFP-ras)s883, which outlines individual cardiac myocytes with the help of membrane-localized GFP. However, no evidence for morphological alterations was obtained. To ensure that the observed arrhythmia phenotype in the popdc2 morphant was based on a myocardial defect and not caused by defective valve development, live imaging was performed revealing properly formed valves. Thus, in agreement with the data obtained in knockout mice, popdc2 and popdc3 genes in zebrafish are involved in the regulation of cardiac electrical activity. However, both genes are not required for cardiac pacemaking, but they play essential roles in AV conduction. In order to elucidate the biological importance of cAMP-binding, wild type Popdc1 as well as mutants with a significant reduction in binding affinity for cAMP in vitro were overexpressed in zebrafish embryos. Expression of wild type Popdc1 led to a cardiac insufficiency phenotype characterized by pericardial edema and venous blood retention. Strikingly, the ability of the Popdc1 mutants to induce a cardiac phenotype correlated with the binding affinity for cAMP. These data suggest that cAMP-binding represents an important biological property of the Popdc protein family. N2 - Die Popeye domain containing (Popdc) Gene kodieren für eine Familie von Membranproteinen, die vorwiegend in der gestreiften und glatten Muskulatur exprimiert werden und in der Lage sind cAMP zu binden. In Mäusen resultiert der Verlust von Popdc1 oder Popdc2 in einer stressinduzierten Sinusknotendysfunktion, die sich altersabhängig entwickelt. In meiner Dissertation habe ich das Expressionsmuster und die Funktion der Popdc Genfamilie mit Schwerpunkt auf dem popdc2-Gen im Zebrafisch untersucht. Das popdc2-Gen im Zebrafisch wurde ausschließlich in der Herz- und Skelettmuskulatur exprimiert, während popdc3 sowohl in der quergestreiften Muskulatur, als auch im Gehirn exprimiert wurde. Um die Funktion dieser Gene zu untersuchen, wurde die Prozessierung der prä-mRNA mit Hilfe von Morpholinos unterdrückt, die gegen die Spleißdonor bzw. -akzeptorsequenzen von popdc2 und popdc3 gerichtet waren. Das Fehlen von popdc2 im Zebrafisch resultierte in einer Fehlentwicklung der Gesichts- und Schwanzmuskulatur. Im Herzen der popdc2-Morphanten waren ventrikuläre Überleitungsstörungen mit einem 2:1 oder 3:1 Rhythmus zu beobachten. Analysen der Calciumfreisetzung mittels SPIM (selective plane illumination microscopy) in der transgenen Zebrafischlinie Tg(cmlc2:gCaMP)s878, die einen fluoreszierenden Calciumindikator exprimiert, zeigten in den popdc2-Morphanten einen AV-Block bis hin zum kompletten Herzblock. Interessanterweise ergaben vorläufige Analysen in popdc3-Morphanten einen ähnlichen Phänotyp. Um eine mögliche morphologische Ursache der beobachteten AV-Überleitungsstörung zu finden, habe ich die Struktur des AV-Kanals von popdc2-Morphanten mit Hilfe der transgenen Zebrafischlinie Tg(cmlc2:eGFP-rass883) und konfokaler Mikroskopie untersucht. Allerdings konnte ich kein Anzeichen für morphologische Veränderungen erkennen. Um sicherzugehen, dass der beobachtete Rhythmusphänotyp der popdc2-Morphanten nicht auf einer myokardialen Störung beruht oder auf einem Defekt bei der Klappenentwicklung, wurden zusätzlich Lebendaufnahmen angefertigt, die zeigten, dass die Klappen normal entwickelt waren. In Übereinstimmung mit den Daten aus den Knockout-Mäusen haben die popdc2- und popdc3-Gene auch im Zebrafisch eine wichtige Funktion bei der elektrischen Reizleitung im Herzen. Allerdings sind die beiden Gene für die Erregungsbildung im Sinusknoten nicht essentiell, sondern werden für die Signalübertragung im AV-Kanal benötigt. Um die biologische Signifikanz der cAMP-Bindung zu untersuchen, wurden Wildtyp-Popdc1 und Punktmutanten, die eine Reduktion in der cAMP-Bindung aufweisen, nach RNA-Injektion in Zebrafischembryonen überexprimiert. Die Injektion von Wildtyp-Popdc1 induzierte eine embryonale Herzinsuffizienz, die durch perikardiales Ödem und venösen Blutstau charakterisiert war. Die Fähigkeit der Popdc1-Punktmutanten, einen Herzphänotyp nach Überexpression zu induzieren, war direkt korreliert mit der Bindungsaffinität für cAMP. Diese Daten lassen den Schluss zu, dass die Fähigkeit der cAMP-Bindung eine wichtige biologische Eigenschaft der Popdc-Proteinfamilie darstellt. KW - Zebrabärbling KW - Genexpression KW - Herzmuskel KW - Herz KW - Popdc Genfamilie KW - heart KW - popdc gene family Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-49413 ER -