Optical tweezers are a technique in which microscopic-sized particles, including living cells and bacteria, can be non-intrusively trapped with high accuracy solely using focused light. The technique has therefore become a powerful tool in the field of biophysics. Optical tweezers thereby provide outstanding manipulation possibilities of cells as well as semi-transparent materials, both non-invasively and non-destructively, in biological systems. In addition, optical tweezers can measure minute forces (< 10-12 N), probe molecular interactions and their energy landscapes, and apply both static and dynamic forces in biological systems in a controlled manner. The assessment of intermolecular forces with force measuring optical tweezers, and thereby the biomechanical structure of biological objects, has therefore considerably facilitated our understanding of interactions and structures of biological systems. Adhesive bacterial organelles, so called pili, mediate adhesion to host cells and are therefore crucial for the initial bacterial-cell contact. Thus, they serve as an important virulence factor. The investigation of pili, both their biogenesis and their expected in vivo properties, brings information that can be of importance for the design of new drugs to prevent bacterial infections, which is crucial in the era of increased bacterial resistance towards antibiotics. In this thesis, an experimental setup of a force measuring optical tweezers system and the results of a number of biomechanical investigations of adhesive bacterial organelles are presented. Force measuring optical tweezers have been used to characterize three different types of adhesive organelles under various conditions, P, type 1, and S pili, which all are expressed by uropathogenic Escherichia coli. A quantitative biophysical force-extension model, built upon the structure and force response, has been developed. It is found, that this model describes the biomechanical properties for all three pili in an excellent way. Various parameters in their energy landscape, e.g., bond lengths and transition barrier heights, are assessed and the difference in behavior is compared. The work has resulted in a method that in a swift way allows us to probe different types of pili with high force and high spatial resolution, which has provided an enhanced understanding of the biomechanical function of these pili. / Optisk pincett är en teknik i vilken mikrometerstora objekt, inkluderande levande celler och bakterier, beröringsfritt kan fångas och förflyttas med hög noggrannhet enbart med hjälp av ljus. Den optiska pincetten har därmed blivit ett kraftfullt verktyg inom biofysiken, som möjliggör enastående precisions-manipulering av celler och semi-transparenta objekt. Dessutom kan denna manipulation göras intracellulärt, dvs. utan att fysiskt öppna eller penetrera cellernas membran. Den optiska pincetten kan även mäta mycket små krafter och interaktioner (< 10-12 N) samt applicera både statiska och dynamiska krafter i biologiska system med utmärkt precision. Optisk pincett är därför en utmärkt teknik för mätning av intermolekylära krafter och för bestämning av biomekaniska strukturer och dess funktioner. Vissa typer av bakterier har specifika vidhäftningsorganeller som kallas för pili. Dessa förmedlar vidhäftningen till värdceller och är därför viktiga vid bakteriens första kontakt. En djupare förståelse av pilis uppbyggnad och biomekanik kan därmed ge information, som kan vara vital i framtagandet av nya mediciner som förhindrar bakteriella infektioner. Detta är av stor vikt i skenet av den ökande antibiotikaresistensen i vårt samhälle. I denna avhandling presenteras konstruktionen av en experimentell uppställning av kraftmätande optiskt pincett tillsammans med resultat från biomekaniska undersökningar av vidhäftande bakteriella organeller. Kraftmätande optisk pincett har använts för att karakterisera tre olika typer av pili, P, typ 1, och S pili, vilka kan uttryckas av uropatogena Escherichia coli. En kvantitativ biofysikalisk modell som beskriver deras förlängningsegenskaper under pålagd kraft har konstruerats. Modellen bygger på pilis strukturella uppbyggnad samt på dess respons som uppmäts med den kraftmätande optiska pincetten. Modellen beskriver de biomekaniska egenskaperna väl för alla tre pili. Dessutom kan ett antal specifika bindnings- och subenhetsparametrar bestämmas, t.ex. interaktionsenergier och bindningslängder. Skillnaden mellan dessa parametrar hos de tre pilis samt deras olika kraftrespons har jämförts. Detta arbete har dels resulterat i en förbättrad förståelse av pilis biomekaniska funktion och dels i en metod som, med hög noggrannhet, tillåter oss att bestämma ett antal biomekaniska egenskaper hos olika organeller på ett effektivt sätt.

Hydrophobic interactions guide important molecular self-assembly processes such as protein folding. On the macroscale, hydrophobic interactions consist of the aggregation of "oil-like" objects in water by minimizing the interfacial energy. However, the hydration mechanism of small hydrophobic molecules on the nanoscale (~1 nm) differs fundamentally from its macroscopic counterpart. Theoretical studies over the last two decades have pointed to an intricate dependence of molecular hydration mechanisms on the length scale. The microscopic-to-macroscopic cross-over length scale is critically important to hydrophobic interactions in polymers, proteins and other macromolecules. Accurate experimental determination of hydration mechanisms and their interaction strengths are needed to understand protein folding. This thesis reports the development of experimental and analytical techniques that allow for direct measurements of hydrophobic interactions in a single molecule. Using single molecule force spectroscopy, the mechanical unfolding of a single hydrophobic homopolymer was identified and modeled. Two experiments examined how hydrophobicity at the molecular scale differ from the macroscopic scale. The first experiment identifies macroscopic interfacial tension as a critical parameter governing the molecular hydrophobic hydration strength. This experiment shows that the solvent conditions affect the microscopic and macroscopic hydrophobic strengths in similar ways, consistent with theoretical predictions. The second experiment probes the hydrophobic size effect by studying how the size of a non-polar side-chain affects the thermal signatures of hydration. Our experimental results reveal a cross-over length scale of approximately 1 nm that bridges the transition from entropically driven microscopic hydration mechanism to enthalpically driven macroscopic hydration mechanism. These results indicate that hydrophobic interactions at the molecular scale differ from macroscopic scale, pointing to potential ways to improve our understanding and predictions of molecular interactions. The system established in this thesis forms the foundation for further investigation of polymer hydrophobicity.

<p>This study is focused on designing and characterizing protein-based building blocks in order to construct self-assembled nano-structured biomaterials. In detail, this research aims to: (1) investigate a new class of proteins that possess nanospring behaviors at a single-molecule level, and utilize these proteins along with currently characterized elastomeric proteins as building blocks for nano-structured biomaterials; (2) develop a new method to accurately measure intermolecular interactions of self-assembling two or more arbitrary (poly)peptides, and select some of them which have appropriate tensile strength for crosslinking the proteins to construct elastomeric biomaterials; (3) construct well-defined protein building blocks which are composed of elastomeric proteins terminated with self-oligomerizing crosslinkers, and characterize self-assembled structures created by the building blocks to determine whether the elasticity of proteins at single-molecule level can be maintained.</p><p>Primary experimental methods of this research are (1) atomic force microscope (AFM) based single-molecule force spectroscopy (SMFS) that allows us to manipulate single molecules and to obtain their mechanical properties such as elasticity, unfolding and refolding properties, and force-induced conformational changes, (2) AFM imaging that permits us to identify topology of single molecules and supramolecular structures, and (3) protein engineering that allows us to genetically connect elastomeric proteins and self-assembling linkers together to construct well-defined protein building blocks.</p><p>Nanospring behavior of á-helical repeat proteins: We revealed that á-helical repeat proteins, composed of tightly packed á-helical repeats that form spiral-shaped protein structures, unfold and refold in near equilibrium, while they are stretched and relaxed during AFM based SMFS measurements. In addition to minimal energy dissipation by the equilibrium process, we also found that these proteins can yield high stretch ratios (>10 times) due to their packed initial forms. Therefore, we, for the first time, recognized a new class of polypeptides with nanospring behaviors. </p><p>Protein-based force probes for gauging molecular interactions: We developed protein-based force probes for simple, robust and general AFM assays to accurately measure intermolecular forces between self-oligomerization of two or more arbitrary polypeptides that potentially can serve as molecular crosslinkers. For demonstration, we genetically connected the force probe to the Strep-tag II and mixed it with its molecular self-assembling partner, the Strep-Tactin. Clearly characterized force fingerprints by the force probe allowed identification of molecular interactions of the single Strep-tag II and Strep-Tactin complex when the complex is stretched by AFM. We found a single energy barrier exists between Strep-tag II and Strep-Tactin in our given loading rates. Based upon our demonstration, the use of the force probe can be expanded to investigate the strength of interactions within many protein complexes composed of homo- and hetero-dimers, and even higher oligomeric forms. Obtained information can be used to choose potential self-assembling crosslinkers which can connect elastomeric proteins with appropriate strength in higher-order structures. </p><p>Self-assembled nano-structured biomaterials with well-defined protein-based building blocks: We constructed well-defined protein building blocks with tailored mechanical properties for self-assembled nano-structured materials. We engineered protein constructs composed of tandem repeats of either a I27-SNase dimer or a I27 domain alone and terminated them with a monomeric streptavidin which is known to form extremely stable tetramers naturally. By using molecular biology and AFM imaging techniques, we found that these protein building blocks transformed into stable tetrameric complexes. By using AFM based SMFS, we measured, to our knowledge for the first time, the mechanical strength of the streptavidin tetramer at a single-molecule level and captured its mechanical anisotropy. Using streptavidin tetramers as crosslinkers offers a unique opportunity to create well-defined protein based self-assembled materials that preserve the molecular properties of their building blocks.</p> / Dissertation

We use time-lapse AFM to visualize collagen fibrils self-assembly. A solution of acid-solubilized collagen was injected into the AFM fluid cell and fibril formation was observed in vitro. Single fibrils continuously grew and fused with each other until the supporting surface was completely covered by a nanoscopically well-defined collagen matrix. Laterally, the fibrils grew in steps of ~4 nm suggesting a two-step mechanism. In a first step, collagen molecules associated together. In the second step, these molecules rearranged into a structure called a microfibril. High-resolution AFM topographs revealed substructural details of the D-band architecture. These substructures correlated well with those revealed from positively stained collagen fibers imaged by transmission electron microscopy. Secondly, a covalent assembly approach to prepare membrane protein for AFM imaging that avoids crystallization was proposed. High-resolution AFM topographs can reveal structural details of single membrane proteins but, as a prerequisite, the proteins must be adsorbed to atomically flat mica and densely packed in a membrane to restrict their lateral mobility. Atomically flat gold, engineered proteins, and chemically modified lipids were combined to rapidly assemble immobile and fully oriented samples. The resulting AFM topographs of single membrane proteins were used to create averaged structures with a resolution approaching that of 2D crystals. Finally, the contribution of specific amino acid residues to the stability of membrane proteins was studied. Two structurally similar proteins sharing only 30% sequence identity were compared. Single-molecule atomic force microscopy and spectroscopy was used to detect molecular interactions stabilizing halorhodopsin (HR) and bacteriorhodopsin (BR). Their unfolding pathways and polypeptide regions that established stable segments were compared. Both proteins unfolded exactly via the same intermediates. This 3 Molecular Assemblies observed by AFM observation implies that these stabilizing regions result from comprehensive contacts of all amino acids within them and that different amino acid compositions can establish structurally indistinguishable energetic barriers. However, one additional unfolding barrier located in a short segment of helix E was detected for HR. This barrier correlated with a Pi-bulk interaction, which locally disrupts helix E and divides into two stable segments.

During vertebrate development, gastrulation leads to the formation of three distinct germlayers. In zebrafish a central process is the delamination and the ingression of single cells from a common ancestor tissue - that will lead to the formation of the germlayers. Several molecules have been identified to regulate this process but the precise cellular mechanisms are poorly understood. Differential adhesiveness, a concept first introduced by Steinberg over 40 years ago, has been proposed to represent a key phenomena by which single hypoblast cells separate from the epiblast to form the mesendoderm at later stages. In this work it is shown that differential adhesion among the germlayer progenitor cells alone cannot predict germlayer formation. It is a combination of several mechanical properties such as cell cortex tension, cell adhesion and membrane mechanical properties that influence the migratory behavior of the constituent cells.

Hydrophobic interactions guide important molecular self-assembly processes such as protein folding. On the macroscale, hydrophobic interactions consist of the aggregation of "oil-like" objects in water by minimizing the interfacial energy. However, the hydration mechanism of small hydrophobic molecules on the nanoscale (~1 nm) differs fundamentally from its macroscopic counterpart. Theoretical studies over the last two decades have pointed to an intricate dependence of molecular hydration mechanisms on the length scale. The microscopic-to-macroscopic cross-over length scale is critically important to hydrophobic interactions in polymers, proteins and other macromolecules. Accurate experimental determination of hydration mechanisms and their interaction strengths are needed to understand protein folding. This thesis reports the development of experimental and analytical techniques that allow for direct measurements of hydrophobic interactions in a single molecule. Using single molecule force spectroscopy, the mechanical unfolding of a single hydrophobic homopolymer was identified and modeled. Two experiments examined how hydrophobicity at the molecular scale differ from the macroscopic scale. The first experiment identifies macroscopic interfacial tension as a critical parameter governing the molecular hydrophobic hydration strength. This experiment shows that the solvent conditions affect the microscopic and macroscopic hydrophobic strengths in similar ways, consistent with theoretical predictions. The second experiment probes the hydrophobic size effect by studying how the size of a non-polar side-chain affects the thermal signatures of hydration. Our experimental results reveal a cross-over length scale of approximately 1 nm that bridges the transition from entropically driven microscopic hydration mechanism to enthalpically driven macroscopic hydration mechanism. These results indicate that hydrophobic interactions at the molecular scale differ from macroscopic scale, pointing to potential ways to improve our understanding and predictions of molecular interactions. The system established in this thesis forms the foundation for further investigation of polymer hydrophobicity.

Estudos têm demonstrado o potencial da terapia fotodinâmica antimicrobiana (aPDT) na inativação de diferentes células microbianas. No geral, são três as fases de crescimento dos microrganismos: fase lag, exponencial e estacionária. Os objetivos deste estudo foram avaliar a susceptibilidade de células de Candida albicans em diferentes fases de crescimento, submetidas à aPDT, associando azul de metileno (50 &mu;M) e luz de emissão vermelha (&lambda;= 660 nm) e investigar alterações morfológicas, mecânicas e bioquímicas, antes e depois da aPDT, por microscopia eletrônica de varredura, de força atômica e por espectroscopia no infravermelho por transformada de Fourier. Os resultados obtidos sugerem que, em parâmetros letais, células em fase estacionária de crescimento (48 h) são menos susceptíveis à aPDT, quando comparadas àquelas em fases lag (6 h) e ex-ponencial (24 h) de crescimento. Entretanto, em parâmetros subletais, células de 6 h e 48 h mostraram a mesma susceptibilidade à aPDT. Em sequência, os experimentos foram realizados em parâmetros considerados subletais para células crescidas por 6 e 48 h. A avaliação morfológica mostrou menor quantidade de matriz extracelular em células de 6 h comparada àquelas de 48 h. A espectroscopia de força atômica mostrou que células em fase lag perderam a rigidez após a aPDT, enquanto que células em fase estacionária mostraram comportamento in-verso. Ainda, células de 48 h diminuíram sua adesividade após a aPDT, enquanto que células de 6 h e 24 h tornaram-se mais adesivas. Os resultados bioquímicos revelaram que as diferenças mais significativas entre as células fúngicas de 6 h e 48 h ocorreram na região de DNA e carboidratos. A aPDT promoveu mais alterações bioquímicas na região de DNA e carboidratos em células de 6 h e em lipídios e ácidos graxos em células de 48 h. Nossos resultados indicam que a fase de crescimento celular desempenha papel importante no sítio de ação da aPDT em células de C. albicans. / Studies have demonstrated the potential of antimicrobial photodynamic therapy (aPDT) on the inactivation of different microbial cells. Overall, there are three phases of cell growth: lag phase, exponential phase and stationary phase. The objectives of this study were to evaluate the susceptibility of Candida albicans in different growth stages submitted to aPDT, with methylene blue (50&mu;M) and red light (&lambda; = 660 nm) and to investigate morphological, mechanical and biochemical changes before and after aPDT, by scanning electron microscopy, atomic force microscopy and by Fourier transform infrared spectroscopy. The results suggested that with lethal parameters, cells in stationary phase (48 h) are less susceptible to aPDT, compared to those in lag phase (6 h) and exponential phase (24 h). However, in sub-lethal parameters 6 h and 48 h cells showed the same susceptibility to aPDT. The following results were obtained in sub-lethal parameters. The morphological evaluation showed lower amount of extra-cellular matrix at 6 h compared to cells growth for 48 h. The atomic force spectroscopy showed that cells in lag phase lost cell wall rigidity after aPDT, while cells in stationary phase showed a reverse behavior. Furthermore, 48 h cells presented a decrease in their adhesiveness after aPDT, whereas cells growth for 6 h and 24 h become more adhesive. The biochemical evaluation showed that the most significant differences among the fungal cells growth for 6 h and 48 h in DNA and carbohydrates. The aPDT caused more expressive alterations on DNA and carbohydrates in cells growth for 6 h, while cells growth for 48 h presented significant alterations on lipids and fatty acids. Our results indicate that cell growth phase play an important role on the target sites affected by aPDT in C. albicans cells.

No description available.

Surface modification has wide ranging implications in lubrication, microelectromechanical systems (MEMS), colloidal systems and biological membranes. Surface modification plays an important role in stabilizing gold nanoparticles, which have applications in targeted drug delivery and catalysis. A variety of surface modification techniques are used for controlling corrosion and wettability, as well as used extensively to understand the nature of interactions between surfaces. This thesis is mainly focused on understanding the kinetics, film growth and surface modification by long chain molecules physisorbed on a surface. The time evolution of film growth and domain formation of octadecylamine on a mica surface is studied using ex-situ AFM and reflectance FTIR. A novel technique of interface creation is developed to measure the height of the adsorbed film. The results show three distinct regions of film growth mechanism. Region I, corresponds to thin film and the interface height is in the monolayer regime. The transient regime (II) consists of a sharp increase in the film thickness, from 1.5 nm to 25 nm within a time span of 180 s. In the final stage of film growth the film thickness is invariant with time, during which domain coarsening is observed. Domain evolution reveals a non-monotonic variation in the domain size as a function of adsorption time. A three stage mechanism is proposed to explain the domain evolution on the surface. In order to explain the observed film thickness variation, we have developed and tested various models to explain the thin to thick film transition observed in the AFM experiments. A model based on adsorption kinetics is solved to obtain the evolution of the adsorbed film. The model with a two-step adsorption isotherm quantitatively captures the thin to thick film transition observed in the AFM experiments. The statistical thermodynamics of adsorption of long chain molecules on a surface has been studied using a lattice model. The molecules are characterized by backbone chain, either lying parallel or perpendicular to the surface. A square lattice with nearest neighbour interactions and a mean field approximation are used to generate the adsorption isotherms for different molecules as a function of chain length. The molecules change their orientation from a surface parallel to an upright configuration with an increase in chemical potential. A similar transition (with time) in the molecular orientation has been observed in the AFM experiments. The transition between these two orientations is accompanied by an entropy maximum The last part of the thesis is concerned with carbon-carbon interactions. More specifically, we are interested in the interactions between graphite surfaces and their modification in the presence of a lubricant or base oil. Diamond like carbon (DLC) AFM tips and highly oriented pyrolitic graphite (HOPG) have been used for this study. Experiments were carried out by treating HOPG graphite in hexadecane oil at different temperatures. It is observed that pull-off forces on bare graphite are smaller when compared to the treated surface. The magnitude of the pull-off forces increases with the temperature of the hexadecane oil bath. Presence of charged patches responsible for the higher adhesion have been confirmed using surface potential microscopy. Results also confirm the presence of a thin liquid-like hexadecane film at room temperature.

Pregnancy tests and related immunoassays are heavily dependent on specific and non-specific protein adsorption. These interfacial processes are affected by many factors that influence the in situ conformations of interfacially immobilised antibodies. This thesis examines a number of representative features with dual polarisation interferometry (DPI) and neutron reflection (NR), thus combining real-time dynamic monitoring with high interfacial structural resolution. Bovine serum albumin (BSA) was initially used as a model system to compare the surface coverage and thickness measurements of DPI and NR. The results show that DPI and NR provided similar surface coverage data but the measured thicknesses differed at BSA concentrations above 0.1 mg/ml. This discrepancy arose from the adoption of the uniform-layer model used by DPI for data analysis and the greater thickness sensitivity of NR. A model pregnancy immunoassay was built in steps on a silica surface so that the adsorption of each protein could be accurately monitored. Both DPI and NR provided evidence of BSA insertion into the gaps on the surface between the antibody molecules. This suggests that BSA adsorption is an excellent method to block the non-specific adsorption of target antigens to the immunoassay test surface. A magnetic tweezer system was designed and built in order to measure the specific antibody/antigen binding force. The antibodies and antigens were used to immuno-link magnetic beads to the experimental surface before the immuno-links were broken by increasing the attractive force between the magnetic tweezers and beads. The force per antibody/antigen immuno-link was estimated to lie between the values of 13.6 pN and 43.8 pN.Immuno-link detachment as a function of time was investigated. It was found that the immuno-link comprised both a strong and a weak interaction. The dissociation constant of the strong antibody/antigen interaction was found to equal 3E-4 /s and had an interaction length of 0.06 nm. The low population of beads bound by the second, weaker interaction meant that it was not possible to obtain accurate values of the dissociation constant and bond length of the second weaker interaction.