Rozprawy doktorskie na temat „Force spectroscopy- biological application”

Un microscope à champ proche optique basé sur un nouveau senseur de force fonctionnant dans le mode cisaillement a été développé. Il peut être combiné à un microspectrofluorimètre confocal laser pour des applications biologiques et utilisé dans différents modes de fonctionnement. Le mécanisme de détection des forces de cisaillement a été expérimentalement étudié. Il s'avère que l'origine principale de ce mécanisme est le contact intermittent de la sonde avec la surface de l'échantillon qui permet de contrôler la distance pointe-surface. Les paramètres expérimentaux concernant l'imagerie ainsi que les artefacts dus à la géométrie de la sonde sont discutés. Des images en champ proche optique d'un réseau de silicium dans le mode réflexion ainsi que la spectroscopie de structures électroluminescentes dans le mode collection ont été respectivement obtenues. Comme étude préliminaire pour des applications biologiques, la distribution de P-glycoprotéines dans la membrane plasmatique de petites cellules cancéreuses de langue humaine a été étudiée avec une résolution inférieure à la limite de résolution due à la diffraction. Cette distribution n'est pas homogène et se présente sous forme de petits amas. De plus, des spectres de fluorescence ont été obtenus sur des cellules cancéreuses du poumon humain colorées avec la sonde fluorescente JC-1. Des variations dans le spectre de fluorescence ont été mises en évidence avec une résolution verticale de l'ordre de 100 nm. Ces résultats suggèrent que notre système est un outil d'investigation prometteur pour des applications biologiques, capable de fournir des informations dignes d'intérêt permettant de mieux comprendre certains problèmes biologiques
Based on a new force sensor, a shear force scanning near-field optical microscope (ShF-SNOM), that can be operated in the different modes and combined with a confocal laser microspectrofluorometer (CLMF) for biological applications, has been developed. Shear force mechanism was experimentally studied and the knocking mechanism is the main origin responsible for shear force distance control in our system. Experimental parameters concerning the shear force imaging and artifacts due to probe geometric effects are discussed. Shear force and near-field imaging of a silicon grating in the reflection mode, imaging and spectroscopy of electroluminescent structures in the collection mode are demonstrated respectively. As a preliminary study for biological applications, the distribution of P-glycoprotein (P-gp) in the plasma membrane of human small cell lung cancer cells were investigated with sub-diffraction limit resolution. The distribution of P-gp in the cell membrane was found to be not homogenous and cluster formation of P-gp in the membrane was observed. In addition, fluorescence spectra were recorded in a single living cell of human breast adenocarcinoma cells stained with the fluorescent dye JC-1. The variations in fluorescence spectra were measured with vertical resolution of about 100 nm. These results suggest that our system would be a promising tool for biological applications and provide valuable information for understanding some biological problems

Thesis (Ph. D.)--University of Missouri-Columbia, 2005.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on October 18, 2007) Vita. Includes bibliographical references.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007.
Includes bibliographical references.
Mechanical forces play an important role in cell morphology, orientation, migration, adhesion and can even induce apoptosis. The eukaryotic cell is equipped with a dynamic frame, known as the cytoskeleton, that provides the cell's structural integrity in order to sustain and react to such forces. Therefore, understanding the mechanical properties of the cytoskeleton is an important step towards building models describing cell behavior. Filamentous actin (F-actin), as one of the major constituents of the cytoskeleton, has been the target of extensive in vitro studies to determine its mechanical properties in bulk. However, there is still a lack in the understanding of how the molecular interactions between F-actin and the proteins that arrange these filaments into networks regulate the dynamic properties of the cytoskeleton Here we present a novel, single molecule assay to test the rupture force of a complex formed by an actin binding protein (ABP) linking two actin filaments. We readily demonstrate the adaptability of this assay by testing it with two different ABPs: filamin, a crosslinker, and a-actinin, a bundler. We measured rupture forces of 28-73 pN and 30-56 pN for filamin/actin and a-actinin/actin respectively, suggesting that the former is a slightly stronger interaction. Moreover, since no ABP unfolding events were observed at our force levels, our results suggest that ABP unbinding is a more relevant mechanism than unfolding for the temporal regulation of the mechanical properties of the actin cytoskeleton. In addition, we explore the micro-scale properties of F-actin networks reconstituted in vitro.
(cont.) Using imaging and microrheology techniques we characterized the effects of filament length and degree of crosslinking on the structural arrangement and mechanical properties of F-actin networks. We found that the mechanical properties of these networks are length-scale dependent. Also, when probed with active methods, the F-actin networks exhibited strain hardening followed by a gradual softening at forces -30 pN, in good agreement with the single molecule rupture force of 28-73 pN. Thus, with the combination of single molecule and network studies, we can expand the knowledge-base on the regulation and control of the cellular machinery starting from the molecular building blocks.
by Jorge M. Ferrer.
Ph.D.

The focus of this dissertation is on refining atomic force micrscopy (AFM) methods and data analysis routines to measure the viscoelastic mechanical properties of soft polymer and biological materials in relevant fluid environments and in vivo using a range of relevant temperatures, applied forces, and loading rates. These methods are directly applied here to a several interesting synthetic and biological materials. First, we probe poly(n-butyl methacrylate) (PnBMA), above, at and below its glass transition temperature in order to verify our experimental procedure. Next, we use AFM to study the viscoelastic properties of coating materials and additives of silicone-based soft contact lenses in a tear-like saline solution. Finally, a major focus in this dissertation is determining the fundamental mechanical properties that contribute to the excellent sensitivity of the strain sensing organs in a wandering spider (Cupiennius salei) by probing under in vivo conditions. These strain-sensing organs are known to have a significant viscoelastic component. Thus, the cuticle of living spiders is directly investigated in near-natural environments (high humidity, temperatures from 15-40 °C). The main achievements of these studies can be summarized through the following findings: We suggest that full time-temperature-modulus relationships are necessary for the understanding of soft materials systems, and present a practical method for obtaining such relationships. These studies will have a direct impact on both scientists in the metrology field by developing practical experimental procedures and data analysis routines to investigate viscoelastic mechanical properties at the nanoscale, and future materials scientists and engineers by showing via spider mechanosensory systems how viscoelasticity can be applied for functional use in sensing technology.

The utilisation of near-infrared Raman spectroscopy for the discrimination of cancers and pre-cancers from normal tissue in the acro-digestive tract has been evaluated. A commercially available Raman microspectrometer has been modified to provide optimum throughput, sensitivity and fluorescence suppression for epithelial tissue measurements. Laser excitation at 830nm was demonstrated to be optimum. High quality (SN ratio 15-20) NIR-Raman spectra have been acquired from oesophageal and laryngeal tissues in time scales under 30 seconds. Pathological groupings covering the full range of normal and neoplastic tissues in the organs of interest have been studied. Both fresh (snap frozen) and formalin fixed tissue samples were investigated, firstly to indicate whether tissue-types can be distinguished in vivo and secondly to demonstrate the use of Raman spectroscopy as a tool for classification in the pathology lab. Results using multivariate statistical techniques to distinguish between spectra from specimens exhibiting different tissue pathologies have been extremely promising. Cross-validation of the spectral predictive models has shown that three groups of larynx tissue can be separated with sensitivities and specificities of between 86 and 90% and 87 and 95% respectively. Oesophageal prediction models have demonstrated sensitivities and specificities of 84 to 97% and 93 to 98% respectively for a three-group consensus model and 73 to 100% and 92 to 100% for an eight-group consensus model. Epithelial tissues including stomach, tonsil, endometrium, bladder and prostate have been studied to identify further tissues where Raman spectroscopy may be employed for detection of disease. Spectra were similar to those obtained from oesophagus and larynx, although sufficiently different for distinct discriminant models to be required. This work has demonstrated the generic nature of Raman spectroscopy for the detection and classification of cancers and pre-cancerous lesions in many tissues. The evidence provided by this study indicates that utilisation of Raman spectroscopy for non-invasive detection and classification of disease is a distinct possibility. Potential difficulties in the transferability from in vitro to in vivo have been evaluated and no significant barriers have been observed. However, further in vivo probe development and optimisation will be required before 'optical biopsy' with Raman spectroscopy can become a reality.

The utilisation of near-infraredR aman spectroscopyfo r the discrimination of cancersa nd pre-cancers from normal tissue in the acro-digestive tract has been evaluated. A commercially available Raman microspectrometehr as been modified to provide optimum throughput, sensitivity and fluorescence suppression for epithelial tissue measurements. Laser excitation at 830nmw as demonstratedto be optimum. High quality (SN ratio 15-20) NIR-Raman spectrah ave been acquired from oesophageaal nd laryngeal tissues in time scales under 30 seconds. Pathological groupings covering the full range of normal and neoplastic tissues in the organs of interest have been studied. Both fresh (snap frozen) and formalin fixed tissue samples were investigated,f irstly to indicate whether tissue-typesc an be distinguishedi n vivo and secondlyt o demonstrateth e use of Raman spectroscopya s a tool for classificationi n the pathology lab. Results using multivariate statistical techniques to distinguish between spectra from specimens exhibiting different tissue pathologies have been extremely promising. Crossvalidation of the spectral predictive models has shown that three groups of larynx tissue can be separated with sensitivities and specificities of between 86 and 90% and 87 and 95% respectively. Oesophageal prediction models have demonstrated sensitivities and specificities of 84 to 97% and 93 to 98% respectively for a three-group consensus model and 73 to 100% and 92 to 100% for an eight-groupc onsensusm odel. Epithelial tissues including stomach, tonsil, endometrium, bladder and prostate have been studiedt o identify further tissuesw hereR amans pectroscopym ay be employedf or detection of disease.S pectraw ere similar to those obtainedf rom oesophagusa nd larynx, although sufficiently different for distinct discriminant models to be required. This work has demonstratedth e genericn atureo f Ramans pectroscopyfo r the detectiona nd classification of cancersa nd pre-cancerousle sionsi n many tissues.T he evidencep rovided by this study indicatest hat utilisation of Ramans pectroscopyfo r non-invasived etectiona nd classification of diseaseis a distinct possibility. Potentiald ifficulties in the transferabilityf rom in vitro to in vivo have been evaluated and no significant barriers have been observed. However, further in vivo probe development and optimisation will be required before 'optical biopsy' with Ramans pectroscopyc anb ecomea reality.

The atomic force microscope (AFM) provides a powerful instrument for investigating and manipulating biological samples down to the subnanometer scale. In contrast to other microscopy methods, AFM does not require labeling, staining, nor fixation of samples and allows the specimen to be fully hydrated in buffer solution during the experiments. Moreover, AFM clearly compares in resolution to other techniques. In general, the AFM can be operated in an imaging or a force spectroscopy mode. In the present work, advantage was taken of this versatility to investigate single biomolecules and biomolecular assemblies. A novel approach to investigate the visco-elastic behavior of biomolecules under force was established, using dextran as an example. While a molecule tethered between a solid support and the cantilever tip was stretched at a constant velocity, the thermally driven oscillation of the cantilever was recorded. Analysis of the cantilever Brownian noise provided information about the visco-elastic properties of dextran that corresponded well to parameters obtained by alternative methods. However, the approach presented here was easier to implement and less time-consuming than previously used methods. A computer controlled force-clamp system was set up, circumventing the need for custom built analogue electronics. A commercial PicoForce AFM was extended by two computers which hosted data acquisition hardware. While the first computer recorded data, the second computer drove the AFM bypassing the manufacturer's microscope control software. To do so, a software-based proportional-integral-differential (PID) controller was implemented on the second computer. It allowed the force applied to a molecule to be held constant over time. After tuning of the PID controller, response times obtained using that force-clamp setup were comparable to those of the recently reported analogue systems. The performance of the setup was demonstrated by force-clamp unfolding of a pentameric Ig25 construct and the membrane protein NhaA. In the latter case, short-lived unfolding intermediates that were populated for less than 10 ms, could be revealed. Conventional single-molecule dynamic force spectroscopy was used to unfold the serine:threonine antiporter SteT from Bacillus subtilis, an integral membrane protein. Unfolding force patterns revealed the unfolding barriers stabilizing structural segments of SteT. Ligand binding did not induce new unfolding barriers suggesting that weak interactions with multiple structural segments were involved. In contrast, ligand binding caused changes in the energy landscape of all structural segments, thus turning the protein from a brittle, rigid into a more stable, structurally flexible conformation. Functionally, rigidity in the ligand-free state was thought to facilitate specific ligand binding, while flexibility and increased stability were required for conformational changes associated with substrate translocation. These results support the working model for transmembrane transport proteins that provide alternate access of the binding site to either face of the membrane. Finally, high-resolution imaging was exploited to visualize the extracellular surface of Cx26 gap junction hemichannels (connexons). AFM topographs reveal pH-dependent structural changes of the extracellular connexon surface in presence of HEPES, an aminosulfonate compound. At low pH (< 6.5), connexons showed a narrow and shallow channel entrance, which represented the closed pore. Increasing pH values resulted in a gradual opening of the pore, which was reflected by increasing channel entrance widths and depths. At pH > 7.6 the pore was fully opened and the pore diameter and depth did not increase further. Importantly, coinciding with pore gating a slight rotation of the subunits was observed. In the absence of aminosulfonate compounds, such as HEPES, acidification did not affect pore diameters and depths, retaining the open state. Thus, the intracellular concentration of taurine, a naturally abundant aminosulfonate compound, might be used to tune gap junction sensitivity at low pH.

Raman spectroscopy has experienced a true "renaissance" since the development of Fourier Transform Raman instrumentation. The objective of this thesis is to illustrate the versatility of FT-Raman spectroscopy for the investigation and characterisation of biological molecules and systems. In addition to the positive attributes possessed by FT-Raman spectroscopy for such analysis, inherent shortcomings of the technique are discussed. This report serves as a concise reference for any researcher or analytical scientist considering FT-Raman spectroscopy for the analysis of biomolecular systems.

Diese Dissertation konzentriert sich auf die Untersuchung multivalenter Wechselwirkungen zwischen Hämagglutinin (HA) sowie Neuraminidase (NA) zweier Stämme des Influenzavirus (H1N1 und H3N2) und dem zellulären Liganden Sialinsäure (SA) unter Verwendung von Rasterkraftmikroskopie und Einzelmolekülkraftspektroskopie (SMFS). Bindungskräfte sowie Dissoziations- und Assoziationskinetiken, zusammen mit den intermolekularen Potentiallandschaften wurden, nach bestem Wissen erstmalig, auf Einzelmolekülebene mittels SMFS quantifiziert. Zu diesem Zweck wurden mono- und multivalente SA-Liganden (SAPEGLA und dPGSA) eingesetzt. Abweichungen der experimentellen Kraftspektren vom klassischen Kramers-Bell-Evans-Modell vorhergesagten Verhalten wurden durch das Friddle-Noy-De Yoreo-Model berücksichtigt. NA beider Virusstämme zeigte trotz ähnlicher Bindungskräfte eine stabilere Bindung mit SA als HA und dissoziierte 3 – 7 mal langsamer. Es wird vermutet, dass die höhere Stabilität die geringere Oberflächendichte von NA auf der Virushülle im Vergleich zu HA ausgleicht. Die Bindungskräfte eines SAPEGLA-Clusters nehmen mit der Anzahl der Bindungen und die Dissoziationskinetik folgt dem theoretisch vorhergesagten Trend. Die Dissoziationsrate von NA ist etwa 6-mal höher ist als ihre katalytische Rate, weshalb Mehrfachbindungen zur Spaltung von SA erforderlich sind. Die Dissoziationsrate von N1 in der gleichen Größenordnung wie die von H3 und es wird vermutet, dass derartige Ähnlichkeiten die Übertragbarkeit des Virus begünstigen. Darüber hinaus wird gezeigt, dass die thermische Stabilität von HA-dPGSA höher ist als von HA-SAPEGLA und im Bereich von 3 - 4 Einzelbindungen liegt, was für NA-dPGSA nicht beobachtet werden konnte. Daher bindet dPGSA spezifisch und kooperativ multivalent an HA. Kompetitive Bindungstests zeigen, dass SMFS zum Screening von antiviralen Inhibitoren verwendet werden und Zugang zu deren Design auf Einzelmolekülebene liefern könnte.
This thesis focuses on studying multivalent interactions between influenza virus hemagglutinin (HA) as well as neuraminidase (NA) of two viral strains (H1N1 and H3N2) and the cellular ligand sialic acid (SA) by using scanning force microscopy and single molecule force spectroscopy (SMFS). Unbinding forces as well as dissociation and association kinetics together with the free energy landscapes were, to the best knowledge for the first time, individually quantified on the single molecule level using SMFS. To this extent, designed synthetic monovalent (SAPEGLA) and multivalent (dPGSA) SA displaying ligands were employed. Surprisingly, the experimental force spectra did not show the log-linear trend predicted by the classical Kramers-Bell-Evans model, but rather follow the more recent Friddle-Noy-De Yoreo model. NA of both viral strains forms a more stable bond with SA than HA, and dissociates 3 to 7 times slower. It is reasoned that the higher stability compensates for the lesser amount of NA compared to HA that is typically found on the viral envelope. The unbinding forces of the cluster of SAPEGLA increased gradually with the number of bonds in the cluster and the dissociation kinetics follow the theoretically predicted trend. The dissociation rate of NA was found to be about 6 times higher than its catalytic rate, indicating that multiple bonds are needed for cleavage of SA. The dissociation rate of N1 is on the same order as that of H3, suggesting that these similarities between the two strains favor transmissibility. The thermal stability of the HA-dPGSA bond is higher than the HA-SAPEGLA reaching that of three to four single bonds, proving specificity and cooperativity. Such an enhancement could not be observed for the binding of NA. This thesis also shows that SMFS could be used as a tool to screen antiviral inhibitors in competitive binding assays, which may contribute insight into the design of antiviral inhibitors on the single molecule level.

The object of this thesis is to present work done using spatio-temporal image correlation spectroscopy (STICS), a technique that uses fluorescence intensity fluctuations in a microscopy image time series to calculate a complete space-time correlation function in order to measure transport dynamics in cells. The time evolution of this correlation function gives information on the magnitude and direction of a flow of fluorescent particles sampled in the image series. First, a new application of STICS to plant cell biology is shown. In dividing plant cells, delivery of new cell wall material to the forming cell plate requires intricate coordination of secretory vesicle trafficking and delivery. In this work, STICS is used to measure vesicle dynamics during plant cell division. It was discovered that vesicle transport to the plane of division occurs in three phases, each with its characteristic flow patterns and range of velocities, which directly reflect the rate of growth of the forming cell plate. The second part of this thesis presents the extension of the STICS technique to a third spatial dimension. The development of this new technique, called 3D STICS, allows the study of transport dynamics in three dimensions, which is more relevant in tissues and non adherent cells which are inherently 3D. Computer simulations were performed to test the accuracy and precision of the technique under a range of parameters such as particle density of immobile and moving populations; and number of images, velocity and resolution in the third spatial dimension. A comparison between values of velocities in a 2D plane recovered using STICS and its new 3D version is also presented.
L'objet de cette thèse est de présenter des travaux faits à l'aide de la spectroscopie par corrélation spatiotemporelle d'images (STICS), une technique qui utilise les fluctuations d'intensité dans une série d'images capturées à l'aide d'un microscope par fluorescence pour calculer la fonction complète de corrélation spatiotemporelle, et ainsi mesurer la dynamique du transport de protéines à l'intérieur de cellules vivantes. L'évolution temporelle de cette fonction de corrélation donne de l'information sur la direction et la vitesse d'un flot de particules fluorescentes présentes dans la série d'images. Tout d'abord, une nouvelle application de la technique en biologie végétale est présentée. Lors de la division cellulaire végétale, le transport du matériel membranaire nécessaire à la formation de la plaque cellulaire requiert une grande précision dans la coordination du transport et de la livraison des vésicules de sécrétion. Dans cette thèse, STICS est utilisée pour mesurer la dynamique de ces vésicules pendant la division cellulaire végétale. Les résultats obtenus révèlent l'existence de trois phases dans le transport des vésicules de sécrétion au site de division cellulaire, chacune présentant une échelle de vitesse et des motifs de mouvement caractéristiques qui se reflètent dans le taux de croissance de la plaque cellulaire. Dans un deuxième temps, le développement de STICS pour inclure l'analyse de la troisième dimension spatiale est présenté. Cette nouvelle technique, appelée STICS 3D, permet l'étude de dynamiques en trois dimensions, ce qui est plus pertinent que la version deux-dimensionnelle pour les tissus et les cellules non adhérentes, qui ont un environnement intrinsèquement 3D. Des simulations par ordinateur ont été effectuées pour déterminer l'exactitude, la précision et les limites de la technique pour un éventail de paramètres comme la vitesse, le nombre d'images et la résolution dans la troisième dimension spatiale ainsi que la densité des populations immobiles et en mouvement. Une comparaison entre les résultats obtenus avec STICS et la nouvelle version 3D de la technique est également présentée.

In 2018 ESA and NASA plan to send the ExoMars rover to the Martian surface. This rover is planned to have a suite of analytical equipment that includes a Raman spectrometer. In this context, an evaluation of Raman spectroscopy as an analytical tool for interplanetary studies is investigated. The preparation techniques for appropriate inorganic and organic mixtures are interrogated. Methods are investigated to optimize the homogeneity of over 50 samples involving mineral phases; calcite, gypsum and goethite and selected organic biomolecular systems; anthracene, naphthalene and beta-carotene. From mixtures produced of these organic and inorganic materials differences between homogeneity of the samples is observed. Different mixing techniques are investigated to reduce this, however all the samples display variation on a micron scale. To resolve this issue a grid system of 9 points is implemented on solid samples and solutions are used to produce standards. The standards are devised using a range of instrument validation parameters for comparison between commercially available spectrometers and the prototype instrument. From these standards a prototype instrument is optimized for data acquisition and an evaluation procedure for instrument performance is established. The prototype Raman spectrometer is evaluated to match the specifications of the spectrometer on board ExoMars rover. A range of astrobiological relevant samples are interrogated; geological samples, biomarkers, cellular systems and bio-geological inclusions. From these samples detection of organics is observed to be only possible, with Raman spectroscopy where organics are localised in high concentrations, upon grinding and mixing geological inclusions Raman spectroscopy is unable to detect the organic components.

In the past years, metabolomics has progressed greatly, providing a reliable and high-throughput approach particularly feasible for the study of complex biological systems. Indeed, thanks to the development of powerful analytical methods capable of screening a large number of chemical compounds in a sample, nowadays metabolomics plays an important role in gaining biological insights toward the influence of internal (genetic and developmental) and external (environmental) factors on phenotypes. The work presented in this Ph.D. thesis shows examples of applications of NMR-based metabolomics to the study of bivalves, facing challenges of interest in both food and environmental sciences. The outcome of such studies yield insights, at molecular level, into several aspects concerning the impact of different storage conditions on shellfish quality and the effect of natural and anthropogenic environmental stressors on bivalves’ metabolic profiles. Firstly, the effect of different cold storage conditions on the hydrosoluble chemical components of Mytilus galloprovincialis (Lamarck, 1819) was investigated for the first time by NMR-based metabolomics. The data revealed substantial time-related changes in the metabolic profiles of mussels stored at 0 °C and 4 °C. The observed biochemical modifications were in good agreement with the microbiological quality of samples, reflecting changes in their microbial loads. These results confirmed the potential use of metabolomics as a reliable method to assess seafood freshness. Secondly, a metabolomic approach was also applied to study the effect of short-term exposure to heavy metals on two different clams’ species: Ruditapes decussatus (Linnaeus, 1758) and Ruditapes philippinarum (Adams & Reeve, 1850). Heavy metals are considered to be among the most harmful pollutants that can contaminate marine environments. The toxicity of trace metals gives rise from their persistent nature which lead to environmental accumulation. Given the high accumulation rates associated with their filter feeding attitude, bivalves mollusks are considered to be feasible monitoring organisms and are widely used in biomonitoring programs. R. decussatus and R. philippinarum, two bivalves’ species widely distributed along the Italian coasts, were selected in this Ph.D. project for assessing lead and zinc effects on their metabolic profiles. The results evidenced a sensible short-time metabolic response upon metal exposure, pointing out a main variability in the content of amino acids and organic osmolytes in relation to both metal nature and bivalve species. These findings show that NMR-based metabolomics has the required sensitivity and specificity to gain insights into the biochemical consequences arising upon heavy metals exposure, providing thus a useful tool for the identification of putative biomarkers as fast and sensitive indicators of contaminant-induced stress. Finally, 1H NMR-based metabolomics was applied with the aim to assess the effects of seasonal change on Ruditapes decussatus metabolic profile. The results of the present study demonstrated that the combined use of advanced multivariate statistical techniques with NMR spectroscopy is a feasible approach to discriminate specimens of R. decussatus according to the sampling season. Moreover, the sensitivity of this analytical tool allowed the individuation of those metabolites whose relative amount significantly varied according to seasonal change (alanine and glycine), paving the way for further investigations that would contribute to achieve additional insights on bivalves’ bio-ecological framework.

Actin filaments serve as a hub of focal adhesion-based mechanotransduction network in a wide variety of cell types. In this role filaments and networks of filaments are exposed to forces in a range of directions and magnitude. Due to the complexity of the adhesome circuit, many questions remain open, including identification of the fundamental mechanosensitive elements and regulation of interconnected elements. One possibility is that actin itself is mechanosensitive and applied forces regulate the interactions between actin and actin regulatory proteins. To address this question we must be able to characterize the force response of individual actin filaments, the mechanical properties of individual filaments and the mechanical properties of individual actin filament crosslinks. This thesis presents new technologies that are capable of addressing such questions. The design and optimization of a dual-beam optical trap, the workhorse technology of single-molecule force spectroscopy is presented first. The capabilities of force spectroscopy are then expanded in the subsequent chapters. The first development is a new instrument that enables us to probe the energy landscapes of weak, short-lived interactions as well as the strongest, longest-lived interactions. Stable, multiplexed femtoNewton force spectroscopy using particle tracking tools for tether diagnostics is demonstrated. We also show that these capabilities can be used to actively characterize the torsional rigidity of actin cross-links. Finally, the development of an elongational flow hydrodynamic trap, simple tape-based microfluidics and a multiplexed microfluidic microscopy assay are developed and applied to study the role of tension in the regulation of actin filament severing by severing proteins cofilin and gelsolin. Our results using hydrodynamic trapping, a surface-free force spectroscopy tool, in concert with the results of our curvature-sensitive experiments suggest that actin is sensitive to the magnitude and direction of an applied force. Ongoing and future studies will provide mechanistic insight to this observation.
Engineering and Applied Sciences - Applied Physics

Amyloids are self-assembled protein materials containing beta-sheets.  While most studies focus on amyloids as the pathogen in neurodegenerative disease, there are instances of "functional" amyloids used to preserve life.  Functional amyloids serve as an inspiration in materials design.  In this study, it is shown that wheat gluten (WG) and gliadin:myoglobin (Gd:My) amyloid morphology can be varied from predominantly fibrillar at low polypeptide concentration to predominantly globular at high polypeptide concentration as measured at the nanometer scale using atomic force microscopy (AFM).  The ability to control the morphology of a material allows control of its properties.  Fourier transform infrared (FTIR) spectroscopy shows that at low concentration, fibrils require interdigitation of methyl groups on alanine (A), isoleucine (I), leucine (L), and valine (V).  At higher concentration, globules do not have the same interdigitation of methyl groups but more random hydrophobic interactions.  The concentration dependence of the morphology is shown as a kinetic effect where many polypeptides aggregate very quickly through hydrophobic interactions to produce globules while smaller populations of polypeptides aggregate slowly through well-defined hydrophobic interactions to form fibrils.    
Functional amyloids also provide a means of creating a low energy process for composites. Poor fiber/matrix bonding and processing degradation have been observed in previous WG based composites.  This study aims to improve upon these flaws by
implementing a self-assembly process to fabricate self-reinforced wheat gluten composites. These composites are processed in aqueous solution at neutral pH by allowing the fibers to form in a matrix of unassembled peptides.  The fiber and the matrix are formed from the same solution, thus the two components create a compatible system with ideal interfacial interaction for a composite.  The fibers in the composite are about 10 microns in diameter and can be several millimeters long.  It has been observed that the number of fibers present along the fracture surface influences the modulus of the composite. In this study, self-assembled wheat gluten composites are formed and then characterized with 3-point bend (3PB) mechanical testing, scanning electron microscopy (SEM), and Fourier transform infrared (FTIR) spectroscopy.

Master of Science

Ce travail concerne l'étude de molécules d'ADN avec un microscope à force atomique utilisé en « contact intermittent ». L'objectif poursuivi est de réaliser des mesures locales d'interaction et de mécanique sur une chaîne d'ADN. Le microscope est sensible aux forces d'interaction entre une pointe de dimension nanométrique et l'objet ou la surface à analyser. En mode dynamique, la pointe oscille au voisinage de la surface à des fréquences de quelques centaines de kilohertz et des amplitudes de l'ordre de la dizaine de nanomètres. Ce comportement peut devenir non-linéaire selon l'intensité de l'interaction et des conditions d'oscillation. Varier ces conditions en termes d'amplitude ou de fréquence constitue une opportunité de contrôler cette interaction. Nous mettons ainsi en évidence la possibilité d'imager une seule molécule d'ADN selon différents modes d'interaction : pur attractif, pur répulsif et un mélange des deux. Par ailleurs, le microscope de force dynamique n'est pleinement efficace que dans le cadre de la maîtrise de l'interaction de la pointe avec l'objet. La qualité de l'échantillon et celle de la pointe doivent être contrôlées à l'échelle des mesures. Des expériences préliminaires ont amené à retenir des substrats de silice greffés avec des molécules de silanes. Concernant les molécules d'ADN, trois types de séquences ont été étudiés. Une longue chaîne de 2500 paires de bases (pb) comportant une succession de séquences non périodique, une séquence de 450pb constituée d'Adénine sur un mono-brin et de Thymine sur le brin complémentaire et une chaîne de même longueur, mais constituée d'une alternance d'Adénine et de Thymine sur chaque mono-brin. Ces échantillons sont intéressants par la diversité des caractéristiques mécaniques attendues. L'exploitation expérimentale des différents régimes permet de révéler des informations géométriques, mécaniques et physico-chimiques sur les conformations des molécules.

The traditional boundary between hard sciences (physics and mathematics) and soft sciences (chemistry and biology) is progressively fading away as the complexity inherent in the biological world is understood and mapped out thanks to a joint attack on two fronts. On the one side more quantitative experiments allow to investigate the details of the atomic structures of biological molecules and to measure with greater precision the laws of interaction among dierent molecules; on the other side, the massive introduction of information technology in the management and catalogation of the multitude of molecular components found inside a cell is allowing to gain deep insights in the complex dynamic equilibrium that regulates the network of interactions amog different molecules. The work described in this thesis concerns the first side of the battlefield: the development of new techniques to allow quantitative measurements of biologically relevant quantities. The work consisted in the design, construction and validation of three different experiments dealing with proteins and DNA mechanics. Key components of the cellular microcosmos, DNA and proteins are large macromolecules that constantly interact and accomplish most of the tasks needed by the cell to survive. The first part of the thesis summarises the known properties of these molecules and introduces the motivations driving the designed experiments. Proteins catalyse chemical reactions in the cell and their threedimensional conguration gives each of them its specic function. The connections between structural and chemical properties of a protein are a subject largely unexplored.The second part of the thesis describes an experiment based on single molecule fluorescence microscopy designed to explore the dynamics of fluctuations of catalytic activity of a single enzyme. The experiments described in this part have not yet given the hoped results. However part of the preliminary considerations done when building these setup were used to write the article F. Mosconi et al. "Some nonlinear challenges in biology", Nonlinearity 21 (2008) T131-T147. DNA stores the genetic information needed by the cell to accomplish its tasks, and such information must be physically stored, read, written and restored in different times during the cell cycle. The importance of a proper knowledge of its mechanical properties is fundamental if its interaction with proteins is to be understood. The third part of this thesis describes two different experiments based on the magnetic tweezers micro-manipulation technique, that attempt to measure some not yet entirely characterised mechanical properties of DNA. The two experiments presented in this part gave interesting results. A new determination of the biologically relevant parameter C, the twist modulus of DNA was obtained developing a novel type of analysis of data collected using the standard magnetic tweezers apparatus. Also, a new type of "soft" magnetic tweezers that allows the simultaneous application of an external force and an external torque has been developed and validated to measure the torque response of a DNA molecule. The results described in this part of the thesis are summarised in two papers that are ready to be submitted.

Les propriétés mécaniques des cellules adhérentes ont une importance capitale pour l'ensemble de leurs fonctions : division, migration, différenciation, etc. De plus, on sait désormais qu'elles sont très sensibles aux caractéristiques mécaniques de leur substrat, auquel elles sont ancrées par l'intermédiaire des intégrines. Ces récepteurs transmembranaires lient indirectement le cytosquelette d'actine intracellulaire aux protéines de la matrice extracellulaire.
Nous avons conçu un dispositif de pinces optiques contrôlées par une boucle de rétroaction, qui permet d'appliquer aux cellules une force locale constante, via des microbilles liées aux intégrines.
Nous pouvons ainsi mesurer la fonction de fluage de chaque cellule et en tirer une estimation de sa rigidité. Des observations simultanées en épifluorescence permettent par ailleurs d'évaluer les effets de l'application de la force sur la répartition d'actine locale.
Nous avons constaté que les cellules se rigidifient sous l'application prolongée d'une force, tout en gardant le même comportement rhéologique : une fonction de fluage en loi de puissance du temps, J(t) = At^(alpha), où A décroît aux temps longs. Cette rigidification est couplée à un recrutement d'actine au niveau des contacts et au sein du réseau cytsoquelettique (jusqu'à plusieurs µm du point d'application de la force). De plus, les dynamiques de ces deux phénomènes semblent fortement corrélées. Ce travail présente une évaluation de la dynamique de renforcement cellulaire sous contrainte, et ouvre des perspectives prometteuses vers l'élucidation des phénomènes intervenant dans la mécanotransduction.

La plupart des réactions chimiques dans la cellule sont catalysées par des protéines dont la configuration tridimensionnelle assure leur spécificité fonctionnelle. L'information génétique nécessaire à la cellule pour assurer son fonctionnement est contenue dans l'ADN. La relation entre les propriétés structurelles et fonctionnelles d'une protéine est encore mal connue et une connaissance détaillée des propriétés mécaniques de l'ADN est indispensable si l'on veut comprendre les interactions ADN-protéines. Nous avons développé des techniques permettant de mesurer quantitativement des grandeurs d'intérêt biologique comme le module de torsion de l'ADN. Nous avons mis au point un dispositif expérimental visant à explorer, par fluorescence, la dynamique des fluctuations d'activité d'une enzyme isolée ainsi qu’un second dispositif expérimental basé sur une technique de pinces magnétiques. Nous montrons que l'on peut appliquer à l'ADN un couple calibré en même temps qu'une force externe.

In reconstructive surgery, tissues are routinely transferred to repair a defect caused by trauma, cancer, chronic diseases, or congenital malformations. Surgical transfer intrinsically impairs metabolic supply to tissues placing a risk for ischemic complications such as necrosis, impaired healing, or infection. Pre-surgical induction of angiogenesis in tissues (preconditioning) limits ischemic complications and improves outcomes but very few preconditioning strategies have successfully been translated to clinical practice. The first goal of our research was to improve current standard of care in reconstructive surgery by developing a translational technique that can effectively and safely increase the vascularization of soft tissues. To achieve this goal, we optimized, using preclinical animal models resembling clinical needs and scenarios in a controlled setting, a method that adopts non-invasive external suction (External Volume Expansion, EVE) to precondition tissues through the induction of hypoxia-mediated angiogenesis. Using a sequential approach in a rodent model we determined the parameters of application (frequency, suction levels, duration, and interfaces) that fine-tune the balance of enhanced angiogenesis, attenuation of hypoxic tissue damage, and length of treatment. The optimized parameters of application (short, cyclical stimulations at moderate suction) almost doubled tissue vascular density after only 5 days of treatment. Our outcomes also showed that the use of micro-deformational interfaces of treatment retain the biological effectiveness of EVE while further reducing the cutaneous damage by distributing forces across the stimulated tissue. Our model confirmed that the optimized technique significantly improves the survival of transferred soft tissues (+20-30%), such as adipose tissue grafts, and can achieve the same beneficial outcomes in animal models of pathologic cutaneous vascularization, such as the one occurring in the skin of patients affected type-2 diabetes. We assessed that EVE retains a beneficial effect on the vascularization and proliferation (adipogenesis) of soft tissues when used both as a pre-conditioning method (before surgeries) and as a post-conditioning method (after surgeries) As a second goal of our research we integrated the knowledge on the application of EVE on soft tissues, to the use of a shelf-ready, bio-mimetic, decellularized allograft adipose matrix (AAM) with the aim of developing an innovative and minimally-invasive strategy for in vivo regeneration of soft tissues. In an animal model we tested the potential of a human-derived, injectable AAM to regenerate soft tissues when used in combination with EVE. This strategy significantly improved long-term volume retention (50-80% higher) and histological quality of reconstructed tissues compared to current standard of care (adipose grafts). The AAM induced both adipogenesis and angiogenesis. Combined use of the AAM and adipose grafts mitigated efficacy. Our studies suggest that EVE can improve the outcomes of reconstructive surgeries by safely and promptly enhance vascularity of soft tissues, in addition to its edema-/mechanically-induced adipogenic effect (confirmed by our study). EVE's use with an AAM, instead, can synergistically and effectively induce in vivo soft tissue regeneration. These translational principles are ready to be translated to clinical trials and, if outcomes will be confirmed, they could establish the basis for a novel therapeutic paradigm in reconstructive and regenerative surgery for the benefit of a large number of patients.
La chirurgia ricostruttiva si basa sul trasferimento di tessuti da un distretto corporeo ad un altro al fine di riparare un difetto tissutale causato da un trauma, un tumore, una malattia cronica, o una malformaizoen congenita. Questo trasferimento chirurgico compromette la vascolarizzazione (e quindi il support metabolico) dei tessuti trasferiti, mettendoli a rischio per complicanze ischemiche quali la necrosi, laguarigione inefficace delle ferite, o la sovrainfezione batterica. L'induzione di fenomeni angiogenici nei tessuti prima della chirurgia (pre-condizionamento) limita le complicanze ischemiche e migliora I risultati chirurgici; tuttavia, pochissime strategie di pre-condizionamento sono oggi disponibili nella pratica clinica. Il primo obiettivo di questa ricerca era di migliorare gli attuali standard in chirurgia ricostruttiva attraverso lo sviluppo di tecniche traslazionali in grado di aumentare la vascolarizzazione dei tessuti in maniera efficace e sicura. Al fine di raggiungere tale obiettivo abbiamo ottimizzato, usando modelli preclinici animali rappresentativi di condizioni cliniche controllate, un metodo che adopera una stimolazione meccanica esterna non invasiva tramite pressione negativa (Espansione Volumetrica Esterna, EVE) per precondizionare I tessuti attraverso l'induzione di fenomeni angiogenici causati da una ischemia transitoria. Tramite questa strategia di ottimizzazione sequenziale in un modello murino abbiamo definite i parametri di trattamento ottimali di EVE (frequenza, livelli di pressione, durata, interfaccia di trattamento) in grado di bilanciare l'induzione di angiogenesis con l'attenuazione del danno ischemico causato ai tessuti, e con la durata di trattamento. L'ottimizzazione di EVE (brevi, cicliche stimulazioni a suzione moderata) ha dimostrato la capacità di raddoppiare la densità vascolare dei tessuti stimulati dopo solo 5 giorni di trattamento. I nostri risultati hanno anche dimostrato che l'uso di interfacce di trattamento a micro-deformazione garantisce il mantenitmento degli stessi effetti biologici di EVE ma allo stesso tempo reduce il danno cutaneo causato ai tessuti tramite la distribuzione delle forze meccaniche su tutto il tessuto stimulato. I nostri modelli sperimentali hanno confermato che l'ottimizzazione di EVE permette di aumentare significativamente (+20-30%) la sopravvivenza dei tessuti trasferiti (ad esempio il tessuto adiposo), e che gli stessi effetti possono essere osservati in modelli di vascolarizzazione cutanea patologica (ad esempio la cute di soggetti affetti da diabete di tipo 2). Inoltre, abbiamo confermato che EVE induce la vascolarizzazione e la proliferazione (adipogenesi) dei tessuti molli sia quando utilizzara come metodo di pre-condizionamento (prima della chirurgia) dei tessuti sia quando utilizzata come metodo di post-condizionamento (dopo la chirurgia). Come secondo obiettivo di questa ricerca abbiamo integrato le conoscenze acquisite sull'applicazione di EVE ai tessuto molli all'uso di una matrice adiposa allogenica (AAM) -ottenuta tramite decellularizzazione di tessuto adipose umano, caratterizzata da proprietà bio-mimetiche, e realizzata in una formulazione iniettiabile "pronta all'uso" - con lo scopo di sviluppare una strategia innovativa e mini-invasiva per la rigenerazione in vivo di tessuto molli. In un modello animale abbiamo testato il potenziale della AAM di rigenerare i tessuti molli quando utilizzata in combinazione con EVE. Questa strategia ha portato ad un significativo aumento volumetrico (+50-80% a 12 settimane) ed un miglioramento della struttura istologica dei tessuti molli ricostruiti in comparazione ai risultati ottenuti con le terapie standard attuali (innesti di tessuto adiposo). Abbiamo evidenziato come la AAM sia in grado di indurre sia fenomeni adipogenici che fenomeni angiogenici: l'applicazione combinate di AAM e innesti di tessuto adiposo, invece, mitigano I risultati ottenibili con l'uso esclusivo della AAM. In conclusion, i nostril studi suggeriscono che EVE è in grado di migliorare i risultati ottenibili in chirurgia ricostruttiva attraverso un incremento, sicuro e rapido, della vascolarizzazione dei tessuto molli, in aggiunta all'efftto adipogenico (mediato da stimolazione meccanica diretta ed edema dei tessuti) gia descritto nella precedente letteratura e qui confermato dai nostril risultati. L'utilizzo di EVE con l'AAM, invece, può, efficacemente e sinergisticamente, indurre fenomeni rigenerativi dei tessuto molli in vivo. Questi principi traslazionali sono pronti per essere validati in trial clinici e, qualora I loro risultati venissero confermati, potrebbero porre le basi per lo sviluppo di nuovi paradigm terapeutici in chirurgia ricostruttiva e in chirurgia rigenerativa, per il beneficio di un grande numero di pazienti

Degenerative disorders of the rotator cuff tendons account for nearly 75% of all shoulder pain, causing considerable pain and morbidity. Given the strong correlation between age and tendinopathy, and unprecedented population aging, these disorders will become increasingly prevalent. Improved understanding of tendon degeneration will guide the development of future diagnostic and treatments, and is therefore urgently needed. However, the aetiology and pathology of rotator cuff tendinopathy remain unclear. The complicated mechanical environment of the rotator cuff is hypothesised to influence the susceptibility of the tendons to degeneration and tearing. Studies have reported biological adaptations in torn cuff tendons indicative of increased compressive loading within the tendon. The material adaptations of healthy and degenerative cuff tendons are largely unreported but will provide further insight into the role of the mechanical environment in rotator cuff aetiology and pathology. This thesis examined the material adaptations of healthy and diseased tendons to explore the role of mechanical loading in rotator cuff pathology. The material adaptations of healthy animal tendons, and healthy and delaminated human cadaveric rotator cuff tendons, in response to different loading environments were characterised. The effects of age, tears, steroid injection and subacromial decompression surgery on the structural adaptations of human cuff tendons were also studied, as was the effect of tendon cell proliferation on the mechanical properties and degradation behaviour of collagen scaffolds. Loading environment significantly affected the structural adaptations of healthy tendons. Regions exposed to compressive and shear strains exhibited thinner fibres, shorter crimp lengths and thinner, less aligned fibrils compared with regions exposed to tensile strains alone. In healthy rotator cuff tendons, the inhomogeneous loading environment produced topographically inhomogeneous structural adaptations. The tendons of a delaminated rotator cuff exhibited less topographical variation in properties and thinner, less aligned fibrils compared with healthy cuff tendons. Torn cuff tendons exhibited thinner fibrils and shorter crimp lengths compared with control samples. These adaptations were identifiable early in the disease progression, and neither steroid injection nor subacromial decompression surgery significantly influenced these adaptations at seven weeks post‐treatment. Significant correlations between decreasing dimensions and increasing tear size were found when age was included as a confounding factor, reflecting the importance of age and tear size in determining the material properties of tendons. Tendon cell proliferation influenced the mechanical properties and degradation behaviour of the collagen scaffolds, emphasising the integral role of cells in the functional adaptation of biological materials. These results demonstrate the effect of mechanical environment on the material adaptations of tendons. They also indicate the importance of the complicated mechanical environment experienced by the rotator cuff tendons in predisposing the tendons to degeneration and tearing. The observed material adaptations of degenerative and torn tendons suggest that rotator cuff pathology is associated with increased levels of compressive and/or shear strains within the tendon. These changes begin early in the disease progression and neither steroid injection nor sub‐acromial decompression surgery are capable of reversing the changes in the timeframe investigated. These findings highlight the urgent clinical need for pre‐rupture diagnostic techniques for the detection of early pathological changes in the rotator cuff. They also emphasize the requirement for new intervention strategies that restore the healthy mechanical environment and reverse early pathological adaptations in order to prevent catastrophic failure of the tendons.

In the search for the conformation of extant or extinct life in an extraterrestrial setting the detection of organic molecular species which may be considered diagnostic of life is a key objective. These molecular targets comprise a range of distinct chemical species, with recognisable spectroscopic features. This project aims to use these features to develop an in-situ molecular specific Raman spectroscopic methodology which can provide structural information about the organic–inorganic interface. The development of this methodology identified a surface enhanced Raman spectroscopic technique, that required minimal sample preparation, allowed for the detection of selected organic species immobilised on an inorganic matrix and was effective for quantities below those which conventional dispersive Raman spectroscopy would detect. For the first time spectral information was gained which allowed analysis of the organic–inorganic interface to be carried out, this gave an insight into the orientation with which molecules arrange on the surfaces of the matrices. Additionally a method for the detection of organic residues intercalated into the interlamellar space of smectite type clays was developed. An evaluation of the effectiveness of uni and multivariate methods for the analysis of large datasets containing a small number of organic features was also carried out, with a view to develop an unsupervised methodology capable of performing with minimal user interaction. It has been shown that a novel use of the Hotellings T2 test when applied to the principal component analysis of the datasets combined with SERS allows identification of a small number of organic features in an otherwise inorganic dominated dataset. Both the SERS and PCA methods hold relevance for the detection of organic residues within interplanetary exploration but may also be applied to terrestrial environmental chemistry.

In the search for the conformation of extant or extinct life in an extraterrestrial setting the detection of organic molecular species which may be considered diagnostic of life is a key objective. These molecular targets comprise a range of distinct chemical species, with recognisable spectroscopic features. This project aims to use these features to develop an in-situ molecular specific Raman spectroscopic methodology which can provide structural information about the organic–inorganic interface. The development of this methodology identified a surface enhanced Raman spectroscopic technique, that required minimal sample preparation, allowed for the detection of selected organic species immobilised on an inorganic matrix and was effective for quantities below those which conventional dispersive Raman spectroscopy would detect. For the first time spectral information was gained which allowed analysis of the organic–inorganic interface to be carried out, this gave an insight into the orientation with which molecules arrange on the surfaces of the matrices. Additionally a method for the detection of organic residues intercalated into the interlamellar space of smectite type clays was developed. An evaluation of the effectiveness of uni and multivariate methods for the analysis of large datasets containing a small number of organic features was also carried out, with a view to develop an unsupervised methodology capable of performing with minimal user interaction. It has been shown that a novel use of the Hotellings T2 test when applied to the principal component analysis of the datasets combined with SERS allows identification of a small number of organic features in an otherwise inorganic dominated dataset. Both the SERS and PCA methods hold relevance for the detection of organic residues within interplanetary exploration but may also be applied to terrestrial environmental chemistry.

Breast cancer in its advanced stage has a high predilection to the skeleton. Currently, treatment options of breast cancer-related bone metastasis are restricted to only palliative therapeutic modalities. This is due to the fact that mechanisms regarding the breast cancer celI-bone colonisation as well as the interactions of breast cancer cells with the bone microenvironment are not fully understood, yet. This might be explained through a lack of appropriate in vitro and in vivo models that are currently addressing the above mentioned issue. Hence the hypothesis that the translation of a bone tissue engineering platform could lead to improved and more physiological in vitro and in vivo model systems in order to investigate breast cancer related bone colonisation was embraced in this PhD thesis. Therefore the first objective was to develop an in vitro model system that mimics human mineralised bone matrix to the highest possible extent to examine the specific biological question, how the human bone matrix influences breast cancer cell behaviour. Thus, primary human osteoblasts were isolated from human bone and cultured under osteogenic conditions. Upon ammonium hydroxide treatment, a cell-free intact mineralised human bone matrix was left behind. Analyses revealed a similar protein and mineral composition of the decellularised osteoblast matrix to human bone. Seeding of a panel of breast cancer cells onto the bone mimicking matrix as well as reference substrates like standard tissue culture plastic and collagen coated tissue culture plastic revealed substrate specific differences of cellular behaviour. Analyses of attachment, alignment, migration, proliferation, invasion, as well as downstream signalling pathways showed that these cellular properties were influenced through the osteoblast matrix. The second objective of this PhD project was the development of a human ectopic bone model in NOD/SCID mice using medical grade polycaprolactone tricalcium phosphate (mPCL-TCP) scaffold. Human osteoblasts and mesenchymal stem cells were seeded onto an mPCL-TCP scaffold, fabricated using a fused deposition modelling technique. After subcutaneous implantation in conjunction with the bone morphogenetic protein 7, limited bone formation was observed due to the mechanical properties of the applied scaffold and restricted integration into the soft tissue of flank of NOD/SCID mice. Thus, a different scaffold fabrication technique was chosen using the same polymer. Electrospun tubular scaffolds were seeded with human osteoblasts, as they showed previously the highest amount of bone formation and implanted into the flanks of NOD/SCID mice. Ectopic bone formation with sufficient vascularisation could be observed. After implantation of breast cancer cells using a polyethylene glycol hydrogel in close proximity to the newly formed bone, macroscopic communication between the newly formed bone and the tumour could be observed. Taken together, this PhD project showed that bone tissue engineering platforms could be used to develop an in vitro and in vivo model system to study cancer cell colonisation in the bone microenvironment.

La cucurbitacine E et l'érythrodiol sont des triterpènes naturels oxygénés ayant respectivement un squelette tétra et pentacyclique. Ils sont reconnus pour leurs diverses propriétés biologiques. Dans ce travail de thèse, nous étudions leur interaction avec les membranes des vésicules lipidiques dans le but de mieux comprendre leur pharmacodynamie. Nous avons préparé des liposomes en absence et en présence de la cucurbitacine E et de l'érythrodiol par les techniques d'évaporation en phase inverse suivie d'une extrusion, d'hydratation du film lipidique et d'injection d'éthanol. Les caractéristiques physicochimiques des vésicules lipidiques incorporant ou non la molécule triterpénique ont été étudiées par des techniques adéquates. Les analyses de la cucurbitacine E et de l'érythrodiol par la chromatographie liquide à haute performance ont montré que leurs taux d'incorporation dans les liposomes sont élevés. Les mesures de taille obtenues par la diffusion dynamique de la lumière ont démontré que les liposomes incorporant les triterpènes présentent une taille moyenne inférieure à celle des liposomes témoins. Les images obtenues par la microscopie électronique à transmission ont confirmé la formation de vésicules sphériques. Les mesures des dimensions des vésicules observées par la microscopie à force atomique (AFM), ont révélé que les liposomes incorporant la cucurbitacine E sont plus hauts et résistent mieux à la force exercée par la pointe AFM que les liposomes témoins. Par ailleurs, les liposomes incorporant l'érythrodiol sont plus fragiles que les liposomes témoins et ont tendance à éclater en bicouches lipidiques à la surface du support. Les courbes thermiques obtenues par la calorimétrie différentielle à balayage ont permis de conclure que la cucurbitacine E est localisée à l'interface polaire-apolaire de la membrane liposomiale alors que l'érythrodiol s'insère entre les chaînes acyles des phospholipides et aboutit à la formation des domaines hétérogènes au niveau de la membrane. La cinétique de libération de la sulforhodamine B, mesurée par la spectroscopie de fluorescence, a révélé que la membrane liposomiale devient, en présence de la cucurbitacine E, plus perméable à la sulforhodamine B incorporée dans la phase aqueuse interne. L'ensemble des résultats suggère que la cucurbitacine E et l'érythrodiol interagissent avec la membrane lipidique et affectent ses propriétés physico-chimiques. Leur effet sur la membrane ne semble pas être similaire. Des études ultérieures impliquant d'autres triterpènes sont envisagées pour identifier le (s) motif (s) structural (aux) et les paramètres physico-chimiques régissant leur interaction et localisation membranaire
Cucurbitacin E and erythrodiol are natural oxygenated triterpenes having respectively, a tetra and pentacyclic skeleton. They are known for their numerous biological properties. In this thesis, we studied their interaction with the membranes of lipid vesicles to better understand their pharmacodynamics. We have prepared liposomes in the absence and presence of cucurbitacin E and erythrodiol using the reverse phase evaporation technique followed by extrusion, the hydration of lipid film and the ethanol injection techniques. The physicochemical characteristics of lipid vesicles incorporating or not the triterpenic molecules were investigated by appropriate techniques. The determination of cucurbitacin E and erythrodiol in the vesicles by high performance liquid chromatography showed high incorporation efficiencies of both triterpenes. Size measurements obtained by dynamic light scattering showed that liposomes incorporating triterpenes were smaller than empty liposomes. The images obtained by transmission electron microscopy confirmed the formation of spherical vesicles. Measurements of vesicles dimensions by atomic force microscopy (AFM) demonstrated that liposomes incorporating cucurbitacin E were higher and more resistant to the force exerted by the AFM tip than the blank liposomes. Liposomes incorporating erythrodiol were more fragile and tend to break up into lipid bilayers on the mica surface. Results obtained by differential scanning calorimetry suggested that cucurbitacin E is localized at the polar-apolar interface of the liposomal membrane while erythrodiol is inserted between the acyl chains of the phospholipids leading to the formation of heterogeneous lipid domains. The release kinetics of the sulforhodamin B encapsulated into the aqueous phase and measured by fluorescence spectroscopy revealed that the liposomal membrane becomes in the presence of cucurbitacin E, more permeable to this probe. The overall results suggest that cucurbitacin E and erythrodiol affect differently

Tese de doutoramento, Ciências Biomédicas (Ciências Funcionais), Universidade de Lisboa, Faculdade de Medicina, 2012
The use of the knowledge and technology generated by nanomedicine research is slowly gaining its way into the clinical areas. Nanomedicine can be defined as the application of nanotechnologies in medicine, to monitor, diagnose and cure diseases, by using molecular knowledge of the human organism to maintain and improve human health at the molecular scale. In this thesis, we proposed to use force spectroscopy based on atomic force microscopy, a nanotechnology approach, to understand the single-molecule mechanisms involved in two different biomedical relevant systems: (i) Fibrinogen interaction with human blood cell membranes (erythrocytes and platelets) receptors. Increased plasma fibrinogen levels are associated with increased erythrocyte aggregation in cardiovascular diseases. With our results, we identified a specific binding between fibrinogen and an erythrocyte integrin receptor with a β3 or β3-like subunit. This interaction decreases significantly upon erythrocyte aging. We presume that young erythrocytes are the main contributors to the cardiovascular diseases associated with increased fibrinogen content in blood. (ii) Dengue virus (DENV) capsid (C) protein interaction with lipid droplets or lipoproteins. Lipid droplets (LDs) and/or lipoproteins may play an important role on Dengue virus pathogenesis. Our results could explain the key processes occurring in vivo on the Dengue virus replication. We found that DENV C N-terminal region is crucial for the interaction with LDs and very low density lipoproteins (VLDL), requiring the presence of potassium and intrinsic protein(s). Also, pep14-23, a peptide based on the DENV C N-terminal conserved region, inhibits the DENV C-LDs interaction. These findings may be important for the development of C protein-targeted treatments for DENV infections. The main findings described on both of these studies contribute to the establishment of atomic force microscopy as a biomedical tool used to improve knowledge on the molecular mechanisms associated with the development of diseases and their effective treatment.
Durante as últimas décadas, a Nanomedicina tem vindo a se tornar numa área científica emergente e em elevada expansão. O termo “Nanomedicina” é definido como a aplicação de técnicas nanotecnológicas em medicina, através do conhecimento, ao nível molecular, do organismo humano para manutenção e melhoria da saúde humana. Através da nanotecnologia é possível criar e desenvolver equipamentos que permitam a análise de sistemas in vivo ao nível molecular. Nos últimos anos, o grau de aceitação ao nível clínico do uso destas nanotecnologias tem vindo a aumentar, contudo, ainda há muito a desenvolver na área da aplicação da Nanomedicina na monitorização, diagnóstico e possível cura de doenças. Atualmente, as grandes empresas farmacêuticas estão mais recetivas à aplicação destas novas tecnologias, mas há sempre a considerar a relação custo/beneficio, bem como os eventuais riscos associados à sua aplicação. Neste trabalho foi utilizada, maioritariamente, a técnica de espectroscopia de força através de um microscópio de força atómica, para identificar interações específicas entre moléculas, conhecer possíveis mecanismos de desencadeamento da doença e apontar possíveis alvos de terapêutica e diagnóstico de duas patologias distintas, doença cardiovascular e infeção pelo vírus da Dengue. Estas duas diferentes aplicações do microscópio de força atómica são exemplos de como é possível a utilização deste equipamento como método de diagnóstico e caracterização funcional aplicado à área da Nanomedicina: Parte I: Estudo da interação do fibrinogénio com um recetor de membrana eritrocitário, na saúde e na doença. O aumento dos níveis de fibrinogénio conduz a alterações das propriedades reológicas do sangue, tais como o aumento da viscosidade plasmática, da agregação eritrocitária e da trombogénese, juntamente com alterações da reatividade vascular e da integridade endotelial. Essas alterações intensificam as complicações na circulação sanguínea periférica em patologias cardiovasculares. A agregação eritrocitária tem adquirido um elevado interesse na avaliação do risco de eventos cardiovasculares primários ou secundários, uma vez que é principalmente influenciada pelo aumento dos níveis plasmáticos de fibrinogénio e de lípidos. É nestas condições clínicas e na fase não aguda da doença que se torna mais premente a determinação da agregação eritrocitária como indicador do risco cardiovascular associado. Um estudo detalhado das interações entre eritrócitos e moléculas de fibrinogénio, de modo a compreender as consequências do seu aumento em patologias cardiovasculares, revelou-se assim necessário. O nosso primeiro trabalho demonstrou a ligação entre moléculas de fibrinogénio e um recetor anteriormente desconhecido na membrana eritrocitária, com afinidade comparável mas menor tempo de vida que a ligação fibrinogénio-plaqueta. A interação fibrinogénio-plaqueta, essencial na coagulação, depende de um recetor da membrana plaquetária, a glicoproteína αIIbβ3 (GP αIIbβ3). O recetor eritrocitário que identificámos não é tão fortemente influenciado pela presença de cálcio nem de eptifibatide, um inibidor específico da GP αIIbβ3, mas a sua inibição na presença de eptifibatide indica que o recetor é semelhante à integrina αIIbβ3. Os resultados obtidos com sangue de doentes com trombastenia de Glanzmann (GT, uma doença hemorrágica hereditária rara causada por deficiência na GP αIIbβ3) mostraram uma deficiência também na interação fibrinogénio-eritrócito. A correlação com os dados genéticos demonstrou que uma das unidades do recetor do fibrinogénio nos eritrócitos é um produto da expressão do gene da β3. O estudo seguinte, efetuado com o intuito de relacionar a interação fibrinogénioeritrócito com o processo de senescência celular, indicou que o recetor para o fibrinogénio na membrana no eritrócito pode desaparecer, ficar “mascarado” ou tornar-se disfuncional com o processo in vivo de senescência celular. Os resíduos de ácido siálico parecem também ter uma função importante como promotores da interação fibrinogénio-eritrócito. A diminuição desta interação durante o processo de senescência poderá estar largamente associada à progressiva depleção de ácidos siálicos na população de eritrócitos mais velhos. Sabendo que a população de eritrócitos mais novos se liga com maior afinidade a moléculas de fibrinogénio, pudemos concluir que a população de eritrócitos mais jovens poderá ter a maior contribuição para as doenças cardiovasculares associadas ao aumento dos níveis plasmáticos de fibrinogénio. O vírus da Dengue é o responsável pelo maior número de casos de febre hemorrágica em todo o mundo, sendo estimado que cause mais de 20 mil mortes por ano. Não existe atualmente uma vacina ou tratamento específico para a Dengue, devido (em parte) à falta de conhecimento sobre os mecanismos moleculares de alguns passos importantes do ciclo de vida viral. O vírus da Dengue é do género Flavivirus, família Flaviviridae, à qual também pertencem os vírus da febre-amarela, do Nilo Ocidental e da hepatite C. Apesar de resultarem em patologias e sintomatologias diferentes, os Flaviviridae infetam o fígado e os epitélios vasculares do hospedeiro, podendo também infetar outros órgãos. Nestas infeções, a estiatose hepática é causada pela disfunção geral do metabolismo lipídico, observando-se um aumento do número e tamanho de corpúsculos lipídicos intracelulares, uma desordem da estrutura membranar, bem como a alteração dos níveis e composição das lipoproteínas plasmáticas. Foi recentemente descoberto que a proteína da cápside (C) do vírus da Dengue interage com os corpúsculos lipídicos intracelulares, influenciando a replicação viral. Também se sabe que o vírus da hepatite C forma lipo-viro-partículas, uma conjugação entre lipoproteínas e viriões, sendo mais infeciosas que os próprios vírus livres. Estas evidências mostram a importância dos sistemas lipídicos no ciclo de vida destes vírus, embora os mecanismos moleculares subjacentes à interação da proteína C do vírus da Dengue com estes sistemas lipídicos ainda sejam desconhecidos. O nosso estudo focou-se na importância da interação da proteína da cápside do vírus da Dengue com sistemas lipídicos do hospedeiro (corpúsculos lipídicos e/ou lipoproteínas), que influenciam vários passos importantes do ciclo de vida deste vírus. Através da aplicação da técnica de espectroscopia de força, os resultados obtidos permitiram concluir que a interação da proteína C com corpúsculos isolados de linhas celulares é forte, específica, dependente do ião potássio e de proteína(s) intrínseca(s) dos corpúsculos. De seguida, desenvolveu-se um péptido (pep14-23), homólogo de um segmento da região N-terminal da proteína C do vírus da Dengue, que concluiu-se ter a capacidade de inibir a interação entre a proteína C e os corpúsculos. Tal desenvolvimento permitirá, no futuro, otimizar esta inibição com péptidos derivados do pep14-23, testando a sua atividade anti-viral contra o vírus da Dengue e outros Flavivirus. As interações da proteína C com as lipoproteínas plasmáticas humanas VLDL e LDL foram também testadas, observando-se interação específica apenas com VLDL. A dependência do ião potássio, tal como nos corpúsculos lipídicos, e a especificidade na ligação às VLDL levou a inferir que a interação depende de proteína(s) presentes nas VLDL. Interessantemente, a apolipoproteína E, uma das proteínas das VLDL (e que não está presente nas LDL), tem semelhança estrutural e de sequência com a perilipina 3 (TIP47), uma das proteínas mais abundantes nos corpúsculos lipídicos. Surge então a possibilidade de estes serem os alvos moleculares da proteína C em cada um dos sistemas lipídicos. Este tipo de interação sugere também que o vírus da Dengue, tal como o da hepatite C, pode formar lipo-viro-partículas, o que explicaria alguns dos sintomas da doença.
FP7-PEOPLE International Research Staff Exchange Scheme (IRSES; project MEMPEPACROSS, European Union); Fundação para a Ciência e a Tecnologia – Ministério da Ciência, Tecnologia e Ensino Superior (FCT-MCTES, projetos CONC-REEQ/140/2001, PTDC/SAU-OSM/73449/2006, PTDC/QUI-BIQ/112929/2009, PTDC/QUI/69937/2006 e PTDC/QUIBIQ/119509/2010); Fundação Calouste Gulbenkian (FCG, Portugal; Project Research in Life Sciences Frontiers 2010 e Programa de Estímulo à Investigação 2009) e FCT-CAPES Portugal-Brazil (Coordenação de Aperfeiçoamento de Pessoal a nível Superior)

碩士
國立臺灣大學
醫學工程學研究所
104
Biosensors that use microwave resonators are compact, easy to operate, low-cost, and capable of real-time monitoring. These advantages, combined with eliminating the need for sample pretreatment, make microwave biosensors the object of widespread attention. However, due to their relatively large size and the expensive laboratory instruments necessary for conducting measurements, they cannot be integrated into general consumer electronic products. With current development in information technology and popularity of portable smart devices, miniature biosensors have become a development trend for the future. In this study, a microwave biosensor system was developed using the technology and process of integrated circuit （IC） and printed circuit board （PCB） to measure, by automatic feedback control, sample-induced capacitance and impedance which correspond respectively to real and imaginary parts of relative permittivity. The device exhibited small size, low power consumption, low operating frequency, label-free detection, high sensitivity, and low cost. In addition, the signal to noise ratio increased with the integration interval. Function validation and sample analysis were performed on the biosensor system. In order to increase the convenience of the detection process, an automated a graphical user interface was developed. In summary, the described system is capable of rapid sample analysis and when integrated into smart device platforms, is a potentially valuable tool for IOT service.

Hot Electron Nanoscopy and Spectroscopy (HENs) is a current-sensing AFM technique recently developed in our lab, which have proven a new kind of response on conduction at the nanometer scale, casting a new light for the comprehension of electronic states in nanomaterials. Direct imaging of DNA structure has long been investigated, with the development of HENs technology, more structural information about DNA could be revealed by simultaneous measurements of height, phase, Raman signal, and conductivity. With the aim of applying it for the first time on biological molecules, customized double-stranded DNA sequences, including thiol-modified oligonucleotides are designed to create preferential conductive paths through the basis as a benchmark system for the technique on biomolecules. This work aims to a final goal to characterize hot-electron current between gold tip and thiol modified DNA which ideally is covalently bonded to the gold surface and optimized for the application. In this work, high density of DNA absorbed by SERS active gold surface with atomic flat islands has been prepared for HENs application. The samples have been characterized by AFM, SKPM and Raman Spectroscopy, as non-destructive and controlled interactive image analysis. High-resolution images of DNA have been acquired, S-S and Au-S bonding of DNA anchored on SERS active gold substrate are also visible with Surface-enhanced Raman and Tip-enhanced Raman signals. A submolecular feature has also been found in both topographical and electrical results. Herein, we report the synthesis and characterization steps to obtain the optimized operation standard.

M. Tech. Chemistry
Recently, traditional electro-thermal atomic absorption spectrometry has lost its popularity to other multi-element spectrometric techniques. Limited linear determination range, which is usually two orders of magnitude, is one of the reasons for the loss. As a result, tens of calibration standards and diluted samples have become an indispensable part of the electro-thermal atomic absorption spectrometric analysis, particularly if numerous samples containing a broad concentration range of analytes are to be analysed. The objective of this work was to establish the mechanism of absorption signals for high concentrations of analyte atoms in the absorption volume. Secondly, to employ the findings for HR-CS ET-AAS data quantification, within a broad range of analyte concentrations. It was expected that the sought algorithm would permit the use of the most sensitive analytical lines or those less prone to interferences, independent of the concentration of the respective analyte in the sample.