Phencyclidine (PCP) blocks glutamate-activated postsynaptic currents

AbstractPhencyclidine (PCP) was tested on the metathoracic tibialis muscles of Locusta migratoria. In physiological solution, the peak amplitude of the excitatory postsynaptic currents (EPSCs) evoked by nerve stimulation was linearly related to membrane potential between −50 and −150 mV. The decay time constant of the EPSC (τEPSC) was exponentially dependent on voltage and decreased with hyperpolarization. The membrane potential change required to produce an e-fold change in τEPSC was 315 mV. PCP (5–40 μM) produced a concentration-dependent depression of both EPSC peak amplitude and τEPSC. A slight nonlinearity in the current-voltage relationship could be discerned at high concentrations of PCP. The shortening of the decay time constant of EPSC (τEPSC) occurred without significant change in the voltage sensitivity observed under control conditions. Under all experimental conditions, the decay of the EPSCs remained a single exponential of time. Fluctuation analysis indicated that 5 μM PCP shortens the lifetime of the glutamate-activated channels by 25.7 ± 3%. PCP (10–80 μM) did not induce desensitization of the glutamate receptors. These results suggest that PCP interacts with the open conformation of ion channels activated by the glutamate receptor.PhencyclidineGlutamate receptorLocustNeuromuscular transmissionExcitatory postsynaptic currentChannel blacke

Conservation of core complex subunits shaped the structure and function of photosystem I in the secondary endosymbiont alga Nannochloropsis gaditana

Photosystem I (PSI) is a pigment protein complex catalyzing the light-driven electron transport from plastocyanin to ferredoxin in oxygenic photosynthetic organisms. Several PSI subunits are highly conserved in cyanobacteria, algae and plants, whereas others are distributed differentially in the various organisms. Here we characterized the structural and functional properties of PSI purified from the heterokont alga Nannochloropsis gaditana, showing that it is organized as a supercomplex including a core complex and an outer antenna, as in plants and other eukaryotic algae. Differently from all known organisms, the N. gaditana PSI supercomplex contains five peripheral antenna proteins, identified by proteome analysis as type-R light-harvesting complexes (LHCr4-8). Two antenna subunits are bound in a conserved position, as in PSI in plants, whereas three additional antennae are associated with the core on the other side. This peculiar antenna association correlates with the presence of PsaF/J and the absence of PsaH, G and K in the N. gaditana genome and proteome. Excitation energy transfer in the supercomplex is highly efficient, leading to a very high trapping efficiency as observed in all other PSI eukaryotes, showing that although the supramolecular organization of PSI changed during evolution, fundamental functional properties such as trapping efficiency were maintained

Going ultra deep to unravel the secret recipe of biofuel

Microalgae are of great importance in ecology, biochemistry, and biotechnology. Nevertheless, just a few genomes have been sequenced so far, most of them by Sanger sequencing. While next generation sequencing techniques have revolutionized genome resequencing, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Microalgae contain fewer repetitive regions in their 30–100 Mb genomes than genomes of mammals or higher plants and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a draft sequence of the Nannochloropsis gaditana genome that was obtained by a combination of SOLiD and Roche 454 sequencing. Mate-Pair SOLiD sequencing of genomic DNA to 250-fold coverage and an additional 7-fold coverage by single-end 454 sequencing resulted in 15 Gb of raw sequence data. Reads were assembled to a 32 Mb draft version (N50 of 50kb) with the a pipeline of tools evolved in our group. 167 scaffolds were produced accounting for 18.7Mb. Our study supports the expectation that for typical microalgae, de novo assembly of genomes from short sequence reads alone is feasible, cheap and efficient; that a mixture of SOLiD and 454 sequencing substantially improves the assembly; and that the resulting data can be used for comparative studies and to provide a valuable framework to plan the application of recombinant techniques. Furthermore a whole transcriptome analyses was carried out to identify, characterize and catalogue all the transcripts expressed, exploiting the great potential of RNA-Seq using the SOLiD platform to determine the correct gene annotation, the identification and characterization of the splicing patterns and to obtain the structure of genes, also defining—at single nucleotide resolution—the transcriptional boundaries of genes and the expressed Single Nucleotide Polymorphisms (SNPs). Important technical innovations were also introduced in this work that allowed a precise mapping of the transcription start to support a robust prediction of the regulatory region on the genome sequence. RNA-Seq was also used to study the differential expression of transcripts in cultures able to accumulate substantially different amounts of lipids, in order to obtain insights on lipid metabolism of N.gaditana. The study was carried on using as input sequences for the SOLiD run both polyadenylated mRNA enriched fractions and ribo-depleted RNA samples that allowed to recovery also the plastidial mRNA. Our results shows how data obtained from a single SOLiD run, applying specific ad hoc variation on the standard protocols, provide enough coverage to support a valuable annotation of a completely new genome and to provide all the information necessary to underline the main features of pathways of interest if different biological samples are compared. The N. gaditana sequencing project fulfilled the two important aims of assembling a draft of the genome sequence using sole next generation short reads and providing a careful genome annotation, with the goal of a better understanding of N. gaditana biology and the idea of improving its value as a model organism for biotechnological applications related to biofuel production.Nonostante le microalghe rivestano una particolare importanza per l’ecologia, la biochimica e le biotecnologie, solo poche specie sono state sequenziate a tutt’oggi e per lo più nell’era del sequenziamento di tipo Sanger. Mentre i sequenziatori di nuova generazione hanno fornito straordinari mezzi al risequenziamento di genomi di singoli individue per cui il genoma della specie di riferimento era già disponibile, il sequenziamento di genomi eucariotici completamente nuovi, utilizzando sequenze corte, presenta ancora grandi difficoltà. Le principali difficoltà si riscontrano in fase di assemblaggio e sono principalmente dovute alle notevoli dimensioni dei genomi eucariotici e alla presenza di regioni ripetute. Le microalghe, nei loro genomi, grandi in genere dalle 30Mb alle 100Mb, contengono poche regioni a bassa complessità e costituiscono pertanto degli interessanti organismi sui quali sperimentare il sequenziamento ex novo utilizzando esclusivamente i sequenziatori di seconda generazione. In questo lavoro, descriviamo il sequenziamento e l’assemblaggio del genoma della microalga Nannochloropsis gaditana, ottenuto utilizzando le sequenze prodotte dal 454 della Roche e dal SOLiD. Il sequenziamento di ‘mate-pairs’ utilizzando il SOLiD ha prodotto una copertura di sequenza di circa 250 volte la grandezza del genoma, mentre il sequenziamento 454 è stato utilizzato per produrre una ulteriore copertura di 7 volte con sequenze di media lunghezza. Le sequenze sono state assemblate in una versione preliminare non del tutto finita di 32Mb, dove 18.7Mb sono state incluse in 167 grandi scaffolds. Il 50% degli scaffolds ottenuti è più grande di 50Kb, mentre un terzo del genoma è stato assemblato in circa 20 contigs. Il nostro lavoro conferma la previsione che le tecniche di nuova generazione sono adatte al sequenziamento del genoma di una microalga e consentono di ottenere risultati utili in un tempo più breve di quelli tradizionali e ad un minor costo. I dati ottenuti potranno esser utilizzati per analisi comparative del genoma e saranno anche un importante prerequisito per l’applicazione di tecniche ricombinati alla microalga di interesse biotecnologico. Inoltre, durante questo progetto, sono state portate avanti anche diverse analisi del trascrittoma tramite sequenziamento, finalizzate alla produzione di una lista di geni utile all’annotazione del genoma e all’identificazione di geni differenzialmente espressi in condizioni di accumulo di lipidi. Vengono presentate, in questo lavoro, anche alcune innovazioni tecniche che hanno permesso di sfruttare al meglio il sequenziamento SOLiD per produrre un’accurata annotazione della struttura del trascrittoma e una più completa analisi dei geni differenzialmente espressi codificati negli organelli. I risultati dimostrano che una sola corsa SOLiD su campioni preparati in modo adeguato, è sufficiente per l’annotazione accurata di un genoma completamente nuovo e per l’individuazione di geni differenzialmente espressi in condizioni di interesse.

Vers une analyse à l'échelle du système de la sécurité des systèmes embarqués

This thesis is dedicated to the improvement of dynamic analysis techniques allowing the verification of software designed for embedded systems, commonly called firmware. It is clear that the increasing pervasiveness and connectivity of embedded devices significantly increase their exposure to attacks. The consequences of a security issue can be dramatic not least in the economical field, but on the technical stage as well. Especially because of the difficulty to patch some devices. For instance, offline devices or code stored in a mask rom which are read only memory programmed during the chip fabrication. For all these reasons, it is important to thoughtfully test firmware program before the manufacturing process. This thesis presents analysis methods for system-wide testing of security and hardware components. In particular, we propose three impvrovements for partial emulation. First, Inception a dynamic analysis tool to test the security of firmware programs even when mixing different level of semantic (e.g., C/C++ mixed with assembly). Second, Steroids a high performance USB 3.0 probe that aims at minimizing the latency between the analyzer and the real device. Finally, HardSnap a hardware snapshotting method that offers higher visibility and control over the hardware peripherals. It enables testing concurently different execution paths without corrupting the hardware peripherals state.Cette thèse se consacre à l'amélioration des techniques d'analyse dynamiques permettant la vérification de logiciels conçus pour des systèmes embarqués, couramment appelé micrologiciel. Au vu de l'augmentation significative de la connectivité des appareils électroniques, les préoccupations concernant leur sécurité s'intensifient. Les conséquences d'une faille de sécurité sur ces appareils peuvent impliquer des répercussions économiques non négligeables et des difficultés techniques importantes pour appliquer un correctif. C’est le cas notamment des amorceurs de code qui sont généralement stockés sur des mémoires mortes et intégrées dans les couches physiques qui constituent le microcontrôleur. Par conséquent, l’analyse de code source spécifique aux systèmes embarqués pendant la phase de production des micro-contrôleurs est cruciale. Cette thèse présente des techniques d'analyse afin de tester la sécurité de composants logiciel et matériel à l'échelle du système. En particulier, nous nous intéressons aux techniques de test basé sur l'émulation partielle dont nous améliorons les capacités avec trois nouvelles approches. Premièrement, Inception un outil d’analyse dynamique permettant d’appliquer des méthodes de tests exhaustifs (exécution symbolique) sur le code source de micrologiciel même lorsque ce dernier dépend de code plus bas niveau (exemple, code binaire ou assembleur). Deuxièmement, une sonde haute performance basé sur le protocol USB 3.0 afin de réduire la latence lors des communications entre l'outil d'analyse et le vrai matériel. Troisièmement, HardSnap une méthode permettant de générer des instantanés des périphériques matériel afin d'augmenter le contrôle et la visibilité lors de l'exécution symbolique. Cet outil permet de réaliser une exploration concurrente de plusieurs chemins d'exécution sans inconsistance

L’Égypte antique dans la bande dessinée

La première chose qui frappe si l'on cherche à définir l'image que la BD (francophone) donne de la civilisation pharaonique c'est, me semble-t-il, le nombre impressionnant d'albums que l'Égypte, plus que toute autre civilisation disparue, a inspirés. A y regarder de plus près, c'est en fait le nombre de scénaristes et de dessinateurs attirés par le monde égyptien qui est remarquable, car si le monde gréco-romain n'est pas en reste pour le nombre d'albums publiés, la plupart de ceux-ci sont l'..

Oceani e clima

Il percorso didattico che presentiamo si compone di quattro esperienze nelle quali si utilizzano conoscenze e competenze di chimica di diverso livello per studiare fenomeni naturali interessanti e rilevanti per l’educazione alla cittadinanza. Nelle varie esperienze le reazioni chimiche, l’acidità, il pH e gli equilibri chimici sono messi al servizio dello studio della ripartizione dell’anidride carbonica tra atmosfera e oceani in diverse condizioni, dei meccanismi di feedback climatico che coinvolgono gas serra e oceani, degli effetti dell’aumento della disponibilità di anidride carbonica sulla chimica degli oceani e sui viventi che li abitano. Le esperienze che presentiamo sono molto intuitive, possono essere usate in contesti didattici diversi e consentono di lavorare anche sul concetto di modello sperimentale