<p>Proteins are specialized molecules that catalyze most of the reactions that can sustain life, and they become functional by folding into a specific 3D structure. Despite their importance, the question, "how do proteins fold?" - first pondered in in the 1930's - is still listed as one of the top unanswered scientific questions as of 2005, according to the journal Science. Answering this question would provide a foundation for understanding protein function and would enable improved drug targeting, efficient biofuel production, and stronger biomaterials. Much of what we currently know about protein folding comes from studies on small, single-domain proteins, which may be quite different from the folding of large, multidomain proteins that predominate the proteomes of all organisms.</p><p>In this thesis I will discuss my work to fill this gap in understanding by studying the unfolding and refolding of large, multidomain proteins using the powerful combination of single-molecule force-spectroscopy experiments and molecular dynamic simulations.</p><p>The three model proteins studied - Luciferase, Protein S, and Streptavidin - lend insight into the inter-domain dependence for unfolding and the subdomain stabilization of binding ligands, and ultimately provide new insight into atomistic details of the intermediate states along the folding pathway.</p> / Dissertation

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第22397号 / 薬科博第119号 / 新制||薬科||13(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 髙倉 喜信, 教授 山下 富義, 教授 小野 正博 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

[pt] A combinação de alta densidade, locais seletivos de nucleação e controle da distribuição de tamanho de nanoestruturas semicondutoras tem acelerado o desenvolvimento de dispositivos ópticos e eletrônicos. Para construir estruturas satisfazendo essas necessidades, várias combinações de técnicas deposição de pontos quânticos e nanolitografia foram desenvolvidas. A nanolitografia por AFM foi aplicada em diversos materiais abrindo uma possibilidade para fabricar dispositivos opto-eletrônicos.Nesta tese de Doutorado, apresentamos um estudo sistemático de crescimento de nanoestruturas de InAs em buracos produzidos na superfície (100) de substratos de InP por nanoindentação com o AFM. Para isto, a ponta precisa exercer uma força no InP que produz deformações plásticas na superfície. A pressão aplicada entre a extremidade da ponta de AFM e a superfície da amostra pode ser variada de modo controlado através do ajuste de alguns parâmetros operacionais do microscópio tais como setpoint, raio da ponta e constante de mola do cantilever. A habilidade para controlar a forma do padrão indentado assim como a natureza dos defeitos cristalinos permite controlar o crescimento seletivo de InAs por epitaxia em fase de vapor de metais orgânicos. Também é apresentada a fabricação de nanoestruturas de InAs/InP alinhadas em uma dimensão. A nanoindentação é produzida pelo arraste da ponta do AFM sob força constante ao longo das direções <100> e <110> do InP. Observamos que o número e o tamanho das nanoestruturas nucleadas são dependentes da distância entre as linhas litografadas. Esses resultados sugerem que o mecanismo de crescimento das nanoestruturas de InAs não é governado por degraus atômicos gerados durante a indentação. Os dados sugerem que, a densidade de defeitos induzidos mecanicamente, tais como discordâncias e fraturas, é o responsável pelo número de nanoestruturas nucleadas. / [en] The combination of high density, site selective nucleation, and size distribution control of semiconductor nanostructures has become a challenge in the development of effective optical and electronic devices. In order to build structures satisfying these requirements, various combinations of quantum dot deposition and nanolithography techniques have been developed. The AFM nanolithography technique has been applied on several materials opening a possibility to fabricate opto-electronic devices. In this Phd Thesis, we present a systematic study of growth of InAs nanostructures on pits produced on (100) InP by nanoindentation with the AFM. For that purpose, the AFM tip needs to exert a force on the InP that produces plastic deformation on the surface. The applied pressure between the very end of the AFM tip and the sample surface may be varied in a controlled way by adjusting some of the microscope operational parameters like set point, tip radius and cantilever normal bending constant. The ability to control the shape of the indentation pattern as well as the nature of the crystalline defects allows control of the selective growth of InAs by metal organic vapor phase epitaxy. We also report the fabrication of one-dimensional arrays of InAs/InP nanostructures. The nanoindentation is produced by dragging the AFM tip under constant force of the substrate, along the <100> and <110> InP crystallographic directions. We have observed that the number and the size of nucleated nanostructures are dependent on the distance between the lithographed lines. These results suggest that the growth mechanism of the InAs nanostructures on the pits produced by AFM on InP is not governed by the number of atomic steps generated during the scratching. Instead, the data suggests that, the density of mechanically induced defects, like dislocations and cracks, are responsible for the number of nucleated nanostructures.

Esta tese apresenta o estudo inédito da obtenção e caracterização de filmes finos de xiloglucanas obtidas de duas fontes diferentes, Hymenaea courbaril (HXG) e Tamarindus indica (TXG) sobre lâminas de Si/SiO2 e lâminas modificadas com grupos amino, assim como a aplicação desses filmes na imobilização da lectina concanavalina A (Con A) por medidas de elipsometria e microscopia de força atômica (AFM). A diferença na estrutura fina de cada XG influenciou na espessura e morfologia de seus filmes finos. Os oligômeros XXXXG presentes apenas na HXG favorecem as interações com a superfície, assim como já observado para interação entre XG-celulose [Lima e Buckeridge, 2001]. A imobilização de Con A foi realizada sobre filmes de carboximetilcelulose (CMC) de HXG, TXG e poli(metacrilato de metila) (PMMA), lâminas de Si/SiO2 e partículas poliméricas decoradas com polissacarídeo PMMA/CMC. A adsorção de Con A foi mais pronunciada sobre os filmes de CMC. As camadas de Con A formadas sobre HXG e TXG apresentam potencial utilização no reconhecimento e detecção de carboidratos para desenvolvimento de diagnósticos para algumas doenças, por não comprometerem o sítio de ligação específica da lectina na adsorção. No caso das partículas PMMA/CMC cobertas com uma camada de Con A, a alta estabilidade coloidal do sistema confere às partículas a possibilidade de explorar a propriedade de aglutinação específica da lectina no reconhecimento de carboidratos. A enzima hexoquinase (HK) foi imobilizada sobre dois diferentes tipos de partículas poliméricas decoradas com polissacarídeo, PMMA/CMC e poliestireno/quitosana (PS/CH). Na primeira, HK perdeu drasticamente sua atividade catalítica, ao contrário do observado quando a mesma enzima foi imobilizada sobre PS/CH, onde a HK manteve aproximadamente 50% de sua atividade enzimática, podendo ser armazenada em temperatura e pressão ambientes por 1 mês e ser reutilizada até 3 vezes. A estrutura da camada de hidratação e a composição química de cada um dos polissacarídeos (CMC ou CH) na superfície das partículas são diferentes, provocando diferenças na conformação das moléculas de HK quando imobilizadas sobre cada tipo de partícula. Medidas de forças de adesão utilizando as partículas poliméricas coladas na extremidade de cantilevers de AFM (Colloidal Probe Technique) permitiram medir as espessuras dessas camadas hidratadas de polissacarídeos nas superfícies das partículas PMMA/CMC e PS/CH, da ordem de (20 &#177; 10) nm e (35 &#177; 11) nm, respectivamente. Essas medidas de força também evidenciaram o potencial dos dois sistemas estudados (PMMA/CMC - Con A e PS/CH - HK) como métodos qualitativos para a detecção dos açúcares manose, glicose e frutose. / This thesis presents the study on thin film formation and characterization of xyloglucans extracted from two different sources, Hymenaea courbaril (HXG) and Tamarindus indica (TXG) onto Si/SiO2 wafers or amino-terminated wafers, as well as the application of these films as substrates for the immobilization of the lectin concanavalin A (Con A) by means of ellipsometry and atomic force microscopy (AFM). The difference in the fine structure of each XG exerted influence on film thickness and morphology. The oligomers XXXXG present only in HXG favor the interactions with the surfaces, as already observed for the interaction between XG-cellulose [Lima and Buckeridge, 2001]. Con A was immobilized onto films of carboxymethylcellulose (CMC), HXG, TXG and poly(methyl methacrylate) (PMMA), Si/SiO2 wafers and on polysaccharide decorated polymeric particles PMMA/CMC. The adsorption of Con A was more pronounced on CMC films. Con A layers formed on HXG and TXG present potential application in the recognition and detection of carbohydrates for diagnostic development, once the specific sugar binding site is not involved in the adsorption process. In the case of polymeric particles PMMA/CMC covered with a Con A layer, the high colloidal stability of the system confers to particles the possibility to explore the lectin property of specific agglutination with carbohydrates. The enzyme hexokinase (HK) was immobilized onto two different types of polysaccharide decorated polymeric particles, PMMA/CMC and polystyrene/chitosan (PS/CH). In the first one, HK drastically lost its catalytic activity. The same enzyme immobilized onto PS/CH kept approximately 50% of its enzymatic activity, even after storing under room temperature and pressure for 1 month. Moreover upon immobilizing HK could be reused up to 3 times. The structure of the hydration layer and the chemical composition due to the presence of each polysaccharide (CMC or CH) on the polymeric particle surface provided different environment, generating differences in the conformation of immobilized molecules of HK on each particle. Measurements of adhesion forces using the polymeric particles glued on the extremity of AFM cantilevers (Colloidal Probe Technique) allowed to measure the thicknesses of these hydrated polysaccharide layers on the surfaces of the PMMA/CMC and PS/CH particles as (20 &#177; 10) nm and (35 &#177; 11) nm, respectively. These force measurements had also evidenced the potential of the two studied systems (PMMA/CMC - Con A and PS/CH - HK) as qualitative methods for detection of the sugars mannose, glucose and fructose.

by Lee, Chun-Sing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 98-105). / Abstracts in English and Chinese. / Acknowledgements --- p.2 / Abstract --- p.3 / Table of Contents --- p.7 / List of Figures --- p.10 / List of Tables --- p.13 / Chapter Chapter 1 --- Introduction --- p.14 / Chapter 1.1. --- Ion implantation --- p.14 / Chapter 1.2. --- Scope of the thesis --- p.15 / Chapter Chapter 2 --- Background Theory --- p.17 / Chapter 2.1. --- Ion stopping --- p.17 / Chapter 2.2. --- The energy-loss process --- p.17 / Chapter 2.3. --- Kinematics of binary elastic collision --- p.20 / Chapter 2.4. --- Nuclear and electronic stopping --- p.21 / Chapter 2.5. --- Radiation Damage --- p.22 / Chapter 2.6. --- Spikes --- p.24 / Chapter 2.7. --- Topography of ion bombarded surface --- p.26 / Chapter Chapter 3 --- Equipment Reviews --- p.31 / Chapter 3.1. --- Metal Vapour Vacuum Arc Ion Source Implanter --- p.31 / Chapter 3.2. --- Atomic Force Microscopy --- p.34 / Chapter 3.3. --- Rutherford Backscattering Spectrometry --- p.37 / Chapter 3.4. --- X-ray Diffraction --- p.40 / Chapter Chapter 4 --- Study of Ion Beam Implanted Germanium by Atomic Force Microscopy and Rutherford Backscattering Spectrometry --- p.43 / Chapter 4.1. --- Introduction --- p.43 / Chapter 4.2. --- Experiments --- p.45 / Chapter 4.3. --- Results and discussion --- p.47 / Chapter 4.3.1. --- AFM --- p.47 / Chapter 4.3.2. --- RBS and ion channeling --- p.64 / Chapter 4.4. --- Conclusions --- p.71 / Chapter Chapter 5 --- Ion Beam Synthesised Cobalt Germanide Alloy by Metal Vapour Vacuum Arc Implantation --- p.73 / Chapter 5.1. --- Introduction --- p.73 / Chapter 5.2. --- Experiments --- p.74 / Chapter 5.3. --- Results and discussion --- p.74 / Chapter 5.3.1. --- XRD --- p.74 / Chapter 5.3.2. --- AFM --- p.78 / Chapter 5.3.3. --- RBS and ion channeling --- p.82 / Chapter 5.4. --- Conclusions --- p.87 / Chapter Chapter 6 --- Tip Artifacts in Atomic Force Microscope Imaging of Ion Bombarded Nanostructures on Germanium Surfaces --- p.89 / Chapter 6.1. --- Introduction --- p.89 / Chapter 6.2. --- Experiments --- p.90 / Chapter 6.3. --- Results and discussion --- p.90 / Chapter 6.4. --- Conclusions --- p.95 / Chapter Chapter 7 --- Conclusions --- p.96 / Bibliography --- p.98 / Publications --- p.105

No description available.

Microbial infections of medical implants occur in 10% of the more than 20 million surgical procedures carried out annually in the United States. The additional treatments required to address these infections generate more than $11 billion in additional patient costs, increase recovery time, and decrease overall patient quality of life. As the population ages, the number of necessary and voluntary surgical procedures increases; The rate of infection increases proportionately. While treatments are available, the biofilm mode of growth confers resistance to antimicrobial therapies up to 500 times greater than that of planktonic microbes. Currently, the only guaranteed method of removing an established microbial implant infection is through surgical excision of the implant and surrounding tissues. While removing the original infection, additional colonization and pathogenesis may take place. This research explores the a priori assumption that a medical implant infection cannot occur unless a microbial cell is capable of adhering to the implant surface. From that assumption, the following sections will focus primarily on identifying the necessary and sufficient factors influencing microbial adhesion, discretizing those factors into measurable quantities, and developing methods by which those factors may be mitigated or eliminated. Following is a brief summary of each major topic treated within this research period. Development of a Benchmark System: We have characterized the interactions between Pseudomonas aeruginosa ATCC 10145 and Candida parapsilosis ATCC 90018 using a novel method of cellular immobilization, which emphasizes minimal chemical modification of the cell surface. This research describes the very different force-separation interactions seen between C. parapsilosis and both a common medical implant material (viz., silicone rubber) and a nascent P. aeruginosa biofilm grown on the same material. This study was the first step in developing an ab initio technique which may be used to determine the relative affinity of a microbial cell for an implant material surface. The Role of the Substrate: Microbial adhesion to a medical implant device involves two major components, being the microbe itself, and the substrate to which it adheres. Each of the two has specific and unique surface chemical and textural characteristics which, when combined, allow for microbial colonization and subsequent infection. The goal of this study was to identify correlations between the adhesive strength of Staphylococcus epidermidis to a variety of chemically and texturally distinct substrates, and common surface characterization parameters (e.g., surface roughness and water contact angle). Relationships to adhesive strength did not demonstrate statistically significant or consistent trends. To extend upon the correlation parameters, we have employed a Discrete Bonding Model, which characterizes the surface texture according to Mandelbrot fractal theory. Correlations between the adhesive strength and the observational scale show stronger relationships, indicating a significant contribution of the surface texture to a microbe's ability to colonize a surface. Finding a Surface That Cannot Be Touched: Historically, AFM force-separation curves demonstrating only repulsive behavior on extension of the piezoactuator have been largely ignored, in terms of quantitative modeling of the interactions. In bacterial systems, such behavior describes the majority of the force profiles recorded by the instrument. As a result of the former lack of study, the latter data sets have remained unanalyzed and unanalyzable. Building on existing mathematical models, we have developed an analytical method by which the point of zero separation between a surface (viz., the microbial cell wall) coated with a polymer brush and an AFM probe may be quantitatively identified.

"Bacterial infections persist as a public threat due to the ease by which bacteria adapt to commonly used antibiotics. In addition, bacteria on surfaces develop protective communities called biofilms that hinder the ability of antibiotics to completely eliminate the pathogens. The rapid development of bacterial resistance to antibiotics has made pharmaceutical companies reluctant to fund new antibiotics research. Hence, novel approaches to prevent and treat infections are needed. The development of infections can be divided into three steps: adhesion, invasion and multiplication. Antibiotics target at the latter two step and are prone to bacterial resistance as passive strategies. Bacterial adhesion to host cells/implanted medical devices is the first step leading to following invasion and multiplication. However, fundamental understanding of bacterial adhesion process is still lacking. The current studies are aimed to systematically investigate biological interactions between pathogenic bacteria and host cell, proteins and biomaterials with both macro and micro scale approaches. The macro scale methods include bacterial adhesion assay, viability studies, and thermodynamic modeling. The micro scale methods include direct adhesion force measurements, ultra surface visualization via atomic force microscopy (AFM) and surface structure modeling. Our work combines experiments and modeling aimed at understanding the initial steps of the bacterial adhesion process, focusing on two case studies: 1) Mechanisms by which cranberry can prevent urinary tract infections through interfering with bacterial adhesion; and 2) Design of anti-adhesive and antimicrobial coatings for biomaterials. We make direct adhesion force measurements between bacteria and substrates with an atomic force microscope (AFM), and combine such experiments with thermodynamic calculations, in order to develop a set of tools that allows for the prediction of whether bacteria will attach to a given surface. These fundamental investigations of the bacterial adhesion process help elucidate the underlying mechanisms behind bacterial adhesion, thus leading to improved clinical outcomes for a number of biomedical applications. "

"The adhesion of bacteria to surfaces has been analyzed in terms of surface charge, surface energy, and the characteristics of polymers on bacteria, to understand the factors that control bacterial adhesion. Pseudomonas aeruginosa has received a great deal of interest because it is responsible for a variety of chronic bacterial infections such as airway infections in cystic fibrosis patients and ulcerative bacterial keratitis in soft contact lens users. Over the past few years, force measurement techniques such as atomic force microscopy (AFM) have made it possible to examine interactions between colloidal particles and surfaces. In the present study, the AFM was used to study the interactions between each of two Pseudomonas aeruginosa strains with proteins. Topographical images and force cycles of bacterial cells and proteins were analyzed. Bovine serum albumin (BSA) and concanavalin A (Con A) were the model proteins chosen to represent protein molecules that might affect bacterial adhesion. In addition, the role of LPS structure in bacterial adhesion was investigated. The magnitude of adhesive forces for two P. aeruginosa stains was not statistically significant when they interact with silicon. Although it is not clear if the pull-off distances are accurate representatives of the absolute length of bacterial surface molecules, the trend indicates that the surface molecules of strain AK1401 are shorter than those of strain PAO1. The semi-rough strain AK1401 was more hydrophobic than the smooth strain PAO1, according to the water contact angle measurements. However, surface free energy components and zeta potential values were not significantly different for both strains. Zeta potential of bacterial cells decreased when they were suspended in HEPES/DTT buffer instead of ultrapure water. The AFM results demonstrate the importance of nano-scale interactions between proteins and bacterial cells. Our results show that the lipid A and core oligosaccharides are the most important molecules influencing the interactions of P. aeruginosa with protein molecules. The interactions of P. aeruginosa with model proteins in our study were weak. Therefore, the role of protein molecules may be inadequate for the purpose of enhancing subsurface delivery for bioremediation. Our results suggest that the semi-rough mutant, AK1401, can adhere to the protein receptors of the epithelial cells or protein coated implants stronger than the smooth strain, PAO1, and therefore can cause serious infections."

"Polyelectrolyte multilayer (PEM) thin films prepared via the Layer-by-Layer (LbL) deposition technique are of special interest in this research. The purpose of this study is to replace current mechanical closure systems, based on hook-and-loop type fasteners (i.e. Velcro), with PEM thin film systems. The technique is simple, cheap, versatile and environmental friendly; as a consequence a variety of thin films can be easily fabricated. By proposing PEMs as non-mechanical and nanoscopic molecular closures, we aim to obtain hermetic sealing, good adhesive strength, and peel off ease. Atomic force microscopy (AFM) and colloidal probe techniques were used to characterize the morphology, roughness and adhesive properties of PEMs. AFM measurements were conducted in air, necessarily requiring careful control of ambient humidity. PEMs were formed by consecutive deposition of polyanions and polycations on a charged polyethylene terephthalate (PET) solid surface, the result of which was stable nanostructured films. By systemically varying the parameters of PEM build-up process: different combinations of polyelectrolytes, different numbers of bilayers (polyanion/polycation pairs), and miscellaneous types and concentrations of salts (NaCl, NaBr and NaF salts at 0.5 M and 1.0 M concentrations), the adhesion and morphology of PEMs were thoroughly investigated. The PEM thin films specifically investigated include poly(ethyleneimine) (PEI), poly(styrene sulfonate) (PSS), poly(allylamine hydrochloride) (PAH), poly(acrylic acid) (PAA), and poly(diallydimethylammonium chloride) (PDADMAC). Silica colloidal probes were utilized in the investigation, some of which were functionalized with COOH and/or coated with PEI-PSS. Silica colloidal probes were used in order to quantify interaction forces on the PEMs. A functionalized silica colloidal probe (a probe with COOH surface chemistry) and a silica colloidal probe coated with PEI-PSS were used to simulate PEM-PEM interactions. The results suggest that adhesion in the PEMs depend on the number of layers, the salt concentration and the salt type used during the build-up process, the environmental conditions where the adhesion force measurements were made, and the choice of probe. "