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1
VILLA, RICCARDO. « The Lpt multiprotein machinery for LPS transport in Gram-negative bacteria:molecular details of the Lpt interactome ». Doctoral thesis, Università degli Studi di Milano-Bicocca, 2012. http://hdl.handle.net/10281/27959.
Texte intégralRésumé :
The hallmark of Gram-negative bacteria is their cell envelope, which is composed of two membranes, the inner or cytoplasmic membrane (IM), and the outer membrane (OM), separated by a compartment (the periplasm) that contains a thin peptidoglycan layer. Lipopolysaccharide (LPS) is the major component of the OM, and it acts as a selective barrier together with the OM proteins (OMPs), preventing the entry of many toxic molecules into the cell. Despite the structure and composition of OM have been elucidated in pivotal studies in the 50s and in the 70s, the factors required for the assembly of this organelle have only recently been identified.
LPS, once it is synthesized in the cytoplasm, has to be translocated through out the cell envelope. Seven essential proteins cooperate in a unique fashion to extract the macromolecule from the IM and deliver it in the outer leaflet of the OM. LptBCFG form the IM complex that empowers the translocation process by ATP hydrolysis (Narita and Tokuda, 2009), LptDE constitute a complex embedded in the OM that finally flips LPS across the OM and deliver it to its final destination (Chng et al., 2010a; Freinkman et al., 2011), and LptA is a periplasmic protein that contacts both the IM and OM complexes (Sperandeo et al. 2007; Tran et al., 2008).
Notably, LptC is single-pass IM protein with a large periplasm-protruding region.
LptC single mutants were obtained in this work by random-mutagenesis, and used in vivo and in vitro experiments to characterize two regions of the protein that distinctly interact with LptA and the IM protein complex LptBFG, respectively.
Chimera versions of LptC, either missing the transmembrane (TM) sequence, or with the IM anchor substituted by a heterologous sequence, were additionally constructed to this purpose.
Moreover, Both LptA and LptC were previously demonstrated to bind LPS in vitro, here it is presented a rapid bioinformatic tool which has been implemented to discover the molecular determinants of LptA for the interaction with Lipid A, the main component of LPS.
Genetic evidences previously obtained in our laboratory together with the presented data strongly support the LPS transport machinery model defined as the trans-envelope complex by Chng and coworkers (Chng et al., 2010a): indeed LptA interacts both with the IM and the OM protein complexes (LptBCFG and LptDE respectively), bridging them together. In support of this model, a phylogeny and structural motif conservation analysis of the Lpt components suggests that the unique structural domain retained in these proteins—despite the low sequence similarity—is the key to make possible the interaction between all the Lpt components.
2
Benedet, M. « CHARACTERIZATION OF THE LIPOPOLYSACCHARIDE TRANSPORT MACHINERY USING AN ESCHERICHIA COLI/PSEUDOMONAS AERUGINOSA HYBRID SYSTEMAND ESCHERICHIA COLI LPTC MUTANTS ». Doctoral thesis, Università degli Studi di Milano, 2015. http://hdl.handle.net/2434/277362.
Texte intégralRésumé :
Characterization of the lipopolysaccharide transport machinery using an Escherichia coli/Pseudomonas aeruginosa hybrid system and Escherichia coli lptC mutants
The lipopolysaccharide (LPS) transport (Lpt) is an essential process for the biogenesis of the outer membrane (OM) in Gram negative bacteria such as Escherichia coli and Pseudomonas aeruginosa. OM, the first bacterial defense against harsh environment and antimicrobial compounds, is composed by an inner leaflet of phospholipids, an outer leaflet of LPS, and outer membrane proteins.
Lpt system, originally identified in E. coli, is a protein machine responsible for LPS delivery from the inner membrane, where it is synthesized, to the outer membrane. It is composed of seven proteins, LptA through LptG, which form a complex spanning the IM and OM. LptBCFG are located at the IM, LptA in the periplasm, and LptDE at the OM. The lpt genes are evolutionarily conserved and appear to play an essential role in most Proteobacteria.
To understand how LPS is delivered to the OM crossing the hydrophilic periplasmic space, we focused on the role of the periplasmic components LptC and LptA by two main approaches:
i) comparison between E. coli and P. aeruginosa Lpt systems to search for conserved features shared by the Lpt machines of different Gram negative species. By plasmid shuffling technique we showed that P. aeruginosa lptCAB genes can complement E. coli ΔlptCAB mutants, thus indicating that the Pseudomonas proteins can interact with both the other proteins of the Lpt machine and the LPS of E. coli. Although E. coli and P. aeruginosa LptC and LptA proteins exhibit limited sequence identity and similarity, their 3D conformation is conserved. This suggests that LptA and LptC overall structure rather than their amino acid sequence may play a major role in Lpt assembly and LPS recognition and transport;
ii) analysis of E. coli lptC mutants in order to elucidate the role of LptC in the system. E. coli appears to tolerate several mutations in lptC, including deletion of the trans membrane domain; moreover the lethality of the lptC C-terminal region deletion can be suppressed by an appropriate expression of lptB. We thus tested whether E. coli could tolerate the lack of LptC, which was thought to be essential. By plasmid shuffling we obtained viable mutants missing lptC. Genome sequencing of such mutants revealed single amino acid substitutions at the R212 residue of the IM component LptF (lptFSupmutants); in complementation tests, lptFSup mutants suppress lethality of LptC conditional expression mutants. These data show that a specific mutation in LptF can compensate the lack of LptC and suggest that LptC may serve as a chaperon of the Lpt machine assembly and/or activity rather than an essential structural component.
3
Bodewits, Karin. « Biosynthesis pathway & ; transport of endotoxin : promising antibacterial drug targets in the Burkholderia cepacia complex (BCC) ». Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/5791.
Texte intégralRésumé :
Burkholderia cepacia complex (Bcc) species are opportunistic pathogens in patients with cystic fibrosis (CF), which are able to cause lethal infections. The Bcc are inherently resistant to most classes of antibiotics, which makes successful treatment problematic. Lipid A (also known as endotoxin), the hydrophobic anchor of lipopolysaccaride (LPS), is the bio-active component of LPS. One of several unique characteristics of the lipid A of the Bcc, is the permanent attachment of 4-amino-4-deoxy-L-arabinose (L-Ara4N) to the lipid A molecule. Also, the genes involved in L-Ara4N biosynthesis are necessary for viability in B. cenocepacia. Here we present research on lipid A biosynthesis, modi cation, and transport in the Bcc and highlight promising antimicrobial targets. The synthetic antibiotic CHIR-090 is an inhibitor of LpxC, an enzyme involved in the lipid A biosynthetic pathway. I investigated the activity of CHIR-090 against the Bcc and found that sensitivity to this antibiotic was both species- and strain-specific. CHIR-090 displayed MICs between 0.1 and 12.5 μg/ml against a panel of B. multivorans, the most prevalent Burkholderia species in CF. The species- and strain-specific sensitivity towards CHIR-090 was further explored and a strong correlation was found between the presence of a unique open reading frame, named LpxC2, in resistant species. To address the problem of multiple drug-resistance of the Bcc, we investigated the activity of the pyridoxal 50-phosphate (PLP)-dependent enzyme inhibitor cycloserine (CS) against the Bcc. CS is used as a second line of defense against M. tuberculosis. The activity of the D-enantiomer of CS (DCS) against the Bcc was tested and displayed MICs between 2 and 128 μg/ml and acted bactericidal towards the Bcc. Additionally, DCS inhibition of recombinant ArnB from B. cenocepacia J2315, a PLP-dependent enzyme necessary for viability in the Bcc, was studied. ArnB was inhibited reversibly by DCS. ArnB was further explored as a promising drug-target in the Bcc, but only CS has been identified as an inhibitor so far. In this thesis it was attempted to find the reason why is L-Ara4N modification of lipid A necessary for viability in B. cenocepacia. Therefore, two proteins were characterised, which are involved in lipid A transport: LptA, the periplasmic lipid A binding protein, and LptB, the cytoplasmic ATP-ase. LptA was found to be able to bind both modified and unmodified lipid A in vitro and therefore is not L-Ara4N specific. Furthermore, LptA could bind deep-rough-, rough-, and smooth- LPS, similar to that described for Escherichia coli LptA. The kinetic parameters of LptB were determined in vitro (kcat = 5.71 min-1 and KM = 0.88 mM), and were comparable to E. coli LptB. The ATP-ase activity of LptB was not influenced by the presence of any forms of LPS (modified or non-modified). Therefore, we concluded that both B. cenocepacia J2315 LptA and LptB are not L-Ara4N specific.
4
SESTITO, STEFANIA ENZA. « LPS-binding proteins : interaction studies with natural and synthetic ligands ». Doctoral thesis, Università degli Studi di Milano-Bicocca, 2015. http://hdl.handle.net/10281/67756.
Texte intégralRésumé :
L’obiettivo di questa tesi è elucidare alcuni aspetti dell’interazione tra proteine che legano il lipopolisaccaride (LPS) batterico e il loro ligando naturale o ligandi di sintesi.
LptC (Lipopolysaccharide transport C) è una proteina batterica che appartiene al sistema di trasporto Lpt, un sistema di 7 proteine essenziali che trasportano l’LPS sulla membrana esterna dei batteri Gram negativi dopo la sua biosintesi. Sebbene molti elementi della biosintesi dell’LPS siano stati elucidati, il preciso meccanismo di trasporto è ancora poco chiaro. Poiché LptC può essere considerata come proteina modello del sistema Lpt, in quanto presenta lo stesso folding delle altre proteine ed è la prima ad essere localizzata nel periplasma, abbiamo sviluppato ed ottimizzato un saggio di binding in vitro per studiare la sua interazione con l’LPS. Abbiamo ottenuto, per la prima volta, dettagliate informazioni sui parametri termodinamici e cinetici dell’interazione LptC-LPS. Abbiamo infatti dimostrato che in vitro il binding LptC-LPS è irreversibile con una Kd dell’ordine del μM. Considerando le analogie strutturali tra LptC e la proteina eucariotica CD14, appartenente al sistema recettoriale del TLR4, in modo analogo è stata studiata l’interazione di LptC con la molecola sintetica IAXO-102, un noto ligando di CD14. È emerso che IAXO-102 condivide lo stesso sito di legame dell’LPS e che l’interazione con la proteina è irreversibile con un’affinità inferiore a quella LptC-LPS. IAXO-102 può dunque essere considerato un prototipo per lo sviluppo di una nuova generazione di antibiotici che ha come target la biogenesi dell’LPS.
L’LPS è in grado di interagire con molte altre proteine, tra le quali quelle del sistema dell’immunità innata (TLR4, CD14, MD-2). Il riconoscimento dell’LPS da parte di questi recettori induce una forte risposta infiammatoria che termina con la produzione di citochine pro-infiammatorie e fattori immunomodulatori. Questa reazione infiammatoria è utile all’organismo, ma quando si manifesta in modo eccessivamente potente e sregolato induce sepsi, processi infiammatori e sindromi autoimmuni per le quali non è ancora disponibile un trattamento farmacologico. Una possibile soluzione al problema consiste nella ricerca e nello sviluppo di composti in grado di modulare questa eccessiva attivazione. Nella seconda parte di questo lavoro, sono riportate le caratterizzazioni biologiche di alcuni composti di sintesi con caratteristiche chimiche differenti. Di tutti i composti è stata valutata la tossicità mediante saggio dell’MTT e l’attività modulatoria del pathway del TLR4 utilizzando cellule HEK stabilmente trasfettate con i geni del TLR4, CD14 ed MD-2. Ulteriori caratterizzazioni sono state effettuate sui composti più promettenti, effettuando saggi in vitro su cellule HEK trasfettate con il complesso umano o murino TLR4•MD-2 e saggi in vivo. Infine, abbiamo investigato la possibile correlazione tra le note proprietà anti-infiammatorie di alcuni composti naturali, come i composti fenolici presenti nell’olio di oliva, e il pathway del TLR4. L’obiettivo di questo lavoro è duplice: individuare un lead compound come possibile modulatore del TLR4, ma anche discriminare quali caratteristiche chimiche siano importanti per ottenere questo effetto. Inoltre, le informazioni ottenute potrebbero essere estremamente utili per guidare il rational design di altri modulatori del TLR4.The purpose of this work is the elucidation of some aspects of the interaction between lipopolysaccharide (LPS) binding proteins and their natural ligand or synthetic compounds. LptC (Lipopolysaccharide transport C) is a bacterial protein belonging to Lpt complex, a molecular machinery composed of 7 essential proteins involved in the transport of LPS to the outer membrane in Gram negative bacteria after its biogenesis. Although many elements of LPS biosynthesis have been clarified, the precise mechanism of transport is still not completely understood. Since LptC can be considered as a model protein of Lpt complex, sharing the same folding of other proteins and being the first one in the periplasm, we have developed and optimized an in vitro binding assay to study its interaction with LPS. We have obtained, for the first time, detailed information about the thermodynamic and kinetic parameters of LptC-LPS binding. We have shown that the in vitro LptC-LPS binding is irreversible with a Kd of the order of μM. Considering the structural similarities between LptC and the eukaryotic protein CD14, belonging to TLR4 receptor system, the binding between LptC and the synthetic molecule iaxo-102, a known ligand of CD14, has been investigated. It is evident that iaxo-102 shares the same binding site of LPS and that the binding is irreversible with an affinity lower than that LptC-LPS. So, iaxo-102 can be considered as a lead compound for the development a new generation of antibiotics targeting the biogenesis of LPS. LPS also binds to other proteins, such as those of innate immunity TLR4, CD14 and MD-2. The LPS recognition by these receptors induces the production of pro-inflammatory cytokines and immunomodulators that trigger the inflammatory and immune responses. These reactions are useful for the organism, but when TLR4 activation is too strong or not well regulated induces sepsis, inflammation and autoimmune syndromes, which still lack a pharmacological treatment. A possible solution to solve this problem consists in the research and development of compounds which modulate this excessive activation. In the second part of thesis work, the biological characterization of some synthetic compounds, with different chemical features, have been reported. All compounds have been screened for their toxicity using MTT assay, and their modulatory activity on TLR4 pathway by using HEK cells stably transfected with TLR4, CD14 and MD-2 genes. The best compounds have been further characterized by in vitro assays on HEK cells transfected with the human or murine complex TLR4·MD-2 and in vivo studies. Finally, the possible correlation between the known anti-inflammatory properties of some natural compounds, such as the phenolic compounds of olive oil, and TLR4 activity has been investigated. The aim of this study is double: to find a lead compound active on TLR4 pathway, but also to discriminate which chemical features are important to obtain this effect. In addition, the information obtained could be very useful to guide the rational design of other TLR4 modulators.
5
CIARAMELLI, CARLOTTA. « Synthesis and characterization of new small-molecule ligands of LPS binding proteins ». Doctoral thesis, Università degli Studi di Milano-Bicocca, 2015. http://hdl.handle.net/10281/77016.
Texte intégralRésumé :
Lo scopo del presente lavoro è la progettazione, la sintesi e la caratterizzazione di nuove small molecules, attive come ligandi di LPS (lipopolisaccaridi)-binding proteins. Gli LPS, o endotossine batteriche, sono macromolecole anfifiliche ubiquitarie sulla membrana esterna dei batteri Gram-negativi. Le proteine che legano gli LPS studiate nel corso di questo progetto di tesi di dottorato appartengono a due categorie: le proteine batteriche di trasporto Lpt e il sistema recettoriale TLR4, che comprende anche i co-recettori LBP, CD14, MD2.
Le proteine Lpt, e in particolare la proteina LptC, sono responsabili del meccanismo di esportazione del LPS alla superficie cellulare, che è uno step fondamentale della via biosintetica dell’LPS. Pertanto, la biogenesi dell’LPS rappresenta un target ideale per lo sviluppo di nuovi antibiotici contro i batteri Gram-negativi. Inoltre, le strutture delle proteine Lpt sono state risolte, ma il meccanismo di trasporto è ancora da elucidare.
Nel presente lavoro di tesi sono stati utilizzate diverse tecniche per studiare l'interazione tra LPS e LptC, con particolare attenzione agli studi di interazione via NMR. Inoltre, un nuovo LPS fluorescente è stato prodotto ed è stato utilizzato come tool per studi di interazione LPS-LptC con tecniche di fluorescenza. Sono state anche sviluppate alcune nuove molecole sintetiche. Questi glicolipidi sono stati progettati e sintetizzati per ottenere ligandi di LptC e, in prospettiva, potenziali antibiotici contro i batteri Gram-negativi.
Il Toll-like receptor 4 (TLR4), il recettore dell'immunità innata, riconosce l’LPS aiutato da altre proteine (LBP, CD14 e MD-2) ed è responsabile dell'induzione della risposta infiammatoria. Molecole sintetiche in grado di modulare l'attività dei recettori dell’immunità innata sono un potente mezzo per studiare il sistema recettoriale TLR4 e hanno grande interesse farmacologico come adiuvanti vaccinali (agonisti), agenti antisepsi e anti-infiammatori (antagonisti).
L’attività biologica di glicolipidi con una funzione amminica (IAXO-102) come antagonisti del TLR4 è stata chiaramente dimostrata dal nostro gruppo di ricerca. La sintesi di molecole derivate da IAXO-102, che mantengano l'attività biologica del precursore, è stato un obiettivo di questo lavoro. In particolare, sono state portate a termine le sintesi di sonde fluorescenti, utilizzate per studi di interazione, derivati zwitterionici e molecole dimeriche. Nei nostri laboratori sono stati ottenuti anche antagonisti anionici del TLR4 con una struttura chimica più simile a Lipide A. Lo scopo di questo lavoro è stato valutare, tramite esperimenti NMR, la loro capacità di legare co-recettore dell'immunità innata MD-2.
Il carattere anfifilico degli analoghi sintetici del lipide A sintetizzati finora è spesso associato ad una bassa solubilità in acqua e a scarsa biodisponibilità. Invece, i composti attivi sul TLR4 di origine naturale hanno una migliore solubilità e biodisponibilità. La modifica chimica di queste strutture è molto utile per modulare l'attività biologica e per migliorare la specificità nei confronti del target. Di conseguenza, in una fase successiva di questo lavoro di tesi, è stata intrapresa la sintesi di nuove molecole con strutture chimiche ispirate ai modulatori naturali del TLR4. Recentemente è stato dimostrato che alcuni composti fenolici estratti da olio di oliva hanno una buona attività come antagonisti del TLR4. Pertanto, la sintesi di alcuni analoghi di queste molecole è stata eseguita per ottenere nuovi potenziali antagonisti del TLR4, con una migliore solubilità in acqua e una ridotta tossicità.The purpose of this work is the design, synthesis and characterization of new small molecules, active as ligands of two different lipopolysaccharide (LPS)-binding proteins. LPS, or bacterial endotoxin, is an amphiphilic macromolecule ubiquitous on the outer membrane of Gram-negative bacteria. The LPS binding proteins studied during this thesis project belong to two classes: the bacterial proteins of the Lpt transport machinery and the mammalian TLR4 receptor system, including the co-receptors LBP, CD14, MD-2. Lpt proteins, and in particular the protein LptC, are responsible for the export mechanism of LPS to the cell surface of Gram negative bacteria, which is a fundamental step of the LPS biosynthetic pathway. Therefore, the LPS biogenesis represents an ideal target for development of novel antibiotics against Gram-negative bacteria. Moreover, the structures of Lpt proteins have been elucidated, but very little is known about the mechanism of LPS transport. In this thesis work different techniques were used to study the interaction between LPS and LptC, particularly NMR binding studies. Moreover, a new fluorescent LPS was produced and it was used as a tool to perform LPS-LptC interaction studies with fluorescence techniques. Some new synthetic molecules were also developed during this thesis. Glycolipidic small molecules were designed and synthesized in order to obtain LptC ligands and, in perspective, potential antibiotics against Gram-negative bacteria. Toll-like receptor 4 (TLR4), the innate immunity receptor, recognizes LPS, helped by other proteins (LBP, CD14 and MD-2), and it is responsible for the induction of inflammatory responses. Synthetic small molecules able to modulate innate immunity receptors activity are a powerful mean to study the TLR4 receptor system and have great pharmacological interest as vaccine adjuvants (agonists), antisepsis and anti-inflammatory agents (antagonists). Antagonist activity on TLR4 receptor system of amino glycolipids (IAXO-102) was clearly demonstrated by our research group. The synthesis of molecules derived from IAXO-102 which retain the biological activity of the precursor was a target of this work. In particular, the synthesis of fluorescent probes, used for binding studies, zwitterionic derivatives and dimeric molecules were performed. Anionic TLR4 antagonists with a chemical structure more similar to Lipid A were also obtained in our labs. The aim of this work was the evaluation via NMR binding experiments of their ability to bind the innate immunity co-receptor MD-2. The amphiphilic character of the synthetic lipid A analogues synthesized so far is often associated with low water solubility and poor bioavailability. In this respect, the natural TLR4-active compounds have better solubility and bioavailability. The chemical modification of these structures is very helpful to modulate their biological activity and to enhance target specificity. Consequently, in a later stage of this work, the synthesis of new small molecules with chemical structures inspired to natural TLR4 modulators was pursued. Very recently it was found that some phenolic compounds from olive oil extracts presented a good activity as TLR4 antagonists. The synthesis of some analogues of these molecules was performed to obtain new potential TLR4 antagonists with better water solubility and reduced toxicity.
6
Garner, Ronald Aaron. « Oxidative Assembly of the Outer Membrane Lipopolysaccharide Translocon LptD/E and Progress towards Its X-Ray Crystal Structure ». Thesis, Harvard University, 2014. http://nrs.harvard.edu/urn-3:HUL.InstRepos:13064991.
Texte intégralRésumé :
Lipopolysaccharide (LPS) is the glycolipid that comprises the outer leaflet of the Gram-negative outer membrane (OM). Because it is essential in nearly all Gram-negative species, and because it is responsible for making these bacteria impervious to many types of antibiotics, LPS biogenesis has become an important area of research. While its biosynthesis at the cytoplasmic face of the inner membrane (IM) is well studied, the process by which it is removed from the IM, transported across the aqueous periplasmic compartment, and specifically inserted into the outer leaflet of the OM is only beginning to be understood. This transport process is mediated by the essential seven-protein LPS transport (Lpt) complex, LptA/B/C/D/E/F/G. The OM portion of the exporter, LptD/E, is a unique plug-and-barrel protein complex in which LptE, a lipoprotein, sits inside of LptD, a β-barrel integral membrane protein. LptD is of particular interest, as it is the target of an antibiotic in Pseudomonas aeruginosa.
Part I of this thesis investigates how the cell forms the two non-consecutive disulfide bonds that connect LptD's C-terminal β-barrel to its N-terminal soluble domain. These disulfides, one of which is almost universally conserved among Gram-negatives, are essential for cell viability. Here, we show that an intermediate oxidation state with non-native disulfide bonds accumulates in the absence of LptE and in strains defective in either LptE or LptD. We then demonstrate that this observed intermediate is on-pathway and part of the native LptD oxidative folding pathway. Using a defective mutant of DsbA, the protein that introduces disulfide bonds into LptD, we are able to identify additional intermediates in the LptD oxidative folding pathway. We ultimately demonstrate that the disulfide rearrangement that activates the LptD/E complex occurs following an exceptionally slow β-barrel assembly step and is dependent on the presence of LptE.
Part II describes work towards obtaining X-ray crystal structures of the LptD N-terminal domain and LptD/E complex. Expression construct and purification optimization enabled the production of stable LptD/E in quantities that make crystallography feasible. Numerous precipitants, detergents, and additives were screened, ultimately resulting in protein crystals that diffract to a resolution of 3.85 Å.Chemistry and Chemical Biology
7
Xue, Mingyu. « Identification and Characterization of Intermediates during Folding on the β-Barrel Assembly Machine in Escherichia coli ». Thesis, Harvard University, 2014. http://dissertations.umi.com/gsas.harvard:11594.
Texte intégralRésumé :
β-barrel membrane proteins play important structural and functional roles in Gram negative bacteria and in mitochondria and chloroplasts of eukaryotes. A conserved machine is responsible for the folding and insertion of β-barrel membrane proteins but its mechanism remains largely
unknown. In E. coli, a five protein β-barrel assembly machine (Bam) assembles β-barrel proteins into the outer membrane (OM). Among all β-barrel membrane proteins in E. coli
, the β-barrel component of the OM LPS translocon is one of
only two essential β-barrels, the other being the
central component of the Bam machinery itself. The OM LPS translocon, which consists of OM β-barrel protein LptD (lipopolysaccharide transport) and OM lipoprotein LptE, is responsible for the final export of LPS molecules into the outer leaflet of the OM, resulting in an asymmetric bilayer that blocks the entry of toxic molecules such as antibiotics. This thesis describes the characterization of the biogenesis pathway of the OM LPS translocon and its folding and insertion
into the OM by the Bam machinery.
An in vivo S35-Methionine pulse-labeling assay was developed to identify intermediates along the biogenesis of the OM LPS translocon. Seven intermediates were identified along the
pathway. We show that proper assembly of the OM LPS translocon involves an oxidative disulfide bond rearrangement from a nonfunctional intermediate containing non-native disulfides. We also found that the rate determining step of OM LPS translocon biogenesis is β-barrels folding process by the Bam machinery.
Using in vivo chemical crosslinking, we accumulated and trapped a mutant form of LptD on BamA, the central component of the Bam machinery. We extended the S35-Methionine pulse-labeling method to allow chemical crosslinking of substrates on the Bam complex and trapped LptD while it was being folded on the Bam machine. We demonstrated that the interaction between LptD and BamA is independent of LptE, while that between LptD and BamD, the other
essential component of the Bam complex beside BamA, is LptE dependent. Based on these findings, we proposed a model of Bam-assisted folding of the OM LPS translocon in which LptE
templates the folding of LptD.Chemistry and Chemical Biology
8
Pandey, Sundar. « Novel Role of Pseudomonas Aeruginosa LptD Operon ». FIU Digital Commons, 2018. https://digitalcommons.fiu.edu/etd/3734.
Texte intégralRésumé :
Pseudomonas aeruginosais an opportunistic pathogen that infects cystic fibrosis (CF) patients contributing to their high morbidity and mortality. P. aeruginosaundergoes a phenotypic conversion in the CF lung, from nonmucoid to mucoid, by constitutively producing a polysaccharide called alginate. These mucoid strains often revert to nonmucoid in vitrodue to second-site suppressor mutations. We hypothesized that mapping these mutations would lead to the identification of novel genes involved in alginate production. In a previous study, a mucoid strain, PDO300 (PAOmucA22), was used to isolate suppressors of alginate phenotype (sap). One of the uncharacterized nonmucoid revertants, sap27, is the subject of this study. The mucoid phenotype in sap27was restored by pMO012217 from a minimal tiling path cosmid library. The cosmid pMO012217 harbors 18 P. aeruginosaopen reading frames (ORF). The cosmid was mutagenized with a transposon to map the contributing gene. It was mapped tolptD(PA0595) encoding lipopolysaccharide transport protein. E. coliLptD transports lipopolysaccharide to the outer leaflet of the outer membrane. The Alg+phenotype was restored upon complementation with P. aeruginosa lptDalone, suggesting that sap27likely harbor a chromosomal mutation inlptD. Sequencing analysis of sap27showed the presence of a mutation not in lptDbut in algO, which encodes a periplasmic protease protein. This suggests LptD is able to bypass analgO mutation by positively regulating alginate production. The lptD is a part of a three-gene operon lptD-surA-pdxA. SurA is an essential protein for survival in starvation and a major chaperone protein for all outer membrane proteins and PdxA is a NAD-dependent dehydrogenase and is involved in the vitamin B6biosynthetic pathway. Pyridoxal 5’-phosphate (PLP) is the active form of vitamin B6.P. aeruginosagrown in a media supplemented with PLP increased production of pyocyanin, a virulence factor. The PLP and aromatic amino acids are synthesized from a common precursor chorismic acid. We demonstrated an increase in pyocyanin production when the bacteria were cultured supplemented by the aromatic amino acids phenylalanine. We concluded that the lptDoperon plays a role in the P. aeruginosavirulence by regulating alginate and pyocyanin production.
9
Martins, José Antônio. « Vocoder LPC com quantização vetorial ». [s.n.], 1991. http://repositorio.unicamp.br/jspui/handle/REPOSIP/261389.
Texte intégralRésumé :
Orientador : Fabio ViolaroDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Eletrica
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Resumo: Neste trabalho são descritos os princípios do vocoder LPC, sendo mostrados os métodos para cálculo dos parâmetros do mesmo. Também são apresentados os resultados de simulações de vocoders LPC usando quantização escalar, quantização vetorial e interpolação dos parâmetros quantizados. Inicialmente foi projetado um vocoder LPC não quantizado, o qual serviu de padrão para a avaliação dos vocoders quantizados. Usando a quantização escalar dos coeficientes razão log-área foi obtido um vocoder à taxa de 2200 bit /s, assegurando uma boa qualidade e alta inteligibilidade da voz sintetizada. Com o uso da quantização vetorial obteve-se um bom desempenho em taxas da ordem de 1000 bit/s. Essas taxas foram reduzidas em 50% com o uso da interpolação linear, transmitindo apenas os parâmetros dos quadros ímpares. Assim, conseguiu-se vocoders com taxas ao redor de 500 bit/s, apresentando voz sintetizada com degradação em relação aos sistemas anteriores, mas ainda assegurando uma boa inteligibilidade
Abstract: Not informed.
Mestrado
Eletronica e Comunicações
Mestre em Engenharia Elétrica
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Titecat, Marie. « Evaluation d’une nouvelle classe d’antibiotiques : les inhibiteurs de LpxC ». Thesis, Lille 2, 2016. http://www.theses.fr/2016LIL2S038.
Texte intégralRésumé :
L’émergence et la diffusion de la résistance aux antibiotiques au sein des bactéries à Gram négatif (BGN) sont aujourd’hui des enjeux de Santé Publique nationaux et internationaux. La multi-résistance aux antibiotiques concerne non seulement des espèces fréquemment responsables d’infections nosocomiales mais aussi des espèces hautement virulentes comme Yersinia pestis, agent de la peste et du bioterrorisme. Dans ce contexte, la mise au point de nouvelles molécules actives sur d’autres cibles bactériennes est primordiale. La métallo-enzyme LpxC catalyse la première étape irréversible de la biosynthèse du lipide A, constituant majeur de la membrane externe des bactéries à Gram négatif. Des inhibiteurs de LpxC sont ainsi développés depuis une vingtaine d’années mais leur spectre sur les BGN était initialement limité aux entérobactéries et leur activité partielle sur P. aeruginosa. Dans ce travail nous avons participé à l’optimisation de la structure chimique de ces molécules grâce à une approche dynamique des interactions enzymes/inhibiteurs utilisant la résonance magnétique nucléaire (RMN). Cette technique a permis l’élaboration d’un nouvel inhibiteur de LpxC, le LPC-058, caractérisé par une forte affinité pour l’enzyme (Ki = 3,5 ± 0,2 pM). Nous avons évalué in vitro l’activité antibiotique du LPC-058 et de trois autres composés (CHIR-090, LPC-011 et LPC-087) vis-à-vis de 369 souches cliniques responsables d’infections nosocomiales aux profils de résistance variés. Le LPC-058 présentait le plus large spectre d’activité en particulier sur A. baumannii et les valeurs de CMI les plus basses (CMI90 = 0,12 mg/L pour les entérobactéries et 0,5 mg/L pour P. aeruginosa). Il était bactéricide vis-à-vis de souches multi-résistantes et son action était synergique avec les C3G, l’imipénème, l’amikacine et la ciprofloxacine vis-à-vis de souches de K. pneumoniae, P. aeruginosa et A. baumannii productrice de carbapénémases, respectivement KPC-2, VIM-1 et OXA-23. Le LPC-058 présentait néanmoins une forte fixation protéique et, in vivo, son volume de distribution était limité au compartiment sanguin (Vd = 1,1 L/kg). Nous avons évalué son activité in vivo dans un modèle murin de peste bubonique car il s’agit de l’une des infections les plus virulentes pour l’homme. Nous avons obtenu une survie de 87 % après 5 jours de traitement à la posologie de 10 mg/kg q8h par voie veineuse. Le LPC-058 occasionnant des diarrhées chez le rongeur, nous avons évalué un de ses dérivés, le LPC-B, caractérisé par une moindre fixation protéique, un plus grand volume de distribution et l’absence d’effets secondaires chez la souris, même à fortes doses. Nous avons démontré qu’à la posologie de 200 mg/kg par voie veineuse, cet antibiotique était aussi efficace que la doxycycline (traitement de référence de la peste). L’ensemble de ces travaux souligne le rôle potentiel des inhibiteurs de LpxC dans la prise en charge des infections par des bactéries multi-résistantes ou hautement virulentesAntimicrobial resistance among Gram-negative bacteria (GNB) has become a national and international public health concern. Resistant strains are involved in nosocomial diseases and in highly virulent infections, such as plague caused by Yersinia pestis, a potential biological terrorism agent. In this context the development of new antimicrobial compounds efficient on new bacterial targets is critical. LpxC metallo-enzyme catalyzes the first commitment step of the lipid A biosynthesis, a major component of the Gram negative cell wall. LpxC inhibitors have been developed for twenty years but their activity was restricted to enterobacteria and weak against Pseudomonas aeruginosa. In this study, we have collaborated in the chemical optimization of the compounds thanks to a dynamic approach of enzyme/inhibitor interactions brought by nuclear magnetic resonance (NMR). This technology enabled the development of LPC-058, a new inhibitor, showing a high potency against LpxC (Ki = 3.5 ± 0.2 pM). We studied the in vitro efficacy of LPC-058 and three other compounds (CHIR-090, LPC-011 and LPC-087) against 369 clinical strains responsible for nosocomial infections with various antibiotic resistance profiles. In this part, LPC-058 displayed the broadest spectrum of efficacy, even on Acinetobacter baumannii with the lowest MIC values (MIC90 = 0.12 mg/L against enterobacteria and 0.5 mg/L against P. aeruginosa). It showed bactericidal activity against multi-resistant strains and synergistic activity in association with third generation cephalosporins, imipenem, amikacin and ciprofloxacin against carbapenemase producing Klebsiella pneumoniae, P. aeruginosa et A. baumannii strains (respectively KPC-2, VIM-1 and OXA-23). However, LPC-058 was constrained by strong protein interactions and a small volume of distribution (Vd = 1.1 L/kg). In vivo efficacy was studied in a murine model of bubonic plague. A 87% survival rate was obtained after five days of 10 mg/kg q8h intravenous administration. As LPC-058 treatment was associated to diarrheas in mice, we evaluated another derivate, LPC-B, characterized by a larger volume of distribution, minor protein fixation and less side effects, even for a high dose posology. We demonstrated a comparable efficacy between 200 mg/kg LPC-B treatment and doxycyclin administration (recommended in plague treatment). This work highlights the potential use of LpxC inhibitors in the management of infections caused by multi-resistant or highly virulent Gram-negative bacteria
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Chauvet, Wilfried. « Étude des filtres LPTV numériques : applications aux communications numériques ». Toulouse, INPT, 2004. http://ethesis.inp-toulouse.fr/archive/00000273/.
Texte intégralRésumé :
Ce travail de thèse propose en premier lieu une étude théorique des filtres LPTV numériques. Le problème de la représentation de ces filtres en numérique est abordé. Une relation d'équivalence avec les bancs de filtres à décimation maximale est en outre définie. Ces résultats permettent de présenter une méthode pour construire des filtres LPTV inversibles. Dans un second temps, l'intérêt de l'utilisation des filtres LPTV numériques pour les communications numériques est considéré. Après avoir recensé les applications existantes, nous proposons alors plusieurs applications des filtres LPTV. Nous proposons notamment l'utilisation de ces filtres pour la réduction du PAPR (Peak to Average Power Ratio) dans le cas d'un système OFDM. Dans un contexte d'accès multiple, nous proposons aussi une méthode pour construire une famille de filtres LPTV orthogonaux. Cette méthode est alors utilisée pour réaliser de l'accès multiple par étalement de spectreThis work is divided into two main parts. The first one is devoted to a theoretical study of digital LPTV filters. Emphasis is first laid on different representations of LPTV filters. In addition, Maximally Decimated Filter Banks are related to LPTV filters. Through these results, we propose a construction method to achieve invertible LPTV filters. The second part is then devoted to applications of LPTV filters in a digital communications framework. After describing a state of the art, we propose then the use of LPTV filters for Peak to Average Power Ratio Reduction in an OFDM context. In a multiuser system framework, we also propose a method to achieve orthogonal LPTV filters. We then take the most of these results to achieve a Spread Spectrum Multiuser System. Performances of this system are considered in term of Bir Error Rate
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Chauvet, Wilfried Lacaze Bernard Roviras Daniel. « Étude des filtres LPTV numériques applications aux communications numériques / ». Toulouse : INP Toulouse, 2006. http://ethesis.inp-toulouse.fr/archive/00000273.
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Chaladauskas, Mindaugas. « GSM LPC komponento realizavimas ir tyrimas ». Master's thesis, Lithuanian Academic Libraries Network (LABT), 2010. http://vddb.laba.lt/obj/LT-eLABa-0001:E.02~2010~D_20100813_113026-64398.
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Kiekvienas, kuris kuria aparatūrinę įrangą, nori, tai atlikti kiek įmanoma greičiau ir už kuo mažesnius kaštus. Gaminys turi greitai patekti į rinką, nes egzistuojanti konkurencija yra labai didelė. Tai galima padaryti naudojant specialią programinę įrangą, vadinamus aukšto lygio sintezės įrankius. HLS įrankiai automatiškai generuoja HDL RTL aprašą bei padeda projektuotojams turėti visas galimas projekto architektūras. HLS įrankiai naudoja algoritminį C aprašą kaip įvesties duomenis. Čia atsiranda galimybė PĮ inžinieriams taip pat projektuoti aparatūrinę įrangą. Nors atrodo, jog HLS technologija yra labai gera, bet šie įrankiai šiandien plačiai nėra naudojami. Turi būti surasti problemu sprendimai. Šioms problemoms spręsti atliekamas eksperimentas. GSM LPC algoritmas rankiniu būdu perrašomas iš C kalbos aprašo į VHDL RTL. Eksperimentas paaiškina aukšto lygio sintezės problemas. Norint, kad HLS įrankiai būtų plačiai naudojami, aukšto abstrakcijos lygio C/C++ aprašas turi būti rašomas su atitinkamais apribojimais. Neturi būti naudajami rodyklės tipo kintamieji, rodyklės tipo kintamųjų aritmetikos, rekursijos, sudėtingų operacijų, dinaminio atminties rezervavimo. Projektuotojai turi mąstyti taip kaip aparatūrinė įranga.Everyone who develops hardware wants to do this as fast as possible and for low costs. Time to the market must be shortened because the competition is very substantial. This can be done by using special development software called high level synthesis tools. HLS tools automatically generate HDL RTL code and helps developers to get all possible architectures of project. HLS tools use an algorithmic C code as input information. There is the possibility for software engineers to develop hardware too. It seems that HLS is very good technology, but HLS tools are not widely used today. It must be found the reasons of this problem and opportunities how this problem can be solved. An experiment is made by solving this problem. A GSM LPC algorithm is written by hand from C description to VHDL RTL. This experiment explains problems of high level synthesis. With the purpose HLS tools to use widely, high level of abstraction (C/C++) code must be written with restrictions. There must be no pointer variables and pointer arithmetic, no recursion, no difficult operations, no dynamic memory allocation. Engineers have to think like hardware.
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Watson, Kathleen. « LPTA versus tradeoff : how procurement methods can impact contract performance ». Thesis, Monterey, California : Naval Postgraduate School, 2015. http://hdl.handle.net/10945/45958.
Texte intégralRésumé :
Approved for public release; distribution is unlimitedEarly in the procurement planning process, the acquisition team determines the method of proposal evaluations that will be used during source selections. The most frequently used methods are lowest price technically acceptable (LPTA) and tradeoff. LPTA is typically used when requirements are well-defined and participants in the evaluation have sufficient knowledge to confidently choose a technically acceptable proposal. Conversely, tradeoff-based evaluations are employed when the evaluators are not as certain about the requirements and utilize non-cost factors to negotiate with offerors. For requiring offices, LPTA can be an easier method if the requirements are well-defined and commercially available, but they may find performance and deliverables after award to be acceptable rather than outstanding. Those using a tradeoff method may find that, by clearly defining expectations during negotiations, offerors are better able to deliver on the expectations of the acquisition team and the customer. The goal of this project is to determine whether or not there is a relationship in U.S. Army contracts between the method of procurement (LPTA versus tradeoff source selection methods) and the quality of the contract outcomes, to be measured by evaluation of Contract Performance Assessment Reports System reports.
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Petersson, Johanna. « Tvångsvårdens ingenmansland : Samspelet mellan LVM och LPT ». Thesis, Örebro universitet, Institutionen för juridik, psykologi och socialt arbete, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:oru:diva-87725.
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Rosén, Aksel, et David Hjertberg. « Personcentrerad vård under LPT : En kvalitativ intervjustudie ». Thesis, Linnéuniversitetet, Institutionen för hälso- och vårdvetenskap (HV), 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:lnu:diva-104636.
Texte intégralRésumé :
Bakgrund: Patienters delaktighet i sin vård ses idag som en självklarhet i Sverige och personcentrerad vård (PCV) är ett verktyg för att öka delaktigheten. Genom att lyssna till patientens berättelse, sträva efter ett jämbördigt partnerskap och dokumentera det som framkommer får sjuksköterskan möjlighet att med patienten skapa en hållbar och långsiktig effekt av vården. Inom tvångsvården enligt Lagen om psykiatrisk tvångsvård (LPT) ges sjukvården rätten och skyldigheten att vårda patienten mot deras vilja när det anses vara befogat för att skydda patientens liv och hälsa. Enligt LPT är syftet med tvångsvården att förmå patienten till att ta emot vården som erbjuds frivilligt. Syfte: Syftet med denna studie är att undersöka erfarenheter hos sjuksköterskor i psykiatrisk vård av att arbeta med PCV med patienter som vårdas enligt LPT. Metod: Studien har genomförts med en kvalitativ semistrukturerad intervju. Detta har sedan analyserats med en kvalitativ innehållsanalys. Resultat: Studiens resultat uppvisar erfarenheter av att främja patientberättelsen men även hinder för denna. Det framkommer även erfarenheter av partnerskap som bedrivs under LPT. Partnerskapet främjas till stor del av sjuksköterskans bemötande. Till sist framkommer erfarenheter av dokumentationens betydelse för PCV som bedrivs enligt LPT. Slutsats: Utifrån den fakta som läggs fram rörande PCV och LPT i bakgrunden är dessa två begrepp relativt komplicerade att förena. I resultatet av denna studie kan det konstateras att delaktighet kan främjas PVC i vård som bedrivs enligt LPT, som i sin tur främja delaktighet. Förhållandet mellan begreppen PCV och delaktighet blir i det närmsta cirkulärt där de stödjer varandra och skapar uppbyggande symbios Kliniska implikationer: Baserat på resultatet framkommer en tilltro till att PCV kan vara positiv i bemärkelsen att det skapas ökad delaktighet mellan patient och sjuksköterska i vård som bedrivs enligt LPT.Background: Patients' participation in their care is today seen as a matter of course in Sweden and person-centred care (PCV) is a tool to increase participation. By listening to the patient's story, striving for an equal partnership and documenting what emerges, the nurse has the opportunity to create a sustainable and long-term effect of care with the patient. In coercive care under the Compulsory Psychiatric Care Act (LPT), healthcare is given the right and obligation to care for the patient against their will when this is considered justified to protect the patient's life and health. According to LPT, the purpose of coercive care is to induce the patient to receive the care offered voluntarily. Aim: The aim of this study is to investigate the experiences of nurses in psychiatric care of working with PCV with patients being cared for according to LPT. Method: The study has been conducted with a qualitative semi-structured interview. This has then been analyzed with a qualitative content analysis. Results: The results of the study show experiences in promoting the patient's story but also obstacles to it. There is also experience of partnerships conducted under LPT. This partnership is largely promoted by the nurse's treatment. Finally, experience of the importance of documentation for PCVs carried out under LPT emerges. Conclusion: Based on the facts presented regarding PCV and LPT in the background, these two concepts are relatively complex to reconcile. In the results of this study, it can be concluded that participation can be promoted PVC in care conducted according to the LPT, which in turn promotes participation. The relationship between the concepts of PCV and participation becomes almost circular where they support each other and create constructive symbiosis. Clinical implications: Based on the results, there is a belief that PCV can be positive in the sense that increased participation between patient and nurse is created in care conducted according to LPT.
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Martin, Vincent. « Contribution des filtres LPTV et des techniques d'interpolation au tatouage numérique ». Phd thesis, Toulouse, INPT, 2006. http://oatao.univ-toulouse.fr/7478/1/martinvincent.pdf.
Texte intégralRésumé :
Les Changements d'Horloge Périodiques (PCC) et les filtres Linéaires Variant Périodiquement dans le Temps (LPTV) sont utilisés dans le domaine des télécommunications multi-utilisateurs. Dans cette thèse, nous montrons que, dans l'ensemble des techniques de tatouage par étalement de spectre, ils peuvent se substituer à la modulation par code pseudo-aléatoire. Les modules de décodage optimal, de resynchronisation, de pré-annulation des interférences et de quantification de la transformée d'étalement s'appliquent également aux PCC et aux filtres LPTV. Pour le modèle de signaux stationnaires blancs gaussiens, ces techniques présentent des performances identiques à l'étalement à Séquence Directe (DS) classique. Cependant, nous montrons que, dans le cas d'un signal corrélé localement, la luminance d'une image naturelle notamment, la périodicité des PCC et des filtres LPTV associée à un parcours d'image de type Peano-Hilbert conduit à de meilleures performances. Les filtres LPTV sont en outre un outil plus puissant qu'une simple modulation DS. Nous les utilisons pour effectuer un masquage spectral simultanément à l'étalement, ainsi qu'un rejet des interférences de l'image dans le domaine spectral. Cette dernière technique possède de très bonnes performances au décodage. Le second axe de cette thèse est l'étude des liens entre interpolation et tatouage numérique. Nous soulignons d'abord le rôle de l'interpolation dans les attaques sur la robustesse du tatouage. Nous construisons ensuite des techniques de tatouage bénéficiant des propriétés perceptuelles de l'interpolation. La première consiste en des masques perceptuels utilisant le bruit d'interpolation. Dans la seconde, un schéma de tatouage informé est construit autour de l'interpolation. Cet algorithme, qu'on peut relier aux techniques de catégorisation aléatoire, utilise des règles d'insertion et de décodage originales, incluant un masquage perceptuel intrinsèque. Outre ces bonnes propriétés perceptuelles, il présente un rejet des interférences de l'hôte et une robustesse à diverses attaques telles que les transformations valumétriques. Son niveau de sécurité est évalué à l'aide d'algorithmes d'attaque pratiques.
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Martin, Vincent Chabert Marie Lacaze Bernard. « Contribution des filtres LPTV et des techniques d'interpolation au tatouage numérique ». Toulouse : INP Toulouse, 2007. http://ethesis.inp-toulouse.fr/archive/00000326.
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Lindell, Jessica. « Domarrollen i LPT-mål : ett terapeutiskt juridiskt perspektiv ». Thesis, Stockholms universitet, Juridiska institutionen, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-95724.
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Stenström, Elin. « Samsjuklighet och rättssäkerhet : Samspelet mellan LVM och LPT ». Thesis, Örebro universitet, Institutionen för juridik, psykologi och socialt arbete, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:oru:diva-76627.
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Glodic, Nenad. « Sensitivity of Aeroelastic Properties of an Oscillating LPT Cascade ». Licentiate thesis, KTH, Kraft- och värmeteknologi, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-123504.
Texte intégralRésumé :
Modern turbomachinery design is characterized by a tendency towards thinner, lighter and highly loaded blades, which in turn gives rise to increased sensitivity to flow induced vibration such as flutter. Flutter is a self-excited and self-sustained instability phenomenon that may lead to structural failure due to High Cycle Fatigue (HCF) or material overload. In order to be able to predict potential flutter situations, it is necessary to accurately assess the unsteady aerodynamics during flutter and to understand the physics behind its driving mechanisms. Current numerical tools used for predicting unsteady aerodynamics of vibrating turbomachinery components are capable of modeling the flow field at high level of detail, but may fail in predicting the correct unsteady aerodynamics under certain conditions. Continuous validation of numerical models against experimental data therefore plays significant role in improving the prediction accuracy and reliability of the models. In flutter investigations, it is common to consider aerodynamically symmetric (tuned) setups. Due to manufacturing tolerances, assembly inaccuracies as well as in-service wear, the aerodynamic properties in a blade row may become asymmetric. Such asymmetries can be observed both in terms of steady as well as unsteady aerodynamic properties, and it is of great interest to understand the effects this may have on the aeroelastic stability of the system. Under certain conditions vibratory modes of realistic blade profiles tend to be coupled i.e. the contents of a given mode of vibration include displacements perpendicular and parallel to the chord as well as torsion of the profile. Current design trends for compressor blades that are resulting in low aspect ratio blades potentially reduce the frequency spacing between certain modes (i.e. 2F & 1T). Combined modes are also likely to occur in case of the vibration of a bladed disk with a comparatively soft disk and rigid blades or due to tying blades together in sectors (e.g. in turbines). The present investigation focuses on two areas that are of importance for improving the understanding of aeroelastic behavior of oscillating blade rows. Firstly, aeroelastic properties of combined mode shapes in an oscillating Low Pressure Turbine (LPT) cascade were studied and validity of the mode superposition principle was assessed. Secondly, the effects of aerodynamic mistuning on the aeroelastic properties of the cascade were addressed. The aerodynamic mistuning considered here is caused by blade-to-blade stagger angle variations The work has been carried out as compound experimental and numerical investigation, where numerical results are validated against test data. On the experimental side a test facility comprising an annular sector of seven free-standing LPT blades is used. The aeroelastic response phenomena were studied in the influence coefficient domain where one of the blades is made to oscillate in three-dimensional pure or combined modes, while the unsteady blade surface pressure is acquired on the oscillating blade itself and on the non-oscillating neighbor blades. On the numerical side, a series of numerical simulations were carried out using a commercial CFD code on a full-scale time-marching 3D viscous model. In accordance with the experimental part the simulations are performed using the influence coefficient approach, with only one blade oscillating. The results of combined modes studies suggest the validity of combining the aeroelastic properties of two modes over the investigated range of operating parameters. Quality parameters, indicating differences in mean absolute and imaginary values of the unsteady response between combined mode data and superposed data, feature values that are well below measurement accuracy of the setup. The findings of aerodynamic mistuning investigations indicate that the effect of de-staggering a single blade on steady aerodynamics in the cascade seem to be predominantly an effect of the change in passage throat. The changes in steady aerodynamics are thereby observed on the unsteady aerodynamics where distinctive effects on flow velocity lead to changes in the local unsteady pressure coefficients. In order to assess the overall aeroelastic stability of a randomly mistuned blade row, a Reduced Order Model (ROM) model is introduced, allowing for probabilistic analyses. From the analyses, an effect of destabilization due to aero-asymmetries was observed. However the observed effect was of moderate magnitude.QC 20130610
Turbokraft
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Pereira, Wesley. « Modifying LPC parameter dynamics to improve speech coder efficiency ». Thesis, McGill University, 2001. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=32970.
Texte intégralRésumé :
Reducing the transmission bandwidth and achieving higher speech quality are primary concerns in developing new speech coding algorithms. The goal of this thesis is to improve the perceptual speech quality of algorithms that employ linear predictive coding (LPC). Most LPC-based speech coders extract parameters representing an all-pole filter. This LPC analysis is performed on each block or frame of speech. To smooth out the evolution of the LPC tracks, each block is divided into subframes for which the LPC parameters are interpolated. This improves the perceptual quality without additional transmission bit rate. A method of modifying the interpolation endpoints to improve the spectral match over all the subframes is introduced. The spectral distortion and weighted Euclidean LSF (Line Spectral Frequencies) distance are used as objective measures of the performance of this warping method. The algorithm has been integrated in a floating point C-version of the Adaptive Multi Rate (AMR) speech coder and these results are presented.
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Attina, Dubesset Virginie. « La langue française parlée complétée (LPC) : production et perception ». Grenoble INPG, 2005. https://tel.archives-ouvertes.fr/tel-00384080.
Texte intégralRésumé :
Le code LPC est un augment manuel qui permet au sourd de désambiguîser la parole visible sur le visage et d'acquérir la phonologie de la langue. Nous avons étudié la production de ce code, encore inexplorée, par une technique de suivi des mouvements labiaux et manuels de quatre code uses professionnelles. Notre résultat comportemental majeur est que, contre toute attente, le geste de la main précède le geste des lèvres. Notre hypothèse est que ce système a été recodé en termes neuralement compatibles pour le contrôle des gestes des voyelles et des consonnes dans la LPC et la parole. Ainsi le contrôle des contacts vocaliques manuels va se trouver en phase avec celui des contacts consonantiques visibles, de façon à ce que, quelle que soit la durée de la syllabe CV, l'aboutissement du stroke de la main se produise dans la phase de tenue de l'attaque consonantique. L'incorporation de la main et de la face dans un espace de contrôle neural commun peut être ainsi pleinement réalisée dans la LPCCued speech is a manual method that allows deaf people to disambiguate the phonological information through the visual channel. Its efficiency for phonological speech acquisition is well established. But a study of cued speech production is lacking. Therefore the aim of this work is to investigate the temporal organization of french cued speech production on four experienced cuers. Our main behavioural result is that the hand gesture anticipates the lip gesture. It is hypothesized that the found pattern of coordination results from the neural compatibility between movement control of consonants and vowels in cued speech and visible speech. Thus the vocalic manual contact control is in-phase with the consonantal contact control of visible speech. This phasing is maintained regardless the variability of the syllable duration : the manual stroke is released du ring the consonant stop phase. This way, in cued speech, hand and face are completely incorporated in a common neural control space
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Andersson, Lisa. « Tvångsvård. Finns det en gråzon mellan LVM och LPT ? » Thesis, Umeå universitet, Statsvetenskapliga institutionen, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-160167.
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På hösten 2018 riktades blickarna mot vården i Sverige. Inte vilken del av vården som helst utan den som berör två av de kanske mest utsatta grupperna i samhället, missbruksvården och psykiatrin. I kölvattnet efter Uppdraggransknings dokumentärer om Sanne beslutade regeringen att det behövdes en (ny) översyn av huvudmannaskapet vid samsjuklighet av missbruk och psykisk sjukdom. (Ny) är denna översyn eftersom Missbruksutredningen 2011 lämnade över sitt slutbetänkande ”Bättre insatser vid missbruk och beroende” som pga. det höga antalet av samsjuklighet yrkade på ett lagförslag där Lag om vård av missbrukare i vissa fall (LVM) och Lag om psykiatrisk tvångsvård (LPT) skulle bli en och samma lag. Regeringen avslog det yrkandet. Hur upplever läkarna, som möter dessa patienter dagligen, att det är att ha två huvudmän för den här patientgruppen? Uppsatsens syfte är att ta reda på hur specialistläkarna inom psykiatri och beroendevården på Vrinnevisjukhuset i Norrköping uppfattar gråzonen mellan LVM och LPT. Detta har gjorts genom intervjuer med läkare som arbetar på Beroendekliniken och på psykiatriska kliniken på Vrinnevisjukhuset i Norrköping. Uppfattningen bland läkarna är att det finns ett behov av ett bättre samarbete mellan de olika instanserna och att strukturerna för hur man ska ha detta samarbete måste vara tydligt. Läkarna anser att lagarna bör fortsatt vara åtskilda men att det kunde vara önskvärt med en och samma huvudman för att minska risken för att patienter hamnar mellan stolarna.
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Velička, Valdas. « Formančių išskyrimo metodų tyrimas ». Master's thesis, Lithuanian Academic Libraries Network (LABT), 2009. http://vddb.library.lt/obj/LT-eLABa-0001:E.02~2009~D_20090630_094841-66393.
Texte intégralRésumé :
Šiame darbe apžvelgėme pagrindinius kalbos atpažinimo požymius, naudojamus kalbos atpažinimo metoduose. Daugiausiai dėmesio skirta formantinių požymių išskyrimo metodams, o vienam iš metodų panaudota programinė įranga. Ji, remdamasi tiesinės prognozės modelio (LPC) parametrais, apskaičiuoja charakteringo polinomo kompleksines šaknis ir iš jų suranda kalbos signalo formančių dažnius. Darbe yra pateiktas aprašas kaip naudotis programa, prieduose įdėti atskirai ištartų žodžių kalbos garsų formantinių požymių tyrimo rezultatai.In this work survey main speech recognition methods as well as features commonly used in speech recognition were discussed in the work. Main attention was paid to formant feature extraction methods. One of the methods feature extraction in software was developed. It is based on linear prediction coding (LPC) parameters and calculates complex roots of characteristic polynomial. These roots are used for formant frequencies determination. Program user guide is presented in the work description. Investigation results of formant features for various speech sounds are presented in the appendices.
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Luo, Qinghua, Yu Peng, Wei Wan, Tao Huang, YaNing Fan et Xiyuan Peng. « Evaluation of FLDPC Coding Scheme for Adaptive Coding in Aeronautical Telemetry ». International Foundation for Telemetering, 2015. http://hdl.handle.net/10150/596396.
Texte intégralRésumé :
ITC/USA 2015 Conference Proceedings / The Fifty-First Annual International Telemetering Conference and Technical Exhibition / October 26-29, 2015 / Bally's Hotel & Convention Center, Las Vegas, NVThe aeronautical telemeter channel is characterized by Multipath interference, Doppler shift and rapid changes in channel behavior. In addition to transmission error during aeronautical telemeter, transmission losing also exists. In this paper, we investigate the correction of transmission error and processing of telemetry transmission losing, and propose an adaptive coding scheme, which organic combines Fountain code and low density parity check (LDPC) code. We call it fountain LDPC (FLDPC) coding. In the coding scheme, The LDPC code is explored to perform transmission error correction, while, the problem of transmission losing is resorted to fountain code. So FLDPC is robust for transmission losing and transmission error. Moreover, without knowing any of these the channel information, FLDPC can adapt the data link and avoid the interference through adjusting the transmission rate. Experimental results illustrated that a signification improvement in transmission reliability and transmitting efficiency can be achieved by using the FLDPC coding.
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Baer, Michael R. « CORRELATION BETWEEN THE LANGUAGE PROCESSING TEST AND THE PROMOTING THE EMERGENCE OF ADVANCED KNOWLEDGE : EQUIVALENCE MODULE ». OpenSIUC, 2016. https://opensiuc.lib.siu.edu/theses/1951.
Texte intégralRésumé :
The present study evaluated the possibility of correlation between the results of the Language Processing Test (LPT3) and the Promoting the Emergence of Advanced Knowledge Relational Training System – Equivalence Module (PEAK-E). Thirteen participants with language disabilities were administered both assessments, and results indicated a significant linear correlation (r = 0.9268, p < 0.1) between the two measures. Implications for a greater understanding of human language and cognition are discussed.
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Carr, Raymond C. « Improvements to a pitch-synchronous linear predictive coding (LPC) vocoder ». Thesis, University of Ottawa (Canada), 1989. http://hdl.handle.net/10393/5954.
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Ko, Choi-lam, et 高彩霖. « Effect of the atherogenic phospholipid, lysophosphatidylcholine (LPC),on endothelial senescence ». Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B44658965.
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Ireton, M. A. « An investigation into LPC based analysis by synthesis speech coding ». Thesis, University of Manchester, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.238646.
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Yehia, Hani Camille. « Análise de funções de erro em sistemas de codificação LPC ». Instituto Tecnológico de Aeronáutica, 1993. http://www.bd.bibl.ita.br/tde_busca/arquivo.php?codArquivo=1733.
Texte intégralRésumé :
A estimação dos coeficientes preditores, em sistemas de codificação de voz baseados em LPC, e realizada través de minimização do valor esperado de uma função do erro de predição. Normalmente utiliza-se uma função quadrática, pois com isso minimiza-se a energia do erro de predição, alem de ser possível uma solução analítica para o problema. Entretanto, não e necessariamente verdade que a minimização da energia do erro de predição esteja associada a maximização do fator de qualidade (ponderado pela curva de resposta do ouvido humano) do sinal decodificado. Neste trabalho, funções de erro alternativas são analisadas e, a seguir, a possibilidade de se adotar um erro de predição relativo e testada. Os resultados obtidos indicam que, apesar do erro quadrático apresentar um bom desempenho quando comparado as funções de erro alternativas, a utilização de um erro de predição relativo pode ser útil em algumas situações.
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Calixto, Mendoza Mario Alejandro. « La Segunda Vuelta Electoral y Reelecci��n Inmediata : un an��lisis para M��xico ». Thesis, Universidad de las Am��ricas Puebla, 2010. http://catarina.udlap.mx/u_dl_a/tales/documentos/lpt/calixto_m_ma/.
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Morales, González Oswaldo. « El Roldán narcotraficante : cultura popular en los narcocorridos ». Thesis, Universidad de las Américas Puebla, 2012. http://catarina.udlap.mx/u_dl_a/tales/documentos/lpt/morales_g_o/.
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Burgess, R. Alan. « Elucidating Molecular Interactions of Shigella Type Three Secretion System Components Critical for Pathogenesis ». DigitalCommons@USU, 2018. https://digitalcommons.usu.edu/etd/6968.
Texte intégralRésumé :
Shigella spp. are Gram-negative, non-motile bacterial pathogens that are the causative agent of bacillary dysentery in humans. During infection, Shigella utilize a complex type three secretion system (T3SS) to inject effector proteins and take over host cell functions. With the rise of multi-antibiotic resistant Shigella strains, the T3SS is a promising alternative therapeutic target. While the needle and syringe-like apparatus of the T3SS has been extensively studied in Shigella, several components and mechanisms of this system remain unclear. The research presented here addresses two major knowledge gaps in the current understanding of the T3SS ATPase, Spa47, and the initial host-pathogen interaction at the tip of the apparatus. In this work, high resolution crystal structures of Spa47 guided the creation of an oligomer model which suggested ATP hydrolysis may be supported by specific side chain contributions from adjacent protomers within the complex. Mutagenesis experiments targeting predicted active site residues and the oligomerization domain revealed that active site residues alone are not responsible for Spa47 oligomerization while protein oligomerization is crucial for ATPase activity. Together with in vivo experiments, we show that ATP hydrolysis and proper Spa47 oligomer formation is critical for T3SS apparatus formation, effector secretion, and overall Shigella virulence. Additionally, we have combined the Langmuir Blodgett technique with fluorescent microscopy to visualize the interaction between key T3SS tip proteins with defined artificial phospholipid membranes. These membranes were generated using Langmuir Blodgett which provided control over lipid phase and composition. Lipid phase and protein localization were monitored using lipophilic dyes and selective fluorescent protein labeling. These experiments suggest a differential interaction between the tip protein IpaB with the membrane components cholesterol and sphingomyelin based on IpaB oligomerization. IpaC, another T3SS tip protein, was found to destabilize membranes when alone, but was stabilized in the presence of IpaB. These experiments suggest that IpaB confers IpaC stability within membranes and that tip protein localization is dependent on lipid phase and composition. Overall, these new insights into the T3SS ATPase and tip proteins provide a more complete understanding of Shigella virulence that will aid in future endeavors to identify alternative therapeutic targets for treatment.
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Lundstedt, Emily. « Lipopolysaccharide structure and LptFG modulate the activity of the LptB2 ATPase ». The Ohio State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu1594998769457759.
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McCree, Alan V. « A new LPC vocoder model for low bit rate speech coding ». Diss., Georgia Institute of Technology, 1992. http://hdl.handle.net/1853/15053.
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Rochette, Denis. « Etude et réalisation d'un vocodeur à dictionnaire LPC 800 BITS/S ». Grenoble 2 : ANRT, 1986. http://catalogue.bnf.fr/ark:/12148/cb37600727b.
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Peters, Richard Alan II. « A LINEAR PREDICTION CODING MODEL OF SPEECH (SYNTHESIS, LPC, COMPUTER, ELECTRONIC) ». Thesis, The University of Arizona, 1985. http://hdl.handle.net/10150/291240.
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Patureau-Hatchett, Micah. « Counselors' Perceptions of Training, Theoretical Orientation, Cultural and Gender Bias, and Use of the Diagnostic and Statistical Manual of Mental Disorders-IV-Text Revision ». ScholarWorks@UNO, 2008. http://scholarworks.uno.edu/td/847.
Texte intégralRésumé :
Counselor educators and counseling practitioners today reflect the future direction of the counseling profession; therefore, their opinions are important when discussing how professional counselors can reconcile the basic philosophies of humanistic counseling with the practical advantages and ethical and philosophical disadvantages that appear to be coexistent when discussing the diagnosis of clients and the Diagnostic and Statistical Manual of Mental Disorders IV-Text Revision (DSM). This study sought to provide a reflective and concise description of the current perceptions of licensed professional counselors in reference to their training, their practice, and their dispositions about diagnosis and utilization of the DSM despite its theoretical grounding in the medical model and its chronic problems with gender and cultural bias—all in direct opposition to counseling's humanistic, multicultural model of practice. Results of this study suggested that more training in DSM/diagnosis led to participants' higher perception of their ability to diagnose and utilize the DSM; however, participants' perceptions were split on whether or not training should include psychopharmacology. Results also suggested that LPCs most frequently occurring ethical dilemma in relation to diagnosis involved the reimbursement requirements of insurance/managed care companies; however, they strongly disagreed that diagnosing clients conflicted with their counseling professional identity. Participants strongly agreed that they were multiculturally competent; however, those participants who indicated that they diagnose using a multicultural or wellness perspective did not agree that the DSM does not adequately present disorders in such a way as to allow LPCs to diagnose culturally diverse and female clients accurately.
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Blanc, Grégory, Alexandre Skrzypczak et Jean-Guy Pierozak. « BLIND EQUALIZATION WITH LDPC CODE : TO QUASIERROR FREE TRANSMISSIONS IN TELEMETRY ». International Foundation for Telemetering, 2016. http://hdl.handle.net/10150/624248.
Texte intégralRésumé :
In a telemetry system, it has been frequently proved that multipath channels and transmission
noise are the most critical sources of distortion. While equalization allows a strong limitation of
the multipath effects, the noise impact can be efficiently reduced if forward error correction is
used. This paper proves that the combination of blind equalization and a powerful FEC like
LDPC strongly improves bit error rates for the SOQPSK modulation. We also prove that a LDPC
code is able to fully correct the residual errors that may persist at the equalizer output. In other
terms, the combination of equalization and LDPC code enables quasi-error free transmissions in
various channel scenarios that represent the various phases of a telemetry mission.
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Lehtinen, Joel, et William Apelqvist. « Restspänningsanalys av sätthärdat stål ». Thesis, Örebro universitet, Institutionen för naturvetenskap och teknik, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:oru:diva-86098.
Texte intégralRésumé :
Denna rapport behandlar restspänning i stålet 16MnCrS5 som uppkommer efter sätthärdning med traditionell uppkolning med släckning i olja och lågtrycksuppkolning med släckning i gas. Rapporten har ett fokus på restspänningar men behandlar även materialegenskaper generellt. I rapporten utförs en karaktärisering av materialet där författarna tittar på, kolhalt, kolprofil, ythårdhet (Rockwell C), kärnhårdhet (Vickers10kg) och hårdhetsprofil (Vickers 1kg) samt analys av mikrostruktur. Även resultat för FWHM kommer tas upp som visat sig kunna relateras till hårdheten hos materialet. Diskussionen handlar om att förklara restspänningen i materialet med hjälp av resultatet från karaktäriseringen. Lågtrycksuppkolning har visat sig inte ge inre oxidation vid sätthärdning, medan provbitar som sätthärdats med traditionell sätthärdning har uppvisat inre oxidation. Den inre oxidationen kan bidra till tryckrestspänningar vid ytan på materialet. Hög andel restaustenit har generellt visat sig vara en bidragande faktor till dragrestspänningar främst vid ytan men även inne i sätthärdningsskiktet. Resultatet har också visat att prover som värmebehandlats med lågtrycksuppkolning och har hög halt restaustenit vid ytan kan också ha hög nivå av dragrestspänning vid ytan.This report addresses residual stress in the steel 16MnCrS5 that occurs after carburization with traditional carburization with quenching in oil and low-pressure carburization with quenching in gas. The report focuses on residual stresses but also deals with material properties in general. The report characterizes the material by looking at the, carbon content, carbon profile, surface hardness (Rockwell C), core hardness (Vickers10kg) and hardness profile (Vickers 1kg) as well as analysis of the microstructure. Low pressure carburization has been shown not to produce internal oxidation, but samples heat treated with traditional carburization has exhibited internal oxidation. High proportion of residual austenite has also been shown to be a contributing factor to tensile residual stresses, mainly at the surface but also within the curing layer. The result have also shown that residual austenite at the surface have been associated with high levels of tensile residual stress at the surface.
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Hussain, Rubina. « Study of the correlation of LSP and LPC frequencies for vowel phonemes ». Thesis, McGill University, 1994. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=26390.
Texte intégralRésumé :
The objective of this thesis is to provide an analysis of Linear Predictive Coding (LPC) frequencies and Line Spectrum Pair (LSP) frequencies. While it is generally known that LSP frequencies congregate about LPC formants, this study will deal with how LSP and LPC frequencies correlate. The work done in this study is intended to enhance the recognition of vowel phonemes by providing observations on the similarities between LPC and LSP frequency distribution. New information regarding exact LSP and formant relationships for different vowel phonemes is provided. Specifically, this study divides vowel and diphthong phonemes into categories, or types. These types pertain to specific patterns in the way LSP frequencies relate to the closest corresponding formant, F1, F2, or F3. In drawing relationships between formants and their corresponding LSPs, another indicator is made possible such that, when incorporated within other statistical or knowledge based techniques, it may serve to enhance the effectiveness of recognition systems.The results of this thesis indicate that it is possible to divide the sixteen vowel phonemes into nine types. The experiment produced a 75% success rate when the test set was applied.
The first part of this thesis provides the theoretical background for the basic understanding of line spectrum pairs and the acoustic-phonetics related to the formation of vowel sounds. The second part of this thesis provides details on how the experiment was conducted and the results of the analysis. Explanations are also provided for these results.
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Jin, Yi-Xuan. « A HIGH SPEED DIGITAL IMPLEMENTATION OF LPC SPEECH SYNTHESIZER USING THE TMS320 ». Thesis, The University of Arizona, 1985. http://hdl.handle.net/10150/275309.
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YEPMO, HENANG VIANNEY. « Model education in the POD space for loss prediction in LPT cascades ». Doctoral thesis, Università degli studi di Genova, 2021. http://hdl.handle.net/11567/1034809.
Texte intégralRésumé :
The present work deals with post-processing by means of Proper Orthogonal Decomposition (POD) of data obtained from experimental investigations on Low-Pressure Turbine (LPT) cascades. The aim is to provide a strategy useful to coarsening design space grids and hence, reduce the data acquisition time during experimental investigations or high-fidelity simulations.
The experimental work has been conducted in a wind tunnel installed at the Laboratory of Aerodynamics and Turbomachinery of the University of Genova. The experimental procedure consists in surveying the flow upstream and downstream of the cascade, aiming to evaluate the total pressure drop through the cascade, and hence the overall losses. Specifically, effects on losses of design parameters such as freestream turbulence intensity, vane solidity, incoming wake parameters (velocity defect, turbulent content and reduced frequency), flow Reynolds number and incidence flow angle have been evaluated.
Then, POD has been applied to data constituted by total pressure loss coefficient distributions in the pitchwise direction under different conditions in order to:
• highlight the loss trend vs the design space parameter variation
• identify the minimum number of requested conditions in the design space, i.e., the number of independent conditions useful for the education of a predictive model
These objectives have been achieved due to the POD capability in providing compact subspaces able to reproduce the same loss trend observed in the physical space. Moreover, in the POD subspace it has been possible to define a smooth kernel with the number of independent conditions in order to estimate the loss distribution of the remaining conditions. Hence, a model has been educated by means of Least Squares Method (LSM) on the POD design space coefficients choosing the predictors as low order polynomials of the independent variables.
Results will show good agreement between measured and modelled loss distribution varying each design space parameter, showing that the model is able to predict loss distribution within the uncertainty measurement. Furthermore, keeping the same strategy for the education of the kernel on another dataset characterizing the loss distribution of two cascades with different geometrical parameters, loss distribution and magnitude are still well captured. Thus, analysis show that the rank of the problem is much lower than the tested conditions, and consequently a reduced number of tests are really necessary. This technique could be useful to reduce the number of scale-resolving simulations or detailed experiments without significant loss of accuracy.
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MORÈ, NICCOLÒ. « Lipopolysaccharide transport and peptidoglycan remodeling : two related processes in Escherichia coli ». Doctoral thesis, Università degli Studi di Milano-Bicocca, 2018. http://hdl.handle.net/10281/198942.
Texte intégralRésumé :
Il rivestimento dei batteri Gram negativi consiste in una membrana interna (IM) e una membrana esterna (OM) separate da uno spazio periplasmatico contenente un sottile strato di peptidoglicano (PG) ancorato alla OM tramite la lipoproteina di Braun (LPP). Mentre la IM è costituita da un doppio strato di fosfolipidi, la OM è una membrana lipidica asimmetrica, contenente fosfolipidi nel foglietto interno, e un lipide complesso il Lipopolisaccaride (LPS) nel foglietto esterno.
Il LPS sintetizzato nel citoplasma, viene traslocato sul lato periplasmatico della IM e preso in carico dal complesso multiproteico Lpt (LPS transport), composto in Escherichia coli da sette proteine essenziali (Lpt ABCDEFG) che si occupano del suo trasporto fino al raggiungimento della sua sede finale, la OM.
Analisi biochimiche hanno dimostrato che le sette proteine Lpt formano un complesso “transenvelope” che connette IM e OM e studi di tipo genetico suggeriscono che esse operino in concerto come un singolo macchinario. Infatti, la deplezione di un qualsiasi componente del complesso Lpt causa lo stesso fenotipo, ovvero l’accumulo del LPS nel versante periplasmatico della IM, la decorazione del LPS con acido colanico e la formazione di una IM anomala, con una densità intermedia tra la IM e la OM.
Nel nostro laboratorio è stata condotta l’analisi differenziale del proteoma delle membrane totali di E. coli in deplezione di LptC, per studiare la risposta globale al blocco del trasporto del LPS. Tra le proteine il cui livello cambia nel confronto tra il ceppo non depleto e il depleto sono state trovate proteine coinvolte nella biogenesi e nel rimodellamento del PG.
Lo scopo di questa tesi è stato lo studio della correlazione tra il blocco del LPS e il rimodellamento del PG.
Inizialmente è stata analizzata la struttura del PG in deplezione di LptC. Questa analisi ha evidenziato che in questa condizione la struttura del PG varia sia per composizione che per tipo di legami crociati tra i filamenti glicanici adiacenti.
Nei batteri Gram negativi il legame tra i filamenti glicanici è generalmente un legame diretto 3-4, che si forma tra il gruppo aminico del diaminoacido in posizione 3 di un tetrapeptide e il gruppo carbossilico della D-alanina in posizione 4 del tretrapeptide adiacente. Il legame 3-4 avviene ad opera delle D,D transpeptidasi PBP. Un altro tipo di legame crociato presente nel PG è quello tipo 3-3 che si forma tra il gruppo aminico del diaminoacido in posizione 3 ed il gruppo carbossilico del diaminoacido presente nel tetrapeptide del filamento glicanico adiacente ed è catalizzato da L,D-transpeptidasi .
In E. coli, sono noti cinque enzimi con attività L,D-transpeptidasica, di cui tre (LdtA, LdtB, LdtC) ancorano la lipoproteina più abbondante della OM (lipoproteina di Braun) al PG e due (LdtD, LdtE) catalizzano il legame crociato 3-3. La delezione di tutti questi geni, singolarmente o in combinazione, non presenta nessun fenotipo, suggerendo che in condizioni normali questo legame è dispensabile.
Per studiare la correlazione tra il legame crociato 3-3 e il blocco del trasporto del LPS abbiamo creato mutanti arabinosio dipendenti per alcuni dei componenti del sistema Lpt deleti contemporaneamente per i geni che esprimono le L,D-transpeptidasi LdtD e LdtE.
In precedenza, nel nostro laboratorio è stato dimostrato che la deplezione di uno qualsiasi dei geni lpt causa la formazione di cellule filamentose e l’arresto della crescita ma non la lisi cellulare. Invece, nei mutanti ΔldtDΔldtE, in deplezione dei geni lpt, oltre alla formazione di cellule filamentose si osserva la formazione di un setto anomalo e la lisi cellulare. Questi dati suggeriscono che il rimodellamento del peptidoglicano a seguito della formazione di legami 3-3 potrebbe essere una forma di riposta al danno alla membrana esterna.The cell envelope of Gram-negative bacteria is a complex multi-layered structure consisting of a cytoplasmic and an outer membrane (CM and OM), which delimit the periplasm containing a thin layer of peptidoglycan (PG) called the sacculus. The primary function of the OM is to establish a selective permeability barrier that enables the cell to maintain favourable intracellular conditions even in hash environments and the lipopolysaccharide (LPS) layer greatly contributes to this peculiar property. The integrity of the PG mesh is essential to protect the cell from bursting due to its turgor and maintain the shape of the cell. OM and PG are synthetized and assembled by multiprotein machineries that need to be finely coordinated as imbalanced growth of these layers may compromise structural integrity of the cell. In order to gain more insight in the mechanism by which the cells coordinate the growth of these two layers, we analysed the PG composition when the biogenesis of OM is compromised due to the block of LPS transport. In this work we shown that when OM is impaired, E. coli cells remodel PG architecture by increasing the non-canonical 3- 3 cross-linkage. We can assume that this is a salvage mechanism to prevent cell lysis when OM is damaged.
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Mračko, Peter. « Analýza Parkinsonovy nemoci pomocí segmentálních řečových příznaků ». Master's thesis, Vysoké učení technické v Brně. Fakulta elektrotechniky a komunikačních technologií, 2015. http://www.nusl.cz/ntk/nusl-221394.
Texte intégralRésumé :
This project describes design of the system for diagnosis Parkinson’s disease based on speech. Parkinson’s disease is a neurodegenerative disorder of the central nervous system. One of the symptoms of this disease is disability of motor aspects of speech, called hypokinetic dysarthria. Design of the system in this work is based on the best known segmental features such as coefficients LPC, PLP, MFCC, LPCC but also less known such as CMS, ACW and MSC. From speech records of patients affected by Parkinson’s disease and also healthy controls are calculated these coefficients, further is performed a selection process and subsequent classification. The best result, which was obtained in this project reached classification accuracy 77,19%, sensitivity 74,69% and specificity 78,95%.
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Vrba, Václav. « Robustní detekce klíčových slov v řečovém signálu ». Master's thesis, Vysoké učení technické v Brně. Fakulta elektrotechniky a komunikačních technologií, 2014. http://www.nusl.cz/ntk/nusl-220670.
Texte intégralRésumé :
The master thesis is divided into two parts theoretical and practical. The theoretical part is focused on methods of analysis and detection of speech signals. In the practical part the system for isolated word recognition was created in Matlab. The system is speaker independent separately for men and women. Also two speech databases were created for further use in the aircraft cockpit. Tests and evaluations were performed even with added noise.
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Söderström, Rose Marie, et Sedvall Kim Jüllig. « Läkemedel och tvång : en empirisk studie om patienters upplevelser av läkemedelsadministrering under LPT ». Thesis, Röda Korsets Högskola, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:rkh:diva-143.
Texte intégralRésumé :
Läkemedelshantering såsom administrering av medicin till patienter är en av de dagliga omvårdnadshandlingarna för en sjuksköterska. För en patient som vårdas under Lagen om Psykiatrisk Tvångsvård (LPT) (SFS, 1991:1128) kan upplevelser av att få medicin vara blandade. I sjuksköterskans omvårdnadsarbete är det viktigt att se patientens unika upplevelser av situationen. Syftet med denna studie är att belysa patienters upplevelser av läkemedelsadministrering då de vårdas under LPT. Metoden är av kvalitativ natur där empirin samlats in med hjälp av intervjuer som har analyserats med manifest innehållsanalys. Resultatet visar blandade upplevelser hos patienter då Acceptans och Välbefinnande blandat med Fruktan, Resignation, Utsatthet, Ovilja och Brist på information framkommit. Av resultatet drogs Slutsatsen att kommunikation mellan patient och sjuksköterska är viktig samt att Brist på kommunikation kan leda till lidande för patienten.
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Bankel, Anna, et Ulrika Jönsson. « Hela havet stormar : En rättsfallsstudie om tillämpningen av LVM och LPT vid samsjuklighet ». Thesis, Linnéuniversitetet, Institutionen för socialt arbete (SA), 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:lnu:diva-60088.
Texte intégralRésumé :
This article is about the application of the two coercive legislations, Care of Abusers (Special Provisions) Act (LVM) and the Compulsory Psychiatric Care Act (LPT) when individuals suffer from comorbidity. The authority who files the application for coercive interventions differs between the legislations. In LVM the social welfare board files the application and in LPT the chief psychiatrist. Earlier research has shown that the individuals of the comorbidity group do not get their needs met in either of the legislations. It has also occurred that concrete arguments which relate to statutory criteria are missing. Our aim was to gain knowledge about how the court argued for these criteria, especially in cases where comorbidity exist. Another aim was to see which legislation that gave legal impact. Finally, we wanted to examine the roles of the chief psychiatrist and the social welfare board in court negotiations. We decided to study twenty court cases from Administrative court, ten cases from LVM and ten from LPT where the same individual had been committed to coercive care according to both legislations, this to capture the comorbidity. We interpreted the documents using discourse analysis and selected two concepts, inspired by Foucault and Fairclough, that we thought could be fruitful for our analysis. The result shows that there is more often argumentation about if the legal criteria in LVM are fulfilled compared to LPT where the argumentation is less informative. This result may have been influenced by the secrecy of Law. We also found out that the legislation that was given legal impact depended on the authorities assess of which need that was most urgent. The last finding was that the chief psychiatrist`s opinion was never questioned by the court, but the statements made by the social welfare board was.
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Brunqvist, Klara, et Stephanie Hansson. « Beredande av vård enligt LVM alternativt LPT för personer med både missbruk och psykisk ohälsa : En studie av rättskällor ». Thesis, Ersta Sköndal högskola, Institutionen för socialvetenskap, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:esh:diva-4021.
Texte intégralRésumé :
I förarbeten till lagarna LVM och LPT framkommer det att det finns en problematik kring vården av personer som lider av både missbruk och psykisk ohälsa. Det framgår att samarbetet mellan psykiatri och socialtjänst i vissa avseende är att anse som bristfälligt och att brukarna faller mellan stolarna. Syftet med studien har varit att, genom granskning av rättskällorna, ta reda på och tydliggöra vad som ska ses som gällande rätt, för om en person med både missbruk och psykisk ohälsa ska få vård enligt LVM eller LPT. Metoden som har använts är en rättsvetenskaplig metod där fokus legat på granskning av rättskällorna. En rättsfallsstudie har även gjorts för att bredda bilden av hur rekvisiten för lagstiftningarna tillämpas. Resultatet har analyserats och redovisats utifrån den rättsdogmatiska metoden. Rättsfallen har tematiserats och redovisats i diagram. Studien visar att det finns gällande rätt kring ämnet, men att gällande rätt varierar beroende på personens situation. Huvuddraget i resultatet är att LPT vid allvarlig psykisk ohälsa bör gå före LVM. Vidare framkommer det att det inte finns något hinder, för att under en kortare period, vårdas enligt båda lagstiftningarna samtidigt.In the preparatory works to the laws of LVM and LPT it has been pointed out that there is a problem regarding the care of persons that both suffer from an addiction as well as mental illness. It appears that the cooperation between psychiatry and social services is deficient and that the users are falling through the cracks. The purpose of the study has been, through review of the sources of law, to find out and define what is to be considered a correct, application of the law for a person that suffers from an addiction as well as a mental illness. Is it to be admitted to compulsory care and treatment according LVM or LPT? The method and material used for this purpose is the sources of law. To broaden the picture a study of court cases has also been made, regarding how the legislation is applied by a lower administrative court. The results has been analyzed and reported through the legal method. Court cases has been thematized and presented in different diagrams. The study shows that the outcome of the law varies depending on the person’s situation. The main feature of the result is that if someone is suffering from serious mental illness, LPT should be chosen instead of LVM. Further it is possible, during a shorter time, to be treated according to both LVM and LPT simultaneously.