Na primeira parte desta dissertação, nos concentramos na utilização de uma abordagem baseada na remoção oxidativa controlada de sementes/núcleos promovida pela adição de HCl na síntese poliol de nanofios de prata (Ag). Isto possibilitou um controle fino sobre a espessura dos nanofios gerados na faixa de de 65 a 765 nm, no qual as larguras obtidos apresentaram um aumento linear em função do aumento na concentração de HCl utilizado na reacção. Embora a largura também se mostrou dependente de outros parâmetros experimentais, tais como a concentração de AgNO3 e polivinilpirrolidona (PVP) e temperatura, remoção oxidativa promovida por HCl possibilitou o controle sobre uma faixa de tamanhos mais ampla. Também investigamos as propriedades ópticas dos nanofios de Ag em função do seu tamanho e sua aplicabilidade como substratos para detecção atraves do fenômeno de espalhamento Raman intensificado por superfície (SERS). Na segunda parte deste trabalho, nanotubos de Ag-Au, Ag-Pt e Ag-Pd contendo morfologias de superfície controladas foram obtidos através da reação de substituicão galvânica entre os nanofios de Ag e íons AuCl4-, PtCl62-, e PdCl42-, respectivamente. Nesse caso, superfícies lisas foram obtidas a 100 oC enquanto superfícies rugosas foram observadas a temperatura ambiente. Mostramos ainda que superfícies lisas também puderam ser obtidas usando-se uma solução saturada de NaCl como solvente durante a reação galvânica. Na terceira parte deste trabalho, investigamos o efeito da excitação da ressonância plasmônica de superfície (SPR) nos nanofios de Ag sobre a atividade catalítica frente a redução do 4-nitrofenol. Contudo, nossos resultados mostraram um queda da atividade catalítica pela excitação SPR, mostrando que a excitação do SPR também pode levar a efeitos detrimentais sobre a atividade catalítica dependendo do mecanismo de reação estudado e natureza de intermediários na etapa determinante da velocidade de reação. Como as propriedades de nanoestruturas metálicas são fortemente dependentes de tamanho, forma e composição, acreditamos que os resultados apresentados aqui relatados podem ter implicações importantes para o design de nanomateriais unidimensionais com características/propriedades desejadas para aplicações em diversas áreas, incluindo óptica e catalise. / In the first part of this thesis, we focused on the utilization of an approach based on controlled oxidative etching for the removal of seeds/nuclei promoted by the addition of HCl in the polyol synthesis of silver nanowires (Ag). This allowed a precise control over the width of the Ag nanowires in the 65-765 nm range. In this case, the widths showed a linear increase with the HCl concentration employed in the reaction. While the width was also dependent on other experimental parameters such as the concentration of AgNO3 and polyvinylpyrrolidone (PVP) as well as the temperature, oxidative etching promoted by HCl enabled the control over a wider range of sizes. We also investigated the optical properties of Ag nanowires as a function of their size and their applications as substrates for surface enhanced Raman scattering (SERS). In the second part of this work, Ag-Au, Ag-Pt, and Ag-Pd nanotubes displaying controlled surface morphologies were obtained by galvanic replacement reaction between the Ag nanowires and AuCl4-, PtCl62-, e PdCl42- ions, respectively. In this case, while smooth surfaces were obtained at 100 °C, rough surfaces were observed at room temperature. We also showed that smooth surfaces could be obtained by using a saturated solution of NaCl as the solvent during the galvanic reaction. Finally, in the third part of this study, we investigated the effect of surface plasmon resonance (SPR) excitation on Ag nanowires over the catalytic activity towards the 4-nitrophenol reduction. However, our results indicated a drop in catalytic activity with SPR excitation, showing that the SPR excitation can also lead to detrimental effects on the catalytic activity depending on the nature of the chemical reaction mechanism and intermediates in the rate-determining step of the reaction. As properties in metallic nanostructures are strongly dependent on size, shape and composition, we believe that the results reported here may have important implications for the design of one-dimensional nanomaterials design with desired features/properties for applications in various fields that include optics and catalysis.

Hop (Hsp70-Hsp90 Organizing Protein) is a co-chaperone of two major molecular chaperones, Hsp70 and Hsp90, and acts by transferring substrates from Hsp70 to Hsp90. Although under normal conditions Hop is predominantly localized within the cytosol, Hop has been detected in the nucleus under certain conditions including cell cycle arrest. A putative nuclear localization signal (NLS) has been identified within Hop, which overlaps with the TPR2A domain (previously shown to be critical for Hop-Hsp90 interactions). Hop is phosphorylated in vitro by two cell cycle kinases, namely, casein kinase II (CKII) at S189 and cdc2-kinase at T198; both residues are found upstream of the putative NLS and TPR2A domain. Mimicking phosphorylation at either phosphorylation site appeared to affect the subcellular localization of Hop. The aim of this study was to characterize Hop with respect to its phosphorylation status in vivo, as well as its subcellular localization pattern under heat stress and determine how these properties affected its interaction with Hsp90 as a co-chaperone. Dephosphorylation of proteins under normal and heat shock conditions changed the isoform composition of Hop, providing strong evidence that Hop was phosphorylated in vivo. Surface plasmon resonance (SPR) and glutatione-S-transferase (GST) co-precipitation studies showed that a cdc2-kinase phosphorylated mimic of Hop disrupted Hop-Hsp90 binding. A full length Hop-EGFP construct, as well as substitution mutants of the predicted NLS residues within the Hop-EGFP construct, were transfected into baby hamster kidney (BHK)-21 cells in order to establish the subcellular localization of Hop under heat stress and to test whether predicted residues were critical for nuclear localization of Hop. Under normal conditions, both Hop-EGFP and the NLS mutants were predominantly cytosolic, but when the cells were subjected to heat stress, Hop and its NLS-mutants were localized to both the cytosol and the nucleus. SPR and GST co-precipitation studies showed that substitution of the residues within the major arm of the putative NLS abrogated Hop-Hsp90 interactions. The data obtained from this study, showed for the first time, that Hop was phosphorylated in vivo and suggested that phosphorylation of Hop by cdc2-kinase could inhibit Hop-Hsp90 interactions. Moreover, these results suggested that the subcellular localization of Hop was dependent on stress levels of the cell, particularly heat stress. We propose that the nuclear localization of Hop may be primarily regulated by stress and secondarily by cell cycle arrest. The major arm of the putative NLS did not affect the localization of Hop directly, but was shown to be critical for Hop-Hsp90 binding in vitro. The results of this study suggested that binding of Hop to Hsp90 sequestered Hop within the cytosol and that Hsp90 acted as a cytosolic retention factor for Hop. Both phosphorylation of Hop, and its subcellular localization, appeared to be intimately related to its interaction with Hsp90 as a co-chaperone.

La découverte de la photoluminescence du silicium sous sa forme nanométrique a ouvert la voie de l’utilisation du silicium dans les composants optoélectroniques. Cependant cette photoluminescence reste trop peu efficace et de nombreuses recherches portent aujourd’hui sur l’amélioration des propriétés optiques du silicium. Ce travail de thèse s’intéresse particulièrement à l’utilisation de plasmons de surface localisés afin d’améliorer les propriétés optiques de nanocristaux de silicium. Grâce au contrôle de tous les paramètres géométriques des nanocristaux de silicium et des nanoparticules métalliques lors de la fabrication des échantillons, il a été possible d’étudier les phénomènes physiques du couplage entre ces deux objets. Une modification de l’émission des nanocristaux de silicium en fonction de la distance, de la taille et de la nature des nanoparticules métalliques a été étudiée. Grâce au développement de différentes techniques de caractérisation optique, il a été possible de montrer que la photoluminescence des nanocristaux de silicium était modifiée à la fois spectralement et spatialement par les plasmons de surface localisés. Ce travail montre que grâce aux plasmons de surface localisés il est possible de grandement améliorer la photoluminescence des nanocristaux de silicium et ainsi il est possible d’imaginer de nouveaux composants optoélectroniques à base de silicium et de plasmons / The discovery of photoluminescence of nanometric silicon paves the way to use silicon in optoelectronic devices. However this photoluminescence remains low and a lot of works aim at improving silicon optical properties. In this dissertation we study localized surface plasmons to improve optical properties of silicon nanocrystals. Thanks to the control of all geometrical parameters of silicon nanocrystals and metallic nanoparticles during the fabrication process, the coupling process between these two objects has been studied. The modification of silicon nanocrystals emission as a function of the distance, the size and the nature of metallic nanoparticles has been investigated. Thanks to the development of experimental optical characterization techniques we showed that silicon nanocrystals photoluminescence is modified both spectrally and spatially by localized surface plasmons. This work shows that it’s possible to enhance silicon’s optical properties and thus to devise optoelectronic devices with silicon and plasmons

One in 3,000 people in the US are born with cystic fibrosis (CF), a genetic disorder affecting the reproductive system, pancreas, and lungs. Lung disease caused by chronic bacterial and fungal infections is the leading cause of morbidity and mortality in CF. Identities of the microbes are traditionally determined by culturing followed by phenotypic and biochemical assays. It was first thought that the bacterial infections were caused by a select handful of bacteria such as S. aureus, H. influenzae, B. cenocepacia, and P. aeruginosa. With the advent of PCR and molecular techniques, the polymicrobial nature of the CF lung became evident. The CF lung contains numerous bacteria and the communities are diverse and unique to each patient. The total complexity of the bacterial infections is still being determined. In addition, only a few members of the fungal communities have been identified. Much of the fungal community composition is still a mystery. This dissertation addresses this gap in knowledge. A snap shot of CF sputa bacterial community was obtained using the length heterogeneity-PCR community profiling technique. The profiles show that south Florida CF patients have a unique, diverse, and dynamic bacterial community which changes over time. The identities of the bacteria and fungi present were determined using the state-of-the-art 454 sequencing. Sequencing results show that the CF lung microbiome contains commonly cultured pathogenic bacteria, organisms considered a part of the healthy core biome, and novel organisms. Understanding the dynamic changes of these identified microbes will ultimately lead to better therapeutical interventions. Early detection is key in reducing the lung damage caused by chronic infections. Thus, there is a need for accurate and sensitive diagnostic tests. This issue was addressed by designing a bacterial diagnostic tool targeted towards CF pathogens using SPR. By identifying the organisms associated with the CF lung and understanding their community interactions, patients can receive better treatment and live longer.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22452号 / 工博第4713号 / 新制||工||1736(附属図書館) / 京都大学大学院工学研究科材料化学専攻 / (主査)教授 田中 勝久, 教授 三浦 清貴, 教授 藤田 晃司 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

The immunoglobulin superfamily (IgSF) is a large protein superfamily of membrane and soluble proteins that influence recognition, binding, and adhesion. Among members of this family are cell adhesion molecules (CAMs), which form cell-cell contact points that play key roles in development, cell polarization, and cellular fate. Cadherins (CADs) are calcium-dependent proteins of the adherens junction (AJ), and polarize epithelium and endothelium. The tight junction (TJ) is a multiprotein junctional complex whose function is to control the permeability of the paracellular pathway. At the membrane level, TJs are composed of three types of proteins: claudins (CLDNs), occludin (OCLN) and junctional adhesion molecules (JAMs). JAMs are members of the IgSF while CLDN and OCLN are 4-α-helix membrane proteins. Although JAMs are part of the TJ and reside in the same ultrastructure, they are similar to CADs in their secondary, tertiary, and quaternary protein structure. Crystallographic studies of CADs in the presence of calcium yielded trans interactions that resulted in cell-cell contacts. In the absence of calcium, CADs form cis interactions that do not form cell-cell interactions. The crystal structure of JAM-A, has a quaternary organization of a cis dimer. In spite of the many similarities, a link between CADs and JAMs remains unclear. Beyond this point, the association between JAMs, CLDNs, and OCLN in the TJ is vaguely understood. The JAM family (JAM-A, -B, -C and 4) and their tissue-specific distribution indicate that they are key to understanding the TJ’s function and the interplay with the AJ. JAM-A has been used as a prototype for the other three members of the family, but based on current evidence we hypothesized that these proteins may display unique properties to support TJ’s function in a given tissue. Are JAMs affected by calcium just as CADs? Do CLDNs and OCLN make direct contact with JAMs? Do JAMs coordinate the interplay between TJ and AJ? We designed a strategy based on recombinant proteins and biophysical methods to answer these questions. First, we fused the extracellular domain of each JAM to maltose-binding protein (MBP). Our results indicate that JAM proteins have similar secondary structures, but unique tertiary structures. Surface Plasmon Resonance experiments showed that JAM proteins favored heterotypic compared to homotypic interactions. Second, we addressed the effects of cations (Ca2+, Mg2+, Cu2+, Fe2+, Fe3+, and Zn2+) on JAM-A. The exposure of JAM-A to the resulted in changes in its secondary, tertiary structure, and homotypic binding affinity. Finally, we addressed whether cations had an effect on the other TJ components and if there is an interplay with E-CAD. We determined that in the assembly of a simple TJ and AJ, JAM-A and E-CAD are calcium-dependent, while CLDN1 and OCLN are calcium independent. We conclude that TJ components such as CLDN1 and OCLN may work as anchors to maintain cell-cell interactions while JAM-A and E-CAD would be regulated by cations in order to accommodate other homeostatic functions.

The human bone marrow vasculature constitutively expresses both E-selectin and P-selectin where they interact with the cell-surface glycan moiety, sialyl Lewis x, on circulating hematopoietic stem/progenitor cells (HSPCs) to mediate the essential tethering/rolling step. Although several E-selectin glycoprotein ligands (E-selLs) have been identified, the importance of each E-selL on human HSPCs is debatable and requires additional methodologies to advance their specific involvement. The first objective was to fill the knowledge gap in the in vitro characterization of the mechanisms used by selectins to mediate the initial step in the HSPCs homing by developing a real time immunoprecipitation-based assay on a surface plasmon resonance chip. This novel assay bypass the difficulties of purifying ligands, enables the use of natively glycosylated forms of selectin ligands from any model cell of interest and study its binding affinities under flow. We provide the first comprehensive quantitative binding kinetics of two well-documented ligands, CD44 and PSGL-1, with E-selectin. Both ligands bind monomeric E-selectin transiently with fast on- and off-rates while they bind dimeric E-selectin with remarkably slow on- and off-rates with the on-rate, but not the off-rate, is dependent on salt concentration. Thus, suggest a mechanism through which monomeric selectins mediate initial fast-on and -off binding to capture the circulating cells out of shear-flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to slow rolling significantly. The second objective is to fully identify and characterize E/P-selectin ligand candidates expressed on CD34+ HSPCs which cause enhanced migration after intravenous transplantation compared to their CD34- counterparts. CD34 is widely recognized marker of human HSPCs but its natural ligand and function on these cells remain elusive. Proteomics identified CD34 as an E-selL candidate on human HSPCs, whose binding to E-selectin was confirmed using some static and flow-based assays. E-selectin binds to CD34 with an affinity comparable to the well-described E-selLs CD44/HCELL and PSGL-1. CD34 knockdown resulted in faster-rolling velocities compared to control cells especially at and above three dyne/cm2. CD34 is the first selectin ligand since PSGL-1 reported to bind E-/P-/L-selectins and likely plays a key role in directing the migration of human HSPCs to the bone marrow.

Title: Multifunctional biomolecular assemblies for parallelized analysis of biomolecular interactions Author: Markéta Bocková Department / Institute: Institute of Physics, Charles University Supervisor of the doctoral thesis: Prof. Jiří Homola, Ph.D., DSc., Institute of Photonics and Electronics, The Czech Academy of Sciences Abstract: Surface plasmon resonance (SPR) biosensors represent the most advanced optical method for the direct, real-time monitoring of biomolecular interactions without the need for labelling. This doctoral thesis aims to advance the SPR biosensor method and to expand its utility in the investigation of biomolecular interactions. This encompasses activities on two major fronts of SPR biosensor research - immobilization methods and biosensing methodologies. Methods for the immobilization of biomolecules were researched with the aim of enabling the immobilization of a broad range of biomolecules on the SPR biosensor surface in a spatially controlled manner. The development of novel biosensing methodologies was pursued in order to address the current limitations of SPR biosensors associated with non-specific adsorption and limited analyte transport, and thus to improve the accuracy and robustness of SPR biosensor measurements. Finally, advances in the development of immobilization...

Lignin is one of the main components of wood and plants that acts as a kind of glue providing mechanical strength. It is a main polymer component composed from three phenolic structures, i.e. p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) units. It currently draws a lot of attention due to its eco-friendly. Recently, it has been shown that it is possible to produce lignin nanoparticles, small spherical particle that are composed out of lignin, that could possibly be used to replace the hazardous silver nanoparticles that are today used frequently in numerous applications. Lignin nanoparticles could potenitally also be used as functional coatings, as well as biologically degradable adhesives and float switches. Five samples, of nanoparticles, were investigated in this study. The first contained pure lignin nanoparticles, the second pure silver nanoparticles, and the three remaining samples contained lignin-coated silver nanoparticles, extracted from acetone, tetrahydrofuran (THF), and dimetylformamid (DMF) solvents. All samples were characterized using spectroscopic methods, e.g. infrared- and dark-field imaging, as well as UV-Vis-, fluorescence-, and Raman spectroscopy. In this thesis it was shown that lignin-coated silver nanoparticles exhibit surface plasmon resonance which induces a heat effect upon infrared irradiation. To identify the phenolic structures of lignin, UV-Vis spectroscopy was used. It was found that the spectra of the samples exhibited several intense bands. The objective of the UV-Vis spectroscopy was to examine the absorbance characteristics of the lignin-coated silver nanoparticles. Possible surface plasmon resonance wavelengths were determined, and two of the phenolic structures were identified. In this study, Raman spectroscopy was used to define characteristic bands of the samples. This was done to investigate if the lignin nanoparticles have the same characteristics as bulk lignin. Raman spectroscopy provide structural information of lignin. Furthermore, p-hydroxyphenyl, guaiacyl and syringyl structures could be identified with an excitation wavelength of 532nm. A comparison of the spectra of the lignin-containing samples indicated the the Raman features of the specimens were similar meanwhile almost no signs of silver were present, which might show that the particles were fully covered with lignin. Main lignin bands were identified and assigned. The fluorescent properties of the nanoparticles were investigated by obtaining emission spectra for blue-, green- and UV light excitation. The spectra were deconvoluted into their Gaussian components. Emission spectra were obtained for blue-, green- and UV light excitation. It was found that the fluorescence, after UV light exposure, increased with time of exposure. Dark-field microscopy was used to generate light scattering images of the particles. As a result, optical images with different colors (white, yellow, blue and red) could be revealed. The color information, that is related to the size of the particles, was used to estimate ratios of the different particle sizes. The lignin-coated silver nanoparticles, extracted from acetone, exhibited a strong surface plasmon resonance effect, which could be due to the absorbance at 463nm. The lignin-coated silver nanoparticles, extracted from DMF, exhibited a medium surface plasmon resonance effect, which could be due to the absorbance at 362nm. The lignin-coated silver nanoparticles, extracted from the THF solvent, exhibited a weak surface plasmon resonance effect, which could be due to the absorption at 379-380nm. The pure lignin- and silver nanoparticles merely showed bulk heating but no surface plasmon resonance effect could be detected. / Lignin är en av huvudbeståndsdelarna av trä och plantor som fungerar likt ett lim som ger mekanisk styrka. Lignin är en biopolymer, som består av tre fenylgrupper: p-hydroxifenyl (H), guaiacyl (G) och syringyl (S). På senaste tid har det visat sig att det är möjligt att tillverka lignin nanopartiklar, det är små sfäriska partiklar som är helt gjorda av lignin, som skulle kunna ersätta de miljöfarliga silver nanopartiklarna som i nuläget används i många olika tillämpningar. Lignin nanopartiklar kan potentiellt också användas som funktionella ytbeläggningar, såväl som biologiskt nedbrytbara lim och flottörer. Fem prover, av nanopartiklar, undersöktes i denna studie. Det första provet innehöll lignin nanopartiklar, det andra silver nanopartiklarna, och de tre återstående proverna innehöll ligninbelagda silver nanopartiklar, extraherade från aceton, tetrahydrofuran (THF) och dimetylformamid (DMF). Alla prover karakteriserades med hjälp av spektroskopiska metoder: infraröd- och mörkfältavbildning, liksom UV-Vis-, fluorescens- och Ramanspektroskopi. I denna avhandling visades att ligninbelagda silver nanopartiklar uppvisar ytplasmonsresonans, vilket inducerar en värmeeffekt vid infraröd bestrålning. För att identifiera ligninets fenylgrupper användes UV-Vis-spektroskopi. Det visade sig att spektra från proverna uppvisade flera intensiva band. Målet med UV-Vis-spektroskopin var att undersöka absorptionsegenskaperna hos de ligninbelagda silvernanopartiklarna. Möjliga ytplasmonresonansvåglängder bestämdes och två av fenylgrupperna identifierades. I denna studie användes Ramansspektroskopi för att definiera karakteristiska band för proverna. Detta gjordes för att undersöka om lignin nanopartiklarna har samma egenskaper som bulk lignin. Ramanspektroskopi ger information om ligninets struktur. Vidare identigierades p-hydroxifenyl-, guaiacyl- och syringylstrukturerna med en excitationsvåglängd på 532nm. En jämförelse av spektra för de lignininnehållande proverna indikerade att provernas Raman-kännetecken var liknande medan nästan inga tecken på silver fanns, vilket kan visa att partiklarna var täckta med lignin. Huvudsakliga ligninband kunde identifieras. Fluorescensegenskaperna, hos nanopartiklarna, undersöktes genom de erhållna emissionspektra efter exponering av blå-, grön- och UV-ljus. De erhållna spektra dekonvoluterades till dess gaussiska komponenter. Det visade sig att fluorescensen, efter exponering av UV-ljus, ökade med exponeringstiden. Mörkfältmikroskopi användes för att generera bilder på partiklarna. De resulterade i bilder med olika färger (vitt, gult, blått och rött) som motsvarade olika partikelstorlekar och geometrier. På så sätt kunde färhållandena mellan de olika partikelstorlekarna uppskattas. De ligninbelagda silver-nanopartiklarna, extraherade från aceton-lösningen, uppvisade en stark ytplasmonresonanseffekt, vilket kan bero på absorptionen (från absorptionsspektrat) vid 463nm. De ligninbelagda silver-nanopartiklarna, extraherade från DMF-lösningen, uppvisade en medelstark ytplasmonresonanseffekt, vilket kan bero på absorptionen vid 362nm. De ligninbelagda silver-nanopartiklarna, extraherade från THF-lösningen, uppvisade en svag ytplasmonresonanseffekt, vilket kan bero på absorptionen vid 379-380nm. De rena lignin- och silver-nanopartiklarna uppvisade endast uppvärmning men ingen ytplasmonresonanseffekt.

A system was developed to perform real-time biological threat agent (BTA) detection with a small autonomous unmanned aerial vehicle (UAV). Biological sensors just recently reached a level of miniaturization and sensitivity that made UAV integration a feasible task. A Surface Plasmon Resonance (SPR) biosensor was integrated for the first time into a small UAV platform, allowing the UAV platform to collect and then quantify the concentration of an aerosolized biological agent in real-time. A sensor operator ran the SPR unit through a groundstation laptop and was able to wirelessly view detection results in real time. An aerial sampling mechanism was also developed for use with the SPR sensor. The collection system utilized a custom impinger setup to collect and concentrate aerosolized particles. The particles were then relocated and pressurized for use with the SPR sensor. The sampling system was tested by flying the UAV through a ground based plume of water soluble dye. During a second flight test utilizing the onboard SPR sensor, a sucrose solution was autonomously aerosolized, collected, and then detected by the combined sampling and SPR sensor subsystems, validating the system\'s functionality. The real-time BTA detection system has paved the way for future work quantifying biological agents in the atmosphere and performing source localization procedures. / Master of Science