Identifikation von Zielgenen des Transkriptionsfaktors SHOX in der Gliedmaßenentwicklung

Das Gen SHOX (short stature homeobox gene) codiert für einen Transkriptionsfaktor, der für eine korrekte Entwicklung der Gliedmaßen notwendig ist. So führt eine SHOX-Defizienz im Menschen zu verschiedenen Kleinwuchssyndromen, bei denen neben genereller Verkürzung insbesondere des Zeugopodiums (Unterschenkel und Unterarm) auch Fehlbildungen des Handgelenks auftreten können (Leri-Weill-Dyschondrosteosis, Langer-Syndrom, Idiopathischer Kleinwuchs, Turner-Syndrom). Bisher ist mit BNP nur ein SHOX-Zielgen beschrieben worden, doch die molekularen Konsequenzen dieser Regulation sind noch unklar. Ziel der vorliegenden Arbeit war, weitere von SHOX regulierte Gene zu identifizieren, um die Rolle von SHOX während der Gliedmaßenentwicklung besser zu verstehen. In Microarrays und verschiedenen Zellkulturexperimenten wurde ein stark positiver Einfluss von SHOX auf die Expression von FGFR3 und RUNX3 gefunden. Beide Gene codieren für bekannte Faktoren in Gliedmaßenentwicklung und Knochenbildung. Luciferase-Reportergen-Assays in mehreren Zelllinien zeigten einen stark aktivierenden Effekt von SHOX auf den erweiterten FGFR3-Promotor. Auf den RUNX3-Promotorbereich wirkte SHOX ebenfalls moderat aktivierend. ChIP- und EMSA-Ergebnisse lassen vermuten, dass die Regulation von RUNX3 indirekt ist, wäh¬rend SHOX direkt an den erweiterten Promotor von FGFR3 binden kann. Zur Untersuchung dieser Regulationen in der Gliedmaßenentwicklung wurden Experimente in mehreren Modellsystemen zur Chondrozytendifferenzierung durchgeführt. Für RUNX3 konnte in diesen Systemen keine reproduzierbare, SHOX-abhängige Regulation gezeigt werden; möglicherweise ist die in den Zelllinien gefundene Regulation in anderen Geweben oder zu anderen Entwicklungszeitpunkten relevant. FGFR3 wurde jedoch in sogenannten Hühner-Micromass-Kulturen, bei denen embryonale Mesenchymzellen aus den sich entwickelnden Gliedmaßen zu Chondrozyten differenzieren, durch virusvermittelte SHOX-Überexpression herunterreguliert. Diese negative Regulation konnte mittels qRT-PCR und in situ-Hybridisierung nachgewiesen werden und würde eine Erklärung für die sich überwiegend ausschließenden Expressionsmuster beider Gene in den Extremitäten von Hühnerembryonen bieten. Wenn man davon ausgeht, dass die gefundene negative Regulation vor allem im Zeugopodium stattfindet, wie es anhand des Expressionsmusters von SHOX anzunehmen ist, ergibt sich außerdem eine mögliche Erklärung für die bei FGFR3- und SHOX-Mutationen beobachteten Phänotypen im Menschen. So führen FGFR3-Gain-of-Function-Mutationen durch vorzeitige Schließung der Wachstumsfugen zu Zwergwuchs, bei dem vor allem die Knochen des Stylopodiums (Oberschenkel und Oberarm) betroffen sind, während eine SHOX-Defizienz eine Verkürzung insbesondere des Zeugopodiums auslöst. Eine SHOX-Defizienz würde entsprechend der in dieser Arbeit formulierten Hypothese, dass SHOX FGFR3 im Zeugopodium negativ reguliert, zu einer erhöhten Menge von FGFR3 in Ulna und Radius sowie Tibia und Fibula führen, was dort eine schnellere Schließung der Wachstumsfuge und damit die relative Verkürzung dieser Knochen bedingen würde. Andererseits würde SHOX generell die Expression von FGFR3 im Zeugopodium niedriger halten als im Stylopodium, weshalb FGFR3-Gain-of-Function-Mutationen in Humerus und Femur einen stärkeren Effekt zeigen. Klinische Daten aus Patienten, die Mutationen in FGFR3 als auch in SHOX tragen, unterstützen diese Hypothese

Mathe-App als Aktivierungsunterstützung beim Studienstart

Dieser Werkstattbericht beschreibt, wie eine breitere Aktivierung und selbstreguliertes Lernen beim Start an der Hochschule durch Einbindung einer Mathe-App unterstützt wird. Die App kann an jedem Ort genutzt werden, auch in normalen Klassenräumen, im Beispiel durch 400 Teilnehmer/innen des Brückenkurses. Durch ausführliche bedarfsorientierte Erklärungen unterstützt sie bei der Betreuung aktiver Übungsphasen unter heterogenen Bedingungen und nahtlos beim Weiterüben zu Hause. Der App-Ansatz zeigt eine breite Akzeptanz und kann durch die Mobilität Lernsettings begleiten, welche PC-basierte E-Learning-Lösungen nicht adressieren. Die Inhalte für den Übergang Schule-Studium orientieren sich an dem hochschulübergreifenden COSH Mindestanforderungskatalog Mathematik. 10.11.2014 | Eva Decker & Barbara Meier (Offenburg

Dynamic Production of Soluble Extracellular Polysaccharides by Streptococcus mutans

Caries development in the presence of Streptococcus mutans is associated not only with the production of extracellular water-insoluble polymers but also is based on water-soluble polysaccharides. The aim of this study was the evaluation of a novel glucan-specific Lectin assay for monitoring water-soluble EPS produced by S. mutans during several growth periods in different media. S. mutans cultures were grown for 24 h, 48 h, and 144 h in medium deficient of sucrose (A) and medium supplemented with 5% sucrose (B). Microtiter well plates were coated with cell-free supernatants followed by the addition of labeled Concanavalin-A and enzyme substrate. The substrate reactions were kinetically detected at 405 nm. The validation of the assay was performed using carbohydrates dextran, xanthan, and sucrose as reference. This new Concanavalin-A-based assay showed the highest sensitivity for dextran and revealed that the glucan production of S. mutans reached its maximum at 144 h in medium B according to bacterial maturation

The ICF forms a useful framework for classifying individual patient goals in post-acute rehabilitation

OBJECTIVE: Timely goal setting in close collaboration with the patient is essential to successful rehabilitation. We therefore sought to identify goals of patients in early post-acute rehabilitation as predictors of improved functioning. DESIGN: We conducted a prospective multi-centre cohort study in 5 early post-acute rehabilitation facilities. PATIENTS: Patients with musculoskeletal, cardiopulmonary and neurological conditions were recruited between May 2005 and August 2008. METHODS: A semi-structured questionnaire was used to identify patient goals and to assess improvement in overall functioning. Patients' goals were coded according to the International Classification of Functioning, Disability and Health (ICF). By means of a mixed effects model we examined the association between goal attainment and improved functioning. RESULTS: A total of 116 patients gave 546 statements, of which 426 were linked to 74 ICF categories, which were assigned to the existing comprehensive post-acute ICF Core Sets. Improvements in walking, recreation and leisure, pain, and transfer were the most frequently reported goals. In multivariable analysis patients' goal attainment was not a predictor for improved overall functioning. CONCLUSION: The ICF can be used to identify and structure patients' goals. Patients' perspective should be considered in the rehabilitation process

Medium optimization for biomass production of three peat moss (Sphagnum L.) species using fractional factorial design and response surface methodology

Peat moss (Sphagnum) biomass is a promising bioresource of renewable material to substitute peat in growing media. For sustainable production on a large scale, the productivity of Sphagnum mosses has to be increased by optimizing culture conditions. Optimization was achieved using experimental design to determine concentrations of eight factors leading to highest biomass yield. We improved an established Sphagnum medium by reducing the concentrations of NH$_{4}$NO$_{3}$, KH$_{2}$PO$_{4}$, KCl, MgSO$_{4}$, Ca(NO$_{3}$)$_{2}$, FeSO$_{4}$ and a microelement solution up to 50%. Together with sucrose concentrations of 16 g L$^{-1}$ for Sphagnum fuscum and 20 g L$^{-1}$ for Sphagnum palustre and Sphagnum squarrosum, moss productivities were enhanced for all tested species in shake flasks. Further upscaling to 5 L photobioreactors increased the biomass yield: 15 g freshweight resulted in about 630 g for S. fuscum (50-fold), 580 g for S. palustre (40-fold) and 400 g for S. squarrosum (25-fold) in 24 days

Quantitative promoter analysis in Physcomitrella patens: a set of plant vectors activating gene expression within three orders of magnitude

BACKGROUND: In addition to studies of plant gene function and developmental analyses, plant biotechnological use is largely dependent upon transgenic technologies. The moss Physcomitrella patens has become an exciting model system for studying plant molecular processes due to an exceptionally high rate of nuclear gene targeting by homologous recombination compared with other plants. However, its use in transgenic approaches requires expression vectors that incorporate sufficiently strong promoters. To satisfy this requirement, a set of plant expression vectors was constructed and equipped with either heterologous or endogenous promoters. RESULTS: Promoter activity was quantified using the dual-luciferase reporter assay system. The eight different heterologous promoter constructs tested exhibited expression levels spanning three orders of magnitude. Of these, the complete rice actin1 gene promoter showed the highest activity in Physcomitrella, followed by a truncated version of this promoter and three different versions of the cauliflower mosaic virus 35S promoter. In contrast, the Agrobacterium tumefaciens nopaline synthase promoter induced transcription rather weakly. Constructs including promoters commonly used in mammalian expression systems also proved to be functional in Physcomitrella. In addition, the 5' -regions of two Physcomitrella glycosyltransferases (i.e. α1,3-fucosyltransferase and β1,2-xylosyltransferase) were identified and functionally characterised in comparison to the heterologous promoters. Furthermore, motifs responsible for enhancement of translation efficiency – such as the TMV omega element and a modified sequence directly prior the start codon – were tested in this model. CONCLUSION: We developed a vector set that enables gene expression studies, both in lower and higher land plants, thus providing valuable tools applicable in both basic and applied molecular research

Use of endogenous signal sequences for transient production and efficient secretion by moss (Physcomitrella patens) cells

BACKGROUND: Efficient targeting to appropriate cell organelles is one of the bottlenecks for the production of recombinant proteins in plant systems. A common practice is to use the native secretory signal peptide of the heterologous protein to be produced. Though general features of secretion signals are conserved between plants and animals, the broad sequence variability among signal peptides suggests differing efficiency of signal peptide recognition. RESULTS: Aiming to improve secretion in moss bioreactors, we quantitatively compared the efficiency of two human signal peptides and six signals from recently isolated moss (Physcomitrella patens) proteins. We therefore used fusions of the different signals to heterologous reporter sequences for transient transfection of moss cells and measured the extra- and intracellular accumulation of the recombinant proteins rhVEGF and GST, respectively. Our data demonstrates an up to fivefold higher secretion efficiency with endogenous moss signals compared to the two utilised human signal peptides. CONCLUSION: From the distribution of extra- and intracellular recombinant proteins, we suggest translational inhibition during the signal recognition particle-cycle (SRP-cycle) as the most probable of several possible explanations for the decreased extracellular accumulation with the human signals. In this work, we report on the supremacy of moss secretion signals over the utilised heterologous ones within the moss-bioreactor system. Though the molecular details of this effect remain to be elucidated, our results will contribute to the improvement of molecular farming systems