1,720,956 research outputs found
Isolation and characterisation of the biological repeating unit of cepacian, the exopolysaccharide produced by Burkholderia cepacia complex bacteria
No full textThe repeating unit of cepacian, the exopolysaccharide produced by the majority of the Burkholderia cepacia complex microorganisms, was isolated from inner bacterial membranes and investigated by mass spectrometry. The data obtained led to the determination of its structure, thus disclosing the oligosaccharidic sequence synthesised by bacteria, and gave also information on the biosynthetic pathway of cepacian repeating unit, thanks to the presence of saccharidic intermediates shorter than the repeating unit. In addition, from zero to four acetyl substituents were detected on the repeating unit before the polymerisation process which takes place in the periplasmic space. The procedure used can be conveniently exploited as an alternative route to the structural determination of bacteria exopolysaccharides, particularly useful for complex repeating units
Inhibition of cathelicidin activity by bacterial exopolysaccharides
No full textThe interaction of bacterial exopolysaccharides, produced by opportunistic lung pathogens, with antimicrobial peptides of the innate primate immune system was investigated. The exopolysaccharides were produced by Pseudomonas aeruginosa, Inquilinus limosus and clinical isolates of the Burkholderia
cepacia complex, bacteria that are all involved in lung infections of cystic fibrosis patients. The effects of the biological activities of three orthologous cathelicidins from Homo sapiens sapiens, Pongo pygmaeus (orangutan) and Presbitys obscurus (dusky leaf
monkey) were examined. Inhibition of the antimicrobial activity of peptides was assessed using minimum inhibitory concentration assays on a reference Escherichia coli strain in the presence and absence of exopolysaccharides, whereas complex formation
between peptides and exopolysaccharides was investigated by means of circular dichroism, fluorescence spectroscopy and atomic force microscopy. Biological assays revealed that the higher the
negative charge of exopolysaccharides the stronger was their inhibiting effect. Spectroscopic studies indicated the formation of molecular complexes of varying stability between peptides and exopolysaccharides, explaining the inhibition. Atomic force
microscopy provided a direct visualization of the molecular complexes. A model is proposed where peptides with an a-helical conformation interact with exopolysaccharides through electrostatic and other non-covalent interactions
CONFORMATIONAL STUDIES OF THE CAPSULAR POILYSACCHARIDE PRODUCED BY NEISSERIA MENINGITIDIS GROUP A
No full textThe effect of different cations on the conformational and morphological properties of the capsular polysaccharide
produced by Neisseria meningitidis group A was investigated. Circular dichroism studies
showed that the presence of Na+, NH4+ or Ca2+ ions induced different local conformations of the polysaccharide
chain through interactions with the phosphodiester group bridging the saccharide residues in the
polymer chain. Atomic force microscopy experiments confirmed that the morphology of the polysaccharide
chains was different depending on the nature of the counterion. Ammonium ions were associated
with the presence of single polymer chains in an elongated conformation, whereas sodium ions favored
the folding of the chains into a globular conformation. The addition of calcium ions produced the aggregation
of a limited number of globular polysaccharide chains to form a ‘toroidal-like’ structure
Relazioni struttura-funzione di polisaccaridi prodotti da patogeni opportunisti
No full text2008/2009Argomento di questa tesi di dottorato sono i polisaccaridi (sia capsulari, CPSs, sia esopolisaccaridi, EPSs), prodotti da batteri e rilasciati nell’ambiente circostante, ed il loro ruolo nelle infezioni batteriche. Sono stati presi in considerazione batteri opportunisti responsabili di infezioni polmonari in pazienti affetti da fibrosi cistica (CF). I risultati riportati in questa tesi di dottorato comprendono diverse linee di ricerca e contribuiscono a chiarire le diverse caratteristiche chimiche e biologiche dei polisaccaridi batterici presi in considerazione.
La prima linea di ricerca riguarda due EPSs strutturalmente simili prodotti da Inquilinus limosus, un batterio recentemente isolato dalle secrezioni polmonari di pazienti con CF. Si è voluto valutare se i due EPSs fossero in grado di assumere conformazioni differenti e quindi ipoteticamente producessero diverse attività biologiche. Le proprietà conformazionali sono state valutate mediante misure di dicroismo circolare ed immagini ottenute mediante microscopia a forza atomica. Studi di modellistica molecolare sono stati effettuati dalla Dr.ssa M. Kuttel (Dip. di Chimica, Univ. di Cape Town). I due polimeri esibiscono effettivamente differenti conformazioni elicoidali (strutture secondarie), che vengono stabilizzate da legami idrogeno e caratterizzate dalla presenza di gruppi piruvato carichi esposti sulla superficie esterna dell’elica. Questo risultato conferma l’ipotesi che la biosintesi di più specie polisaccharidiche può risultare un mezzo utile per il batterio per diversificare i propri strumenti di difesa da possibili minacce esterne.
La seconda linea di ricerca ha preso in considerazione l’esopolisaccaride cepaciano, prodotto dalla maggioranza dei ceppi appartenenti al complesso della Burkholderia cepacia (Bcc). L’unità ripetitiva (RU) di questo EPS è stata isolata dalla membrana interna batterica, dove si trova legata a un trasportatore lipidico prima della polimerizzazione. Durante il lavoro di questa tesi, è stato sviluppato un protocollo per isolare l’unità ripetitiva e la sua RU biologica è stata determinata mediante analisi ESI-MS. Questo studio, che nasce dalla collaborazione con la Dr.ssa C. Lagatolla (Dip. di Scienze della Vita, Univ. di Trieste), impegnata nella ricerca dei geni coinvolti nella biosintesi del cepaciano, ha inoltre permesso di determinare lo schema di acetilazione della stessa, utile per la possibile comprensione del ruolo biologico dei gruppi acetili, e di investigare alcuni passi del processo biosintetico dell’unità ripetitiva del polisaccaride.
L’utilizzo di enzimi in grado di degradare il polisaccaride cepaciano è utile per demolire la matrice esopolisaccaridica presente attorno ai batteri e consentirne l’utilizzo in sinergia con terapie antibiotiche. In precedenza era stato trovato un enzima, prodotto da un ceppo ambientale di Bacillus, con attività liasica verso il cepaciano. Il suo isolamento per un futuro sequenziamento è stato oggetto di questa tesi. Questo enzima è in grado di idrolizzare una catena laterale dello scheletro polisaccaridico del cepaciano, così lasciando intatta la natura polimerica dell’EPS. Benché l’eliminazione di questa catena laterale provochi una decisa diminuzione della viscosità, probabilmente dovuta alla perdita di aggregazione, la ricerca di altri enzimi in grado di idrolizzare lo scheletro polimerico a frammenti a basso peso molecolare sarà oggetto di ricerche future.
La quarta linea di ricerca ha coinvolto lo studio dell’interazione tra diversi EPSs con tre peptidi antimicrobici del sistema dell’immunità innata, appartenenti alla famiglia delle catelicidine. Negli esperimenti sono stati utilizzati esopolisaccaridi prodotti da Pseudomonas aeruginosa, Inquilinus limosus e ceppi clinici del Bcc. L’inibizione dell’attività dei peptidi è stata valutata tramite saggi di minima concentrazione inibente su un ceppo di Escherichia coli di riferimento in presenza o meno degli EPSs. La formazione di complessi tra peptidi ed EPSs è stata studiata per mezzo di misure di dicroismo circolare, di spettroscopia di fluorescenza e di microscopia a forza atomica. E’ stato infine proposto un modello in cui peptidi strutturati ad α-elica interagiscono con gli esopolisaccaridi mediante interazioni elettrostatiche e non covalenti.
Infine, la ricerca ha coinvolto il polisaccaride capsulare prodotto da Neisseria meningitidis gruppo A, usato per sviluppare un vaccino coniugato dal Prof. N. Ravenscroft (Dip. di Chimica, Univ. di Cape Town). In base alle sue osservazioni sulla scarsa reattività del polisaccaride durante il processo di derivatizzazione necessario per la coniugazione con le proteine, è stata formulata l’ipotesi di una possibile aggregazione del polisaccaride capsulare in presenza di ioni Ca2+, derivanti dal processo di purificazione dello stesso. Per capire e risolvere questo problema, le proprietà conformazionali e morfologiche del CPS in presenza di ioni NH4+, Na+ e Ca2+ sono state studiate nel laboratorio di Trieste, mediante tecniche quali dicroismo circolare, spettroscopia di fluorescenza, viscosimetria e microscopia a forza atomica. La presenza di ione ammonio porta le catene polimeriche ad una conformazione più allungata, mentre lo ione sodio favorisce il ripiegamento delle catene in una conformazione globulare. L’addizione di calcio produce aggregazione di un limitato numero di polisaccaridi globulari per formare una struttura ‘toroidal-like’. Questi risultati giustificano la bassa solubilità del polimero in forma calcio e offrono una spiegazione della sua bassa reattività nella preparazione del coniugato proteico.This PhD thesis focuses on polysaccharides (both capsular, CPSs, and exopolysaccharides, EPSs) produced by bacteria and released in the external environment as well as on their role in bacterial infections. For this, opportunistic bacteria involved in lung infections in cystic fibrosis (CF) patients were investigated. The results reported in the PhD thesis involved several topics and contributed to clarify different biological and chemical characteristics of bacterial polysaccharides.
The first topic of the research has taken in account two structurally similar EPSs produced by a bacterium recently isolated from respiratory secretions of CF patients, Inquilinus limosus. The working hypothesis was that the two EPSs assume different conformations and therefore exert different biological activity. The conformational properties of these two EPSs were investigated by circular dichroism (CD) and atomic force microscopy (AFM) techniques, whereas the molecular modelling studies were carried out by Dr. M. Kuttel (Dept. of Chemistry, Univ. of Cape Town). The two polymers were shown to exhibit different helical conformations, stabilised by hydrogen bonding and characterised by charged pyruvate groups on the external helical surface. These findings confirming that the biosynthesis of two polymeric species might offer multiple tools to protect bacteria from external threats.
The second part of the research involved cepacian, the EPS produced by the majority of bacteria belonging to the Burkholderia cepacia complex (Bcc). In this case, the isolation of its repeating unit (RU) from the internal bacterial membrane where it is bound to a lipid carrier prior to the EPS polymerisation was performed. A procedure to isolate cepacian repeating units was developed and the structure of the biological RU was determined by ESI-MS analysis. At the same time, the RU was useful to determine the acetylation scheme of cepacian, which could be connected with the biological role of acetyl groups, and to obtain information on its biosynthetic sequence. All the data obtained were considered in connection with the genetic investigation on the cepacian biosynthesis, carried out by Dr. C. Lagatolla (Dept. of Life Sciences, Univ. of Trieste).
The third topic involved the search and identification of enzymes that can degrade cepacian in order to find biochemical tools able to degrade the EPS matrix present around bacteria and possibly used in synergy with antibiotic therapies. One lyase was found produced by Bacillus spp. and its isolation and sequencing is in progress. This lyase cleaves one of the lateral chains of cepacian backbone, thus living intact the polymeric nature of the EPS. Although the elimination of this lateral chain produces a marked lowering of the cepacian viscosity probably due to loss in aggregation ability, the search of different enzymes able to cleave the EPS backbone into oligosaccharides is still active.
The fourth topic of the research focused on the interactions between different bacterial EPSs and three antimicrobial peptides, belonging to the cathelicidin family of primate’s innate immune system, to investigate the possible EPSs protective role towards bacterial cells. The experiments involved EPSs produced by Pseudomonas aeruginosa, Inquilinus limosus and clinical isolates of the Burkholderia cepacia complex. The inhibition of the peptides activity was assessed by minimum inhibitory concentration assays on a reference Escherichia coli strain in the presence and in the absence of EPSs. The complex formation between peptides and EPSs was investigated by means of CD, fluorescence spectroscopy and AFM. As a result, a model was proposed where peptides with a α-helical conformation interact with the EPSs backbone through electrostatic and non-covalent interactions.
The last issue of the research involved the capsular polysaccharide produced by Neisseria meningitidis group A; this CPS is used to develop a protein conjugate vaccine by Prof. N. Ravenscroft (Dept. of Chemistry, Univ. of Cape Town). He observed that the derivatisation process, necessary prior to protein conjugation, yielded less product, in terms of protein-conjugate, than expected, and he thought that this was due to CPS aggregation in the presence of Ca2+, used in the purification steps. In order to solve this problem, the conformational and morphological properties of the CPS, in the presence of NH4+, Na+ and Ca2+ cations, were investigated in the Trieste laboratory. The study was carried out using different techniques such as circular dichroism, fluorescence spectroscopy, viscosity measurements and atomic force microscopy. It was shown that ammonium ions were associated with the presence of single polymer chains in an elongated conformation, whereas sodium ions favoured the folding of chains into a globular conformation. The addition of calcium ions produced the aggregation of a limited number of globular polysaccharide chains to form a ‘toroidal-like’ structure. This effect justified the polymer low solubility in the presence of calcium ions and offered an explanation of the polymer low reactivity in the preparation of the protein conjugate.XXII Ciclo198
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
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
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