Target genes of ß-catenin in early mouse limb development

Untersuchungen der letzten Jahre führten zum ß-catenin/Wnt-Signalweg als möglichen Schlüssel zur Kontrolle der Differenzierung von mesenchymalen Zellen zu Knorpelzellen. Stabilisierung von ß-catenin in der frühen Gliedmaßenanlage scheint die Expression des Transkriptionsfaktors Sox9 und damit die Differenzierung in Knorpelzellen zu inhibieren. Es ist jedoch unklar wie diese Kontrolle von Sox9 durch ß-catenin/Wnt erfolgt. Diese Diplomarbeit ist Teil eines Projektes das zum Ziel hat den Mechanismus dieser Kontrolle aufzuklären. Dazu sollen ß-catenin/Wnt-regulierte Gene der frühen Gliedmaßenentwicklung identifiziert und auf einen möglichen Einfluss auf die Sox9-Expression getestet werden. Am Beginn dieser Diplomarbeit standen bereits Daten einer Genexpres-sionsstudie zur Verfügung. Nach Analyse dieser Daten wurden 48 mögliche Kanditatengene mittels In-situ-Hybridisierung bzw. RT-PCR validiert. Es konnten so 10 Gene identifiziert werden, welche in der Gliedmaßenanlage von Embryonen, die konditionell stabilisiertes ß-catenin expremieren, hochreguliert sind. Darunter befanden sich die Transkriptionsfaktoren Tcf4 (Itf2), Irx5 und Fra2 (Fosl2). Die Promotoren dieser Gene wurden in Luciferase Reporter Assays auf ihre ß-catenin/Wnt Abhängigkeit untersucht. Weiteres wurden diese Gene in Luciferase Assays mit einem Sox9-Promoter-Reporterkonstrukt getestet. Um Hinweise auf die Funktion der Gene in vivo zu erhalten wurden die kodierenenden Regionen der Gene in einen Vektor kloniert, welcher eine transiente Überexpression in transgenen Embryos erlauben sollte. Für das Gen Tcf4 wurde eine erste Runde von Injektionen durchgeführt. Diese lieferten jedoch nur einen transgenen Embryo, welcher jedoch einen Phenotyp in der Gliedmaßenanlage zeigte. Durch das zeitlich bedingte Ende der Arbeit waren leider keine weiteren Analysen möglich. Zusammenfassend wurden in dieser Arbeit eine Anzahl möglicher Zielgene des ß-catenin/Wnt-Signalweges identifiziert. Für zwei davon Irx5 und Tcf4 ist eine direkte Abhängigkeit vom ß-catenin/Wnt Signalweg sehr wahrscheinlich. Weiters hatten beide Gene das Potential die Sox9-Expression zumindest in vitro zu inhibieren. Jedoch wären weitere Untersuchungen notwendig um eine mögliche Funktion dieser Gene in der Knorpelzellendifferenzierung bzw. Gliedmaßenentwicklung aufzudecken.In recent years Wnt-Signaling has been implicated to play an important role in the control of differentiation of mesenchymal cells to chondrocytes. A key observation has been that stabilization of β-catenin in the early limb bud mesenchyme represses differentiation of mesenchymal cells into skeletal precursors. Probably this is mediated by transcriptional repression of the transcription factor Sox9, which is a master regulator in chondrocyte differentiation. The object of this thesis was to identify putative mediators of this Sox9 repression. Target genes of canonical Wnt signaling in limb bud development were identified and tested for their potential to repress Sox9. To do so I used gene expression data already available. I validated 48 candidate genes suggested by the data analysis using in situ hybridisation and semi-quantitative RT-PCR, respectively. 10 genes could be confirmed as being positively regulated in the limbs of ß-catenin gain of function mice. For three of them, the transcription factors Tcf4 (Itf2), Irx5 and Fosl2 (FRA2), I cloned parts of their promoters into luciferase reporter vectors and tested their response to ß-catenin/Wnt-signaling. Furthermore I cloned their cDNAs and tested their effect on part of the Sox9 promoter in luciferase reporter assays. For Tcf4 and Irx5 I could confirm their positive response to canonical Wnt-signaling, as well as their potential to repress Sox9 in vitro. To gain insight into the in vivo function of these genes, constructs for specific overexpression in the early limb bud mesenchyme have been generated. By the end of the thesis work one round of pronucleus injections has been carried out for the gene Tcf4, which yielded in one transgenic embryo with a strong limb phenotype. Unfortunately further analysis was not carried out due to the fact of the diploma work had ended. In conclusion this work suggests a number of new genes as targets of Wnt/ß-catenin signaling in limb development. For two of them Irx5 and Tcf4 I established a possible direct regulation by the ß-catenin/Wnt-pathway and that they have the potential to inhibit Sox9 at least in vitro. Further studies would now be necessary to examine whether they also excert a negative effect on Sox9 in vivo and to establish their potential functions during limb development

Оценка эффективности комбинированной терапии у больных артериальной гипертензией

Представлены результаты исследования воздействия комбинации антигипертензивного препарата аккупро и психотропного препарата золофта на клинические показатели больных артериальной гипертензией и их психологическое состояние и качество жизни. Показана высокая эффективность комбинированной терапии.The findings of the research of the effect of combination of an antihypertensive drug Accupro and a psychotropic drug Zoloft on clinical parameters in patients with arterial hypertension as well as their mental state and quality of life are presented. A high efficacy of the combined therapy is shown

Entpd5 is essential for skeletal mineralization and regulates phosphate homeostasis in zebrafish

Bone mineralization is an essential step during the embryonic development of vertebrates, and bone serves vital functions in human physiology. To systematically identify unique gene functions essential for osteogenesis, we performed a forward genetic screen in zebrafish and isolated a mutant, no bone (nob), that does not form any mineralized bone. Positional cloning of nob identified the causative gene to encode ectonucleoside triphosphate/diphosphohydrolase 5 (entpd5); analysis of its expression pattern demonstrates that entpd5 is specifically expressed in osteoblasts. An additional mutant, dragonfish (dgf), exhibits ectopic mineralization in the craniofacial and axial skeleton and encodes a loss-of-function allele of ectonucleotide pyrophosphatase phosphodiesterase 1 (enpp1). Intriguingly, generation of double-mutant nob/dgf embryos restored skeletal mineralization in nob mutants, indicating that mechanistically, Entpd5 and Enpp1 act as reciprocal regulators of phosphate/pyrophosphate homeostasis in vivo. Consistent with this, entpd5 mutant embryos can be rescued by high levels of inorganic phosphate, and phosphate-regulating factors, such as fgf23 and npt2a, are significantly affected in entpd5 mutant embryos. Our study demonstrates that Entpd5 represents a previously unappreciated essential player in phosphate homeostasis and skeletal mineralization

Wilms Tumor 1b defines a wound-specific sheath cell subpopulation associated with notochord repair

Regenerative therapy for degenerative spine disorders requires the identification of cells that can slow down and possibly reverse degenerative processes. Here, we identify an unanticipated wound-specific notochord sheath cell subpopulation that expresses Wilms Tumor (WT) 1b following injury in zebrafish. We show that localized damage leads to Wt1b expression in sheath cells, and that wt1b+cells migrate into the wound to form a stopper-like structure, likely to maintain structural integrity. Wt1b+sheath cells are distinct in expressing cartilage and vacuolar genes, and in repressing a Wt1b-p53 transcriptional programme. At the wound, wt1b+and entpd5+ cells constitute separate, tightly-associated subpopulations. Surprisingly, wt1b expression at the site of injury is maintained even into adult stages in developing vertebrae, which form in an untypical manner via a cartilage intermediate. Given that notochord cells are retained in adult intervertebral discs, the identification of novel subpopulations may have important implications for regenerative spine disorder treatments

Revisiting in vivo staining with alizarin red S - a valuable approach to analyse zebrafish skeletal mineralization during development and regeneration

Background The correct evaluation of mineralization is fundamental for the study of skeletal development, maintenance, and regeneration. Current methods to visualize mineralized tissue in zebrafish rely on: 1) fixed specimens; 2) radiographic and μCT techniques, that are ultimately limited in resolution; or 3) vital stains with fluorochromes that are indistinguishable from the signal of green fluorescent protein (GFP)-labelled cells. Alizarin compounds, either in the form of alizarin red S (ARS) or alizarin complexone (ALC), have long been used to stain the mineralized skeleton in fixed specimens from all vertebrate groups. Recent works have used ARS vital staining in zebrafish and medaka, yet not based on consistent protocols. There is a fundamental concern on whether ARS vital staining, achieved by adding ARS to the water, can affect bone formation in juvenile and adult zebrafish, as ARS has been shown to inhibit skeletal growth and mineralization in mammals. Results Here we present a protocol for vital staining of mineralized structures in zebrafish with a low ARS concentration that does not affect bone mineralization, even after repetitive ARS staining events, as confirmed by careful imaging under fluorescent light. Early and late stages of bone development are equally unaffected by this vital staining protocol. From all tested concentrations, 0.01 % ARS yielded correct detection of bone calcium deposits without inducing additional stress to fish. Conclusions The proposed ARS vital staining protocol can be combined with GFP fluorescence associated with skeletal tissues and thus represents a powerful tool for in vivo monitoring of mineralized structures. We provide examples from wild type and transgenic GFP-expressing zebrafish, for endoskeletal development and dermal fin ray regeneration

Putting crystals in place - the regulation of biomineralization in zebrafish

In humans the skeleton has a number of crucial functions: It provides protection and mechanical support to the body, is an important metabolic organ and represents the place of adult hematopoiesis. There are a number of human diseases related to the muscoskeletal system; prominent examples are osteoporosis and osteoarthritis. Osteoporosis is characterized by a gradual reduction of bone mass due to an imbalance between bone formation and resorption, which leads to an increased risk of fracture. In osteoarthritis, loss of joint cartilage tissue with subsequent inflammation and formation of bone spurs leads to chronic pain and loss of joint flexibility. These diseases represent a major burden to the health systems in an ever-aging society. Another aspect related to skeletal biology is the regulation of bio-mineralization. Ectopic (displaced) mineralizations can occur in most soft tissues and are a burden for patients with systemic mineral imbalance such as in chronic kidney disease, but are also seen as a consequence of rare, monogenetic diseases, injury or aging. Tissues of the cardiovascular system are particularly inclined to ectopic mineralization. Such ectopic mineralization in the cardio-vasculature correlates with severe clinical symptoms such as myocardial infarction. In recent years it has become clear that, mechanistically, bio-mineralization is a process that has to be actively inhibited as a default state. This inhibition must be released in a rigidly controlled manner in order for mineralization to occur in skeletal elements. A central aspect of this concept is the tightly controlled balance between phosphate, a constituent of the biomineral hydroxyapatite, and pyrophosphate, a physiochemical inhibitor of mineralization. The research described in my thesis investigated zebrafish mutants showing defective bone-mineralization. The mutant “no bone” (nob), completely lacks mineralization, and the mutant “dragonfish” (dgf), shows excessive and pathologic mineralization in multiple tissues. The respective mutations could be attributed to the genes entpd5 (nob) and enpp1 (dgf), respectively. The well-established role of enpp1 as an enzyme generating pyrophosphate, allowed us to characterize entpd5, as a previously unknown and critical factor regulating bio-mineralization in zebrafish via the phosphate/pyrophosphate axis. A further key finding was that in response to ectopic mineralization in dgf mutants a rapid cellular response occurs by cells with osteoclastic (mineral resorbing) properties. This finding is of relevance for patients with a disease named generalized-arterial-calcification-of-infancy (GACI), which is caused by mutations in ENPP1. One treatment option for them is bisphosphonates which can inhibit further mineralizations, but are also known for their inhibitory effect on osteoclasts, which is exploited in the treatment of osteoporosis. Hence novel drugs inhibiting mineralization are desirable. We show in a proof-of-concept that dgf mutants can be a useful and easy to evaluate model for testing candidate compounds. Additionally we describe a zebrafish mutant for osterix, a key transcription factor in osteoblasts; and discovered that while the formation of the head skeleton in zebrafish depends on osterix, the first mineralized pattern in the axial skeleton occurs independently of osterix, pointing towards the existence of a distinct population of osteogenic cells that differs from the typical osteoblast

Roboter, Digitalisierung, Industrie 4.0 und deren Auswirkungen auf Gesellschaft und Arbeitswelt : eine Diskursanalyse der Berichterstattung in österreichischen Tageszeitungen von 2010-2016

1569 Artikel fünf verschiedener österreichischer Tageszeitungen, die anhand der Stichworte „robot“, „Industrie 4.0“ und „Digitalisierung & arbeit“ im Zeitraum 2010-2016 aufschienen werden analysiert. Dafür wurden die Ansätze der Diskursanalyse von Keller und Diaz-Bone kombiniert. Im Rahmen der Arbeit werden eine zentrale Rolle der Wissenschaft in der Erstellung von Zukunftsvorstellungen, eine Vielfalt von angeblich von technologischer Arbeitslosigkeit betroffenen Berufsgruppen, Parallelen zur CIM-Debatte (Computer-integrated Manufacturing) in den 1980er-Jahren und eine Schärfung der menschlichen Identität im Vergleich mit Robotern deutlich. Sechs verschiedene Betrachtungsweisen und Einstellungen bezüglich der Technologien werden charakterisiert. Diese spiegeln die Einstellung bezüglich der neuen Technologien und den erwarteten Auswirkungen wieder und sind: ‚politische Aufgabe‘, ‚Chance‘, ‚praktisch-technisch‘, ‚pragmatisch-optimistisch‘, ‚distanziert-skeptisch‘ und ‚beunruhigt-verängstigt‘. Es wird gezeigt, dass die Betrachtungsweisen teilweise in Argumenten bestehender Paradigmen bezüglich der Auswirkungen von Technologien auf Gesellschaften bzw. Organisationen fundiert sind. Diese sind: Der Technologiedeterminismus, das ‚Management of Technology‘-Paradigma und das Paradigma ‚Politisches Interesse‘. Dabei lässt sich erkennen, dass ein gänzliches Hinterfragen, wie es im interpretivistischen Paradigma erfolgt, im medialen Diskurs fehlt. Zusätzlich wird die Häufigkeit des Auftretens der Betrachtungsweisen untersucht. Dieses variiert sowohl zeitlich als auch zwischen den Medien. Zudem wird die Legitimationsfunktion des Technologiediskurses, insbesondere für politisches Handeln und das Vorherrschen der Überzeugung vom Technologiedeterminismus, beleuchtet.Following the approach of “discourse analysis”, 1569 articles from five different Austrian newspapers containing the keywords “robot”, “Industry 4.0” and “digitalization & work” during the period 2010 to 2016, were analyzed in this master thesis. For this purpose, the methodological concepts of Keller and Diaz-Bone for discourse analysis were combined. Insights include the detected importance of science in determining expectations for the future, a wide variety in the professions that are deemed to be replaced by technology, clear parallels with previous discussions about CIM (Computer-integrated manufacturing) in the 1980s, and a new way of clarifying the definition of human identity in comparison to the robot. Six perspectives on recent technological change and its effects on work can be found in this discourse, which reflect the opinion about new technologies and their expected impact. They are: ‘political interest, ‘chance, ‘practical-technical, ‘pragmatic-optimistic, ‘distanced-skeptical, ‘concerned-scared. It is shown that all these perspectives are founded in the argumentations of previously-existing paradigms about technological change and its effects on society and organizations. The occurring paradigms are: ‘Technological Determinism, ‘Management of Technology, and ‘Political Interests. It can be observed that a total questioning of the occurring technological change, as it takes place in the ‘Interpretivist Paradigm, is missing from this media discourse. The prevalence of the six perspectives varies over time as well as between the newspapers in the sample. In the course of this analysis, it became evident that technological discourse is a chance for political legitimization and that technological determinism is dominant in this discourse.Carla Apschner, B.A. (Econ.)Abweichender Titel laut Übersetzung des Verfassers/der VerfasserinZusammenfassungen in Deutsch und EnglischKarl-Franzens-Universität Graz, Masterarbeit, 2017(VLID)229345

Ultras : elements of identity creation, linguistic creativity and the role of English

Jan-Arne ApschnerAlpen-Adria-Universität Klagenfurt, Diplomarbeit, 2018(VLID)527201

Not All Bones are Created Equal - Using Zebrafish and Other Teleost Species in Osteogenesis Research

Developmental osteogenesis and pathologies of mineralized tissues are areas of intense investigations in the mammalian field, but different from other areas of organ formation and developmental biology, zebrafish have been somewhat slow in joining the area of bone research. In recent years, however, genetic screens have provided a number of exciting mutants, and transgenic lines have been developed that permit visualization of osteoblasts and osteoclasts in vivo. We here review some of the recent literature and provide examples where insights from studies in zebrafish have complemented the information available from mammalian models or clinical studies. Furthermore, we provide a comparative overview about different forms of bone within the teleost lineage, and between teleosts and mammal