The role of Ken&Barbie in JAK/STAT signalling during development of Drosophila melanogaster

Cell-cell interactions during embryonic development are crucial in the co-ordination of growth, differentiation and maintenance of many different cell types. To achieve this co-ordination each cell must properly translate signals received from neighbouring cells, into spatially and temporally appropriate developmental responses. A surprisingly limited number of signal pathways are responsible for the differentiation of enormous variety of cell types. As a result, pathways are frequently 'reused' during development. Thus, in mammals the JAK/STAT pathway is required during early embryogenesis, mammary gland formation, hematopoiesis and, finally, plays a pivotal role in immune response. In the canonical way, the JAK/STAT pathway is represented by a transmembrane receptor associated with a Janus kinase (JAK), which upon stimulation by an extra-cellular ligand, phosphorylates itself, the receptor and, finally, the signal transducer and activator of transcription (STAT) molecules. Phosphorylated STATs dimerise and translocate to the nucleus where they activate transcription of target genes. The JAK/STAT pathway has been conserved throughout evolution, and all known components are present in the genome of Drosophila melanogaster. Besides hematopoietic and immunity functions, the pathway is also required during development for processes including embryonic segmentation, tracheal morphogenesis, posterior spiracle formation etc. This study describes Drosophila Ken&Barbie (Ken) as a selective regulator of JAK/STAT signalling. ken mutations identified in a screen for modulators of an eye overgrowth phenotype, caused by over-expression of the pathway ligand unpaired, also interact genetically with the pathway receptor domeless (dome) and the transcription factor stat92E. Over-expression of Ken can phenocopy developmental defects known to be caused by the loss of JAK/STAT signalling. These genetic interactions suggest that Ken may function as a negative regulator of the pathway. Ken has C-terminal Zn-finger domain, presumably for DNA binding, and N-terminal BTB/POZ domain, often found in transcriptional repressors. Using EGFP-fused construct expressed in vivo revealed nuclear accumulation of Ken. Therefore, it is proposed that Ken may act as a suppresser of STAT92E target genes. An in vitro assay, termed SELEX, determined that Ken specifically binds to a DNA sequence, with the essential for DNA recognition core overlapping that of STAT92E. This interesting observation suggests that not all STAT92E sites may also allow Ken binding. Strikingly, when effects of ectopic Ken on the expression of putative JAK/STAT pathway target genes were examined, only a subset of the genes tested, namely vvl, trh and kni, were down-regulated by Ken, whereas some others, such as eve and fj, appeared to be unresponsive. Further analysis of vvl, one of the genes susceptible to ectopic Ken, was undertaken. In the developing hindgut, expression of vvl is JAK/STAT pathway dependent, but remains repressed in the posterior spiracles, despite the stimulation of STAT92E by Upd in their primordia. Importantly, ken is also expressed in the developing posterior spiracles. Strikingly, up-regulation of vvl is observed in these tissues in ken mutant embryos. These imply that while ectopic Ken is sufficient to repress the expression of vvl in the hindgut, endogenous Ken is also necessary to prevent its activation in the posterior spiracles. It is therefore conceivable that ectopic vvl expression in the posterior spiracles of the ken mutants may be the result of de-repression of endogenous STAT92E activity. Another consequence of these observations is a fine balance that must exist between STAT92E and Ken activities. Apparently, endogenous level of Ken is sufficient to repress vvl, but not other, as yet unidentified, JAK/STAT pathway targets, whose presumable activation by STAT92E is required for posterior spiracle development as the embryos mutant for dome, the receptor of the pathway, show severe spiracle defects. These defects are also observed in the embryos mis-expressing Ken. Though it is possible that the posterior spiracle phenotype caused by higher levels of Ken results from a JAK/STAT pathway independent activity, it seems to be more likely that Ken acts in a dosage dependent manner, and extra Ken is able to further antagonise JAK/STAT pathway target genes. While STAT92E binding sites required for target gene expression have been poorly characterised, the existence of genome data allows the prediction of candidate STAT92E sites present in target genes promoters to be attempted. When a 6kb region containing the putative regulatory domains flanking the vvl locus are examined, only a single potential STAT92E binding site located 825bp upstream of the translational start can be detected. Strikingly, this site also includes a perfect Ken binding sequence. Such an in silico observation, though consistent with both Ken DNA binding assay in vitro and regulation of STAT92E target genes in vivo, however, requires further analysis. The JAK/STAT pathway is implicated in a variety of processes during embryonic and larval development as well as in imago. In each case, stimulation of the same transcription factor results in different developmental outcomes. While many potential mechanisms have been proposed and demonstrated to explain such pleiotropy, the present study indicates that Ken may represent another mechanism, with which signal transduction pathways are controlled. Ken selectively down-regulates a subset of potential target genes and so modifies the transcriptional profile generated by activated STAT92E - a mechanism, which may be partially responsible for differences in the morphogenetic processes elicited by JAK/STAT signalling during development.

Molecular mechanisms controlling the precision of chemokine guided cell migration

By the end of the first day of embryonic development, zebrafish primordial germ cells (PGCs) arrive at the site where the gonad develops. In our study we investigated the mechanisms controlling the precision of primordial germ cell arrival at their target. We found that in contrast with our expectations which were based on findings in Drosophila and mouse, the endoderm does not constitute a preferred migration substrate for the PGCs. Rather, endoderm derivatives are important for later stages of organogenesis keeping the PGC clusters separated. It would be interesting to investigate the precise mechanism by which endoderm controls germ cell position in the gonad. In their migration towards the gonad, zebrafish germ cells follow the gradient of chemokine SDF-1a, which they detect using the receptor CXCR4b that is expressed on their membrane. Here we show that the C-terminal region of CXCR4b is responsible for down-regulation of receptor activity as well as for receptor internalization. We demonstrate that receptor molecules unable to internalize are less potent in guiding germ cells to the site where the gonad develops, thereby implicating chemokine receptor internalization in facilitating precision of migration during chemotaxis in vivo. We demonstrate that while CXCR4b activity positively regulates the duration of the active migration phases, the down-regulation of CXCR4b signalling by internalization limits the duration of this phase. This way, receptor signalling contributes to the persistence of germ cell migration, whereas receptor down-regulation enables the cells to stop and correct their migration path close to the target where germ cells encounter the highest chemokine signal. Chemokine receptors are involved in directing cell migration in different processes such as lymphocyte trafficking, cancer and in the development of the vascular system. The C-terminal domain of many chemokine receptors was shown to be essential for controlling receptor signalling and internalization. It would therefore be important to determine whether the role for receptor internalization in vivo as described here (allowing periodical corrections to the migration route) and the mechanisms involved (reducing the level of signalling) apply for those other events, too.

Analysis of the electronic structure, hyperfine structure, and volume isotope shifts in the low lying states of BA I and BA II

Relativistic multi-configuration Dirac Fock (MCDF) wavefunctions coupled to good angular momentum J have been calculated for low lying states of Ba I and Ba II. These wavefunctions are compared with semiempirical ones derived from experimental atomic energy levels. It is found that significantly better agreement is obtained when close configurations are included in the MCDF wavefunctions. Calculations of the electronic part of the field isotope shift lead to very good agreement with electronic factors derived from experimental data. Furthermore, the slopes of the lines in a King plot analysis of many of the optical lines are predicted accurately by these calculations. However, the MCDF wavefunctions seem not to be of sufficient accuracy to give agreement with the experimental magnetic dipole and electric quadrupole hyperfine structure constants.

Nachhaltige Energie für ländliche Entwicklung in dem Sub-Sahara Afrika: interdisziplinäre Herangehensweise und organisatorische Herausforderung

Angesichts der Geschichte der Entwicklungspolitik, ist diese Arbeit darauf ausgerichtet, einige Beobachtungen in Bezug auf die so genannte Entwicklung hervorzuheben; insbesondere auf die andauernde prekäre Situation und Armut in ländlichen afrikanischen Gebieten. Armut ist nach Amartya SEN – weiter präzisiert von J.L. Dubois – die Deprivation von „Fähigkeiten“, die Individuen und lokale Gemeinschaften zu ausgeschlossenen und vergessenen Akteuren des Systems machen. Das nennt Paulo Freire, das Menschen zu „Objekten“ gemacht werden. Es rechtfertigt die starke Annahme, die in dieser Studie getroffen wird, dass vielmehr die Menschen als „Subjekte“ ihrer Veränderung und Entwicklung im Mittelpunkt stehen. Die Arbeit zeigt und erklärt in historischer Chronologie, wie die Entwicklungspolitiken und unterschiedliche Beteiligte auf allen Ebenen diese Situation verursachen. Trotz alledem bleiben die Individuen und lokalen Gemeinschaften, die in Symbiose mit ihrer natürlichen Umwelt leben, die reich an verschiedenen Ressourcen und Potentialen ist, als Reaktion darauf und gleichzeitig als Überlebensstrategie zutiefst verbunden mit dem, was sie vor Ort haben, womit sie eine tiefere und intensive Beziehung besitzen, wenn man von ihrer Geschichte, ihrer Kultur und der Handlungslogik ausgeht. Für externe Akteure, die sie über das vorhandene System dominieren und beeinflussen bleiben sie „Objekte“, aber in der Vielzahl ihrer endogenen Initiativen, zeigen sie die Fähigkeit und Substanz, die beweisen, dass sie auf ihrer Ebene das eigentliche Subjekt sind, die dynamischen Akteure. Aber isolierte Initiativen auf spezifische reale Bedürfnisse bei gleichzeitiger Dominierung durch das System mit seiner Marktlogik, führt dies langfristig nur zu dem Zirkulus Vitiosus der Armut. Daher ist eine ganzheitliche Sicht entscheidend für nachhaltige Entwicklung und für die notwendige Veränderung. Es geht nicht nur um die Veränderung des Systems und die Wahl politischer Maßnahmen, sondern genau genommen um das Verhalten der Akteure auf allen Ebenen und die Art der Beziehungen zwischen ihnen allen. Es ist eine Frage des erneuten Überdenkens des Entwicklungspfades, der andere Logik, Visionen, Interessen und Strategien aller Beteiligten, unserer so genannten Akteure einschließt. Ob dies von endogenen Initiativen oder neuen gemeinsamen Projekten ausgeht: man wird in einen Prozess kollektiven Lernens eintreten, den Paul Singer und Clarita Müller-Plantenberg erläutern und entwickeln in dem Konzept der Inkubation und Solidarischen Ökonomie, die Eigeninitiative, Selbstbestimmung und Selbstverwaltung von lokalen Gemeinschaften und die Öffnung für eine Neu-Konzeptualisierung und Institutionalisierung einschließt. So ein Prozess ist nur mit einem interdisziplinären Rahmen möglich. Dieser Rahmen soll auf einer zusätzlicher Kommunikation zwischen den Akteuren und Sozialwissenschaften beruhen und mit jenen, die auf dem Feld der Technologie arbeiten. So können dann technische „Experten“ angesichts eines technischen Projektfehlers, der aufgrund von bestimmten sozialen und kulturellen Realitäten zustande kam sagen, „es ist kein Scheitern ; es war ein Schritt innerhalb eines Lernprozesse der in die technischen Projekte und Studien einbezogen werden muss“. Wir haben das Energiethema gewählt; und insbesondere, Energie für eine nachhaltige ländliche Entwicklung in Subsahara-Afrika, um den Weg von der Theorie in die Praxis zu illustrieren und experimentell auszuprobieren, den Weg von den Beobachtungen zu der Veränderung, wobei Fragen, Annahmen, Strategien und konkrete Aktionen für den Wandel behandelt werden. Wir nennen unseren experimentellen Weg: DRIEE, das heißt auf Deutsch Ländliche Entwicklung und Inkubation von Energieunternehmen. Dabei gehen wir davon aus, dass: - Energie im Allgemeinen auf der internationalen Ebene fast gleichbedeutend mit Elektrizität ist. Heute bestehen die wichtigsten Bedürfnisse nach Energie dort wo die agro-pastorale Produktion, das Kochen, die Nahrungsmittelkonservierung und Verarbeitung …etc. stattfindet. - Diese ländliche Bevölkerung zu etwa 80% der nationalen Wirtschaft ausmacht. Dass sie gleichzeitig aber nur zu weniger als 5% der Energieproduktion Zugang hat, was oft auf Licht reduziert ist und nicht einmal ihrer Produktion zugute kommen kann. - Die Projekte für Energie und Elektrizität vor allem auf die Technologischen Fragen konzentriert sind und weniger auf die Bedürfnisse. Fast die Gesamtheit der Fonds für Energie wird in Bezug auf die Investitionen Infrastruktur der Produktion und Verteilung durch die konventionellen zentralisierten Netze geplant. Angesichts dieser Analysen gehen die in dieser Arbeit vorgenommenen Studien in Gambia und Kamerun von Bestandsaufnahmen und / oder beschreibenden regionalen Analysen aus: - von Bedürfnissen, von Praktiken und lokalen Initiativen von Fragen der Energie, für einzelne Professionen, Haushalte, Gruppen, spezifische Gruppen, wie Frauen, ländliche Gemeinden mit ihren spezifischen Charakteristika. - Von Potentialen: natürliche lokale Energieressourcen, soziokulturelle Ressourcen – so z.B. die empirisch feststellbaren menschliche Ressourcen wie endogenes Wissen und praktische organisatorische Fähigkeiten gegenüber den Problemen der Energie. Dieser experimentelle Schritt von Handlungsforschung (DRIEE) in Kamerun führte zu der Gründung einer Organisation, über die und mit der wir die Logik der Inkubation und Solidarischen Ökonomie einführen. Das ist FERDEDSI, das heißt auf Deutsch „Forum für Erneuerbare Energie – Nachhaltige Entwicklung und Internationale Solidarität“. Zunächst war dies eine Energiegenossenschaft und dann (im Prozess) wurde es zu einer institutionellen Nische von mehreren Mikro Initiativen in ländlichen Gebieten. FERDEDSI ist ein Prozess der Inkubation und ein Inkubator ist also gleichzeitig ein inkubiertes Energieunternehmen aber auch ein Inkubator für lokale Organisationen. Die ersten Aktionen finden in den Departments von Noun und Ménoua in der westlichen Provinz von Kamerun statt. Während der Forschungsperiode findet akademische Austausch statt (Nord-Süd und Süd-Süd), diese ist dabei zu formalen Partnerschaften zu werden, nicht nur zwischen Universitäten sondern genauer lokale Organisationen und Universitäten. Dieser letzte Typ von Partnerschaften, die die solidarische Ökonomie ausmachen ist auch eine Innovation des Prozesses für die afrikanischen Fälle, die dem Beispiel dessen, was in Lateinamerika geschieht, folgen. So kommt es zu gegenseitiger sinnvoller Ausbildung in den internationalen Arbeitsgruppen und Seminaren der Universität.

Characterization of DnmA, the Dnmt2 homolog in Dictyostelium discoideum

Eukaryotic DNA m5C methyltransferases (MTases) play a major role in many epigenetic regulatory processes like genomic imprinting, X-chromosome inactivation, silencing of transposons and gene expression. Members of the two DNA m5C MTase families, Dnmt1 and Dnmt3, are relatively well studied and many details of their biological functions, biochemical properties as well as interaction partners are known. In contrast, the biological functions of the highly conserved Dnmt2 family, which appear to have non-canonical dual substrate specificity, remain enigmatic despite the efforts of many researchers. The genome of the social amoeba Dictyostelium encodes Dnmt2-homolog, the DnmA, as the only DNA m5C MTase which allowed us to study Dnmt2 function in this organism without interference by the other enzymes. The dnmA gene can be easily disrupted but the knock-out clones did not show obvious phenotypes under normal lab conditions, suggesting that the function of DnmA is not vital for the organism. It appears that the dnmA gene has a low expression profile during vegetative growth and is only 5-fold upregulated during development. Fluorescence microscopy indicated that DnmA-GFP fusions were distributed between both the nucleus and cytoplasm with some enrichment in nuclei. Interestingly, the experiments showed specific dynamics of DnmA-GFP distribution during the cell cycle. The proteins colocalized with DNA in the interphase and were mainly removed from nuclei during mitosis. DnmA functions as an active DNA m5C MTase in vivo and is responsible for weak but detectable DNA methylation of several regions in the Dictyostelium genome. Nevertheless, gel retardation assays showed only slightly higher affinity of the enzyme to dsDNA compared to ssDNA and no specificity towards various sequence contexts, although weak but detectable specificity towards AT-rich sequences was observed. This could be due to intrinsic curvature of such sequences. Furthermore, DnmA did not show denaturant-resistant covalent complexes with dsDNA in vitro, although it could form covalent adducts with ssDNA. Low binding and methyltransfer activity in vitro suggest the necessity of additional factor in DnmA function. Nevertheless, no candidates could be identified in affinity purification experiments with different tagged DnmA fusions. In this respect, it should be noted that tagged DnmA fusion preparations from Dictyostelium showed somewhat higher activity in both covalent adduct formation and methylation assays than DnmA expressed in E.coli. Thus, the presence of co-purified factors cannot be excluded. The low efficiency of complex formation by the recombinant enzyme and the failure to define interacting proteins that could be required for DNA methylation in vivo, brought up the assumption that post-translational modifications could influence target recognition and enzymatic activity. Indeed, sites of phosphorylation, methylation and acetylation were identified within the target recognition domain (TRD) of DnmA by mass spectrometry. For phosphorylation, the combination of MS data and bioinformatic analysis revealed that some of the sites could well be targets for specific kinases in vivo. Preliminary 3D modeling of DnmA protein based on homology with hDNMT2 allowed us to show that several identified phosphorylation sites located on the surface of the molecule, where they would be available for kinases. The presence of modifications almost solely within the TRD domain of DnmA could potentially modulate the mode of its interaction with the target nucleic acids. DnmA was able to form denaturant-resistant covalent intermediates with several Dictyostelium tRNAs, using as a target C38 in the anticodon loop. The formation of complexes not always correlated with the data from methylation assays, and seemed to be dependent on both sequence and structure of the tRNA substrate. The pattern, previously suggested by the Helm group for optimal methyltransferase activity of hDNMT2, appeared to contribute significantly in the formation of covalent adducts but was not the only feature of the substrate required for DnmA and hDNMT2 functions. Both enzymes required Mg2+ to form covalent complexes, which indicated that the specific structure of the target tRNA was indispensable. The dynamics of covalent adduct accumulation was different for DnmA and different tRNAs. Interestingly, the profiles of covalent adduct accumulation for different tRNAs were somewhat similar for DnmA and hDNMT2 enzymes. According to the proposed catalytic mechanism for DNA m5C MTases, the observed denaturant-resistant complexes corresponded to covalent enamine intermediates. The apparent discrepancies in the data from covalent complex formation and methylation assays may be interpreted by the possibility of alternative pathways of the catalytic mechanism, leading not to methylation but to exchange or demethylation reactions. The reversibility of enamine intermediate formation should also be considered. Curiously, native gel retardation assays showed no or little difference in binding affinities of DnmA to different RNA substrates and thus the absence of specificity in the initial enzyme binding. The meaning of the tRNA methylation as well as identification of novel RNA substrates in vivo should be the aim of further experiments.

Right to Food, Food Security and Food Aid Under International Law, or the Limits of a right-based approach

The right to food has become a pillar of international humanitarian and human rights law. The increasing number of food-related emergencies and the evolution of the international order brought the more precise notion of food security and made a potential right to receive food aid emerge. Despite this apparent centrality, recent statistics show that a life free from hunger is for many people all over the world still a utopian idea. The paper will explore nature and content of the right to food, food security and food aid under international law in order to understand the reasons behind the substantial failure of this right-centred approach, emphasising the lack of legal effects of many food-related provisions because of excessive moral connotations of the right to be free from hunger. Bearing in mind the three-dimensional nature of food security, the paper will also suggest that all attention has been focused on the availability of food, while real difficulties arise in terms of accessibility and adequacy. Emergency situations provide an excellent example of this unbalance, as the emerging right to receive food aid focus itself on the availability of food, without improving local production and adequacy. Looking at other evolving sectors of international law, such as the protection of the environment, and particularly the safeguard of biological diversity, alternative solutions will be envisaged in order to “feed” the right to food.