Control of self-renewal and cell fate in Drosophila neural stem cell lineages

Eine einzige Stammzelle ist in der Lage eine komplette Zelllinie bestehend aus mehreren differenzierten Tochterzellen zu generieren. Um dies zu ermöglichen muss die Stammzelle das Gleichgewicht zwischen Selbsterneuerung und Differenzierung bewahren. Die Regulierung dieses Gleichgewichts ist sehr komplex und unterliegt einer sehr strengen Kontrolle. Geht dieses Gleichgewicht verloren, können Tumore entstehen. Deswegen ist es wichtig, jene Mechanismen zu verstehen, welchen die komplexe Regulierung dieses Gleichgewichts unterliegt. In dieser Studie untersuchen wir neuronale Stammzellen, sogenannte Neuroblasten, aus dem larvalen zentralen Nervensystem der Drosophila. Neuroblasten teilen sich asymmetrisch und können sich somit selbst erneuern und gleichzeitig differenzierte Tochterzellen generieren. Es wurde ursprünglich angenommen, dass Neuroblasten sehr „einfache“ Zelllinien generieren, welche aus einer Stammzelle (Neuroblast) und einigen wenigen differenzierten Zellen (Ganglion-Mutterzellen und Neuronen) bestehen. Diese Studie beschreibt einen bisher uncharakterisierten Neuroblastentyp, welcher zunächst intermediäre Vorläufernervenzellen generiert aus denen anschließend die Ganglion-Mutterzellen und Neuronen hervorgehen. Aufgrund dieser Vorläufernervenzellen, welche ebenso wie die Neuroblasten die Fähigkeit besitzen sich selbst zu erneuern, können sehr viel mehr Neuronen in kurzer Zeit generiert werden. Jedoch ist diese Art von Neuroblast genetische instabiler und somit sehr viel sensitiver in Bezug auf die Ausbildung von Tumoren. Bevor sich intermediäre Vorläufernervenzellen teilen und Ganglion-Mutterzellen generiern, durchlaufen sie eine Art Reifeprozess. Dieser Reifeprozess zeichnet sich durch die sukzessive Expression zweier Transkriptionsfaktoren aus. Um die Biologie hinter diesen Vorläufernervenzellen besser zu verstehen, führten wir innerhalb der Neuroblast-Zelllinien eine genomweite Mutantenklassifizierung durch, wobei wir uns hierfür die RNA-Interferenz (RNAi) Bibliothek zu Nutze machten. Es konnten Gene identifiziert werden, welche bei Funktionsverlust zu einer vermehrten Anzahl von Neuroblasten führten. Darunter befand sich ebenfalls das bisher unbekannte Gen CG6049/Barricade (barc). Diese Studie befasst sich mit der Charakterisierung von Barc und zeigt dessen wichtige Bedeutung innerhalb der Neurogenese. Wird Barc mittels RNAi herunter reguliert, verweilen intermediäre Vorläuferzellen in einem unreifen Stadium, anstatt den Reifeprozess vollständig zu durchlaufen, um anschließend Neuronen zu generieren. Somit ist Barc ein neu identifizierter Regulator intermediärer Vorläuferzellen. Barc ist ein Kernprotein, welches zwei RNA Erkennungsmotive und ein Barc/Tat-SF1 Motiv besitzt. Wir zeigen, dass, für die Funktion von Barc innerhalb der intermediären Vorläuferzellen, nur ein RNA Erkennungsmotiv notwendig ist. Ebenso demonstrieren wir in vivo, dass Barc mit der DNS interagiert. Barc ist das homologe Gen des humanen Tat-SF1 und dem CUS2 Gen aus Hefe. Beiden Genen konnte eine Rolle in der transkriptionalen Elongation und des RNA-Spleißmechanismus zugewiesen werden. Um festzustellen ob Barc eine ähnliche Funktion ausübt, etablierten wir ein Zellkultursystem, in welchem wir Barc zunächst effizient ausschalten konnten und anschließend eine Bibliothek von ‚short-capped’ RNAs bzw. mRNAs herstellten. Die nähere Untersuchung dieser Bibliotheken wird uns Aufschluss darüber geben, ob und inwiefern Barc eine Rolle in der Regulierung der transkriptionalen Elongation und/oder des RNA- Spleißmechanismus spielt. Ebenso generierten wir ein mutantes barc Allel und verschiedene transgene Barc-Konstrukte, mittels welcher wir die Funktion von Barc detaillierter analysieren können. Mit Hilfe dieser Konstrukte können wir zum einen untersuchen, ob Barc einen Einfluss auf den Zellzyklus oder das Zellschicksal hat, zum anderen helfen sie uns, um Interaktionspartner von Barc zu identifizieren oder um das Barc-DNA Interaktionsmuster zu entschlüsseln.Stem cells need to control the balance between proliferation and generation of differentiated cells in order to produce functional lineages. Studying the mechanisms that regulate this equilibrium is particularly relevant since defects in this balance can lead to tumorigenesis. We use the asymmetrically dividing Drosophila larval neuroblasts as a model to study how stem cells self-renew and form specific lineages. Larval neuroblasts of the central brain were thought to form rather simple lineages composed of a single stem cell (neuroblast) and of a few differentiating cells (ganglion mother cells – GMCs – and neurons). Here, we present a previously uncharacterized type of larval neuroblast that produces transit-amplifying cells, called secondary neuroblasts or Intermediate Neural Progenitors (INPs), which then produce GMCs and neurons. Additionally, we show that the lineages formed by these rare neuroblasts are particularly important because they produce a very large amount of neurons and are very sensitive to tumor formation. Before INPs can divide to produce GMCs, they need to mature – a process characterized by the successive expression of two transcription factors. To get a better understanding of INP biology, we made use of a genome-wide RNA interference (RNAi) screen, conducted specifically in Drosophila larval neuroblast lineages. In this screen, several genes led to overproliferation of progenitors upon knock down. Among them was the previously unstudied gene CG6049/barricade (barc). In this study, we characterize barc and show that it is important in neuroblast lineages for the production of neurons. Additionally, we show that upon barc RNAi knock down, most INPs remain in an immature state, demonstrating that Barc is a novel regulator of INPs. Barc is a nuclear protein composed of two RNA recognition motifs (RRMs) and a Barc/Tat-SF1 motif (BTS). Here, we show that only the second RRM of Barc is dispensable for the function of the protein in larval neuroblast lineages. Additionally, we demonstrate that Barc associates with DNA in vivo. Barc is the homologue of human Tat- SF1 and yeast CUS2, two proteins that are involved in transcription elongation and splicing. To test whether Barc acts in a similar way, we established a cell culture system where we can efficiently knock down Barc and from which we prepared libraries of short capped RNAs and mRNAs. Analysis of these libraries will allow us to determine whether Barc acts by regulating transcription elongation and/or splicing. Finally, we generated a mutant allele and several rescue constructs that will enable us to study the effect of barc on cell cycle and cell fate, to identify the binding partners of Barc and to determine its binding pattern on DNA

Universal cumulants of the current in diffusive systems on a ring

We calculate exactly the first cumulants of the integrated current and of the activity (which is the total number of changes of configurations) of the symmetric simple exclusion process (SSEP) on a ring with periodic boundary conditions. Our results indicate that for large system sizes the large deviation functions of the current and of the activity take a universal scaling form, with the same scaling function for both quantities. This scaling function can be understood either by an analysis of Bethe ansatz equations or in terms of a theory based on fluctuating hydrodynamics or on the macroscopic fluctuation theory of Bertini, De Sole, Gabrielli, Jona-Lasinio and Landim

Spectral 3D Computer Vision -- A Review

Spectral 3D computer vision examines both the geometric and spectral properties of objects. It provides a deeper understanding of an object's physical properties by providing information from narrow bands in various regions of the electromagnetic spectrum. Mapping the spectral information onto the 3D model reveals changes in the spectra-structure space or enhances 3D representations with properties such as reflectance, chromatic aberration, and varying defocus blur. This emerging paradigm advances traditional computer vision and opens new avenues of research in 3D structure, depth estimation, motion analysis, and more. It has found applications in areas such as smart agriculture, environment monitoring, building inspection, geological exploration, and digital cultural heritage records. This survey offers a comprehensive overview of spectral 3D computer vision, including a unified taxonomy of methods, key application areas, and future challenges and prospects

Modeling On-Board Software Dynamic Architecture: A Related Experience using UML-MARTE

International audienceMARTE (Modeling and Analysis of Real-Time and Embedded Systems) is the UML extension profile dedicated to the modeling of Real-time and Embedded Systems (RTES). Standardized by the OMG, UML-MARTE is well accepted in the Model Based Driven Engineering community. However there still exists a big gap to bridge for its use in operational space projects. Some of the identified limiting factors are (1) the high density of the MARTE specification which provides thousands of defined concepts and though requires a deep investment to be correctly handled and understood, (2) the absence of methodology associated to the notation and (3) the lack of experiences relating to the use of MARTE on realistic and operational system in space domain. This paper presents an experience of using UML-MARTE to model the dynamic architecture of an operational space On-Board Software (OBSW) to make a step towards the adoption of UML-MARTE. The modeling methodology adopted in this study is illustrated by a use case based on an operational OBSW. This experience has been conducted in the scope of a R&D study founded by the CNES with the collaboration of Astrium Satellites and Atos

Thermodynamic formalism and large deviation functions in continuous time Markov dynamics

The thermodynamic formalism, which was first developed for dynamical systems and then applied to discrete Markov processes, turns out to be well suited for continuous time Markov processes as well, provided the definitions are interpreted in an appropriate way. Besides, it can be reformulated in terms of the generating function of an observable, and then extended to other observables. In particular, the simple observable $K$ giving the number of events occurring over a given time interval turns out to contain already the signature of dynamical phase transitions. For mean-field models in equilibrium, and in the limit of large systems, the formalism is rather simple to apply and shows how thermodynamic phase transitions may modify the dynamical properties of the systems. This is exemplified with the q-state mean-field Potts model, for which the Ising limit q=2 is found to be qualitatively different from the other cases.Comment: Proceedings of the conference "Work, dissipation, and fluctuations in nonequilibrium physics" held in Brussels 22-25 March 2006. Submitted to CRA

Setting sub-organellar sights: accurate targeting of multi-transmembrane-domain proteins to specific chloroplast membranes

Engineering novel chloroplast functions requires an understanding of how to accurately target proteins to specific chloroplast sub-compartments. This is particularly difficult in the case of membrane proteins where localization can be confounded by multiple membrane types. In an elegant study, Singhal and Fernandez (2017) have now provided greater insight into this challenge by dissecting out the signals that control differential targeting of two related proteins to specific chloroplast membranes. Further development of this information should inform attempts to direct engineered proteins to specific sub-organellar membranes, bringing about desired phenotypic changes

Bile acid sodium symporter BASS6 can transport glycolate and is involved in photorespiratory metabolism in Arabidopsis thaliana

© 2017, American Society of Plant Biologists. All rights reserved. Photorespiration is an energy-intensive process that recycles 2-phosphoglycolate, a toxic product of the Rubisco oxygenation reaction. The photorespiratory pathway is highly compartmentalized, involving the chloroplast, peroxisome, cytosol, and mitochondria. Though the soluble enzymes involved in photorespiration are well characterized, very few membrane transporters involved in photorespiration have been identified to date. In this work, Arabidopsis thaliana plants containing a T-DNA disruption of the bile acid sodium symporter BASS6 show decreased photosynthesis and slower growth under ambient, but not elevated CO2. Exogenous expression of BASS6 complemented this photorespiration mutant phenotype. In addition, metabolite analysis and genetic complementation of glycolate transport in yeast showed that BASS6 was capable of glycolate transport. This is consistent with its involvement in the photorespiratory export of glycolate from Arabidopsis chloroplasts. An Arabidopsis double knockout line of both BASS6 and the glycolate/glycerate transporter PLGG1 (bass6, plgg1) showed an additive growth defect, an increase in glycolate accumulation, and reductions in photosynthetic rates compared with either single mutant. Our data indicate that BASS6 and PLGG1 partner in glycolate export from the chloroplast, whereas PLGG1 alone accounts for the import of glycerate. BASS6 and PLGG1 therefore balance the export of two glycolate molecules with the import of one glycerate molecule during photorespiration

Identification of novel regulatory factor X (RFX) target genes by comparative genomics in Drosophila species

An RFX-binding site is shown to be conserved in the promoters of a subset of ciliary genes and a subsequent screen for this site in two Drosophila species identified novel RFX target genes that are involved in sensory ciliogenesis