1,720,976 research outputs found
Espressione omologa ed eterologa della [FeFe ]-idrogenasi di Chlamydomonas reinhardtii
No full textHydrogenases, key enzymes in hydrogen metabolism in several microorganisms, are considered as a possible future energy source. In particular, the ability of the green alga C. reinhardtii to reduce protons and release hydrogen gas (H2) upon illumination by means of a [FeFe]-hydrogenase represents a phenomenon of great scientific interest, because it holds the promise of generating energy from nature’s most plentiful resources: solar energy and water. However, the catalytic activity is strongly and irreversibly inhibited by the molecular oxygen produced during photosynthesis; furthermore, the algal hydrogenase is expressed at very low levels and only in conditions of strict anaerobiosis. This mutually exclusive nature of O2 and H2 photoproduction cannot be easily overcome and represents an important problem in the development of H2 production biotechnology. The study of the structure-function relationship of [FeFe] hydrogenases, which would help to clarify the molecular mechanisms underlying both H2 production and O2 sensitivity, requires the characterization of purified native and modified proteins. The 3D structure of the [FeFe]-hydrogenase from C. reinhardtii has not been solved, mainly because of the very low levels of protein which can be directly purified from the algae. I overexpressed this enzyme in homologous and heterologous systems to obtain enough protein for biochemical studies. The cyanobacterium Synechococcus PCC 7942, which holds a bidirectional [NiFe]-hydrogenase, is able to produce the [FeFe]-hydrogenase from C. pasteurianum in a catalytically active form, thus suggesting that the [NiFe]-hydrogenase maturation pathway of cyanobacteria may be flexible enough to allow the biosynthesis of functional Fe-only enzyme. I obtained two constructs to stably transform the cyanobacterium Synechocystis sp. PCC 6803 and to enable it to express the C. reinhardtii [FeFe]-hydrogenase. The recombinant strains expressing the algal [FeFe]-hydrogenase were able to release H2 gas amounts 4 to 5 times higher than that of wild type strain (which has only a [NiFe]-hydrogenase). These data open new perspectives about the indispensable presence of HydE, HydF and HydG auxiliary proteins to obtain a correctly folded [FeFe]-hydrogenase. At the same time, I proceeded with the homologous overexpression of the [FeFe]-hydrogenase in C. reinhardtii. Since serious limits of this system are the low amount of protein expressed and the instability of mutant algal strain, I am defining new strategies to solve this problem. I will operate site-direct mutations in critical residues to understand the catalytic mechanism and to improve the hydrogen productivity of the enzyme.Le idrogenasi, enzimi chiave nel metabolismo dell’idrogeno di molti microrganismi, sono considerate una possibile futura fonte di energia. In particolare, la capacità dell’alga verde C. reinhardtii di ridurre protoni e rilasciare idrogeno gassoso dopo illuminazione per mezzo di una [FeFe]-idrogenasi rappresenta un fenomeno di grande interesse scientifico, poiché permetterebbe di ottenere energia dalle due risorse naturali più diffuse: acqua ed energia solare. Tuttavia, l’attività catalitica è fortemente ed irreversibilmente inibita dall’ossigeno prodotto durante la fotosintesi; inoltre, l’idrogenasi algale è espressa a livelli molto bassi e solo in stretta anaerobiosi. Questa natura mutualmente esclusiva della fotoproduzione di idrogeno ed ossigeno rappresenta un importante ostacolo nello sviluppo della produzione biotecnologica di idrogeno. Lo studio della relazione struttura-fuzione della [FeFe]-idrogenasi, che permetterebbe di chiarire i meccanismi molecolari alla base della produzione di H2 e della sesibilità all’O2, richiede la caratterizzazione della proteina nativa e modificata. La struttura 3D della [FeFe]-idrogenasi di C. reinhardtii non è ancora stata ottenuta, principalmente perché le quantità di proteina purificata direttamente dall’alga sono molto basse. Quindi, ho sovraespresso tale enzima in un sistema omologo ed in uno eterologo per ottenere una quantità di proteina sufficiente per condurre studi biochimici. Il cianobatterio Synechococcus PCC 7942, che possiede una [NiFe]-idrogenasi, è in grado di sintetizzare la [FeFe]-idrogenasi di C. pasteurianum in forma attiva, suggerendo che il sistema di maturazione dei cianobatteri sia suffientemente flessibile da permettere la biosintesi di una [FeFe]-idrogenasi funzionale. Nel corso del dottorato ho ottenuto due costrutti per trasformare il cianobatterio Synechocystis sp. PCC 6803 e permettere l’espressione della [FeFe]-idrogenasi algale. I ceppi ricombinanti esprimenti l’enzima di C. reinhardtii sono in grado produrre idrogeno in quantità da 4 a 5 volte superiori rispetto al ceppo wild type (che ha solo una [NiFe]-idrogenasi). Questi dati aprono nuove prospettive circa l’indispensabile presenza delle proteine accessorie HydE, HydF e HydG per ottenere una [FeFe]-idrogenasi correttamente ripiegata. Contemporaneamente, ho condotto l’espressione omologa dell’enzima direttamente in C. reinhardtii. Dal momento che importanti limiti di questo sistema sono dati dal basso quantitativo di proteina espressa e dall’instabilità del ceppo mutante dell’alga, sto attualmente mettendo a punto nuove strategie per risolvere questi problemi. Inoltre, saranno condotte delle mutagenesi sito-specifiche per chiarire i meccanismi catalitici e migliorare la produttività dell’enzim
Crystal structure of alpha;-carbonic anhydrase from the human pathogen Helicobacter pylori
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Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
The cyanobacterium Synechocystis sp PCC 6803 is able to express an active [FeFe]-hydrogenase without additional maturation proteins
No full text[FeFe]-hydrogenases have been claimed as the most promising catalysts of hydrogen bioproduction and
several efforts have been accomplished to express and purify them. However, previous attemps to obtain a functional recombinant [FeFe]-hydrogenase in heterologous systems such as Escherichia coli failed due
to the lack of the specific maturation proteins driving the assembly of its complex active site. The unique exception is that of [FeFe]-hydrogenase from Clostridium pasteurianum that has been expressed in active form in the cyanobacterium Synechococcus PCC 7942, which holds a bidirectional [NiFe]-hydrogenase with a well characterized maturation system, suggesting that the latter is flexible enough to drive the synthesis of a [FeFe]-enzyme. However, the capability of cyanobacteria to correctly fold a [FeFe]-hydrogenase in the absence of its auxiliary maturation proteins is a debated question. In this work, we expressed the [FeFe]-hydrogenase from Chlamydomonas reinhardtii as an active enzyme in the cyanobacterium Synechocystis sp. PCC 6803. Our results, using a different experimental system, confirm that cyanobacteria are able to express a functional [FeFe]-hydrogenase even in the absence of additional chaperones
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
The proton iron-sulfur cluster environment of the [FeFe]-hydrogenase maturation protein HydF from Thermotoga neapolitana
No full text[FeFe]-hydrogenases contain a complex [4Fee4S]-2Fe cluster (H-cluster) and are able to efficiently reduce protons to H2. Due to their potential exploitation for renewable energy production biotechnologies, significant efforts have been put into understanding the mechanisms driving the H-cluster assembly, which involves three conserved proteins. Among them, HydF works as scaffold upon which the H-cluster precursor is synthesized and carrier to deliver it to the hydrogenase, resulting in its activation. A FeS cluster binding sequence (CxHx46-53HCxxC) is conserved in all HydF proteins and should in principle provide four ligands to coordinate the Fe atom. However, we found that alternative metal coordination may exist in different HydF proteins and that only the three cysteines are strictly required, whereas the fourth ligand may vary and is, in any case, readily exchangeable. In this work we analyzed by EPR/HYSCORE the FeS cluster proton environment of HydF fromThermotoga neapolitana to determine the possible role of surrounding residues in the non-cysteinyl iron ligation of the protein
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