Pseudomonas aeruginosa is extremely harmful to immunocompromised individuals. An atomic force microscope was used to measure the surface forces of this bacteria’s exopolymers. These forces were characterized with the AdG force model, which is a function of brush length, probe radius, temperature, separation distance and an indefinite density variable, s. This last parameter could represent the root spacing or mesh spacing of the exopolymers. This study aims to clarify s by obtaining force values as a function of temperature. The data suggest that s represents the mesh spacing. If s is the root spacing it should remain constant regardless of the changing polymer lengths, on the other hand if it is the mesh spacing it will vary with changing temperature, as shown by the data presented in this research. This knowledge will aid in understanding and characterizing how bacteria cause infections.

"American cranberry (Vacciniumm macrocarpon) has been long known for its benefits in maintaining urinary tract health. Clinical trials have shown that drinking cranberry juice can prevent urinary tract infections (UTIs) in various subpopulations that are prone to UTIs, especially women, but the mechanisms by which cranberry acts against uropathogenic bacteria are still unclear. Studies showed that when exposed to cranberry juice or A- PACs, a group of tannins that are unique to cranberry, the adhesion activity and biofilm formation of uropathogenic bacteria were reduced. However, the metabolism of cranberry juice has not be elucidated, therefore further study is needed to find out whether the anti-bacterial components in cranberry could survive the digestive system and reach the urinary tract, and how the components or metabolites remaining in urine act against uropathogenic bacteria. We used atomic force microscopy (AFM) to study the surface adhesion force of uropathogenic E. coli incubated with urine samples that were collected from volunteers after drinking 16 oz. of cranberry juice cocktail (CJC) or water. The urine samples were collected at 0, 2, 4, 6, and 8 hours after CJC or water consumption. When incubated with post-water urine, the adhesion forces of pathogenic bacteria that have fimbriae (E. coli B37, B73, B78, BF1023, CFT 073, and J96) did not change; whereas the adhesion forces of these strains decreased over the 8 hour period after CJC consumption. The control strain that does not have frimbriae, E. coli HB101, showed low adhesion force when incubated with post-water and post-CJC urine. In a human red blood cell agglutination (HRBC) assay, the attachment of pathogenic E. coli to red blood cells was significantly lower after exposed to post-CJC urine, compared to those exposed to post-water urine. These results indicate the anti-bacteria components or metabolites of CJC stay active in urine, and these compounds prevent adhesion of E. coli by reducing fimbriae-mediated adhesion. We also examined the effects of drinking CJC on biofilm formation of uropathogenic bacteria. Female volunteers were given 16 oz. of CJC or placebo, and their urine was collected at 0, 2, 8, 24, and 48 hours after consumption. Bacteria (E. coli B37, CFT073, BF1023, HB101, and S. aureus ATCC43866) were cultured in a mixture of urine and growth media in 96 well microtiters. The biofilm formed was quantified by staining the biofilm dissolved in a solvent with crystal violet and measuring the absorbance at 600 nm. The results showed that biofilm formation was reduced within 24 hours after CJC consumption, and it started to increase after 48 hours, possibly due to the washout of CJC in the system. These studies suggest that CJC can be an effective preventive measure for UTIs as it inhibits adhesion and biofilm formation of uropathogenic bacteria."

"Steric forces related to the lipopolysaccharide (LPS) brush on bacterial surfaces is of great importance in biofilm research. However, the atomic force microscopy (AFM) data, or force curves, produced require extensive analysis to obtain any useful information about the sample. Normally, after several force curves have been measured, the individual curves would be fit to a model for analysis. This process is not only time-consuming, but it is also extremely subjective as it lends itself to user bias throughout the analysis. A Matlab program to analyze force curves from an AFM efficiently, accurately, and with minimal user bias has been developed and is presented here. The analysis is based on a modified version of the Alexander and de Gennes (AdG) polymer model, which is a function of equilibrium polymer brush length, probe radius, temperature, separation distance, and a density variable. The program runs efficiently by cropping curves to the region specified by the model and then fitting the data. Automating the procedure reduces the amount of time required to process 100 force curves from several days to less than two minutes. Accuracy is ensured by making the program highly adjustable. The user can specify experimental constants such as the temperature and cantilever tip geometry, as well as adjust many cropping and fitting parameters to better analyze the data. Additionally, as part of this program, researchers can compare data from related experiments by choosing to plot the calculated fit parameters using either error bars or box plots to quickly identify relationships or trends. The use of this program to crop and fit force curves to the AdG model will allow researchers to ensure proper processing of large amounts of experimental data and reduce the time required for analysis and comparison of data, thereby enabling higher quality results in a shorter period of time. "

Orientadores: Mônica Alonso Cotta, Peter Alexander Bleinroth Schulz / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Física Gleb Wataghin / Made available in DSpace on 2018-09-02T17:54:43Z (GMT). No. of bitstreams: 1 Vicaro_KlausOrian_D.pdf: 6412839 bytes, checksum: d994f5b0a67263a799df2b77a074f96b (MD5) Previous issue date: 2008 / Resumo: Neste trabalho caracterizamos as propriedades elétricas de nanoestruturas semicondutoras de InAs/InP, principalmente quantum dots e quantum wires, obtidas pelo modo de crescimento Stranski-Krastanov com epitaxia por feixe químico (CBE). Medidas de topografia, de condutância elétrica e corrente-voltagem com resolução espacial foram realizadas nas estruturas crescidas utilizando microscopia de força atômica em modo condutivo (C-AFM) com ponta metalizada. Estruturas tipo mesa foram processadas nas amostras usadas em C-AFM e medidas elétricas a temperaturas mais baixas que 273 K foram adquiridas. Transporte por emissão termiônica tridimensional (não-homogêneo) foi observado entre a ponta condutora e as nanoestruturas de InAs. Isso sugere que as vizinhanças da nanoestrutura, formada pela wetting layer (WL), alteram a configuração da altura da barreira, tornando-a dependente da voltagem aplicada na junção metal-semicondutor. Por outro lado, a voltagem de limiar, definida como a voltagem necessária para obter a menor corrente elétrica detetável, varia com o tamanho e forma da nanoestrutura; ela está relacionada com o estado eletrônico da nanoestrutura e também com o gap eletrônico do semicondutor, que é menor nas nanoestruturas maiores. Condução elétrica por hopping e ruído telegráfico aleatório (RTN) foram observados a baixas temperaturas nos dispositivos fabricados via e-beam com dezenas ou centenas de nanoestruturas de InAs/InP. O transporte tipo hopping de Éfros-Shklovskii ocorre a temperaturas mais altas (> 70 K) e polarizações baixas onde a densidade de portadores no dispositivo é baixa e a interação coulombiana forte. Com o aumento da polarização o hopping muda para intervalo variável de Mott em sistemas 2D, e correlacionado com a dimensionalidade da WL ¿o canal de condução. O RTN aparece em temperaturas mais baixas (< 40 K) mas somente nos dispositivos contendo nanoestruturas que permitem o aprisionamento de portadores. Simulações numéricas usando um modelo heurístico mostraram que poucas nanoestruturas podem alterar o transporte elétrico num ensemble com centenas delas / Abstract: In this work we characterized the electrical properties of InAs/InP semiconductor nanostructures, mainly quantum dots e quantum wires, obtained by Stranski-Krastanov growth mode using chemical beam epitaxy (CBE). Topography, electrical conductance, and current-voltage measurements with spatial resolution were performed on the grown structures using atomic force microscopy in conductive mode (C-AFM) with metalized tip. Mesa-like structures were processed on the samples used in C-AFM; electrical measurements at temperatures lower than 273 K were then acquired. Three-dimensional thermionic emission (non-homogeneous) transport was observed between the conductive tip and the InAs nanostructures. This suggests that the nanostructure neighborhood, formed by the wetting layer (WL), changes the barrier height configuration and makes it dependent on the voltage applied to the metal-semiconductor junction. On the other hand, the threshold voltage, defined as the voltage necessary to detect the lowest current level, varies with nanostructure size and shape; it is related to the nanostructure electronic state and also to the semiconductor electronic gap that is smaller for the larger nanostructures. Electrical conductance via hopping and random telegraphic noise (RTN) were observed at low temperatures on the devices fabricated via e-beam with dozens or hundreds of InAs/InP nanostructures. The Éfros-Shklovskii hopping transport occurs at higher temperatures (> 70 K) and low polarizations where the device carrier density is low and the coulombian interaction is strong. Increasing the polarization the hopping changes to the Mott variable range on 2D system, which correlates to the WL dimensionality ¿the conduction channel. The RTN appears in low temperatures (< 40 K) but only in those devices with nanostructures that allow carrier trapping. Numerical simulations using a heuristic model showed that few nanostructures can change the electrical transport in an ensemble with hundreds of them / Doutorado / Física / Doutor em Ciências / 01/13463-1 / FAPESP

<div>The atomic force microscope (AFM), traditionally known as a nanoscale instrument for surface topography imaging and compositional contrast, has a unique ability to investigate buried, subsurface objects in non-destructive ways with very low energy. The underlying principle is the detection of interactions between the AFM probe and the sample subsurface in the presence of an external wave or eld. The AFM is a newcomer to the field of subsurface imaging, in comparison to other available highresolution techniques like transmission or scanning electron microscopy. Nevertheless,</div><div>AFM offers signicant advantages for subsurface imaging, such as the operation over a wide range of environments, a broad material compatibility, and the ability to investigate</div><div>local material properties. These make the AFM an essential subsurface characterization tool for materials/devices that cannot be studied otherwise. </div><div><br></div><div><div>This thesis develops a comprehensive qualitative and quantitative framework underpinning the subsurface imaging capability of the AFM. We focus on the detection of either electrostatic force interactions or local mechanical properties, using 2nd-harmonic Kelvin probe force microscopy (KPFM) and contact-resonance AFM (CRAFM),</div><div>respectively. In 2nd-harmonic KPFM we exploit resonance-enhanced detection to boost the subsurface contrast with higher force sensitivity. In CR-AFM we use the dual AC resonance tracking (DART) technique, in which the excitation frequencies are near one of the contact resonance frequencies. Both techniques take advantage of the maximized response of the cantilever at resonance which improves the signal to noise ratio. These enable high-resolution subsurface mapping on a variety of polymer</div><div>composites.</div></div><div><br></div><div><div>A relevant challenge is the ability to reconstruct the properties of the subsurface objects from the experimental observables. We propose a method based on surrogate</div><div>modelling that relies on computer experiments using nite element models. The latter are valuable due to the lack of analytical solutions that satisfy the complexity of the geometry of the probe-sample system and sample heterogeneity. We believe this work is of notable interest because offers one of few approaches for the non-destructive characterization of buried features with sub-micron dimensions.</div></div>

Collagen II and the proteoglycan aggrecan are key extracellular matrix (ECM) proteins in cartilaginous tissues such as the intervertebral disc (IVD). Given the functional role that these structural and functional proteins have in the IVD, ECM in tissue engineered intervertebral disc (TE IVD) constructs needs to recapitulate native tissue. As such, there is a need to understand the structure and mechanical function of these molecules in native tissue to inform TE strategies. The aims here were to characterise aggrecan and collagen II using atomic force microscopy (AFM), size-exclusion chromatography multi angle light scattering (SEC-MALS), histology, quantitative PCR, nanomechanical and computational modelling in: (i) skeletally immature and mature bovine articular cartilage (AC) and nucleus pulposus (NP), (ii) TE IVD constructs cultured in hypoxia or treated with transforming growth factor beta [TGFÎ23] or growth differentiation factor [GDF6]), and (iii) porcine AC and NP tissue. No variation in collagen II structure was observed although the proportion of organised fibrillar collagen varied between tissues. Both intact (containing all three globular domains) and non-intact (fragmented) aggrecan monomers were isolated from both AC and IVD and TE IVD constructs. Mature intact native NP aggrecan was ~60 nm shorter (core protein length) compared to AC. In skeletally mature bovine NP and AC tissue, most aggrecan monomers were fragmented (99% and 95%, respectively) with fragments smaller and more structurally heterogeneous in NP. Similar fragmentation was observed in skeletally immature bovine AC (99.5%), indicating fragmentation occurs developmentally at an early age. Fragmentation was not a result of enhanced gelatinase activity. Aggrecan monomers isolated from notochordal cell rich porcine NP were also highly fragmented, similar to bovine NP. Application of a computational packing model suggested fragmentation may affect porosity and nutrient transfer. The reduced modulus was greater in AC than NP (497 kPa and 76.7 kPa, respectively) with the difference likely due to the organisation and abundance of ECM molecules, rather than individual structure. Growth factors (GDF6 and TGFÎ23), and not oxygen tension treated TE IVD constructs were structurally (with >95% fragmented monomers), histologically and mechanically (GDF6: 60.2 kPa; TGFÎ23; 69.9 kPa) similar to native NP tissue (76.7 kPa) and there was evidence of gelatinase activity. To conclude, these results show that the ultrastructure of intact aggrecan was tissue and cell dependent, and could be modified by manipulation of cell culture conditions, specifically GDF6 which may play a role in aggrecan glycosylation.

Orientador: José Manoel Balthazar / Banca: Átila Madureira Bueno / Banca: Bento Rodrigues de pontes Junior / Resumo: Desde 1986, quando Binnig et al (1986) criaram o microscópio de força atômica (AFM), esse aparelho se tornou um dos mais importantes microscópios de varredura (SPM), sendo usado para análise de DNA, nanotubos, etc. (Rützel et al, 2006). O AFM tem como componente principal uma microviga, com uma ponteira em uma das extremidades, que vibra próximo de sua frequencia de ressonância para mandar sinais a um fotodetector que traduz esse sinal e gera as imagens da superfície da amostra. O modo de operação tapping é o mais usado, e o comportamento caótico é muito comum nesse modo de operação, por esse motivo, AFM se tornou um assunto muito importante no mundo científico. Nesse trabalho, a microviga é modelada com o uso das equações de Bernoulli, as interações entre ela e a amostra são modeladas usando o potencial de Lennard Jones. Simulações numéricas detectam movimento caótico no sistema, a necessidade de estabilizá-lo nos leva a usar os seguintes métodos: Método do Balanço Harmônico, sincronização de Sistemas Não Lineares, Método das Equações de Estado Dependentes de Riccati (SDRE), Método de Realimentação de Sinal Atrasado. Por fim, a aplicação dos métodos se mostra eficiente, com pequeno erro e fácil implementação / Abstract: Since 1986, when Binnig et al (1986) created the atomic force microscope (AFM), this unit became one of the most important scanning probe microscopes (SPM) being used for DNA analysis, nano tubes, etc. (Rutzel et al, 2006). The AFM has as a main component, a micro cantilever, with a tip at its free end, which vibrates near its resonance frequency to send signals to a photo detector that translates the signal and generates images of the sample surface. The tapping mod of operation is the most widely used and chaotic behavior is very common in this mode, therefore, AFM has become a very interesting subject in the scientific world. In this work, the micro cantilever is modeled using Bernoulli's equation and the interactions between the tip and the sample are modeled using the Lennard Jones potential. Numerical simulations detect chaotic motion in the system and the need to stabilize it leads us to use the following methods, Harmonic Balance Method; Synchronization of Nonlinear Systems; the State Dependent Riccati Equation control method (SDRE); the Method of Feedback Delay. Finally, the application of the methods proved to be effective, with small error and easy implementation / Mestre

This study presents a systematic investigation of two types of soft biomaterials: phospholipid-based microbubbles (MBs) and agarose hydrogels, using atomic force microscopy (AFM) force-distance curves. Microbubbles are used widely in several applications, especially in medical applications, where they are used as ultrasound contrast agents (UCAs) and as vehicles for transporting the drugs and genes to their targets, which is commonly known as drug/gene delivery. Although plenty of attention has been paid to these materials by medical researchers there is a shortage of engineering research on the properties of these materials. The present study tries to address this gap by studying these materials from the engineering perspective; therefore, the aim of this study is to investigate the mechanical properties of MBs and hydrogels. In this research, phospholipid-based microbubbles (MBs), commercially called SonoVue® microbubbles and used as UCAs, were investigated to measure their mechanical properties using an AFM mode of operation called force-distance curves (or force spectroscopy mode); this mode allows for direct mechanical tests to acquire the force-deformation (F-Δ) behaviour of the MBs. The compression tool was a flat (tipless) cantilever moved at constant speed, whereas the variable was MB size. The MBs behaviour was assessed by calculating several mechanical properties, which were the stiffness, Young's modulus (three different models were applied), hysteresis, plasticity, adhesion forces, nonlinearity and instability. The stiffness and the Young's modulus values were measured to be in the same range as found in similar studies. A phenomenon was observed that the local stiffness of the MB increases after each unstable step provided that the MB stays within the linear elastic region. The Young's modulus was calculated applying three models, two for estimating the elastic modulus of the shell and the third for modulus of elasticity of the whole MB. The stretching component of the membrane theory was found to provide the best prediction of the Young's modulus value. To investigate the effect of the tip geometry on the mechanical properties of the MBs, the MBs were studied with different cantilever/tips, including a conical-tipped cantilever. The study concluded that there is no impact of the contact geometry on the mechanical properties of the MBs if the applied forces and the spring constant of the cantilever are the same. The same phenomenon, increasing the local stiffness of the MB after each unstable step, was found however with a higher rate. Hydrogels were also studied in this research using AFM and adopting a nanoindentation technique. The indenter was a conical tip moving toward the sample surface with constant speed and applying similar forces on all samples, where the variable was the gel concentration. In addition to the previous mechanical properties, other properties were investigated, such as hardness, universal hardness and pressure. An effect of the gel concentration on the mechanical properties of the gels was observed. There is a difference in the results compared to those reported in the literature review, where some of the results are in the same range as those found here, while others were either higher or lower, due to the influence of factors such as the indenter geometry, the applied force and the load rate; moreover, it was found that the viscoelastic behaviour of the gels played a significant role.

Being the most abundant protein in the body, by mass, type I collagen provides the building blocks for tissues such as bone, extra-cellular matrix, tendons, cornea, etc[1-3]. The ability of a single protein to create structures with such various mechanical properties is not fully understood. Before one can engineer and assemble a complex tissue, such as cornea, the mechanisms underlying the formation and assembly, mechanical properties, and structure must be investigated and quantified. The work presented herein contains an extensive study of Type I collagen from the molecular to the tissue level. The engineering of collagenous tissues that mimic the mechanical and optical properties of native human cornea have been performed by a number of groups[4-7]. In all of these studies, the corneal-mimicking tissues have been created using a number of methods including repeated flow casting. To date, the ability to create self-assembled corneal tissue has not been achieved. Understanding the mechanisms of formation of native cornea will not only bring us closer to achieving self-assembled transplantable corneal tissue but will also aid in the engineering of all collagenous tissues and other structures comprised of filamentous units. Recently, the study of type I collagen has primarily focused on the tissue, fiber, and fibril scale[2, 8-21]. Grant, et al.[20] measured the elastic modulus of collagen fibrils in various solutions and found that by increasing ion concentration, in the solution around the fibril, the elastic modulus increased. The solution dependent behavior of the elastic modulus of collagen fibrils was measured but the cause of the dependence was unknown. Grant et al. state that due to the complex nature of the interactions between collagen fibrils and aqueous solutions, the exact cause of this effect is difficult to determine. Through work presented herein, not only do we show that this behavior is seen at the molecular level but also quantify the relationship between ionic concentration and molecular stiffness for a variety of ionic species. Studies of collagen mechanics, on the molecular level, are brief[22-26]. The most prominent of these studies in recent years was performed by Sun, et al.[27] wherein a persistence length of 14.5nm, for human type I procollagen, was measured. The persistence length of the molecule, which is a measure of flexibility, is a highly debated topic with quoted values of 14.5nm[27], 57nm[28], 130nm[29], 175nm[30], 308nm[31], and 544nm[32]. The broad range of values indicates that the flexibility of the collagen molecule is a complex question. It became apparent that the disagreement of the persistence length of molecular collagen in the literature may be due to the use of different ionic solutions. To address this, an initial atomic force microscope, AFM, study of the persistence length of molecular collagen diluted in DI water and two ionic solutions was conducted. This study showed that there is a strong solution dependence to the flexibility of the molecule. The ionic solutions presented molecules with a large persistence length, a straightened configuration, while the DI water dilution resulted in a persistence length that was a factor of 10 smaller. Because two different complex ionic solutions in the initial study showed different persistence lengths, an evaluation of the effect of each individual salt was performed. To elucidate the effects of individual ionic species on the conformations and persistence length of Type I collagen varying concentration of monovalent and divalent salts with different cations and anions were tested. It was found that increasing ionic concentration for all species types resulted in a higher persistence length but the rate of change in persistence length as a function of concentration is unique to each species. In 2002 Leikina, et at.[33] suggested that Type I molecular collagen is unstable at body temperature using differential scanning calorimetry. To examine these results, an AFM study was performed that imaged the collagen molecules after being held at body temperature for varying times. The density of molecules deposited onto mica, above a 200nm length cutoff, was calculated and it shows that the number of molecules above 200nm in length decreases with increasing incubation time. These environmental studies were performed with an aim to understanding the role of environment in creating a corneal mimicking tissue. Currently, the most promising method of collagen membrane fabrication for corneal replacement was developed by Tanaka, et al.[4]. This unique repeated flow casting method allows for the manufacturing of transparent collagen membranes with controllable thickness and fibrillar alignment. Using the repeated flow casting technique, orthogonally oriented collagen membranes were created and their optical properties were measured using the Generalized High Accuracy Universal Polarimeter, G-HAUP. When engineering a tissue for the eye, the optical properties of the tissue are of the utmost importance. Appropriately for corneal tissues, the measurements for linear birefringence and linear dichroism were negligible. It was clear, from the literature, that a fundamental understanding of molecular type I collagen was not available. In this work, the mechanical properties and environmentally sensitive behavior of bovine dermal type I molecular collagen is studied. The exploration into the unique behavior of these systems begins with documenting the rich ionic species and concentration dependent flexibility of molecular type I collagen and the temperature dependence on the stability of the molecule is tested. The study concludes with the construction of corneal mimicking tissues using the repeated flow casting method and measuring the complex optical properties of these tissues.

An atomic force microscopy beam-like cantilever is combined with an electrical tuning fork to form a shear force probe that is capable of generating an acoustic response from the mesoscopic water layer under ambient conditions while simultaneously monitoring force applied in the normal direction and the electrical response of the tuning fork shear force probe. Two shear force probes were designed and fabricated. A gallium ion beam was used to deposit carbon as a probe material. The carbon probe material was characterized using energy dispersive x-ray spectroscopy and scanning transmission electron microscopy. The probes were experimentally validated by demonstrating the ability to generate and observe acoustic response of the mesoscopic water layer.