Pattern formation, i.e., the outcome of self-organization, has fascinated scientists for centuries. A large effort was devoted to understand the formation of regular patterns in dissipative structures. More recently, it appears that self-organized structures could also be achieved near equilibrium. There is a great variety of physical and chemical systems that, near equilibrium, exhibit periodic patterns. For instance, stripes or bubbles could be observed in thin films of magnetic garnet, superconducting materials, block copolymers, liquid crystals, phospholipids, and ferrofluids. Wrinkling instability of compressed rigid membranes on soft elastic substrates leads also to the formation of periodic patterns near equilibrium. Since the seminal paper of Bowden et al. (Nature 1998), various systems were proposed to generate nano- and micrometric wrinkles via the application of compressive stresses to multilayers. In addition to its purely fundamental interest, these instabilities also offer a new route to build in a simple, cost-effective, and well-defined way nano- and microstructured surfaces without the use of the traditional, robust techniques developed in the microelectronics industry. In this thesis, we develop a new system, metal-polymer-substrate trilayers, that exhibit wrinkling when heated above the glass transition temperature of the polymer. We explain in detail the mechanism at the origin of wrinkling and expand existing models to obtain a complete description of the relevant parameters that govern both the amplitude and the wavelength of the obtained pattern. In light of this, we show that by playing with the rheological properties of the polymer we are able to control precisely the geometry of the wrinkles. Furthermore, to generate surfaces with a tailor-made buckling pattern, we develop an original variant of the experiments. We tune the boundary conditions at the polymer-substrate interface by chemically patterning the substrates with regions of high and low adhesion. In this way, we obtain patterns with wrinkles being oriented differently above the sticky and the slippery regions. This last result is very surprising since it seems, at first sight, unrealistic to imagine that the chemical nature of the substrate could affect the elastic instability of the skin through a micron-thick polymer film. To explore wrinkled patterns with complex morphologies, we couple the wrinkling instability with solvent diffusion. Molecular diffusion in the polymer layer triggers the transition from an unwrinkled to a wrinkled state, provided that stimuloresponsive mutlilayers are used. The wrinkled pattern obtained is determined by the geometry of the diffusion process. To understand this surprising observation, we explain in detail how the scalar field related to the solvent concentration affects so strongly the elastic instabilities usually determined by the tensorial stress field. This mechanism allows us thus to grant exotic stress distributions which lead to very intriguing patterns (e.g. parallel or radial folds, herringbones). Interestingly, we find that under specific conditions, a hierarchical wrinkled morphology, i.e. pattern of wrinkles branching into generations of ever-higher folds, develops. We study other manifestations of hierarchical structures existing around us. In this frame, we derive a general concept that a plate constrained at one edge (with a fixed wavelength) but free at the opposite one evolves naturally to larger wavelengths to minimize its bending energy. We show theoretically that the evolution results from a compromise between the gain in bending energy and the energetic penalties related to the change of wavelength. We demonstrate the universality of these concepts by showing that our commonplace suspended curtain behaves like nanometer-thick polystyrene films deposited on water and further compressed. We close this thesis by making a short review of the main applications related to wrinkling that are already described in literature and develop in detail one of them, the use of wrinkling to investigate cell contact guidance.

Three-dimensional biomolecule patterned hydrogels provide cellular microenvironments that mimic in vivo conditions. We are particularly interested in the fabrication of materials to spatially control stem cell differentiation towards the creation of tissue analogues. To this end, we have designed a 3D protein patterning system where differentiation factors were immobilized within distinct volumes through two-photon chemistry, which provides 3D control since the excitation volume is limited to the focal point of the laser. Agarose hydrogels were modified with 6-bromo-7-hydroxy-coumarin (Bhc) protected amines or thiols, which upon two-photon excitation are deprotected in defined volumes yielding reactive amines or thiols. Fibroblast growth factor-2 (FGF-2) was immobilized onto agarose-thiol-Bhc through either disulfide bond formation with agarose thiols or the physical interaction between human serum albumin (HSA) and the albumin binding domain (ABD). The use of biological binding pairs also provides mild immobilization conditions, minimizing the risk for bioactivity loss. Similarly, two differentiation factors for retinal stem progenitor cells were simultaneously immobilized: 1) ciliary neurotrophic factor (CNTF); and 2) N-terminal sonic hedgehog (SHH). Maleimide modified binding proteins, such as maleimide-streptavidin; react with exposed thiols, yielding 3D patterns of covalently immobilized streptavidin in agarose hydrogels. Growth factors are then introduced as fusion proteins with binding domains, such as biotin-CNTF, for complexation and thus 3D immobilization. By combining multiple binding systems with two-photon patterning, we were able to simultaneously 3D immobilize proteins towards the creation biomimetic hydrogels.

Three-dimensional biomolecule patterned hydrogels provide cellular microenvironments that mimic in vivo conditions. We are particularly interested in the fabrication of materials to spatially control stem cell differentiation towards the creation of tissue analogues. To this end, we have designed a 3D protein patterning system where differentiation factors were immobilized within distinct volumes through two-photon chemistry, which provides 3D control since the excitation volume is limited to the focal point of the laser. Agarose hydrogels were modified with 6-bromo-7-hydroxy-coumarin (Bhc) protected amines or thiols, which upon two-photon excitation are deprotected in defined volumes yielding reactive amines or thiols. Fibroblast growth factor-2 (FGF-2) was immobilized onto agarose-thiol-Bhc through either disulfide bond formation with agarose thiols or the physical interaction between human serum albumin (HSA) and the albumin binding domain (ABD). The use of biological binding pairs also provides mild immobilization conditions, minimizing the risk for bioactivity loss. Similarly, two differentiation factors for retinal stem progenitor cells were simultaneously immobilized: 1) ciliary neurotrophic factor (CNTF); and 2) N-terminal sonic hedgehog (SHH). Maleimide modified binding proteins, such as maleimide-streptavidin; react with exposed thiols, yielding 3D patterns of covalently immobilized streptavidin in agarose hydrogels. Growth factors are then introduced as fusion proteins with binding domains, such as biotin-CNTF, for complexation and thus 3D immobilization. By combining multiple binding systems with two-photon patterning, we were able to simultaneously 3D immobilize proteins towards the creation biomimetic hydrogels.

This thesis attempted to further understanding of various aspects of the competitive state anxiety response. The specific questions that were addressed in the five studies reponed involve investigations into antecedents of competitive anxiety, temporal patterning, additional dimensions to the anxiety response and relationships with performance. Competitive anxiety was assessed in all of the studies by the Competitive State Anxiety Inventory-2 (CSAI-2) which measures cognitive anxiety, somatic anxiety and self-confidence. The first two studies employed a purely quantitative approach whilst the final three studies incorporated both quantitative and qualitative methodologies. The first study investigated situational factors which predict the CSAI-2 components in the specific / population of middle-distance runners. Cognitive anxiety· was predicted by three factors, 'Perceived Readiness', 'Attitude Towards Previous Performance' and 'Position Goal', whilst self-confidence was predicted by 'Perceived Readiness' and 'External Environment'. None of the factors predicted somatic anxiety. These results suggested that cognitive anxiety and self-confidence share some common antecedents but that there are also factors unique to each. The second study examined the temporal patteming of the CSAI-2 components in the period leading up to competition as a function of gender. Gender has previously been shown to mediate patteming of responses so that antecedents were also examined in an attempt to explain such findings. Results showed that males and females reported differential temporal patteming for cognitive anxiety and self-confidence and that different antecedents predicted these variables. Significant predictors of cognitive anxiety and self-confidence were associated with personal goals and standards in females and interpersonal comparison and winning in males. The third and fourth studies investigated the importance of additional dimensions to the competitive state anxiety response in furthering understanding of the construct. These studies examined the frequency and direction dimensions of anxiety and findings suggested that the intensity alone approach currently employed is restrictive and that important information can be gained from considering these other dimensions. The fifth study focused on the dimensions of intensity and direction of anxiety and their specific relationship with sports performance. Findings revealed that a direction dimension was a better predictor of basketball performance than any of the intensity variables, further suggesting that future anxiety research should measure this dimension.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第17583号 / 工博第3742号 / 新制||工||1570(附属図書館) / 30349 / 京都大学大学院工学研究科材料化学専攻 / (主査)教授 平尾 一之, 教授 田中 勝久, 教授 三浦 清貴 / 学位規則第4条第1項該当

Cell patterning platforms support diverse research goals including tissue engineering, the study of cell physiology, and the development of biosensors. Patterning and interfacing with neurons is a particular challenge, being approached via various bioengineering approaches. Such constructs, when optimised, can inform our understanding of neuronal computation and learning, and ultimately aid the development of intelligent neuroprostheses. A fundamental pre-requisite is the ability to dictate the spatial organization and topography of patterned neuronal cells. This thesis details efforts to pattern neurons using photolithographically defined arrays of the polymer parylene-C, printed upon oxidised silicon wafers. Initial work focused on exploring the parylene-C:SiO2 construct as a wide-ranging cell-patterning platform, assessing cell adhesion from both substrate- and cell-centric perspectives. Next, the LUHMES (Lund Human Mesencephalic) cell line was used to explore the potential for construction of interrogatable, topographically-defined neuronal networks. In isolation, LUHMES neurons failed to pattern and did not show any morphological signs of cellular differentiation. However, in the context of a cellular template (the HEK 293 cell line which was found to pattern reliably), LUHMES were able to adhere secondarily on-chip. This co-culture environment promoted morphological differentiation of neurons. As such, HEK 293 cells fulfilled a role analogous to glia, dictating neuronal cell adhesion and generating an environment conducive to neuronal survival. Neurites extended between islands of adherent cell somata. The geometry and configuration of parylene-C influenced the organisation of neurites. With appropriate designs, orthogonal neuronal networks could be created. The dominant guidance cue for neurite growth direction appears to be a diffusible chemotactic agent. HEK 293 cells were later replaced with slower growing human glioma-derived precursors, extracted during tumour debulking surgery. These primary cells patterned accurately on parylene-C and provided a similarly effective, and longer lasting, scaffold for neuronal adhesion.

This thesis presents the study of organic layers covalently assembled onto carbon surfaces. As a result of their attachment, the properties of carbon surfaces were controllably adjusted so that these surfaces could be used for desired applications. In order that a wide range of properties were imparted onto the carbon surface, many different modifiers were attached and thoroughly characterised. Three applications that the modified carbon surfaces were used for were the subsequent coupling of molecular species, adsorption of protein and assembly of aldehyde/sulfate-functionalised polystyrene (PS) and citrate-capped gold nanoparticles (NPs). Finally, patterning of different organic layers at pre-determined spatially defined locations on the one carbon surface was also investigated. The carbon surfaces used in this work were glassy carbon (GC) and pyrolysed photoresist film (PPF) surfaces. For PPF, methods for the reproducible fabrication of electrochemically suitable surfaces were investigated. The properties of GC and PPF surfaces are very similar apart from the surface roughness. PPF has near atomic smoothness and has RMS roughness values that are approximately four times smaller than GC. The first series of modifier layers attached to the carbon surfaces was via the oxidation of seven different primary amines. The different layers allowed the modulation of the wettability of the surface. Both n-tridecylamine (TDA, monoamine) and 1,12-diaminododecane (DAD, diamine) are able to form multilayers. The stability of TDA and DAD layers were tested by scanning, soaking and sonicating the layers in different media. Changes in the layer were monitored by the probe response of ferrocene monocarboxylic acid (FCA). However, atomic force microscope (AFM) depth profiling experiments showed that changes in the probe response did not indicate cleavage of the covalently attached layer and mechanisms are proposed to account for the changes in the response of the probe. Surface concentrations of the amine modifiers were estimated by the coupling of an electrochemically active species, FCA and nitrobenzoyl chloride (NBC). The electrochemical reduction of the 4-nitrophenylethylamine (NPEA) layer in acid caused the layer to 'shrink'. Surface concentration estimates of NPEA from acid reduction of layers with different thicknesses suggested that only a limited fraction of the p-nitrophenyl groups were reduced in acid. However, in ACN (acetonitrile)/0.1 M [Bu4N]BF4 (tetrabutyl ammonium fluoroborate) the relationship between the concentration of electroactive surface groups and layer thickness was linear. The other series of modifiers that was attached to alter the surface properties was performed by the reduction of aryl diazonium salts. Subsequent coupling reactions of tetraethylene glycol diamine (TGD) to para methylene carboxylic acid phenyl (MCA) and NBC to electrochemically reduced para nitro phenyl (NPh) layers were carried out. Surface concentrations of NPh as estimated from reduction scans was higher when reduction was performed in ethanol/water compared to acid. Four peaks at N1s binding energies were observed in x-ray photoelectron spectroscopy (XPS) spectra for both acid and ethanol/water reduced layers. The ability of attached amine and aryl layers to modulate the adsorption of protein was investigated using fluorescently labelled protein, bovine serum albumin-fluorescein isothiocynate (BSA-FITC) and fluorescence microscopy. TGD, para methyl phenyl (MP), para hexyl phenyl (HP) and para polyethylene glycol phenyl (PEG)-modified GC surfaces promoted protein adsorption relative to as-prepared GC, whereas n-hexylamine (HA) and polyethylene glycol diamine (PGD) layers reduced protein adsorption. The assembly of two types of NPs, aldehyde/sulfate-functionalised PS and citrate-capped gold NPs, onto amine-containing modifiers layers was examined. Citrate-capped gold NPs were synthesised and characterised. The surface coverage of the gold NPs was controlled by using different modifiers of different chemical compositions, tuning the modification conditions and adjusting the immersion time, concentration and pH of gold NP solution. Approaches to creating patterns of modifiers in pre-determined spatially defined locations on GC and PPF surfaces using poly(dimethyl)siloxane (PDMS), poly(vinyl)alcohol (PVA) and thin metal films were investigated. With the "fill-in" approach using PDMS, the smallest pattern of modifiers was the parallel lines with a line width of 20 µm and straight edges and was created by performing electrochemistry in PDMS microchannels which has not been previously investigated. Visualisation techniques, based on optical and scanning electron microscopy, were demonstrated for the molecular patterns.

No description available.

Evaporation and wetting of droplets are a phenomena present in everyday life and in many industrial, biological or medical applications; thus controlling and understanding the underlying mechanisms governing this phenomena becomes of paramount importance. More recently, breakthroughs in the fabrication of new materials and nanomaterials have led to the synthesis of novel nanoscale particulates that dispersed into a base fluid modify the properties of this latter. Enhancement in heat transfer or the self-assembly of the particles in suspension during evaporation, are some of the areas in which nanofluids excel. Since it is a relatively new area of study, the interplay particle-particle, particle-fluid or particle-substrate at the macro-, micro-, and nanoscale is yet poorly understood. This work is an essay to elucidate the fundamental physics and mechanisms of these fluids during free evaporation, of great importance for the manipulation and precise control of the deposits. The evaporative behaviour of pure fluids on substrates varying in hydrophobicity has been studied and an unbalance Young’s force is proposed to explain the effect of substrate hydrophilicity on the pinning and the depinning forces involved during droplet evaporation. On other hand, the addition of nanoparticles to a base fluid modifies the evaporative behaviour of the latter and: a more marked “stick-slip” behaviour is observed when increasing concentration on hydrophobic substrates, besides the longer pinning of the contact line reported on hydrophilic ones when adding nanoparticles. A deposition theory to explain the final deposits observed, for the outermost ring, after the complete vanishing of a 0.1% TiO2-ethanol nanofluid droplet has also been developed. In addition, the evaporation of pinned nanofluid droplets on rough substrates at reduced pressures has been systematically studied. A revisited Young-Lippmann equation is proposed as one of the main findings to explain the enhancement on electrowetting performance of nanoparticle laden fluid droplets when compared to the pure fluid case. On the other hand, of relevant importance is the absence of “stick-slip” behaviour and the more homogeneous deposits found after the complete evaporation of a nanofluid droplet under an external electric field applied when compared to free evaporation of these fluids.

Fibroblast growth factors (FGFs) are critical in many aspects of embryonic development and other cellular functions including apoptosis, cell adhesion, and proliferation. FGF8a, specifically, is known to initiate retinal ganglion cell (RGC) differentiation along with FGF3 early in retinal development (Martinez-Morales et al., 2005b). There has been little research into later roles for FGF8a in eye development. Here we show mRNA expression of fgf8a in the presumptive RGCs of 2 day-old zebrafish, past the time of RGC differentiation (28-48 hours)(Schmitt and Dowling, 1996). In addition, mRNA expression of putative receptor, FGFR1b, was localized outside the retina on the presumptive vasculature. Acerebellar (ace) mutants lacking FGF8a show mispatterned retinal vasculature and a lack of blood flow through the eye at 48 hpf. Further, we looked to see if this lack of blood flow had any effect on the developing neural retina. We found a significant reduction in the size of ace mutant eyes and also a reduction in total cell numbers in the retina starting at 48 hours post fertilization (hpf) suggesting a role for fgf8a in neurovascular signaling. The cause of the small eye phenotype was found to be due to a lack of proliferating cells and not an increase in cell death. We hypothesized if this phenotype was a result of a lack of blood flow to the retina. It has previously been reported that zebrafish survive and develop normally for 7 days without blood flow as the embryo receives nutrients by simple diffusion with its surroundings (Sehnert et al., 2002). To investigate the role that blood flow plays on the developing retina we utilized a silent heart mutant (sih) fish line, which lacks cardiac troponin t resulting in embryos without blood flow, as heart contractility does not initiate. To explore lack of blood flow to the retina as a cause for the observed ace mutant phenotype, sih mutant eye phenotypes were assessed. Retina cell counts from these embryos show a decreased eye diameter and a loss in total retina cell numbers due to lack of proliferation, phenocopying ace mutants. sih mutants also show a mis-patterning of their retinal vasculature with ectopic vessel branches similar to ace mutants. Our data support the small eye phenotype seen in both mutants is a result due to lack of proliferation. After morpholino knock down of the receptor, fgfr1b, we see mispatterend vasculature that phenocopies what we see in ace mutants. These finding led us to hypothesize that FGF8a, secreted by the RGCs, signals through its receptor, FGFR1b, on the retinal vasculature to promote cell growth and development. Further these data suggest that the retinal vasculature subsequently responds by secreting an unknown factor to support the proliferation and maintenance of the RGCs.