Tesis sobre el tema "L18A"

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Peptdeos antimicrobianos possuem em geral cadeias curtas, carga líquida positiva devido ao excesso dos res duos b asicos e são ricos em amino acidos hidrof obicos. Possuem um grande potencial farmacológico, com atividade antimicrobiana modulada por interações eletrost aticas e hidrof obicas. Neste trabalho utilizou-se o pept deo sintético L1A (IDGLKAIWKKV ADLLKNT NH2), que devido aos res duos de acido asp artico e lisinas constituintes de sua cadeia, possui carga liquida +3, em pH neutro. Foram analisadas as insuficiências do pH e da carga de ves culas aniônicas na adsorçãao do pept deo. Este estudo utilizou medidas de potencial eletrocin etico de ves culas e experimentos de titulação monitorados por dicro smo circular (CD). A adsorção do L1A a LUVs de POPC e misturas POPC/POPG em diferentes proporções de POPG, foi medida nos pHs 4, 7 e 10. Os espectros de dicro smo circular dos peptídeos, na presença de vesícula, apresentaram caracter sticas de estruturas helicoidais, enquanto apresentaram estruturas desordenadas em tampão. As frações de hélices obtidas são maiores quando o pept deo adsorve em ves culas com maiores quantidades de POPG, indicando forte contribuição eletrost atica. As constantes aparentes de adsorção dos pept deos as membranas modelo foram calculadas atrav es da elipticidade de CD normalizada em 222 nm, obtidas por titulações de pept deo com ves culas. A a - nidade do pept deo a ves culas aniônicas e signi cativamente maior do que a ves culas zwitteriônicas, o que refor ca a import ancia das interçõeoes eletrost aticas no processo de adsorção do pept deo na bicamada. O efeito conjunto de carga das ves culas e de pH resultaram em signi cativa regulação de carga do pept deo resultando em valores de carga efetiva alta em pH 4,0 devido a protonação dos res duos de asp artico. Em pH 10,0 a pequena carga efetiva calculada deve-se as desprotona ações do N-terminal e das lisinas...
Antimicrobial peptides have in general short chains, positive net charge due to the excess of basic residues and are rich in hydrophobic amino acids. They have pharmacological potential as antimicrobial agents and their activity is modulated by electrostatic and hydrophobic interactions. In this work, we used the synthetic peptide L1A (IDGLKAIWKKV ADLLKNT NH2) that due to its acid and basic residues have an net charge +3, at neutral pH. The e ects of the pH and of the charges of anionic vesicles on the adsorption of L1A were analized. This study used measurement of electrokinetic potential of vesicles and titration experiments monitored by circular dichroism spectroscopy (CD). Adsorption of L1A to POPC and POPC/POPG LUVs in di erent POPG contents were assessed in the pH 4.0, 7.0 and 10.0. Circular dichroism spectra of the peptides in the presence of vesicles showed to features of helical structures; while random coil structures were observed at bu er for all the used pHs. The helical content was evaluated and showed to increase when the peptide adsorbs into vesicles with increasing amounts of POPG, indicating that there is a strong electrostatic contribution. Partition coe cients were calculated through the normalized CD ellipticities at 222 nm and showed that the a nity of the peptide for anionic model membranes is e ectively higher than those found for zwitterionic ones. It reinforces the importance of the electrostatic contribution to the process of peptide-lipid interaction. The coupled e ect of the vesicle charge and pH lead to signi cant charge regulation of the peptide resulting in high values of e ective charge at pH 4.0 due to the deprotonation of aspartic acid residues. At pH 10.0 the estimated e ective charge is small as a consequence of the deprotonation of both N-terminus and the lysines. The electrostatic and interfatial free energies seems to be additive only at pH 4.0, especially for the bilayers with higher content...

Orientador: João Ruggiero Neto
Banca: Alexandre Suman de Araujo
Banca: Fernando Luis Barroso da Silva
Resumo: Peptdeos antimicrobianos possuem em geral cadeias curtas, carga líquida positiva devido ao excesso dos res duos b asicos e são ricos em amino acidos hidrof obicos. Possuem um grande potencial farmacológico, com atividade antimicrobiana modulada por interações eletrost aticas e hidrof obicas. Neste trabalho utilizou-se o pept deo sintético L1A (IDGLKAIWKKV ADLLKNT ���� NH2), que devido aos res duos de acido asp artico e lisinas constituintes de sua cadeia, possui carga liquida +3, em pH neutro. Foram analisadas as insuficiências do pH e da carga de ves culas aniônicas na adsorçãao do pept deo. Este estudo utilizou medidas de potencial eletrocin etico de ves culas e experimentos de titulação monitorados por dicro smo circular (CD). A adsorção do L1A a LUVs de POPC e misturas POPC/POPG em diferentes proporções de POPG, foi medida nos pHs 4, 7 e 10. Os espectros de dicro smo circular dos peptídeos, na presença de vesícula, apresentaram caracter sticas de estruturas helicoidais, enquanto apresentaram estruturas desordenadas em tampão. As frações de hélices obtidas são maiores quando o pept deo adsorve em ves culas com maiores quantidades de POPG, indicando forte contribuição eletrost atica. As constantes aparentes de adsorção dos pept deos as membranas modelo foram calculadas atrav es da elipticidade de CD normalizada em 222 nm, obtidas por titulações de pept deo com ves culas. A a - nidade do pept deo a ves culas aniônicas e signi cativamente maior do que a ves culas zwitteriônicas, o que refor ca a import^ancia das interçõeoes eletrost aticas no processo de adsorção do pept deo na bicamada. O efeito conjunto de carga das ves culas e de pH resultaram em signi cativa regulação de carga do pept deo resultando em valores de carga efetiva alta em pH 4,0 devido a protonação dos res duos de asp artico. Em pH 10,0 a pequena carga efetiva calculada deve-se as desprotona ações do N-terminal e das lisinas...
Abstract: Antimicrobial peptides have in general short chains, positive net charge due to the excess of basic residues and are rich in hydrophobic amino acids. They have pharmacological potential as antimicrobial agents and their activity is modulated by electrostatic and hydrophobic interactions. In this work, we used the synthetic peptide L1A (IDGLKAIWKKV ADLLKNT ���� NH2) that due to its acid and basic residues have an net charge +3, at neutral pH. The e ects of the pH and of the charges of anionic vesicles on the adsorption of L1A were analized. This study used measurement of electrokinetic potential of vesicles and titration experiments monitored by circular dichroism spectroscopy (CD). Adsorption of L1A to POPC and POPC/POPG LUVs in di erent POPG contents were assessed in the pH 4.0, 7.0 and 10.0. Circular dichroism spectra of the peptides in the presence of vesicles showed to features of helical structures; while random coil structures were observed at bu er for all the used pHs. The helical content was evaluated and showed to increase when the peptide adsorbs into vesicles with increasing amounts of POPG, indicating that there is a strong electrostatic contribution. Partition coe cients were calculated through the normalized CD ellipticities at 222 nm and showed that the a nity of the peptide for anionic model membranes is e ectively higher than those found for zwitterionic ones. It reinforces the importance of the electrostatic contribution to the process of peptide-lipid interaction. The coupled e ect of the vesicle charge and pH lead to signi cant charge regulation of the peptide resulting in high values of e ective charge at pH 4.0 due to the deprotonation of aspartic acid residues. At pH 10.0 the estimated e ective charge is small as a consequence of the deprotonation of both N-terminus and the lysines. The electrostatic and interfatial free energies seems to be additive only at pH 4.0, especially for the bilayers with higher content...
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Proteins of the family of LRR-RLKs (receptor-like-kinases with leucine-rich repeats) have a conceptual relevance in signaling events but in plants information regarding function is limited to a few members of this family. The receptors NIK1, NIK2 and NIK3 of Arabidopsis thaliana belong to the sub-family LRRII-RLK and were initially identified by their capacity to interact with the geminivirus NSP protein. In response to an unknown stimulus, NSP-interacting kinases (NIKs) are activated after the formation of dimmers and intermolecular autophosphorylation. The inhibition of autophosphorylation of NIK by NSP and the activation of NIK genes increase the susceptibility to viral infection, suggesting that this protein is involved in a defense pathway against geminivirus infection. The downstream components of this pathway, mediated by the protein NIK, have yet to be identified. In the present study, the biochemical and functional characterization of two ribosomal proteins, L10 and L18 were described, these being capable of interacting with the protein NIK via the yeast two-hybrid system. In vitro studies demonstrated that the protein NIK is capable of phosphorylating the protein L10, but not L18. Of the members of the LRRIIRLK family, the protein NIK2 phosphorylates L10, while NIK3 presents a low capacity for phosphorylation of the substrate. However, the development protein SERK1 does not use L10 as a substrate. Assays of transient expression in tobacco plants, revealed that the L18 protein is located in the cytoplasm, as well as around the nucleus and the nucleoli of some cells. In turn, in 97% of the cells, L10 was localized only in the cytoplasm, although it was also found in the nucleus, in approximately 3% of the observed cells. The transient expression of NIK1 and NIK2 redirects the L10 protein to the nucleus in approximately 30% of the cells. In contrast, NIK3 does not relocalize the L10 protein to the nucleus, and L18 does not change its localization in the presence of the NIKs. In plants infected with TGMV, a change only in the cytoplasmic localization of L10 was observed, accumulating in points of the cytoplasm when not co-localized with NIK. To prove genetically the interactions of L10-NIK and L18-NIK, null alleles for the genes L10 and L18 de Arabidopsis, containing T-DNA insertion, were obtained and inoculated with CaLCuV. The inactivation of the genes L10 and L18 restored the elevated susceptibility phenotype of nik1 and the knockout plants, principally l10, presented severe symptoms and high rates of infection when compared with the wild columbia plants. The results of this work are consistent with a model that places the ribosomal proteins L10 and L18 as functional components of the defense signaling pathway mediated by the protein NIK, L10 being a component immediately downstream of the transmembrane receptor.
As proteínas da família das LRR-RLKs (receptor-like-quinases com repetições ricas em leucina) possuem uma relevância conceitual em eventos de sinalização mas, em plantas, a informação funcional ainda é restrita a alguns membros desta família. Os receptores NIK1, NIK2 e NIK3 de Arabidopsis thaliana pertecem à sub-familia LRRII-RLK e foram inicialmente identificados pela sua capacidade de interagir com a proteína NSP de geminivírus. Em resposta a um estímulo desconhecido, NSP-interacting kinases (NIKs) são ativadas após a formação de dímeros e autofosforilação intermolecular. A inibição da autofosforilação de NIK por NSP e a inativação de genes NIKs aumenta a suscetibilidade à infecção viral, sugerindo que esta proteína estaria envolvida em uma via de defesa contra a infecção por geminivírus. Os componentes downstream dessa via de sinalização, mediada pela proteína NIK, ainda não foram identificados. No presente estudo foi descrita a caracterização bioquímica e funcional de duas proteínas ribossomais, L10 e L18, as quais foram capazes de interagir com a proteína NIK através do sistema de duplo híbrido de leveduras. Estudos in vitro demonstraram que a proteína NIK é capaz de fosforilar a proteína L10, mas não L18. Entre os membros da família LRRII-RLK, a proteína NIK2 fosforila L10, enquanto NIK3 apresenta uma baixa capacidade de fosforilação do substrato. No entanto, a proteína de desenvolvimento SERK1 não utiliza L10 como substrato. Ensaios de expressão transiente, em plantas de tabaco, revelaram que a proteína L18 está localizada no citoplasma, bem como ao redor do núcleo e no nucléolo de algumas células. Por sua vez, em 97% das células, L10 foi localizada apenas no citoplasma, embora tenha sido encontrada no núcleo, em aproximadamente 3% das células observadas. A expressão transiente de NIK1 e NIK2 redireciona a proteína L10 para o núcleo em aproximadamente 30% das células. Em contraste, NIK3 não relocaliza a proteína L10 para o núcleo, e L18 não muda sua localização na presença das NIKs. Em plantas infectadas com TGMV, observou-se mudança apenas na localização citoplasmática de L10, acumulando-se em pontos do citoplasma quando não colocalizada com NIK. Para se comprovar geneticamente as interações de L10-NIK e L18- NIK, alelos nulos para os genes L10 e L18 de Arabidopsis, contendo inserção de T-DNA, foram obtidos e inoculados com o CaLCuV. A inativação dos genes L10 e L18 recapitulou o fenótipo de suscetibilidade aumentada de nik1 e as plantas knockout, principalmente l10, apresentaram sintomas severos e taxa de infecção alta quanto comparados com as plantas selvagens columbia. Os resultados deste trabalho são consistentes com um modelo que posiciona as proteínas ribossomais L10 e L18 como componentes funcionais da via de sinalização de defesa mediada pela proteína NIK, sendo L10 um componente imediatamente downstream ao receptor transmembrana.

Bien que les grains de poussières représentent 1% du milieu interstellaire en masse, leur étude est essentielle pour comprendre le contenu et la structure des nuages interstellaires. Les grains de poussière, après avoir quitté leurs lieux de formation, se dispersent dans le milieu diffus avant d’être rassemblés lors de la formation des nuages moléculaires denses. C’est lors de cette étape qu’ils grossissent, notamment par coagulation, et acquièrent des manteaux de glaces. Ces changements morphologiques modifient également leurs propriétés optiques (absorption, diffusion et émission). Cependant, leur composition comme leur taille et leur forme restent difficiles à déterminer à partir des observations et constituent un problème hautement dégénéré. L’utilisation des longueurs d’onde en émission n’est pas suffisante pour lever cette dégénérescence dans les cœurs denses, lieu de formation des futures étoiles et planètes. En revanche la diffusion peut dominer l’absorption à 3.6 et 4.5 μm, ce phénomène appelé coreshine, est particulièrement utile pour sonder les parties les plus denses des nuages. La présence de coreshine dans plus d’une centaine de nuages de notre Galaxie permet d’éliminer bon nombre de modèles de poussières. La modélisation multi–longueurs d’onde en 3 dimensions est une approche nécessaire pour caractériser la balance entre l’absorption du rayonnement et sa diffusion. Alors que la plupart des travaux se concentrent sur l’absorption et la réémission du rayonnement, la diffusion, souvent délaissée, permet d’apporter une vision complète du transfert de rayonnement dans les nuages denses
Even though dust grains contribute only to 1% of the interstellar medium mass, their study is crucial to understand both the structure and content of interstellar clouds. Dust grains leave their birth places, spread out into the diffuse medium before being gathered together again when dense molecular clouds form. During this last stage, they grow, by coagulation especially, and they acquire ice mantles composed mainly of water. These morphological changes also modify their optical properties (absorption, scattering, and emission). However, it remains a highly degenerate issue to determine their composition, size, and shape from observations. In particular, I will highlight that using wavelengths associated to dust emission is not sufficient to investigate the dense cores, where stars and planets will form. I will show that scattering can dominate the absorption at 3.6 and 4.5 μm, and that this phenomenon called coreshine is a powerful tool to investigate the densest parts of molecular clouds. The coreshine detection in more than one hundred clouds of our Galaxy allows us to eliminate a large number of dust models. Multi–wavelength 3D modeling is mandatory to characterize the balance between the absorption and the scattering of the radiation field. While most of the work about dust focus on absorption and re–emission of the radiation, I will present how scattering, often neglected, brings a complete picture of the radiative transfer inside dense clouds

L'évolution de la prospère famille des standards 802.11 a encouragé le développement des technologies appliquées aux réseaux locaux sans fil (WLANs). Pour faire face à la toujours croissante nécessité de rendre possible les communications à très haut débit, les systèmes à antennes multiples (MIMO) sont une solution viable. Ils ont l'avantage d'accroître le débit de transmission sans avoir recours à plus de puissance ou de largeur de bande. Cependant, l'industrie hésite encore à augmenter le nombre d'antennes des portables et des accésoires sans fil. De plus, à l'intérieur des bâtiments, la déficience de rang de la matrice de canal peut se produire dû à la nature de la dispersion des parcours de propagation, ce phénomène est aussi occasionné à l'extérieur par de longues distances de transmission. Ce projet est motivé par les raisons décrites antérieurement, il se veut un étude sur la viabilité des transcepteurs sans fil à large bande capables de régulariser la déficience de rang du canal sans fil. On vise le développement des techniques capables de séparer M signaux co-canal, même avec une seule antenne et à faire une estimation précise du canal. Les solutions décrites dans ce document cherchent à surmonter les difficultés posées par le medium aux transcepteurs sans fil à large bande. Le résultat de cette étude est un algorithme transcepteur approprié aux systèmes MIMO à rang déficient.

碩士
國立陽明大學
生化暨分子生物研究所
103
β-amyloid peptide (Aβ), which consists of 39-42 residues, is derived from proteolysis of β-amyloid precursor protein (APP). Aggregation of β-amyloid peptide (Aβ) is thought to be an early event in the pathogenesis of Alzheimer’s disease. The molecular mechanism underlying Aβ aggregation is not entirely understood. The process of Aβ aggregation into amyloid fibril involves conformational changes, suggesting that the conformational stability of Aβ play a key role in the aggregation process. Recently, we reported that L17A/F19A replacements could increase the conformational stability of wild-type Aβ40 and familial Alzheimer's disease-linked E22G variant of Aβ40 (Arctic Aβ40 variant), and reduce their α-helix-to-β-strand conversion and fibril formation rates. In this study, the effects of L17A/F19A replacements on the aggregation behavior and secondary structure propensity of D23N variant of Aβ40 (Iowa Aβ40 variant), which is also a familial Alzheimer's disease-linked variant of Aβ40, were characterized by using Nuclear Magnetic Resonance spectroscopy (NMR spectroscopy), Circular Dichroism spectroscopy (CD spectroscopy), Transmission Electron Microscopy (TEM) and Thioflavin T fluorescence assay. The results of kinetic aggregation studies indicated that L17A/F19A replacements can inhibit conformational changes and fibril formation of Aβ40(D23N). NMR structural studies revealed that L17A/F19A replacements mainly increased the α-helical propensity of the residues located in the α/β-discordant segment of Aβ40(D23N). These findings suggested that the α-helical propensity of the α/β-discordant segment is one key factor in governing the aggregation propensity of Aβ. This result may also provide a structural basis toward understanding the molecular mechanism of Aβ aggregation.

碩士
逢甲大學
工業工程學所
92
One of the basic assumptions of Taguchi’s parameter design is that the error variances are not equal for all treatment conditions. Thus, the optimal level combination of parameter is determined by maximizing signal-to-noise ratio for the quality characteristics. On the contrary, one of the basic assumptions of experimental design is that error variance are equal for all treatment conditions. Thus, the factor level combinations whose quality characteristics is significant different from the other factors is found by using ANOVA. If the assumption of the later is true, then there is an alpha risk that Taguchi method may fail to select the optimal level combinations. The purpose of this paper is to investigate the alpha risk of Taguchi method for the-larger-the-better (LTB) type problem using L18 by simulation.