Dissertations / Theses on the topic 'Cell surfaces'

Cell-substrate interactions are of interest in modern biology. The system of surface bound hydrogels is commonly used as a cell culture surface in the field of cell biomechanics. However, the effect gel geometry (thickness, width and length) has on both the mechanics of the gel and the cells behaviour has usually been ignored. It was discovered that a cell differentially spreads and preferentially accumulates at a certain position with respect to the local variation in thickness along a wedge gel (thickness varied from ~50 to 400 µm). This happened although this range of thickness is supposed to be sufficient to prevent the cells from sensing the underlying rigidity of the supporting glass. The mechanical anisotropy of the gel due to its being bound to the substrate was hypothesised to be the cause of the cell behaviour observed. It was later proven that lateral swelling varies exponentially with thickness. The consequences are the decrease in lateral compression and the lateral dilution of gel network density with increasing gel thickness. Both could cause variations of substrate mechanics. The amount of crosslinker, the geometry of the bound area and type of bathing medium all changed the degree of lateral swelling, and thus are contributing factors influencing the lateral mechanics of the swollen gel. Surface bound square gels (50-2000 μm thick) were found to be stiffer with increasing thickness as measured with an atomic force microscope (AFM). This could be due to a change in osmotic pressure. These indentation based measurements of vertical mechanics might be of little relevance with respect to the cellular response though. This was supported with another set of cell experiments on such samples, where the cell did not respond in accordance with the stiffness as measured by AFM. It was therefore implied that the difference in cell behaviour observed on the substrates of different height might be a result of an interplay between the lateral mechanics and the rate of liquid flow though the gel. The x-, y-aspect ratio was also found to influence cell alignment. Cells tended to align randomly on square (aspect ratio: 1:1), and perpendicular to the direction of the long axis of the gels in high aspect ratio (1:4 – 1:11) gels. This preference could be impaired by inhibition of the interaction between actin and myosin II using blebbistatin treatment. This suggests that actomyosin activity is necessary for such the behaviour. The set of studies stressed the importance of x-, y- and z- macrogeometries of surface bound gels as these factors influence mechanical surface anisotropy. These results could have an implication not only in pure cell biology and cell biomechanics but also in regenerative medicine, physiology, wound healing, embryo development, and oncogenesis, wherever cells are in contact with soft biomaterials or orient themselves with respect to mechanical or other features

Cell attachment is vital for the integration of biomaterials in the body. Surface modification using cell adhesive peptides, such as Arginine-Glycine-Aspartic (RGD), has showed promise for enhancing cell adhesion. Cell adhesion on glass and polyethylene glycol (PEGylated) surfaces modified with active RGD and Proline- Histidine-Serine-Arginine-Asparagine (PHSRN) peptides as well as inactive RDG and HRPSN control peptides was investigated in serum free conditions using three cell lines; NIH3T3 fibroblasts, MC3T3 pre-osteoblasts and C2C12 pre-myoblasts. Peptide attachment to glass surfaces was confirmed by x-ray photoelectron spectroscopy and contact angle measurements. Cell attachment and spreading was equivalent on all peptide and fibronectin coated glass surfaces and was significantly higher than on unmodified glass after 3 hours. Cell attachment to the peptide modified glass was reduced in the presence of soluble RGD and RDG peptides, indicating that cell attachment to these surfaces may be integrin mediated, but not specific for RGD. Inhibition of protein synthesis with cycloheximide revealed that endogenous protein synthesis did not influence the specificity of cell attachment to the peptide modified glass surfaces in all cell types within a 3 hour period. However, cycloheximide treatment inhibited cell spreading on the peptide modified glass surfaces, suggesting that proteins synthesis was required for spreading. Long term adhesion studies, within a 24 hour period, showed that all cell lines were able to remain attached to the peptide modified glass surfaces, while C2C12 and MC3T3 cells were also able to form focal adhesions during this period. Cell attachment to peptide modified PEGylated surfaces over a 3 hour period showed that NIH3T3 and C2C12 cells experienced significantly higher levels of cell attachment on the RGD modified surface compared to the other peptides. MC3T3 cells attached to all the peptide modified PEGylated surfaces to the same extent, suggesting that cell attachment to peptide modified PEGlyated surfaces, can be cell type dependent. In conclusion all the peptides were able to promote cell adhesion on glass surfaces in the absence of a PEG linker. In the presence of a PEG linker cellular response to the peptide surfaces was both peptide and cell type dependent.

The primary objective of this study was to identify, develop and characterise a novel bioactive surface capable of binding hepatocytes and enabling the retention of hepatocyte-specific cell function during in-vitro culture. The materials were designed to exploit a unique characteristic of hepatocyte biology, with β-galactose moieties displayed to allow cellular adhesion via the specific asialoglycoprotein receptors (ASGP-R) found on hepatocytes. Hydrogels were created by modifying a commercially available block co-polymer of polyethylene glycol (PEG) and acrylamide, (PEGA) with galactose moieties contained within lactobionic acid (LA), producing a unique bioactive sugar-based gel. A control sugar, D-glucuronic acid (GA), was used as a non-ASGP-R binding control. Monomers used were mono- and bis-acryloamido PEG (Mw=1900 gmol-1), and dimethylacrylamide. The pendant PEGA amine groups were used as ligands to bind to the sugars. The resultant gels were characterised using Fourier Transform Infrared Spectroscopy (FT-IR), protein adsorption, Fmoc-Phe and dansyl chloride labelling. The biocompatibility of the gel surfaces was evaluated using a hepatocyte cell line and the degree of attachment, proliferation, and morphology was characterised using light microscopy, live/dead assays, DNA assays, immunochemical staining, flow cytometry and reverse-transcription polymerase chain reaction (RT-PCR).FT-IR analysis of LA revealed a distinctive band at approximately 1740cm-1 corresponding to carbonyl stretching (C=O) of carboxylic acid. This unique peak disappeared as the galactose moieties within the LA were incorporated into the PEGA gel. A similar trend was also observed with the control GA sugar within the PEGA gel, confirming that the sugars had been integrated into the material. Protein adsorption assays confirmed the non-fouling nature of PEGA. Cell culture experiments showed that hepatocytes attached preferentially to the sugar surfaces, with few cells seen on the PEGA surfaces. It was observed that cells on the PEGA with LA surface were more metabolically active, than the controls and proliferated to a monolayer by day 7 in culture. Immunocytochemical staining of the cells for actin, vinculin and phosphorylated focal adhesion kinase illustrated differences in cell morphology between cells grown on different surfaces. It was determined that the sugar PEGA surfaces maintained some characteristics of hepatocyte functionality e.g. urea synthesis over the course of 7 days. To improve the reproducibility of the surfaces generated, a preliminary investigation of two-dimensional PEG monolayer surfaces as a well defined platform for surface reactions was conducted. These were chemically functionalised in a stepwise manner with the sugars. The number of coupling steps and the choice of solvent were shown to affect the efficiency of the reaction. Further more, the need for careful sample preparation was highlighted as contamination could potentially inhibit the interpretation of the surface chemistry.The overall conclusion of this work is that saccharides within non-fouling surfaces composed of thin layers of PEG-acrylamide hydrogels are able to support hepatocyte attachment and the retention of cell type specific functions in culture. However, this preliminary work has shown that much further research is necessary to elucidate the role that the surface chemistry plays in the attachment of hepatocytes.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2009.
Cataloged from PDF version of thesis.
Includes bibliographical references (p. 123-130).
Molecular sensors are powerful because they make it possible to adapt the measurement to the sample instead of a sample to an instrument. Many reporter are available for measuring the chemical properties of a sample, but no purpose-built molecular sensors exist to report a sample's mechanical properties. To address recent interest in the mechanical coordinate of molecular interactions, we developed a prototype molecular sensor, calibrated its force-fluorescence relationship, and adapted the sensor to a cell adhesion assay. This thesis focuses on the considerations for combining force measurement with the environmental and distance sensitivity offered by fluorescence to measure cell-surface adhesion. We showed that DNA can be used as a scaffold to build a sensor molecule, that fluorescence can be used as a reporter of a threshold force, and that introducing cells to the sensor molecules changes the fluorescence properties. Because Cy3 experiences an enhanced intensity sensitivity when conjugated to DNA, the reporter's FRET signal was occluded and we instead activated the sensor complex as a novel, all-fluorescent means of reporting cell-surface proximity. This method for reporting cell-surface separation is significant because it simplifies measurements in thicker and more complex materials interesting to cell-substrate interaction studies.
by Mariya Barch.
Ph.D.

The modulation of biological interactions with artificial surfaces is a vital aspect of biomaterials research. Protein adsorption is established as an early biological response to implanted materials that influences biocompatibility, hence an understanding of how to direct specific protein and cellular responses is critical for the development of future biomaterials. The effects of protein adsorption and subsequent cellular interactions on a variety of surfaces are investigated. Acrylic-based hydrogels are used as a model system in which to investigate both tear and serum protein adsorption from simple and complex solutions. The effect of surface topography, created by colloidal silica, on serum protein adsorption and conformation as well as cell adhesion is also investigated. Tantalum (Ta) and oxidised polystyrene (PSox) are investigated for their ability to support cell adhesion when precoated with various serum proteins. Protein interactions are examined using a combination of quartz crystal microbalance with dissipation (QCM-D), surface plasmon resonance (SPR), dual polarisation interferometry (DPI) and enzyme-linked immunosorbent assay (ELISA) while cellular interactions are analysed using QCM-D, microscopy and adhesion assays. The QCM-D technique was evaluated for its ability to provide new insight into cell-surface interactions. Most tear and serum proteins were found to adsorb onto the acrylic hydrogels, however, lysozyme was found to absorb into the hydrogel matrix and decrease the hydration, which may lead to an adverse biological response. Fibronectin adsorbed onto nanotextured colloidal silica surfaces was found to be conformationally changed compared to flat controls which is likely to correlate with the reduced endothelial cell adhesion observed on these textured surfaces. Ta and PSox precoated with either serum or fibronectin were shown to support cell adhesion and spreading, while surfaces precoated with albumin were not. QCM-D responses varied between underlying surfaces, protein precoating, ECM deposition, cytoskeletal activity and length of exposure indicating that alterations in cell-material responses are reflected in QCM-D measurements. QCM-D parameters were found to correlate with adhered cell numbers, cell contact area and cytoskeletal activity. The results highlight that characterisation of interfacial interactions with a wide range of analytical techniques is necessary to gain insight into cell-protein-material interactions which can then be utilised in the development of new generations of biomaterials with improved properties designed for specific applications.

As a self-assembled mimetic structure of biological membranes, polydiacetylene liposomes have been studied for the development of platforms for various applications including nano-containers, nano-transporters, and nano-delivery systems for biological-, life- and materials-science applications. Liposomes incorporating amphiphilic polymer poly(10, 12 pentacosadiynoic acid) (PDA) was used as a building block for investigations mimicking cellular reaction and processes at the membrane cell. Changes in local membrane micro-organization and packing as a result of biomolecular and bioparticle reactions and processes at the liposomal membrane were investigated through the use of colorimetric and emission responses of PDA liposomes in solution phase. My dissertation comprises of six chapters. I provide brief overview of each chapter in the following paragraphs: Chapter 1: Introduction. In this chapter, an introduction is given on structure and function of lipid bilayer and multilayer of liposomes from a perspective of shared features with biological membranes. Amphiphilic molecules along with natural lipids at (or higher) critical micelle concentration self-assemble in aqueous medium, thereby, forming a lipid bilayer or multilayer to reduce the free energy of the system. When one of the components of the lipid bilayer is a polymerizable monomer, micelles/liposomes with enhanced mechanical and chemical stability are achieved. The lipid bilayer of liposomes is a boundary that includes at least three different regions: inside aqueous cavity, hydrophobic membrane zone, and membrane-aqueous interfaces. The membrane surface is available for further functionalization. In general, all three regions of the liposomes are utilized for both fundamental and applied studies. For example, the PDA liposomes have been employed for biosensing, drug/protein/nucleic acid transport and delivery and target release, and various probing cellular-like reactions and processes at the membranes. Here, in this chapter, literature on PDA was reviewed for a time period of 2008-2015. Furthermore, emphasis was given to application of PDA liposomes as (bio) sensing elements utilizing colorimetric, fluorescence, and FRET mechanisms. Chapter 2. Polydiacetylene (PDA) liposomes have been accepted as attractive colorimetric bionanosensors. The molecular recognition elements, either embedded within the liposomal membrane or covalent bound at the membrane surface, are available for interaction with biological and chemical analytes. Usually, PDA liposomes perform transduction activity through perturbation of the conjugated polymer backbone, which provides a colorimetric change in solution or solid-state phase. Here, we report that trapping self-quenched fluorescent specie within inner cavity of the liposomes is a simple and effective analytical tool for evaluating biomolecular binding events at the membrane surface. The release of fluorophores in response to the membrane binding event led to amplified emission signal which was utilized for probing reactions at the membrane surface that mimics reactions occurring at the cellular membrane surface. Specifically, a covalent binding on enzyme-substrate reaction resulted in a change of membrane fluidity, thereby releasing inner fluorophore content of the PDA liposomes. Fluorescent markers were loaded at or higher self-quenched concentration in the cavity of the liposome. Amplification of the fluorescence intensity was positively correlated with the concentration of protein added in the solution. The bilayer fluidity alteration also appears to depend on the molecular weight of the protein bound at the membrane. Overall, binding of protein with membrane promoted changes in the local PDA membrane organization and packing that enhanced the membrane permeability. The encapsulated content therefore leaked through “transient pores” formed in the membrane yielding substantial emission amplification. Chapter 3. Inspired by stability of the PDA liposomes, surface functionalization with a variety of molecules and loading within bilayer and inner cavity of the liposomes, we utilized liposomes as biocatalytical nanoreactors. Removable template molecules were embedded in the lipid bilayer and active protein encapsulated in the internal cavity was used for studying the transport properties of liposomes through substrate-enzyme reactions. Detergent Triton X-100 was used to remove a small portion of lipid and template molecules embedded in the membrane. The removal of lipid/template molecules not only affected the membrane fluidity but also provided transient pores in the membrane, allowing transport of substrate for enzymatic oxidation of glucose and 2-deoxy-glucose. Three important biological-relevant properties of cellular membrane: transport, bioavailability, and bio-reactivity of enzyme and substrate were studied. We found that enzyme molecules retained their reactivity when encapsulated within the aqueous inner cavity of the PDA liposomes, and that their activity was comparable to that in the bulk solution. Chapter 4. This chapter introduces studies on (at least partially) answering important questions how and if anchored enzyme activity at the liposome surface is affected through limited diffusion and spatial constraints. A further crucial question was investigated what effect of protein binding at the surface of the liposomes to enzymatic activity was. These relevant questions were important for increasing our fundamental knowledge related to reactions, interactions, and transport processes in biological cellular systems. A functionalized liposome system containing enzyme (Trypsin) covalently attached at the PDA liposome surface was synthesized. Using PDA liposomes as an immobilization scaffold, we evaluated and compared the cleavage behaviors of Trypsin in either immobilized at the membrane surface or in the free form. The covalent binding interaction and tryptic cleavage at the membrane-water interface was monitored by UV-vis and fluorescent spectroscopy, fluorescent anisotropy and spectro-micro-imaging. Trypsin binding at the membrane appeared to be significantly affected the enzymatic activity of the bound enzyme as seen from colorimetric response of the PDA liposomes. Chapter 5. Hierarchical structures support structures with new functionalities, therefore, advances in fabrication and characterization of biomimetic systems based on biological building blocks may present substantial potential rewards in material science. We take advantage of non-covalent forces known in biology for creating spatial organization by assembly tobacco mosaic virus-liposome polymeric hierarchical systems through biotin-streptavidin linkages. The advantage of using the biological thin rods such as TMV is that it can span the whole liposomal membrane allowing us to create microscopic hinge structures that connected liposomes. Our findings through electron and fluorescence microscopy confirmed that SA-TMV motif was able to stay inserted within the lipid bilayer of liposomes which yielded hierarchical structures after binding with Bt-liposomes. These hierarchical structures may find potential applications in targeted load (drug/protein/DNA) delivery, investigations involving virus-cell interactions, and sensing of virus particles. Chapter 6. Conclusions and Future work The present work in this dissertation utilized exploitation of biological self-assembly of small lipid molecules and larger biological-like motifs for enhancing our understanding of reactions and processes occurring at the cellular membrane surface. Overall the following four major studies were accomplished; 1. Sensing through amplified delivery, 2. Triggering an encapsulated bioreactor system at nanometric size, 3. Holding active biological elements when liposomes perform an attachment matrix, 4. Formation of hierarchical structures promoted by self-assembling of biological motifs with mimickers of cell membrane From our findings by mimicking the lipid bilayer of cell structures through liposomal membrane future work holds different ways to contribute in enhancing fundamental understanding of biological behavior. Active transport is an important function of all natural cells, playing important roles in intercellular communication. Liposomes composed of natural and polymerizable lipids may allow investigation involving exocytosis, formation of filopodia, vesicle fusion, budding and reproduction of neural synapses. Our liposome system may also mediate a broader range of highly selective and sensitive detection and sensing of cellular reactions and processes in physiological condition. I hope that this work in collaboration with multiple PIs will contribute to the fields at the interface of biology and material science.

The cardiovascular system of bilaterians developed from a common ancestor. However, no endothelial cells exist in invertebrates demonstrating that primitive cardiovascular tubes do not require this vertebrate-specific cell type in order to form. This raises the question of how cardiovascular tubes form in invertebrates? Here we discovered that in the invertebrate cephalochordate amphioxus, the basement membranes of endoderm and mesoderm line the lumen of the major vessels, namely aorta and heart. During amphioxus development a laminin-containing extracellular matrix (ECM) was found to fill the space between the basal cell surfaces of endoderm and mesoderm along their anterior-posterior (A-P) axes. Blood cells appear in this ECM-filled tubular space, coincident with the development of a vascular lumen. To get insight into the underlying cellular mechanism, we induced vessels in vitro with a cell polarity similar to the vessels of amphioxus. We show that basal cell surfaces can form a vascular lumen filled with ECM, and that phagocytotic blood cells can clear this luminal ECM to generate a patent vascular lumen. Therefore, our experiments suggest a mechanism of blood vessel formation via basal cell surfaces in amphioxus and possibly in other invertebrates that do not have any endothelial cells. In addition, a comparison between amphioxus and mouse shows that endothelial cells physically separate the basement membranes from the vascular lumen, suggesting that endothelial cells create cardiovascular tubes with a cell polarity of epithelial tubes in vertebrates and mammals.

The cardiovascular system of bilaterians developed from a common ancestor. However, no endothelial cells exist in invertebrates demonstrating that primitive cardiovascular tubes do not require this vertebrate-specific cell type in order to form. This raises the question of how cardiovascular tubes form in invertebrates? Here we discovered that in the invertebrate cephalochordate amphioxus, the basement membranes of endoderm and mesoderm line the lumen of the major vessels, namely aorta and heart. During amphioxus development a laminin-containing extracellular matrix (ECM) was found to fill the space between the basal cell surfaces of endoderm and mesoderm along their anterior-posterior (A-P) axes. Blood cells appear in this ECM-filled tubular space, coincident with the development of a vascular lumen. To get insight into the underlying cellular mechanism, we induced vessels in vitro with a cell polarity similar to the vessels of amphioxus. We show that basal cell surfaces can form a vascular lumen filled with ECM, and that phagocytotic blood cells can clear this luminal ECM to generate a patent vascular lumen. Therefore, our experiments suggest a mechanism of blood vessel formation via basal cell surfaces in amphioxus and possibly in other invertebrates that do not have any endothelial cells. In addition, a comparison between amphioxus and mouse shows that endothelial cells physically separate the basement membranes from the vascular lumen, suggesting that endothelial cells create cardiovascular tubes with a cell polarity of epithelial tubes in vertebrates and mammals.

Cell adhesion is the first step of cell response to materials and the extracellular matrix (ECM), and is essential to all cell behaviours such as cell proliferation, differentiation, migration and apoptosis for anchor-dependent cells. Therefore, studies of cell attachment have important implications to control and study cell behaviours. During many developed techniques for cell attachment, the manipulation of surface chemistry is a very important method to control initial cell attachment. To control cell adhesion on a two-dimensional surface is a simple model to study cell behaviours, and is a fundamental topic for cell biology, tissue engineering, and the development of biosensors. From the engineering point of view, the preparation of a material with controllable surface chemistry can help studies of cell behaviours and help scientists understand how surface features and chemistry influence cell behaviours. During the fabrication, the challenge is to create a surface with heterogeneous surface properties in the micro scale and subsequently to guide cell initial adhesion. In order to control cell adhesion in a spatial and temporal manner, a photochemical method to control surface chemistry was employed to control the surface property for cell adhesion in this project. Two photocleavable derivatives of the nitrobenzyl group were tried on two types of surfaces: a model self-assembled monolayer (SAM) with alkanethiol-gold surface and biodegradable chitosan. Reactive functional groups on two different surfaces can be inactivated by covalent binding with these photocleavable molecules, and light can be further introduced into the system as a stimulus to recover their reactivity. By simply applying a photomask with diffe

The objective of this thesis was to investigate and characterize the interaction between blood proteins and different surfaces with emphasis on protein adsorption to bioceramics and model surfaces. Special effort was made to monitor the spontaneous and selective adsorption of proteins from human plasma and to examine the orientation, conformation and functional behavior of single proteins after adsorption.

Five different ceramic biomaterials: alumina (Al₂O₃), zirconia (ZrO₂), hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂) and two glass-ceramics, AP40 (SiO₂-CaO-Na₂O-P₂O₅-MgO-K₂O-CaF₂) and RKKP (AP40 with Ta₂O₃-La₂O₃), were exposed to human plasma and their protein binding capacities and affinities for specific proteins were studied by chromatography, protein assays, two-dimensional gel electrophoresis and Western blotting. The studies showed that all materials adsorbed approximately the same high amount of plasma proteins and that they therefore should be fully covered by proteins in an in vivo setting. The adsorbed proteins were different for most materials which could explain their previously observed different levels of tissue integration in vivo.

Four of the proteins that behaved differently, ceruloplasmin, prothrombin, α₂-HS-glycoprotein and α₁-antichymotrypsin, were selected for characterization with atomic force microscopy and ellipsometry. The studies, which were performed on ultraflat silicon wafers (silica), showed that the proteins oriented themselves with their long axis parallel to the surface or as in case of ceruloplasmin with one of its larger sides towards the surface. All of them had globular shapes but other conformational details were not resolved. Furthermore, prothrombin (none of the others) formed multilayers at high proteins concentrations.

The functional behaviour of the adsorbed proteins, referring to their cell binding and cell spreading capacity on silica and a positive cell adhesion reference surface (Thermanox®), was affected by the underlying substrate. Ceruloplasmin, α₂-HS-glycoprotein and α₁-antichymotrypsin stimulated cell attachment to silica, but suppressed attachment to Thermanox®. Prothrombin stimulated cell attachment to both surfaces. The attachment was in most cases mediated both by cell membrane-receptors (integrins) and by non-specific interactions between the cell and the material.

This thesis showed that the compositional mixture, orientation, conformation and functional behavior of the adsorbed proteins are determined by the properties of the underlying surface and if these parameters are controlled very different cellular responses can be induced.

Embryonic stem cells are of great interest to scientists as they can differentiate into any somatic cell lineage making them excellent candidates for tissue regeneration and cell based treatment therapies. Currently, human embryonic stem cells (hESCs) are cultured using feeder fibroblasts or protein substrates such as matrigel, fibronectin or laminin in conditioned media. hESCs are then subcultured using enzymes to detach them from the culture substrate. However, the use of the xenosupport systems makes the hESCs therapeutic applications difficult due to cross-contamination with animal pathogens from the animal derived feeders, matrix or conditioned media to the hESCs. Moreover, the use of enzymes to recover hESCs can damage these cells. For the mentioned reasons, development of completely synthetic surfaces is desirable for hESCs culture. Thermo-responsive surfaces have been extensively studied for cell culture using the well know thermo-responsive polymer poly (N-isopropylacrylamide) (PNIPAAM) which has a switchable properties across its lower critical solution temperature (LCST) at 32°C. However, more biocompatible polymers that have similar thermo-responsive properties to PNIPAAM and biocompatible properties, such as PEG based materials, have been proposed for use as switchable surfaces. Within this thesis, thermo-responsive copolymer brushes composed of 2-(2- methoxyethoxy) ethylmethacrylate (ME02MA) and oligo (ethylene glycol) methacrylate (OEGMA) that have LCST close to body temperature (37°C) were investigated for use as a cell culture surface for temperature sensitive passaging. Poly (ME02MA-co-OEGMA) brushes were grafted from the surface using atom transfer radical polymerisation (ATRP). Hydroxyl plasma-polymer functionalised glass slides were prepared using plasma polymerisation and then an ATRP initiator was reacted to these surfaces. ATRP of the copolymers was then performed from these initiation sites. These surfaces were characterised using X-ray photoelectron spectroscopy (XPS), Time of Flight Secondary Ion Mass Spectroscopy (ToF-SIMS), atomic force microscopy (AFM) and water contact angle (WCA). Using 3T3 fibroblasts as a model cell type for initial studies, it was demonstrated that these cells adhered and proliferated on the poly (ME02MA-co-OEGMA) thermo-responsive surfaces at 37°C when the polymer brushes are in their hydrophobic state. Subsequent detachment assays were conducted when the temperature was lowered to 20°C i.e. the hydrated conformation of the copolymer brushes. Mouse embryonic stem cells (mESCs) were then cultured on these surfaces following adsorption of fibronectin (to encourage cell attachment) and cultured for 3 days. Passaging experiments were performed for 10 passage cycles and the cells analysed for retention of the undifferentiated stem cell status. These thermoresponsive polymer/fibronectin surfaces are found to be suitable for mESCs culture but evidence of differentiation was observed. Attachment of a novel gelatine based peptide to the poly (ME02MA-co-OEGMA) was also investigated for mESCs culture to avoid the use of fibronectin (which was thought to be a contributing factor to the stem cell differentiation seen). mESCs adhesion was observed both to the peptide adsorbed on TCPS and on the peptide coupled to the thermo-responsive poly (ME02MA-co-OEGMA) surface. This research indicates that these smart stimuli biomaterials have promise as a new generation of culture surfaces for enzyme free cell culture and passage suitable for generating cell populations for clinical applications.

Enzymes are promising stimuli for the development of responsive biomaterials for biomedical applications. Enzymes are inherently present in the biological environment thus cleverly designed materials for biomedical applications may require no external stimuli to ellicit the required material response. They have been targeted as stimuli in self assembly of bulk materials owing to the material changes in chemical composition afforded by the enzyme interaction. The first examples of autonomous self-regulated drug delivery systems have been reported via the development of reversible enzyme responsive materials that undergo a material change regulated by the enzymes in their environment. Although enzyme responsive surfaces have been reported there are no examples of reversible enzyme response surfaces. The surface is the first point of contact between the biological environment and a biomedical device/implant. Improving this interaction will improve the integration of these biomaterials in biological systems and it has been proposed that biomimetic surfaces are a promising method for full biomaterial integration in the biological environment. The body strives towards homeostasis and this is frequently achieved by enzymatic activating and deactivation of proteins in the body. This process is repeatable and reversible. Herein we address the absence of reversible and repeatable synthetic enzyme responsive surfaces towards the improvement of biomaterial integration. We aim to develop a truly autonomous system wherein enzymes present in the environment can interact with the modified surface to mediate a reversible material response. This goal was achieved by modifying surfaces with copolymers that contain the recognition sequence for Casein kinase II and Alkaline phosphatase to undergo enzymatic phosphorylation and dephosphorylation. Co and homo polymers of serine and glutamic acid were synthesised in solution and conformation/composition relationship was determined by analysis with NMR, GPC and FTIR. Polymerisation from the surface with NCA-Glu and NCA-Ser was achieved as characterised by FTIR, ToF SIMS, XPS and WCA. Enzymatic mediated phosphorylation (CKII) and dephosphorylation (AP) was monitored by surface analysis (ToF SIMS), by monitoring ATP to ADP conversion and phosphate cleavage from the surface using luminescence and colorimetric assays. Conformational changes mediated by enzymatic interactions with the surface was monitored indirectly using a FRET system incorporated in the surface modification. The modified surfaces were able to support cell culture and osteogenesis. This project has made advances in several fields, 1) The use of NCA-ROP as a method to modify surfaces with copolymers, in particular for a random/ alternating amino acid sequence. 2) The use of NCA-ROP as a method to develop stimuli responsive surfaces, specifically, this is the first report of an enzyme responsive surface prepared from NCA-amino acid derivatives. 3) The use of enzymes as stimuli, specifically, this is the first report of a reversible enzymatic responsive surface. In this system reversible phosphorlyation and dephosphorylation was monitored via changes in fluorescence output indicative of induced conformational changes.

In the field of bioelectronics various electronic materials and devices are used in combination with biological systems in order to create novel applications within cell biology and medicine. A famous example of a successful bioelectronics application is the pacemaker. Metals are the most common electrical conductors, whereas polymers are generally considered being insulators. However, in the late 1970s it was shown that a special class of polymers with conjugated double bonds, could in fact, after some chemical modifications, conduct electricity. This was the start of the research field known as conducting polymers, and then later on organic electronics, a research area that has grown rapidly during the last decades. Conjugated polymers are also suitable to interact and interface with cells and tissues, as they are generally soft, flexible and biocompatible. In addition, their chemical properties can be tailor-made through synthesis to fit biological requirements and functions. During the last years applications using organic bioelectronics have become numerous. This thesis describes applications based on different conjugated polymers aiming to stimulate and control cell cultures. When culturing cells it is of interest to be able to control events such as adhesion, spreading, proliferation, differentiation and detachment. First, the impact of different polymer compositions and redox states on the adhesion of bacteria and subsequent biofilm formation was investigated. Similar polymer electrodes were also used to steer differentiation of neural stem cells, through changes in the surface exposure of a relevant biomolecule. Controlled delivery of molecules was achieved by coating nanoporous membranes with polymers that swell and contract when changing the redox state. Finally, electronic control over cell detachment using a water-soluble polymer was achieved. When applying a positive potential to this polymer, it swells, cracks and finally detaches, taking the cells that was cultured on top along with it. Together, the work and results presented in this thesis demonstrate a versatile conjugated polymer technology to achieve electronic control of the different growth stages of cell cultures as well as cellular functions.

Over millions of years evolution has optimized the properties of materials via natural selection for many specific purposes. Indeed, natural materials have unique properties which come very close to perfection. Cells, for instance, are able to carry out intricate sequences of chemical reactions that are difficult or impossible to carry out ex vivo, cell membranes are the most complex selective and responsive semipermeable membranes that exist, and animal shells exhibit a clever nanostructure that renders them hard and tough at the same time. In short, materials scientists can learn a lot from nature’s materials. The perfection and performance of nature’s materials not only spark fascination, but also trigger the question as to why certain structures or surfaces exhibit outstanding properties and inspire research towards new materials. While the materials of living nature impressively serve dedicated purposes, they are formed under restricted conditions. For instance, they have to be designed to function under a narrowly defined set of physiological conditions, and can only be composed of building blocks an organism has available. Without these restrictions, material scientists can design entirely new materials or surfaces.

Photochemical control of cell adhesion onto surfaces, a process commonly achieved with caged molecules, has become an important technique in the fabrication of cellular assemblies for biosensors, tissue engineering applications, and studies for cell-cell and cell-substrate interactions. The ability to study cell behaviour at a biomaterial surface requires control of material surface chemistry. In this study the RGD (Arg-Gly-Asp) and RGE (Arg-Gly- Glu) peptide motifs were immobilized by conjugation to surface bound azides to afford cell-receptive modified surfaces. The peptides were synthesized using Frnoc- solid phase peptide synthesis (SPPS) and purified and characterized by high performance liquid chromatography (HPLC). The modified surfaces were characterized using X-ray photoelectron spectroscopy (XPS) and the effect of these surfaces on fibroblast adhesion and spreading were examined at several time points. Our results demonstrate a higher degree of cell attachment and spreading on RGD modified surfaces compared to unmodified and control surfaces. Dynamic control over the interactions between the cells and artificial substrate was investigated by protecting the side chain of the aspartate residue in the RGD peptide with a photolabile protecting group. Combination of the immobilisation techniques, described alongside in situ uncaging (UV light 365nm) has allowed us to develop a photo-addressable surface that will facilitate surface patterning of cell adhesion.

The research presented in this thesis investigates the potential for sputter deposited titanium and calcium phosphate thin films, singularly and in combination, to directly induce osteogenic differentiation in human bone marrow derived mesenchymal stem cells (MSCs). The nature and scale of these substrate driven effects have been compared with osteogenesis induced in MSCs by exposure to biochemical stimulants in the culture media. MSCs hold great promise for use in bone tissue engineering applications, therefore the provision of biomaterials that can direct the osteogenic differentiation of MSCs has significant implications for improving the repair or replacement of damaged or lost bone tissue. RF magnetron sputter deposition was used to create titanium and calcium phosphate thin films from titanium metal and hydroxyapatite powder targets respectively. The distinct chemical and topographical surface properties of each substrate were confirmed using XPS, FTIR, XRD, AFM and water contact angle analysis. The commercially obtained MSCs used for this work were characterised in detail to provide a benchmark data set for correlation with subsequent thin film substrate studies. To determine the previously unreported effects that these types of sputter deposited surfaces have on MSC behaviour, a range of relevant biological assays and detailed quantitative real time PCR gene expression studies were used. In this way, a comprehensive profile of MSC response to each type of surface condition was obtained over an in vitro culture period of up to 28 days. Early stage adhesion and morphology was also examined in order to attain a better understanding of the underlying mechanisms of the attendant MSC behaviour. All of the sputter deposited thin film surfaces directly promoted significant levels of osteogenic differentiation, to varying degrees, without the use of biochemical stimulants. This work shows for the first time, that the topography of the sputtered titanium coatings and the bioactive chemistry of the calcium phosphate coatings can individually direct the differentiation of MSCs towards an osteogenic lineage.

Polymeric hydrogels were used to create bio-smart hydrogels serving multifunctional roles interfacing with cells and enzyme substrates. Their value lies in their use as: i) Stimuli- responsive membranes that directly transmute chemical potential energy into proportionate electrical signals, ii) as biomimetically inspired biocompatible coatings on stents and other implantable bionic devices, iii) as bio receptor hosting membranes for enzyme-based implantable biosensors. Biosensors use oxidoreductase enzymes such as glucose oxidase (GOx) and lactate oxidase (LOx) to confer specificity. Such enzymes may initiate more complex in vivo inflammatory response. In this thesis individual and combined effects of different enzymes (GOx, Superoxide dismutase (SOD), and catalase) were studied to achieve hydrogelenzyme systems, which in theory may mitigate against adverse cell outcomes. The incorporation of enzymes into bioactive hydrogels was investigated, and revealed effects on the growth, viability and attachment of surface dependant RMS13 human muscle fibroblasts and B50 rat neuronal cells. Agarose and p(HEMA)-based hydrogels were prepared with fibrinogen 5% (w/v) to promote integrin-mediated cellular attachment and also with different combinations of glucose oxidase (GOx), catalase (CAT) and superoxide dismutase (SOD). Cell viability was maintained best on catalase hydrogels. The presence of GOx within hydrogels membrane compromised cell viability in both hydrogel types, presumably due to accumulation of H2O2 confirmed by amperometric detection using fabricated platinum needle electrodes. Hydrogels prepared with GOx and CAT showed improved cell viability, further suggesting the negative influence of H2O2. High temperature treatment of the enzyme-hydrogel membranes, resulting in enzyme denaturation, returned all constructs to control levels of viability, confirming the relationship of cell viability with enzyme activity. An additional study was undertaken into the viability and growth of B50 cells on crosslinked protein membranes of fibrinogen and albumin as a potential bioreactor surface. The use of crosslinked fibrinogen to facilitate cell growth within microfluidic channels appears to have been realized. Fabrication and use of miniaturized gold-filled silica recess and inlaid disc electrodes, compared with the use of agarose gels in the recesses was investigated to improve stabilization of an amperometric H2O2 electrode. From this, a microfluidic device with an integrated inner diameter working and counter / reference electrode was fabricated which showed feasibility of more rapid amperometric detection of H2O2 in miniature flow channels.

Thesis (Ph. D. in Medical and Electrical Engineering)--Harvard-MIT Program in Health Sciences and Technology, 2012.
Cataloged from PDF version of thesis.
Includes bibliographical references (p. 108-114).
Adhesion-based cell capture on surfaces in microfluidic devices forms the basis of numerous biomedical diagnostics and in vitro assays. Solid surface microfluidic platforms have been widely explored for biomedical diagnostics since samples can be precisely and reproducibly manipulated under well-defined physicochemical conditions. However, at these small length scales, the fluid dynamics are dominated by the high surface-to-volume ratio and interfacial phenomena limiting device performance at high flow rates. In contrast, cell homing to porous vasculature is highly effective in vivo during inflammation; stem cell trafficking and cancer metastasis. In this work, we demonstrate that fluid-permeable surface functionalized with cell-specific antibodies can promote efficient and selective cell capture in vitro. This architecture might be advantageous due to enhanced transport due to fluid field modification leading to diverted streamlines towards the surface. Moreover, specific cell-surface interactions can be promoted due to reduced shear, allowing gentle cell rolling and arrest. Together, these synergistic effects enable highly effective target cell capture at flow rates over an order of magnitude larger than existing devices with solid surfaces. Additionally, in this study, we overcome a major limitation relevant to porous surfaces due to formation of stagnant layers of cells from non-target background population. These stagnant layers are detrimental to device performance as they act to reduce interaction of the cells with the reactive surface thereby reducing capture efficiency. We theoretically and experimentally understand the mechanisms for formation of the stagnant bioparticle layer in microfluidic devices and define a parameter space for optimal operation of the device over long periods of time. Key insights from these studies, collectively allow us to design a spatially modified microfluidic devices that allow us to isolate cancer lines as low as 5 cells/mL spiked into buffy coat.
by Sukant Mittal.
Ph.D.in Medical and Electrical Engineering

It is thought that stem cells hold promise for use in future therapeutics. One such application is tissue engineering (TE) which aims to repair or replace diseased or damaged organs in vitro. Successful applications of TE, where the tissue is replaced and is functional, could improve a patients’ quality of life. Mesenchymal stem cells (MSCs) are a form of adult stem cell that are a precursor for fat, cartilage and bone cells. Bone is the second most transplanted tissue after blood therefore, enabling TE strategies through provision of high quality bone cells to facilitate bone repair would be beneficial. As MSCs are a precursor to bone, their use is attractive. Additionally, their proliferative potential and immunoregulatory properties make MSCs an ideal candidate cell for TE. MSCs require behavioural cues in vitro that direct phenotype in a targeted way. One method to direct stem cell behaviour is to utilise materials engineering. Static materials (examples include topography, chemistry and stiffness) have been employed but research has now moved towards stimuli responsive technologies to provide dual functionalities for culture and that emulate the properties of the stem cell niche. It is the intention of the work described in this thesis to utilise an enzyme responsive technology to promote MSC self-renewal and stimulate MSC differentiation to bone. Using solid phase peptide synthesis (SPPS) a biomimetic enzyme responsive material was made with the sequence PEG-GPAG↓LRGD tethered to a glass coverslip. Due to enzyme action on the sequence, the PEG cap is removed to create on demand adhesion to the peptide RGD. Further, the surface is designed to be under the control of cell secreted enzymes, rather than in response to enzymes added in by the user. The cell secreted enzymes that were investigated for this thesis were the matrix metalloproteases (MMPs). Here we confirm that the primary MMP secreted by MSCs was the gelatinase MMP-2 and a peptide sequence was designed to be cleaved by this MMP. It is known that redundancy can occur in MMP families and the role of MMP-9 was also investigated. The results show that MMP-9 is as efficient for surface cleavage, although cell supernatant concentration was 100-fold lower. MMP-2 concentration increased at week 3 specifically in response to peptides and so formed the original hypothesis that cleavage occurred at that time point. However, due to the potency of MMP-9 this may not be the case. Due to the limitations of manual synthesis and availability of materials, there was not enough evidence of MSC self-renewal. Further there was some indication of osteogenesis, specifically in response to the sequence at 4-6 weeks, however this is too long in culture to be therapeutically relevant. It may be better in the future to employ an enzyme responsive surface that can guarantee 100% efficiency of cleavage to ensure a synchronised population of end terminal cells.

A major drawback of cartilage tissue engineering is that human mesenchymalstem cells (hMSCs) from osteoarthritic (OA) patients express high levels of typeX collagen. Type X collagen is a marker of late stage chondrocyte hypertrophy, linked with endochondral ossification. It has been shown that a novel plasmapolymer, called nitrogen-rich plasma-polymerized ethylene (PPE:N), is able to inhibit type X collagen expression in committed MSCs. The aim of this study was to determine if the decreased expression of type X collagen, induced by PPE:N, is maintained when MSCs are transferred to pellet cultures, an arrangement of cells which mimics embryonic condensation of mesenchymal stem cells, which results in prehypertrophic chondrocytes. hMSCs were obtained from the bone marrow of donors undergoing total hip replacement for OA. hMSCs were cultured on polystyrene dishes and on two different PPE:N surfaces: high (H) and low (L)pressure deposition for 7 days. Cells were transferred for 7 additional days in serum free media in pellet culture. RNA was extracted using a standard TRIzolprotocol. RNA was subjected to RT-PCR using primers specific for type I and X collagen. As observed in previous studies, type X collagen mRNA level was suppressed when cultured on both H- and L-PPE:N. HPPE:N was more effective in decreasing type X collagen expression than LPPE:N. Results also showed that the decreased type X collagen expression was maintained when cells were removed from the PPE:N surfaces and transferred to pellet cultures. Culturing hMSCs from OA patients on PPE:N surfaces and in pellet culture had however no effect on the level of type I collagen mRNA. The present study confirmed the potential of PPE:N surfaces in suppressing type X collagen expression in hMSCs from OA patients. More importantly, when these cells are transferred to pellet cultures, type X collagen suppression is maintained. These results show a promising future for tissue engineering using autologous hMSCs.
Un inconvénient majeur de l'ingénierie tissulaire du cartilage est dû aux niveaux de collagène de type X élevés exprimés dans les cellules souches mésenchymateuses (CSM) des patients arthritiques. Le collagène de type X est un marqueur de l'hypertrophie avancée des chondrocytes, qui est lié à l'ossification endochondrale. Il a été démontré que l'expression du collagène de type X par les CSM différenciées peut être inhibée en présence des polymères plasma enrichies d'azote (PPE:N, nitrogen-rich plasma-polymerized ethylene). Le but de cette étude était de déterminer si la diminution de l'expression du collagène de type X, induite par PPE:N serait maintenue lorsque les CSM sont transférée dans des cultures culots. Les dernières sont un arrangement de cellules mimant la condensation embryonique des CSM résultant ainsi en des chrondrocytes prehypertrophiques. Les CSM ont été obtenus à partir de la moelle osseuse des donneurs subissant une arthroplastie totale de la hanche. Les CSM ont été cultivées sur des boîtes de polystyrène, ainsi que sur deux surfaces de PPE:N différents; à haute (H) et à faible (L) pression de dépôt pendant 7 jours. Les cellules ont été transférées pour 7 jours supplémentaires dans des milieux sans sérum dans la culture culot. L'ARN a été extrait selon un protocole standard TRIzol. L'ARN a été soumis à la RT-PCR avec des amorces spécifiques pour les collagènes de type I et X. Tel qu'observé dans des études antérieures, la quantité de ARNm de collagène X été supprimée lorsque les CSM était cultivées sur les surfaces HPPE:N et LPPE:N. Le HPPE:N était plus efficace pour diminuer l'expression de collagène de type X que le LPPE:N. De plus, la diminution d'expression du collagène de type X a été maintenue bien que les cellules ont été retirées de la surface PPE:N et transférées à des cultures culots. Cependant, le niveau de ARNm du collagène de type I récupéré dans les patients atteints d'arthrose n'a pas été influencé par les surface PPE:N, ni par la culture culot. Cette étude a établie le potentiel des surfaces PPE:N en supprimant l'expression de collagène de type X par les CSM chez les personnes souffrant d'arthrose. Plus important encore, lorsque ces cellules sont transférées à des cultures culots, la suppression du collagène de type X est maintenue. Ces résultats montrent un avenir prometteur pour l'ingénierie tissulaire en combinaison avec les CSM autologues.

Tissue transglutaminase (TG2) is a widely distributed, protein-cross-linking enzyme that creates high-molecular weight polymers from its substrate proteins. Its substrates in bone are mainly matricellular adhesion proteins: fibronectin (FN), osteopontin (OPN) and bone sialoprotein (BSP), which have been localized to the osteoid and to the pericellular matrix of osteocytes. The aim of this study was to compare in vitro the effects of monomeric versus polymeric FN, OPN and BSP on MC3T3-E1/C4 osteoblast adhesion, proliferation and differentiation. We showed that for each of the three TG2-mediated protein polymers tested, a significant increase in cell adhesion was observed. Also when observed by phase-contrast microscopy, the morphology of the cultured cells demonstrated increased cell spreading on polymerized protein.
Our results show that FN, OPN and BSP, when polymerized by TG2, significantly increase cell adhesion and decrease proliferation of MC3T3-E1/C4 osteoblasts without affecting their ability to differentiate. Decrease in proliferation appears to be modulated by beta1 integrin possible affecting beta 5 activity and its translocation to cell surface. We hypothesize that OPN and BSP polymers aid the maintenance of non-proliferatioe state of osteocytes in bone.

Although the growth of native corneal epithelial cells over the anterior surface of an artificial corneal implant is desired, the growth of these cells on the interface located between the implant and the stromal layer of the host eye tissue (i.e. epithelial cell downgrowth) poses a significant problem to be overcome in developing a suitable implant. In this study the growth factor surface modification of a polymer substrate was examined as a means of inhibiting the proliferation of corneal epithelial cells while promoting corneal stromal cell growth. Two separate studies were conducted in which transforming growth factor-beta1 (TGF-beta1) and transforming growth factor-beta2 (TGF-beta2) respectively, were immobilized via a bifunctional poly (ethylene glycol) (PEG) spacer, MW 3400, to poly(dimethyl siloxane) surfaces (PDMS) that had been aminated by the plasma polymerization of allylamine. The modified surfaces were chemically and biologically characterized. The effect of the surface modification with TGF-beta1 and TGF-beta2 respectively, on interactions with corneal epithelial and corneal stromal cells was examined using in vitro cell culture. (Abstract shortened by UMI.)

Cells in tissues are exposed to extracellular signals that integrate and appropriately translate into specific responses. Receptors at the cell membrane recognize a variety of soluble ligands, extracellular matrix proteins and molecules presented by the neighboring cells. Ligand-receptor recognition event triggers intracellular signal transduction pathways modulating the resulting cell function. Some receptors do not function individually as signaling units but require interactions and associations with other receptors in multimolecular complexes. This process is known as receptor clustering and is an evolutionarily preserved mechanism responsible for the integration of highly complex signals. Increasing evidences suggest that this exceptional integration is subjected to spatially controlled ligand distribution at the nanoscale. Recent developments in highly sophisticated nanofabrication approaches have allowed to experimentally address this detailed spatial regulation on cell signaling. However, it is still unclear how the nanoscale distribution of ligands can impact on the dynamics of receptor activation and signaling processes. Herein we present a nanostructured platform to create patterns of ligands in regular nanosized (< 30 nm) clusters. We based our platform in self-assembled diblock copolymers composed of poly(styrene) (PS) and poly(methyl methacrylate) (PMMA) that tend to segregate into nanodomains. The hexagonal arrangement of the PMMA domains acts as template to be replicated by the ligand distribution. Thanks to the versatile functionalization strategy developed, any amine-bearing molecule can be covalently immobilized. The spatial distribution of ligand was analyzed by Atomic force microscopy (AFM) and stochastic reconstruction microscopy (STORM), unveiling the high level of fidelity between the nanopatterned ligands and the underlying polymeric template. To validate these substrates as platforms for systematic study of receptor clustering processes, an adhesive peptide which promotes focal adhesion formation, was immobilized on the nanopatterned surfaces. While the overall ligand surface density was maintained constant, the spatial distribution of ligands showed a remarkable impact on focal adhesion formation. Cells on nanopatterns showed an increased number of focal contacts, which were, in turn, more matured than those found in cells cultured on randomly presenting ligands. These findings suggest that ligand presentation in a clustered format might promote multivalent ligand-receptor interactions which can help to shed light on receptor oligomerization processes. In addition, the nanopatterned substrates developed were used to investigate the dynamics of the process of Eph receptor assembly into oligomeric clusters upon stimulation with ephrin ligands. It is known that Eph receptor oligomer composition is crucial in the fine-tuning of receptor signaling, as it will trigger intracellular signals feedback which will modulate cell response. Oligomerization processes, which imply resolving the temporal evolution of nanometric size objects in diffusive environments such as cell membranes are beyond the reach of live-imaging tools. We in here resolve the oligomerization kinetics of the Eph receptor in live cells with the required spatial and temporal resolution by using an enhanced version of the Number and Brightness (eN&B) technique, which can discriminate with molecular sensitivity the oligomeric species. The results demonstrated that stimulation through surface-bound ligands with a random distribution was not sufficient to activate the receptor signaling. Conversely, when nanopatterned on our substrates, ligands effectively induced receptor oligomerization. In addition, surface-induced ligand clustering by our nanopatterning approach accelerated the dynamics of receptor oligomerization process when compared to antibody-induced ligand clustering. Such an efficiency was induced even when ligand surface coverage was 9-fold lower in the nanopatterned presentation. Therefore, our ligand presenting platform is thought to induce multivalent ligand-receptor interactions, and might be a useful strategy to precisely tune and potentiate receptor responses. It has promising applications in biotechnology and biomedicine, such as cell culture systems to provide insight into relevant receptor clustering processes and design of new bioactive materials and drug-delivery systems.
En los tejidos, las células reciben múltiples señales tanto de naturaleza física como química del entorno que las rodea. Inmersas en un entorno tridimensional, las células interactúan entre sí y con la matriz proteica que las envuelve. Además, hasta ellas difunden diversos factores solubles que transmiten señales químicas revelantes implicadas en el correcto funcionamiento celular. Ante tan complejo entorno, las células son capaces de reconocer de manera diferencial los estímulos que reciben y responder a todos ellos a través de complejos mecanismos intracelulares de señalización. Recientemente, se han desarrollado herramientas altamente sofisticadas que permiten estudiar el comportamiento celular ante una presentación definida de ligandos. Se ha demostrado que fenómenos tan relevantes como la adhesión, la proliferación o la diferenciación celular son sensibles a la distribución espacial nanométrica de ligandos en superficie. Múltiples receptores celulares, cuando son estimulados por sus correspondientes ligandos, necesitan agruparse y formar clústers que modulan la transmisión de la señal. Desafortunadamente, todavía se desconocen los pormenores de la activación y la dinámica de agregación de los mismos ante las múltiples combinaciones espaciales de ligandos. Por este motivo, este trabajo tiene como objetivo el desarrollo de superficies que permitan la presentación controlada de ligandos en grupos nanométricos para analizar el efecto de los mismos en los procesos de señalización intracelular. Para abordar este ambicioso objetivo, se desarrolló una plataforma a partir de copolímeros en bloque cuya principal particularidad es que se autoensamblan, generando estructuras nanométricas. El copolímero en bloque más utilizado en este ámbito es el compuesto por poliestireno y poli(metil metacrilato) (PS-b-PMMA). En este estudio se utilizaron dos copolímeros en bloque con distinta fracción volumétrica de cada uno de los componentes, de manera que se autoensamblan generando cilindros nanométricos de PMMA inmersos en una matriz de PS. Cuando se depositan en una capa fina sobre un sustrato de silicio o de vidrio, y se controla tanto el grosor de la capa como la energía superficial del sustrato, se puede conseguir que los cilindros se posicionen de forma perpendicular y ordenada sobre la superficie. Para ello, en primer lugar se modificó la energía superficial del sustrato mediante el anclaje de polímeros con una disposición de monómeros aleatoria. Por otro lado, el grosor de la capa fina se controló mediante la concentración de la solución empleada y esta capa fina se sometió a un tratamiento térmico a 220°C en vacío que permite equilibrar las tensiones superficiales del PS y del PMMA. De este modo, se fabricaron dos plataformas nanoestructuradas con patrones circulares compuestos de cilindros de PMMA (21 y 28 nm de diámetro) separados por una matriz de PS. Una vez obtenidas las plataformas nanostructuradas, se diseñó un proceso de funcionalización que permitiera la localización de pequeños grupos de ligandos sobre los dominios nanométricos de PMMA. Para ello, se realizó una hidrólisis superficial de los grupos metilos del PMMA, generando así grupos ácidos más reactivos que posibilitan la unión covalente de cualquier molécula con un grupo amino terminal. En este tipo de moléculas se incluyen todas las proteínas y pequeños péptidos, lo cual pone de manifiesto la gran versatilidad de la estrategia de funcionalización. La caracterización de la disposición espacial de los ligandos se realizó mediante microscopía de fuerzas atómicas, y se corroboró utilizando una novedosa técnica de alta resolución denominada microscopía de reconstrucción óptica estocástica, que permite confirmar el estado de agregación de los ligandos biológicamente activos. Para validar la utilidad de estas superficies nanoestructuradas, primeramente se inmovilizó un conocido ligando de adhesión celular y se monitorizó la respuesta celular, en concreto evaluando la formación de contactos focales. Los resultados demostraron que sobre estas superficies, los fibroblastos se expandían de tal manera que el área ocupada por las células era equivalente en todos los sustratos. En cambio, cuando se analizó en detalle las estructuras macromoleculares que forman los receptores en la membrana celular tras la activación por parte del ligando, se observaron diferencias significativas. El número de contactos focales formados en la superficie donde los grupos de ligandos estaban más separados, era menor que en aquellos cuya distancia entre ligandos era menor. Por otro lado, aquellas superficies donde los ligandos se presentaban en grupos fomentaban la maduración de los contactos focales, revelando de este modo que este proceso puede manipularse utilizando estrategias de presentación de ligandos como la desarrollada en esta tesis. Tras verificar el potencial de nuestras plataformas, se indagó en el proceso de agregación del receptor EphB2 ante ligandos (efrinas) con una distribución nanométrica variada. Para alcanzar la resolución espacio-temporal necesaria y ser capaces de distinguir entre los diferentes oligómeros formados por el receptor, se empleó una innovadora técnica que analiza las fluctuaciones en intensidad de cada uno de los pixeles de imágenes de fluorescencia. En combinación con un modelo matemático, se demostró que la agregación de receptores para formar hexámeros y octámeros impulsa la activación máxima del receptor EphB2. Anteriormente, se había descrito que los ligandos solubles individuales eran incapaces de activar el receptor y de promover su oligomerización. En cambio, la presentación controlada de ligandos en grupos nanométricos, no sólo fomenta la activación del receptor, sino que además acelera la formación de clústers, demostrando nuevamente la efectividad de los ligandos nanoagrupados como moduladores y potenciadores del dinámico proceso de oligomerización. A la vista de los resultados obtenidos, se puede concluir que hemos sido capaces de desarrollar una plataforma nanoestrucuturada mediante copolímeros en bloque para su posterior modificación covalente con ligandos celulares cuya distribución en nanoagregados favorece las interacciones multivalentes con los receptores. De este modo, estas plataformas tienen potenciales aplicaciones a la hora de promover una respuesta concreta de los receptores, en función del tamaño del grupo de ligandos y del espaciado entre ellos. Este tipo de ligandos multivalentes se presentan como una atractiva estrategia para activar los complejos receptor-ligando de manera más potente, y por lo tanto, menos costosa. Por lo tanto, las posibles aplicaciones de estos sistemas de presentación de ligandos comprenden desde aplicaciones biotecnológicas a aplicaciones biomédicas, incluyendo sistemas de cultivo celular, materiales bioactivos y administración de fármacos

This research is based on the hypothesis that by permanently immobilizing Fibronectin (FN) on a metal substrate surface, it will be possible to enhance endothelial cells (EC)/surface interactions. FN was covalently bound onto self assembled monolayers (SAMs) modified gold surfaces with COOH terminal groups. SAMs composed of various surface ratios of X/COOH (X=CH3, OH, NH2) were formed in order to control the surface conformation of FN by modulating the surface charge (potential) and wettability. Human umbilical vein endothelial cell (HUVEC) attachment and proliferation onto such surfaces was investigated. Results have shown that SAM and SAM-protein surfaces are stable with time. An increased HUVEC attachment and proliferation was obtained on FN-free SAMs compared to a bare Au surface. However, the presence of covalently bound FN on the SAM surface further significantly enhanced EC attachment and proliferation rate. The optimum surface for the HUVEC attachment and proliferation was found to be the NH2/COOH surface, which is positively charged and hydrophilic.
Cette recherche est basée sur l'hypothèse qu'en immobilisant la protéine Fibronectin (FN) sur une surface métallique, il serait possible d'améliorer les interactions entre les cellules endothéliales et la surface. Un lien covalent a été fait entre FN et le group terminal de COOH apparentant à une couche unitaire de molécules assemblées (CUMA) sur une surface en or. Les CUMAs étaient composés de plusieurs fraction des groupes terminales X/COOH (X=CH3, OH, NH2) dans le but de modifier la charge et l'hydrophobicité sur la surface afin de contrôler la conformation de FN. L'attachement et la prolifération des cellules provenant d'une veine ombilicale humaine (CVOH) furent investigué sur ces surfaces. Les résultats démontrent que les surfaces constitués de CUMA et CUMA-protéine sont stables avec le temps. Les surfaces CUMA-FN ont éprouvé plus d'activités cellulaires comparées aux surfaces de CUMA. Ces derniers étés plus avantageux que les surfaces d'or. Les surfaces composées des groups NH2/COOH, qui étés de nature chargée et hydrophilique ont obtenu le plus haut niveau d'activités cellulaires.

Cell adhesion to extracellular matrix proteins is critical to physiological and pathological processes as well as biomedical and biotechnological applications. Cell adhesion is a highly regulated process involving initial receptor-ligand binding, and subsequent clustering of these receptors and rapid association with the actin cytoskeleton as focal adhesions are assembled. Focal adhesions enhance adhesion, functioning as structural links between the cytoskeleton and the extracellular matrix and triggering signaling pathways that direct cell function. The objective of this thesis research is to develop a mechanical and biochemical analysis of the adhesion strengthening response. Our central hypothesis was that focal adhesion size and position regulate cell adhesion strength by controlling the distribution of mechanical loading. We engineered micropatterned surfaces to control the size and position of focal adhesions in order to analyze the contributions of these specialized adhesive structures to adhesion strengthening. By applying surface micropatterning techniques, we showed robust control over cell-substrate contact area and focal adhesion assembly. Using a hydrodynamic shear assay to quantify adhesion strength to micropatterned substrates, we observed significant adhesive area- and time-dependent increases in adhesion strength. Complimentary biochemical assays allowed us to probe the role of structural proteins recruited to focal adhesions and examine the structure-function relationships between these adhesive structures and adhesion strength. These findings provide insights into the role of focal adhesions in adhesion strengthening, and may contribute to tissue engineering and biomaterials applications.

ABSTRACT INTERACTION BETWEEN MICRO AND NANO PATTERNED POLYMERIC SURFACES AND DIFFERENT CELL TYPES Ö
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elik, Hayriye Ph.D., Department of Biology Supervisor: Prof. Dr. Vasif Hasirci Co-Supervisor: Dr. Celestino Padeste August 2012, 139 pages Micro and nanopatterned surfaces are powerful experimental platforms for investigating the mechanisms of cell adhesion, cell orientation, differentiation and they enable significant contributions to the fields of basic cell and stem cell biology, and tissue engineering. In this study, interaction between micro and nanopatterned polymeric surfaces and different cell types was investigated. Three types of micropillars were produced by photolithography (Type 1-3), while nanometer sized pillars were produced in the form of an array by electron beam lithography (EBL). Replica of silicon masters were made of polydimethylsiloxane (PDMS). Polymeric [P(L-D,L)LA and a P(L-D,L)LA:PLGA blend] replica were prepared by solvent casting of these on the PDMS template and used in in vitro studies. The final substrates were characterized by various microscopic methods such as light microscopy, atomic force microscopy (AFM) and scanning electron microscopy (SEM). In order to investigate deformation of the nucleus in response to the physical restrictions imposed by micropillars, Type 1 and Type 2 pillars were used. These substrates were covered with pillars with different interpillar distances. While Type 1 is covered with symmetrically (in X-Y directions) distributed pillars, Type 2 pillars were distributed asymmetrically and the inter-pillar distances were increased. Nuclei deformation of five cell v types, two cancer cell lines (MCF7 and Saos-2), one healthy bone cell (hFOB1.19), one stem cell (bone marrow origined mesemchymal stem cells, BMSCs) and one standard biomaterial test cell type, (L929) fibroblasts was examined by using fluorescence microscopy and SEM. The nuclei of Saos-2 and MCF7 cells were found to be deformed most drastically. Nucleus deformation and intactness of nuclear membrane was examined by Anti- Lamin A staining. The interaction of the cells with micropillars was visualized by labelling focal adhesion complexes (FAC). Wettabilities of patterned and smooth surfaces were determined. As the patterns become denser (closer micropillars, Type 1) the hydrophobicity increased. Similar to water droplets, the cells were mostly spread at the top of the Type 1 pillars. The number of cells spread on the substrate surface was much higher on Type 2 patterned films. In order to support these qualitative findings, nucleus deformation was quantified by image analysis. Frequency of nucleus deformation was determined as the ratio of deformed to the total number of nuclei (%). In order to quantify the intensity of nuclei deformation, their circularity was evaluated. In addition to nucleus deformation, alterations in the ratio of cell area-to-nucleus area in response to micropillars were determined by image analysis. The results indicated that cancerous cells were more deformable. The qualitative microscopic evaluation and the data obtained by quantification of the nucleus and cellular deformation were in good agreement. In addition, the findings were consistent with expectations which suggest that cancerous cells are &ldquo
softer&rdquo
. In the second part of the research the force applied by the cells on arrays of micropillars with high aspect ratios (Type 3 substrates) during tugging at the pillars was investigated. Micropillars were produced using P(L-D,L)LA as well as a 60:40 blend of P(L-D,L)LA with PLGA. The blend is a material with lower stiffness than P(L-D,L)LA. The mechanical properties of the two materials were determined by tensile testing of solvent cast films. Deformation of Type 3 micropillars by the cellular tugging force of Saos-2 and L929 was studied by fluorescence and SEM microscopy, both on stiff and softer substrates. Displacements of the centers nodes of the pillars were evaluated from SEM micrographs. On the stiff surface, the two cell types bent the pillars to the same extent. On the other softer substrate (blends), however, the maximum displacements observed with Saos-2 cells were higher than the ones caused on the stiffer substrate or the ones caused by L929 cells. It is reported that stiffness of the substrate can determine stem cell lineage commitment. In order to examine the effects of change of substrate stiffness on osteogenic differentiation of BMSCs, osteopontin (OPN) expression was determined microscopically. It was found that osteogenic differentiation is enhanced when BMSCs are cultured on P(L-D,L)LA Type 3 pillars. vi In the last part of research, arrays of nanopillars whose interpillar distances systematically varied to form different fields were examined in terms of adhesion and alignment in order to determine the differential adhesion of BMSCs and Saos-2 cells. The difference in their adhesion preference on nanopillar arrays was quantified by image analysis. It was observed that BMSCs and Saos-2 cells behaved in an opposite manner with respect to each other on the fields with the highest density of nanopillars. The BMSCs avoided the most densely nanopillar covered fields and occupied the pattern free regions. The Saos-2, on the other hand, occupied the most densely nanopillar covered fields and left the pattern free regions almost unpopulated. It was also found that both BMSCs and Saos-2 cells aligned in the direction of the shorter distance between the pillars. Both BMSCs and Saos-2 cells started to align on the pillars if the distance in any direction was >
1.5 &mu
m. To better understand the effects of chemical and physical cues, protein coating and material stiffness were tested as two additional parameters. After fibronectin coating, the surfaces of P(L-D,L)LA films with the highly dense pillar covered fields, which were avoided when uncoated, were highly populated by the BMSC. Similarly, decreasing the stiffness of a surface which was normally avoided by the BMSCs made it more acceptable for the cells to attach.

The aims of this study were to screen an array of plant families for novel lectins, to isolate candidate lectins whose reactivity suggested may be functionally useful and to assess the reactivity and modulatory effects of the novel lectins on the cells of the gastrointestinal tract (including the immune regions) and lymphocytes. Few of the seed and bulb samples screened had significant levels of lectin. However, the seeds, roots, stem and bark of Morus nigra, the black mulberry tree from the Moraceae plant family, were found to contain particularly high lectin activity. Two new lectins Morus nigra agglutinin-I (MNAI) and Morus nigra agglutinin-II (MNAII) were isolated. They were found to differ significantly from each other in their sugar specificity, subunit structure, amino acid sequence identity, glycosylation and haemagglutinating activity. MNAI has similarities in sugar inhibition characteristics (GalNAc) and amino acid identity to both MPA and jacalin, which also belong to the Moraceae family. MNAI recognises the similar intestinal glycan structures as jacalin and recognises T/Tn blood group antigen, both with and without sialylation. However, it differs significantly from MPA and jacalin in its lymphocyte stimulatory properties. MNAII appears to be novel and did not show amino acid sequence identity with any known proteins contained in the deltamass database. It is inhibitable by α-D-methyl mannoside. It may have an affinity for structures such as some form of N-linked glycans and appears to have low affinity for α2,6 sialylated structures. It labelled glycan structures present on the villus brush border, dome FAE and most M cells of many of the species tested in vitro.

Metal contamination in groundwater aquifers poses risks to human health as well as other life forms. Previous laboratory experiments have demonstrated that bacteria found in geologic settings like aquifers are likely to adsorb metal contaminants and attenuate metal migration. However, as bacteria can also migrate through the groundwater aquifer a better understanding of the combined effect of these two processes is required. The aim of this laboratory study was to a) explore the affinity bacteria exhibit towards metals and porous media of varying composition, b) investigate the effect of mineral and solution composition on the bacterial filtration and c) use the combined data to predict the impact of microbes on metal mobility in porous media. Pantoea Agglomerans was used as a model bacterium while column materials consisted of quartz sand and iron-oxide coated sand (IOCS). Bacteria were characterised using potentiometric titrations to identify the type and concentration of sites present on their bacterial wall. Particular attention was paid to the effect of kinetics of proton and metal adsorption due to the variable contact times that solutions have with bacteria in columns. It was found that increasing the contact time between cell surfaces and protons during potentiometric titrations resulted in less reproducible results. This was due to the release of cell exudates under high pH conditions rather than cell death. Exudates were also found to adsorb protons. Moreover, zinc adsorption onto cell surfaces is higher after 60 to 90 minutes of contact time, while there is a decline in adsorption for longer contact times due to release of cell exudates in the solution. Stability constants for the adsorption of zinc onto cell surface sites, quartz and IOCS materials were determined through batch adsorption experiments, providing a mechanistic explanation of the adsorption process. Reactive transport models incorporating kinetics and surface complexation are developed to describe zinc movement through packed columns. Batch kinetic studies showed that significant Zn sorption to IOCS takes place gradually during the first two hours of contact time. Adsorption continues to take place at a slower rate for an additional 10 hours. This kinetic effect is manifested also during flow-through experiments (column dimensions: length 0.12 m, diameter 0.025 m) with a Darcian velocity 6.1·10-3 cm s-1, which is comparable to natural groundwater flow rates through sand porous media. A pseudo-second order kinetic adsorption model is combined with a numerical advection dispersion model for the first time to predict Zn transport. Model output results are of mixed quality as the model cannot successfully describe contaminant arrival time and breakthrough curve shape simultaneously. Moreover, a mechanistic surface complexation reactive transport model is capable of predicting Zn sorption under varying pH conditions demonstrating the versatility of mechanistic models. However, these models do not account for kinetics and therefore they are not intended to fit the dispersion of the contaminant due to kinetic effects of adsorption. Experiments in mixed zinc/cell systems demonstrate that transport through IOCS is dominated by the adsorption to the porous medium. This is consistent with the batch surface complexation predictions for the system. Adsorption to bacteria is reversible and zinc is stripped from the cells and redistributed onto the IOCS. Adsorption onto cells becomes significant and plays a role in mobile metal speciation only once the column is saturated with zinc.

Le diabète mellitus de type I est une maladie de plus en plus abondante. Cette dernière est caractérisée par la destruction auto-immunitaire des îlots de Langerhans incluant les cellules de type [bêta] qui produisent de l'insuline dans le pancréas endocrinien. Une option de traitement pour les patients atteints de cette maladie est notamment une greffe des îlots de Langerhans. Ce traitement est limité dû au nombre restreint de donneurs d'organes et aussi à la perte de fonctionnalité des îlots suite à la greffe. Les études effectuées tout au long de cette thèse ont pour optique d'adresser ces contraintes par le biais de la science des biomatériaux. La thèse débute avec un survol détaillé des concepts de base et des complexités associés aux interactions de type cellules et surfaces trouvées dans la littérature. II s'agit spécifiquement des interactions physiques et chimiques, des systèmes expérimentaux ainsi que des caractérisations et modifications associés aux interactions entre cellules et surfaces. La première étude de nature expérimentale examine la morphogenèse des cellules progénitrices ductales (PANC-1 cell line) vers des îlots qui produisent des agrégats semblables à des îlots (ILA). Le tout est fait sur des surfaces de carboxyméthyl dextrane (CMD) sur lesquelles le RGD est greffé via un lien covalent. L'expression des marqueurs d'lLAs (cytokeratin-19, Ki67, et E-cadherin) qui peuvent être associés à un changement de phénotype de ces cellules a été évaluée ainsi que la sécrétion et l'expression de l'insuline. La seconde étude de nature expérimentale a pour optique l'immobilisation de la fibronectine (FN) sur les mêmes surfaces de CMD mentionnées auparavant sur lesquelles des cellules ayant un phénotype [bêta] (INS-1 cell line) ont proliféré. Lors du processus d'immobilisation, plusieurs solutions ont été étudiées. L'immobilisation de la fibronectine sur des surfaces de CMD a été validée par la spectrométrie de photoélectrons induits par rayons X. Le mécanisme d'immobilisation a été déterminé par imagerie et mesures de force par microscopie à force atomique, la spectroscopie de dichroïsme circulaire ainsi que par la diffusion dynamique de la lumière. De plus, la croissance des cellules de type INS-1 et la sécrétion d'insuline ont été évaluées. La dernière étude de nature expérimentale visait l'étude de la coculture des cellules endothéliales et des îlots de porc dans un gel de fibrine. L'effet de la présence des cellules endothéliales sur la production d'insuline des îlots a été évalué. De plus, l'apoptose cellulaire en coculture a été évaluée et comparée aux cultures simples.

Biomolecules on cell surfaces play critical roles in diverse biological and physiological processes. However, conventional bulk scale techniques are unable to clarify the density and distribution of specific biomolecules in situ on single, living cell surfaces at the micro or nanoscale. In this work, a single cell analysis technique based on Atomic Force Microscopy (AFM) is developed to spatially identify biomolecules and characterize nanomechanical properties on single cell surfaces. The unique advantage of these AFM-based techniques lies in the ability to operate in situ (in a non-destructive fashion) and in real time, under physiological conditions or controlled micro-environments. First, AFM-based force spectroscopy was developed to study the fundamental biophysics of the heparin/thrombin interaction at the molecular level. Based on force spectroscopy, a force recognition mapping strategy was developed and optimized to spatially detect single protein targets on non-biological surfaces. This platform was then translated to the study of complex living cell surfaces. Specific carbohydrate compositions and changes in their distribution, as well as elasticity change were obtained by monitoring Bacillus cells sporulation process. The AFM-based force mapping technique was applied to different cellular systems to develop a cell surface biomolecule library. Nanoscale imaging combined with carbohydrate mapping was used to evaluate inactivation methods and growth temperatures effects on Yersinia pestis surface. A strategy to image cells in real time was coupled with hydrophobicity mapping technique to monitor the effect of antimicrobials (antimicrobial polymer and copper) on Escherichia coli and study their killing mechanisms. The single spore hydrophobicity mapping was used to localize the exosporium structure and potentially reconstruct culture media. The descriptions of cell surface DNA on single human epithelial cells potentially form a novel tool for forensic identification. Overall, these nanoscale tools to detect and assess changes in cell behavior and function over time, either as a result of natural state changes or when perturbed, will further our understanding of fundamental biological processes and lead to novel, robust methods for the analysis of individual cells. Real time analysis of cells can be used for the development of lab-on-chip type assays for drug design and testing or to test the efficacy of antimicrobials.

In this thesis, the dye molecule cis-bis(isothiocyanato)bis(2,2'-bipyridyl-4,4'-dicarboxylato)-ruthenium(II) (N3) is studied on the rutile TiO2(110) and Au(111) surfaces. The molecules were deposited onto the surfaces using an ultra-high vacuum (UHV) electrospray deposition system. Thermally labile molecules such as N3 cannot be deposited using the typical method of thermal sublimation, so development of this deposition technique was a necessary step for entirely in situ experiments. The geometric and electronic structure of the samples are characterised using core-level and valence band photoemission spectroscopy, x-ray absorption fine structure spectroscopy, density functional theory, resonant x-ray emission spectroscopy and scanning tunnelling microscopy. These reveal that N3 bonds to TiO2(110) by deprotonation of the carboxyl groups of one bi-isonicotinic acid ligand so that its oxygen atoms bond to titanium atoms of the substrate, and one of the thiocyanate groups bonds via a sulphur atom to an oxygen atom of the substrate. N3 bonds to Au(111) via sulphur atoms with no deprotonation of the carboxylic groups, and at low coverages decorates the Au(111) herringbone reconstruction. For N3 on TiO2, a consideration of the energetics in relation to optical absorption is used to identify the main photoexcitation channel between occupied and unoccupied molecular orbitals in this system, and also to quantify the relative binding energies of core and valence excitons. For N3 on Au(111), the energetics show that the highest occupied molecular orbital overlaps with the Au conduction band. The transfer of charge between the N3 molecule and the TiO2(110) and Au(111) surfaces was studied using resonant photoemission spectroscopy and resonant x-ray emission spectroscopy. These techniques, combined with knowledge gained about the geometric and electronic structure, are used to determine the locations and electronic levels of N3 from which charge is readily transferred to the substrate. The core-hole clock implementation of resonant photoemission spectroscopy is used to reveal that electron delocalisation from N3 to TiO2(110) occurs within 16 femtoseconds.

Polyelectrolyte multilayers (PEMs) are attractive surfaces for use in live-cell experiments due to their biocompatibility and ease of fabrication. PEMs of poly(allylamine hydrochloride) and poly(Disperse Red 1 acrylate-co-acrylic acid), an azo polymer, were prepared using a novel dropping method developed to conserve the materials used for film preparation. The PEMs were fabricated using a series of pH conditions as well as a different number of layers, yielding surfaces with varying properties. Embryonic chick heart cells were cultured on the PEM films; depending on film preparation conditions, we were able to selectively direct the morphology of chick heart cell growth. Although cells were expected to produce only confluent monolayers, the cells grew as aggregates of variable size or as confluent monolayers, depending on the surfaces upon which they were grown. Inclusion of the azo polymer affords surfaces with reversible photoresponsive properties; upon irradiation with visible green light at 488 nm, the azobenzene isomerizes. Optical properties of the polymer in solution and as a film were investigated. Aggregate cardiac cell beating were monitored using three different microscope setups: a macroscopic setup, a confocal microscope and a total internal reflection fluorescence (TIRF) microscope. A cellular response to 488 nm laser light on the TIRF microscope was detected in azo polymer films.
Les multicouches de polyélectrolytes (MPE) sont des surfaces intéressantes pour des expériences utilisant des cellules vivantes en raison de leur biocompatibilité et de leur facilité de fabrication. Les MPE de poly(chlorhydrate d'allylamine) et poly(Disperse Red 1 acide acrylate-co-acrylique), un polymère azoïque, ont été préparées en utilisant un nouveau procédé de déposition goutte-à-goutte qui a été mis au point afin de conserver les matériaux utilisés durant la préparation du film. Les MPE ont été fabriquées en variant les conditions de pH ainsi que le nombre de couches déposées, ce qui permet d'obtenir des surfaces présentant des propriétés différentes. Des cellules cardiaques embryonnaires de poussin ont été cultivées sur les films MPE mentionnés ci-haut. Selon les conditions de préparation des films, nous avons été en mesure de diriger sélectivement la morphologie de la croissance des cellules cardiaques de poussin. Bien que nous nous attendions à ce que les cellules ne produisent que des monocouches confluentes, les cellules se sont développées sous forme d'agrégats de taille variable ou des monocouches confluentes en fonction de la surface sur laquelle elles ont été cultivées. L'inclusion du polymère azoïque offre des surfaces ayant des propriétés photosensibles réversibles. Lors d'une irradiation avec un laser qui émet dans le visible à 488 nm (vert), l'azobenzène s'isomérise. Les propriétés optiques du polymère en solution ainsi que sous forme de film ont été étudiées. Les battements des agrégats de cellules cardiaques ont été contrôlés à l'aide de trois configurations de microscopes différents: une installation macroscopique, un microscope confocal et un microscope de fluorescence par réflexion totale interne (TIRF). Une réponse cellulaire à la lumière émise par le laser à 488 nm a été détectée sur le microscope TIRF pour le film de polymère azoïque.