The Fabrication and Applications of Protein Patterns Produced Via Particle Lithography

A novel particle lithography technique with the ability to pattern protein in hexagonal dot arrays was developed. The patterning method consists of a simple three-step procedure: (1) formation of a close-packed polystyrene microsphere monolayer, (2) grafting of a protein-resistant layer of poly(ethylene glycol) (PEG), and (3)selective adsorption of protein into the resulting PEG holes. The diameter and center-to-center spacing of the patterned features was varied simultaneously by changing the diameter of the spheres used in the lithographic mask or independently using a simple heating modification. A combination of the original and modified procedures was used to produce patterns of protein dots with diameters of 450 nm - 9 ìm and center-to-center spacings of 2 - 10 ìm. To demonstrate the applicability of the particle lithography technique, a fluorescent-based immunoassay was created using quantum dot bioconjugates (QDBCs). The millions of protein dot features per patterned substrate served as redundant sampling points that produced a subpicomolar detection limit. Finally, the QDBC patterns were also used to investigate the differences between neutrophil spreading on patterned and homogenously coated anti-PSGL-1 (PL1) surfaces

Light and Single-molecule Coupling in Plasmonic Nanogaps

Plasmonic cavities confine optical fields at metal-dielectric interfaces via collective charge oscillations of free electrons within metals termed surface plasmon polaritons (SPPs). SPPs are confined in nanometre gaps formed between two metallic surfaces which creates an optical resonance. This optical resonance of the system is controlled by the geometry and the material of the nanogap. The focus of this work is to understand and utilize these confined optical modes to probe and manipulate the dynamics of single-molecules at room temperature. In this thesis, nanogap cavities are constructed by placing nanoparticles on top of a metal-film separated by molecular spacers. Such nanogaps act as cavities with confined optical fields in the gap. Precise position and orientation of single-molecules in the gap is obtained by supramolecular guest-host assembly and DNA origami breadboards. The interaction of light and single-molecules is studied in two different regimes of interaction strength. In the perturbative regime molecular light emission from electronic and vibrational states is strongly enhanced and therefore is used for the detection of single-molecules. In this regime the energy states remain unaltered, however profound effects emerge when the gap size is reduced to <1 nm. New hybridized energy states which are half-light and half-matter are then formed. Dispersion of these energies is studied by tuning the cavity resonance across the molecular resonance, revealing the anti-crossing signature of a strongly coupled system. This dressing of molecules with light results in the modification of photochemistry and photophysics of single-molecules, opening up the exploration of complex natural processes such as photosynthesis and the possibility to manipulate chemical bonds.I would like to acknowledge the financial support I received from the Dr. Manmohan Singh scholarship from St. John’s College, University of Cambridge

Label-Free Monitoring of Tumor Models by Surface-Enhanced Raman Scattering

184 p.El objetivo general de la presente tesis se ha centrado en la monitorización de modelos celulares mediante la técnica de espectroscopia de Raman aumentada en superficies (SERS). Las tecnologías desarrolladas en la tesis han perseguido, por un lado, mejorar la recreación del ambiente tumoral a escala de laboratorio, y por otra parte, su integración junto con estructuras plasmónicas para el análisis por SERS de los modelos tumorales creados artificialmente. Más en concreto, se han analizado las alteraciones en la concentración relativa de los metabolitos presentes en el medio extracelular como resultado de la reprogramación metabólica característica de los tumores, la cual permite a su vez un crecimiento descontrolado de dichas células.La disposición conjunta de ambas tecnologías (cultivos celulares en 3D y nanoplasmónica) ofrece un marco único para la identificación de aquellos procesos celulares que se encuentran alterados durante el crecimiento de tumores. Hasta la fecha, la mayoría de las técnicas de laboratorio que se habían empleado para caracterizar ambientes celulares en el laboratorio implicaban procesos invasivos, es decir, quemodifican o incluso desintegraban la muestra para poder analizarla. En contraposición, la espectroscopia Raman había permitido adquirir información sobre la composición del medio celular de una manera mínimamente invasiva. Basada en los fenómenos de dispersión inelástica, la técnica de Raman emplea luz monocromática (generalmente de un láser) para irradiar la muestra bajo análisis, de forma que la interacción entre la muestra y el láser provoca un cambio en la energía de los fotones dispersados, específico de los modos vibraciones de las moléculas irradiadas. Por lo tanto, la luz dispersada y recogida por un detector, permite caracterizar el sistema biológico que ha sido previamente iluminado, sin marcaje previo. Sin embargo, las señales detectadas por dispersión Raman son de manera general muy débiles, por lo que se requiere una intensificación de dichas señales para poder detectar la presencia de metabolitos extracelulares (a bajas concentraciones). En esta tesis se decidió implantar la modalidad conocida como SERS, que hace uso de las propiedades plasmónicas de nanopartículas metálicas (principalmente de oro), las cuales dan lugar a campos eléctricos elevados cuando se iluminan en resonancia con los plasmones superficiales. Como resultado, la señal de Raman de las moléculas adsorbidas sobre dichas superficies metálicas se ve amplificada en varios órdenes de magnitud. Sobre esta base, se han desarrollado en la tesis diferentes plataformas destinadas a combinar sustratos plasmónicos, formados por fijación de nanopartículas de oro sobre estructuras rígidas en 2D, o bien embebidas en redes poliméricas, junto con modelos de células tumorales en crecimiento. La finalidad de la tesis ha sido pues, la monitorización de diferentes procesos celulares en dichos dispositivos mediante SERS, y su posterior interpretación biológica en el ámbito del metabolismo tumoral y la mejora del tratamiento.CICbioGUNE; CICbiomaGUN

Biomimetic substrates for immune cell activation

This thesis describes the fabrication of biomimetic substrates, and their use as tools to probe cellular interactions of key immune cells. Nanoparticles of gold and zinc sulfide have been fabricated, and patterned into nanoarrays. Adaptive (T cell) and innate (NK cell) immune cell responses to nanoscale spacing of ligand-receptor pairs were measured, and the effect of presenting stimulatory ligands on substrates with varying mechanical properties has been tested for T cell responses. The advanced materials in this thesis act to create artificial immune synapses, and probe the effect of these stimuli on engagement and activation of human immune cells. Specifically, block co-polymers were used to form polymer micelles which encapsulate metal ions and form metal or metal compound nanoparticles. Micelles encapsulating metal ions or nanoparticles were formed and deposited onto substrates using Block Co-polymer Micellar Lithography (BCML) to form nanoparticle arrays with controlled inter-particle spacing. Well controlled gold nanoparticle arrays with spacing between 25-150nm have been produced. The technique has been further developed to include fabrication of zinc sulfide particles and nanoarrays. Zinc sulfide nanoparticles showed a unique internal structure with 5nm crystalline domains set in an amorphous matrix and an optical band gap of between 3.88-4.28eV. Nanoparticle arrays were then functionalised with biological ligands, notably antibodies that engage with the NK cell surface receptor CD16, or the T cell TCR/CD3 moiety. The cellular response to these materials was measured, and was sensitive to the nanoscale arrangement of stimulatory ligands; both cell types responded to ligands with 25nm, but not 104nm, inter-ligand spacing. In an alternative approach, spherical PEG hydrogel particles of diameter 5-50μm were formed with controlled rigidity between 3-2000kPa. T cell response as a function of substrate rigidity was tested, and cells showed maximal response to anti-CD3 functionalised substrates with rigidities of 3-5kPa.Open Acces

MicroRNA profile and function in kidney ischaemia and reperfusion injury

PhD ThesisIschaemia and reperfusion injury (IRI) in renal allografts is an important contributing factor to chronic allograft dysfunction. MicroRNAs (miRNA) have been shown to play important roles in cellular adaptation to pathological conditions, including IRI. This study aimed to evaluate changes in miRNA profile following IRI, and how the changes in particular miRNAs may influence renal proximal tubular epithelial cell (PTEC) morphology and function, potentially contributing to the development of chronic allograft dysfunction. To achieve this, several objectives were set. These included: (1) isolation and culture of primary human PTECs, (2) miRNA expression profiling following IRI and selection of candidate miRNA, and (3) in vitro and human pathology validation to explore the molecular mechanism of the candidate miRNA. Primary PTECs were isolated from normal renal tissue. These cells showed features of epithelial cells under light microscope and electron microscope. The cells were also characterised using immunofluorescent staining, which showed positive expression of epithelial cell markers, and negative expression for mesenchymal cell markers. MiRNA profiling using NanoString platform was performed on cell lines (HKC-8 and HK-2) and primary PTECs, which were exposed to either hypoxia or free radicals. Results revealed distinct miRNA signature changes following IRI in cells. However, only a small proportion of microRNAs were found to be significantly up/down-regulated in either cell lines or primary cells, which included miR-21, miR-376, miR-190b, miR-34a, miR-210, miR-363, miR-142 and miR-130b. MiR-21 was shown to be up-regulated in all cells following both type of injuries. Online target prediction analysis also showed miR-21 to be involved in pathways relevant to cellular response to IRI and the development of fibrosis. The role of miR-21 was studied in detail. Up-regulation of miR-21 following ischaemia was shown to suppress SMAD7 and facilitate intra-nuclear localisation of SMAD3. In the presence of exogenous TGF-b1 or hypoxia, over-expression of miR-21 in cells led to an increase in SMAD3 activity. Over-expression of miR-21 also led to phenotypic shift in HKC- 8 cells, characterised as a decrease in E-cadherin and an increase in a-SMA and Collagen-1 expression. Human pathology evaluation confirmed high expression of miR-21 in the tubular cells of severely ischaemic kidneys compared to non-ischaemic kidneys. In conclusion, changes in miRNA profile were observed in acute IRI in the kidney. One of the significantly affected miRNAs was miR-21. MiR-21 up-regulation resulted in sensitisation of tubular cells to TGF-b1, which may be essential in cellular repair processes, but may also contribute to deterioration of long-term organ function.Indonesian Endowment Fund for Educatio

Quantification of cell adhesion strength on artificial surfaces with a microfluidic shear force device

Adhesion strength is a measure to determine the interaction between cells and their environment. Numerous types of devices and coatings are developed in order to meet medical and non medical issues and surface properties can be tuned in order to evoke specific cell response. In this work various properties of solid surfaces were investigated towards their impact on adhesion process and adhesion strength of mammalian cells, which both give information on the cell interaction with the substrate. Therefore a sophisticated assay to observe cell adhesion and measure cell adhesion strength on artificial surfaces was developed. Its capability to measure cell adhesion strength in the order of five magnitudes with a high reliability and quantitative output was applied to synthetic surfaces with different degree of hydration, anisotropic topography, bioactivity and different polarizations. Investigation of fibroblast adhesion on ethylene glycol self assembled monolayers showed that cell adhesion strength is reduced by increasing degree of hydration. At the same time it was found that cell adhesion strength was independent of cell spreading area, in particular when a certain spreading size was reached. This finding may strengthen the zipper detachment mechanism by which the cell detachment occurs after distinct bonds are broken. Another study on hydrogel like polysaccharides confirmed the inability of fibroblasts to attach to hydrated surfaces. At the same time it was found that hematopoietic progenitor cells expressing CD44 receptors overcome the inertness and attach to the coating by shear force induction through a hydrodynamic flow. This finding may explain the fact that the presence of hyaluronan is a prerequisite in the stem cell homing and engraftment process into the bone marrow. Besides receptor ligand interactions more basic surface polarity effects were studied, which have been reported to have a minor impact towards cell adhesion. Here it was shown by investigation of fibroblast adhesion on periodically poled ferroelectric lithium tantalite crystals, that the gradient between two opposite polarities can be sensed by cells but not the polarity itself. The cells do not distinguish the overall polarity of a surface, but avoid placing the nucleus in proximity to the sharp borders in between to inverse polarities as the cells start to spread. Even though this astonishing reaction is unexpected it is not contradictive to the absence of polarity sensing because sensing of a polarity gradient is different from a distinct polarization spread over a large area. Instead of a gradient, anisotropic surface properties can be achieved by directional surface texture. Anisotropically textured poly(p-xylylene) surfaces, which consist of dense packed tilted nanorods, revealed a force directional dependence of fibroblast cell adhesion strength. The hydrodynamic shear force applied with the direction of nanorod tilting revealed a reduced cell adhesion strength compared to force application perpendicular and against the tilting. This finding could be explained by a model which accounts for cell filopodia attaching between nanorods of the surface. In order to prove the filopodia attachment theory more sophisticated imaging, which reveals ultrastructural components, was needed. Therefore, cell preparation protocols were established with special attention to preserve cellular structure to image via X-ray holography under ultrahigh vacuum conditions. The imaging project was conducted in a consortium of researchers and first successful imaging was demonstrated

Domain-sensitive topic management in a modular conversational agent framework

Flexible nontask-oriented conversational agents require content for generating responses and mechanisms that serve them for choosing appropriate topics to drive interactions with users. Structured knowledge resources such as ontologies are a useful mechanism to represent conversational topics. In order to develop the topic-management mechanism, we addressed a number of research issues related to the development of the required infrastructure. First, we address the issue of heavy human involvement in the construction of knowledge resources by proposing a four-stage automatic process for building domain-specific ontologies. These ontologies are comprised of a set of subtaxonomies obtained from WordNet, an electronic dictionary that arranges concepts in a hierarchical structure. The roots of these subtaxonomies are obtained from Wikipedia’s article links or wikilinks; this under the hypothesis that wikilinks provide a sense of relatedness from the article consulted to their destinations. With the knowledge structures defined, we explore the possibility of using semantic relatedness over these domain-specific ontologies as a mean to propose conversational topics in a coherent manner. For this, we examine different automatic measures of semantic relatedness to determine which correlates with human judgements obtained from an automatically constructed dataset. We then examine the question of whether domain information influences the human perception of semantic relatedness in a way that automatic measures do not replicate. This study requires us to design and implement a process to build datasets with pairs of concepts as those used in the literature to evaluate automatic measures of semantic relatedness, but with domain information associated. This study shows, to statistical significance, that existing measures of semantic relatedness do not take domain into consideration, and that including domain as a factor in this calculation can enhance the agreement of automatic measures with human assessments. Finally, this artificially constructed measure is integrated into the Toy’s dialogue manager, in order to help in the real-time selection of conversational topics. This supplements our result that the use of semantic relatedness seems to produce more coherent and interesting topic transitions than existing mechanisms