Tablets are the most common forms of drug administration. They are convenient to administer and easy to manufacture. However, problems associated with the adhesion of the powders to the tableting tools are common. This phenomenon is known as sticking and even though it has been well documented and studied, it remains poorly understood. The many factors that contribute to good performance of the powders make the sticking problem difficult to solve. The goal of this study is to establish a relationship between the properties measured at the nanoscale to the overall tablet mechanical properties, tablet performance and powder pre-processing induced modifications. By using atomic force microscopy (AFM) we aim to develop an analytical method to characterize the mechanical and adhesive properties of the pharmaceutical powders at the nanoscale. Other methodologies such as scanning electron microscopy (SEM), thermal analyses (DSC, TGA) and tablet strength test were also used. The materials used in this study are commonly used excipients, a sticky drug and magnesium stearate (MgSt). Two different approaches offered by AFM were employed: sharp tip imaging and colloidal probe force measurements. Nano-mechanical properties of the materials were evaluated with a sharp tip cantilever showing that higher adhesion correlates with higher tablet cohesion and that both are significantly affected by the presence of MgSt. AFM characterization of the particle surface mechanical properties at the nanoscale was also used to detect the crystallinity and amorphicity levels of the materials. New approaches to presenting such data considering the particle heterogeneity and to track the dynamics of surface recrystallization are revealed. Adhesive interactions between a steel sphere and sticky and non-sticky powders were performed with the colloidal probe technique. Sticky materials presented a higher adhesion against the steel surface, and reveal the mechanism of stickiness. This work thus contributes to the provision of predictability of the performance of formulations at an early stage of the development process. / <p>QC 20170315</p>

Physical forces in the cellular microenvironment play an important role in governing cell function. Forces transmitted through the cell cause distinct deformation of the nucleus, and possibly play a role in force-mediated gene expression. The work presented in this thesis drew upon innovative strategies employing simultaneous atomic force and laser-scanning confocal microscopy, as well as parallel optical stretching experiments, to gain unique insights into the response of eukaryotic cell nuclei to external force. Non-destructive approaches confirmed the existence of a clear anisotropy in nuclear mechanical properties, and showed that the nucleus' mechanical response to extracellular forces is differentially governed by both nuclear and cytoskeletal prestress: nuclear prestress regulates shape and anisotropic deformation, whereas cytoskeletal prestress modulates the magnitude and degree of deformation. Importantly, the anisotropic mechanical response was conserved among diverse differentiated cell types from multiple species, suggesting that nuclear mechanical anisotropy plays an important role in cell function.

Mechanical cues have been recognized to be critically important in the regulation of cells. A myriad of cellular processes including differentiation, proliferation, and gene expression are all affected by physical forces from the extra- and intra-cellular microenvironments. Despite recent advances in nano-technologies, many questions still surround how cells sense and respond to forces. Through a series of studies, we demonstrate how both the structure and inherent mechanical properties of the cell affect their response to mechanical cues. We first develop a methodology to mechanically manipulate cells while simultaneously characterizing their deformations. Using combined atomic force and confocal microscopy techniques and through systematic examination we demonstrate the role of the cytoskeleton and nucleus in the deformability and shape change of epithelial cells. Mechanical properties have been used in recent years to identify diseased states, including cancer. With this in mind, we used HeLa cells as a model and characterized significant deformability of their plasma membrane and underlying cortex. Importantly, we demonstrate and characterize their ability to recover from large shape changes, which we also observed in other epithelial cells. Shape recovery is shown to be rapid and reliant upon the actin cytoskeleton and intracellular fluid flow. Although the nucleus does not contribute significantly to the deformation and recovery of HeLa cells, the importance of nuclear mechanics cannot be forgone. In vitro studies have shown that mechanical forces transmitted through the cell’s cytoskeleton critically affect nuclear mechanics and gene transcription processes. Many others have used simple models and isolated nuclei in an attempt to characterize nuclear properties. Thus, in a subsequent study, we examine the nucleus within intact cells. Nuclear shape change, in response to force, is shown to be complex and cannot be well-characterized by isotropic mechanical properties. Characterization of the mechanics of the cell, as demonstrated through our findings, is crucial in the field of biological physics. The aforementioned studies, written as scientific articles, are presented in the body of this thesis (Chapters 2-5). A review article that focuses on mechanotransduction and relevant examples using AFM as a tool for its examination acts as an introductory chapter.

Living organisms are made up of a multitude of individual cells that are surrounded by biomolecules and fluids. It is well known that cells are highly regulated by biochemical signals; however it is now becoming clear that cells are also influenced by the mechanical forces and mechanical properties of the local microenvironment. Extracellular forces causing cellular deformation can originate from many sources, such as fluid shear stresses arising from interstitial or blood flow, mechanical stretching during breathing or compression during muscle contraction. Cells are able to sense variations in the mechanical properties (elasticity) of their microenvironment by actively probing their surroundings by utilizing specialized proteins that are involved in sensing and transmitting mechanical information. The actin cytoskeleton and myosin-II motor proteins form a contractile (actomyosin) network inside the cell that is connected to the extracellular microenvironment through focal adhesion and integrin sites. The transmission of internal actomyosin strain to the microenvironment via focal adhesion sites generates mechanical traction forces. Importantly, cells generate traction forces in response to extracellular forces and also to actively probe the elasticity of the microenvironment. Many studies have demonstrated that extracellular forces can lead to rapid cytoskeletal remodeling, focal adhesion regulation, and intracellular signalling which can alter traction force dynamics. As well, cell migration, proliferation and stem cell fate are regulated by the ability of cells to sense the elasticity of their microenvironment through the generation of traction forces. In vitro studies have largely explored the influence of substrate elasticity and extracellular forces in isolation, however, in vivo cells are exposed to both mechanical cues simultaneously and their combined effect remains largely unexplored. Therefore, a series of experiments were performed in which cells were subjected to controlled extracellular forces as on substrates of increasing elasticity. The cellular response was quantified by measuring the resulting traction force magnitude dynamics. Two cell types were shown to increase their traction forces in response to extracellular forces only on substrates of specific elasticities. Therefore, cellular traction forces are regulated by an ability to sense and integrate at least two pieces of mechanical information - elasticity and deformation. Finally, this ability is shown to be dependent on the microtubule network and regulators of myosin-II activity.

Ce travail de thèse porte sur les divers aspects de l'application de la microscopie à force atomique (AFM), pour la caractérisation de semi-conducteurs organiques et de réseaux d’ADN, pour des applications en électronique organique et en biologie. Sur ces surfaces molles, le mode de fonctionnement Amplitude modulation de l’AFM a été choisi. Ce choix est argumenté par une étude des processus dissipatifs, réalisée sur un échantillon particulier, une puce à ADN. Nous avons montré l’influence des paramètres expérimentaux d’amplitude sur la qualité des images topographique et de phase. A partir du calcul de l’énergie dissipative, il a été montré que la dissipation sur la puce ADN était principalement induite par une interaction pointe-échantillon de type viscoélastique. L’étude par AFM de la croissance “thickness-driven“ du pentacène a été réalisée afin de relier sa morphologie à la nature du substrat et aux performances électriques pour la réalisation de transistors organiques à effet de champ, OFET (Organic Field EffectTransistor). Déposé sur deux substrats de polymères, le parylène et le benzocyclobutène (BCB), le pentacène a été caractérisé à l’échelle nanométrique pour des épaisseurs de film entre 6 et 60nm. Il a été démontré que les grains créés par le dépôt étaient les plus étendus pour une épaisseur déposée de 30nm. La spectroscopie AFM en mode contact a été utilisée, comme une alternative à la méthode des angles de contact, pour mesurer localement l'énergie de surface. Une énergie de surface minimale caractéristique d’une surface mieux ordonnée a été mesurée pour l’épaisseur de pentacène déposée de 30nm pour les deux substrats. Des méthodes spectrales d'analyse statistique d’images, à base de PSD (Power Spectrum Density), ont été utilisées pour expliquer la morphologie des films de pentacène. En outre, ces modèles ont fourni une description exhaustive non seulement de la surface accessible de l’échantillon, mais aussi de ses propriétés structurales intérieures. Mise en évidence dans les modèles, cette épaisseur critique de 30nm correspond à une transition de la phase orthorhombique à la phase triclinique pour les molécules de pentacène déposées surparylène. De même, une transition polymorphique se produit sur le BCB. Sur des OFET créés à base de pentacène sur BCB, la mobilité la plus importante de 3.1x10-2cm²/Vs correspond à la couche de pentacène de 30nm, ce qui montre l'avantage de l'moléculaire orthorhombique en comparaison du triclinique. L’assemblage moléculaire de structures en X et en Y à base d’ADN a été observé par AFM à l’air et dans deux solutions buffer de Tris et HEPES sur un substrat de mica. Il a été montré que le traitement du mica par des ions Ni2+ augmente la force d’interaction ADN/substrat et réduit la diffusivité des molécules. A l'air, des macromolécules filaires contenant une seule structure double brin sont observées sur le mica non traité et des macromolécules avec une géométrie 2D ramifiée, sur le mica prétraité. Sur une surface non-traitée, l’agitation thermique suffit à déplacer les molécules d’ADN faiblement liées au mica, ce qui conduit à la formation de structures plus simples 1D. L’organisation est différente dans les solutions de Tris et d’HEPES. Dans la solution deTris, contenant des cations Mg2+, les arrangements conduisent à une architecture 2D, bien organisée. Dans la solution d’HEPES, contenant des cations Ni2+, la force ionique est 10 fois plus faible, qui conduit à une rupture des liaisons préalablement formées entre le mica et l'ADN. Cependant, les molécules d'ADN restent les unes près des autres en raison d'une substitution partielle des cations de Mg2+ déjà adsorbés par les cations de Ni2+ de plus grande affinité avec le mica. [...] / This work reports the various aspects of the application of atomic force microscopy (AFM), for the characterization of organic semiconductors and DNA-based arrays, for organic electronics and biological applications. On these soft surfaces, the amplitude modulation AFM mode was chosen. This choice is argued by a study of dissipative processes, performed on a particular sample, a DNA chip. We showed the influence of experimental parameters on the topographic and phase image quality. By calculating the dissipative energy, it was shown that the dissipation on the DNA chip was mainly induced by a viscoelastic tip-sample interaction.The AFM study of the "thickness-driven” pentacene growth was made to link the morphology to the nature of the substrate and to the electrical performance of created pentacene-based Organic Field Effect Transistor (OFET). Deposited on two polymer substrates, parylene and benzocyclobutene (BCB), pentacene has been characterized for nanoscale film thicknesses between 6 and 60nm. It has been shown that the larger grains were created for a deposited thickness of 30nm. Spectroscopic AFM mode was used as an alternative to the method of contact angles, to measure local surface energy. Decrease of surface energy is characteristic of a more ordered surface and was measured for a thickness of 30 nm of pentacene deposited on both substrates. Models of statistical analysis of spectral images, based on the Power Spectrum Distribution (PSD) have been used to explain the morphology of pentacene films. In addition, these models have provided a comprehensive description not only of the accessible surface of the sample, but also of its internal structural properties. Highlighted in the models, the critical thickness of 30 nm corresponds to a transition from the orthorhombic phase to the triclinic phase for pentacene molecules deposited on parylene. Similarly, a polymorphic transition occurs on the BCB. On OFETs, based on pentacene on BCB, the largest mobility of 3.1x10-2 cm²/Vs corresponds to the pentacene layer of 30nm, that shows a better ordering of the orthorhombic molecular packing in comparison with the triclinic packing.The molecular arrangement of X and Y structures based on DNA was observed, by AFM, in air and in two buffer solutions of Tris and HEPES on a mica substrate. It was shown that the treatment of the mica by Ni2 + ions increases the strength of the DNA/substrate interaction and reduces the diffusivity of the molecules. In air, wired macromolecules containing one double-stranded structure are observed on untreated mica and macromolecules with a 2D geometry on pretreated mica. Onto a non-treated, the greater thermal motion of weakly bounded to mica DNA molecules leads to the rupture of intermolecular bonding and the forming structures are more simple and not branched. The organization is different in solutions of Tris and HEPES. In the Tris solution, containing Mg2+ cations, the arrangement leads to a well-organized 2D architecture. In the HEPES solution, containing Ni2+ cations, the ionic strength is 10 times lower, this leads to a breaking of the bonds previously formed between DNA and mica. However, DNA molecules are near each other due to a partial substitution of already adsorbed Mg2 + cations by Ni 2 + cations of higher affinity with the mica. These results show that the two liquids promote a 2D assembly. In air, the networks are not stable and the few observed ones remain in a dendritic structure on the surface of pretreated mica and as a linear macromolecule on the untreated mica.

Therapeutics targeting the PI3K (phosphatidylinositol 3-kinase) and the Ras/MAPK (mitogen-activated protein kinases) pathways have potential as non-toxic treatments for triple-negative breast cancer due to their frequent over-activation in several forms of cancer. Interestingly, the PI3K and Ras/MAPK pathways have been shown to incite cancer dormancy behavior individually and tumorigenic behavior in unison when induced in healthy breast epithelial cells (MCF-10A) in vivo. Tumorigenesis and metastasis are heavily reliant on the specific mechanical and adhesive properties of cells, including decreased stiffness, increased mechanosensitivity, and decreased adhesion. However, the describe cellular behaviors are poorly understood for dormant cancer phenotypes. Understanding the mechanical and adhesive behaviors of MCF-10A cells as a function of PI3K and/or Ras/MAPK pathway over-activation further explores the cross-talk enabling unique dormant and tumorigenic characteristics. Cellular viscoelasticity and adhesion were measured for MCF-10A cells with PTEN (phosphatase and tensin homolog) knockout and activated KRAS (Kristen rat sarcoma viral oncogene homolog) overexpression to activate the PI3K and Ras/MAPK pathways respectively with atomic force microscopy. PTEN knockout alone has no observable influence on cell adhesion but resulted in softer cells with less organized cytoskeleton. Activated KRAS overexpression increased cell stiffness and cell adhesion regardless of PTEN expression level. Moreover, the overexpression of activated KRAS enhanced the sensitivity of cells to the substrate stiffness. The findings suggest that the cancer-associated pathways PI3K and Ras/MAPK regulate cell adhesion and mechanics to promote tumor formation and metastasis. More importantly, the results that signify mutations of different molecular pathways associated with cancer dormancy regulate cell mechanics differently suggests that cell stiffness is a biomarker that detects and differentiates different types of dormant cancers.

Atomic force microscopy (AFM) is widely used to study material properties and domain heterogeneity of polymers. In both quasi-static force spectroscopy and dynamic AFM, challenging complexities such as the presence of different effective tip-surface forces, surface dynamics, and material viscoelasticity can occur on polymer samples. Many models that attempt to link experimental observables to contact mechanics fail to rigorously account for these complexities. This may lead to inaccurate and unreliable predictions, especially when examining soft polymers. Therefore, having access to rigorous models that can facilitate the understanding of the underlying phenomena during tip-surface interaction, explain the observations, and make reliable and accurate predictions, is of great interest. Among the previously developed models, Attard et al. proposed a novel non-Hertzian-based model that has a versatile ability to systematically incorporate different linear viscoelasticity constitutive models and surface adhesive forces. However, the implementation of Attard’s model into the AFM framework is challenging.<div><br></div><div>In a series of studies, we improve the computational speed and stability of Attard’s viscoelastic contact model and embed it into an AFM framework by proposing algorithms for three AFM operational modes: tapping mode, bimodal, and peak force tapping. For each mode, the results are successfully verified/validated against other reliable AFM codes, FEM simulations, and experiments. The algorithms’ predictions illustrate how viscoelasticity and surface adhesive hysteresis of polymeric samples is reflected in AFM observables. However, since Attard’s model does not lead to a closed-form solution for tip-surface interaction force, using that to quantify the surface mechanical properties based on the AFM observables is not straightforward. Therefore, we utilize the data analytics-based approaches such as linear regression and machine learning algorithms to enable the material viscoelasticity and adhesive parameters estimation based on the provided instrument observables.<br></div><div><br></div><div>The set of results reported in this thesis improves the current knowledge about complex phenomena that occur during tip-surface interactions, especially on soft-viscoelastic-adhesive polymers. The introduced “improved Attard’s model” fulfills the need for a continuum mechanics viscoelasticity contact model that rigorously captures the complexities of such samples. The viscoelasticity contact model and the proposed inverse solution algorithms in this thesis facilitate quantitative measurement and discrimination of the surface adhesive and viscoelastic properties based on the acquired nanoscale AFM maps of polymeric samples.<br></div>

京都大学 / 新制・課程博士 / 博士(工学) / 甲第23217号 / 工博第4861号 / 新制||工||1759(附属図書館) / 京都大学大学院工学研究科物質エネルギー化学専攻 / (主査)教授 作花 哲夫, 教授 安部 武志, 教授 佐藤 啓文 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

京都大学 / 新制・課程博士 / 博士(工学) / 甲第23232号 / 工博第4876号 / 新制||工||1761(附属図書館) / 京都大学大学院工学研究科化学工学専攻 / (主査)教授 宮原 稔, 教授 松坂 修二, 教授 山本 量一 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第23336号 / 生博第454号 / 新制||生||60(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 野田 岳志, 教授 片山 高嶺, 教授 高田 穣 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM