Accurate determination of the in vitro kinetic parameters Km (Michaelis constant) and Ki (inhibition constant) is critical for the quantitative prediction of in vivo drug clearance and the magnitude of inhibitory drug interactions. A cause of inaccuracy in vitro arises from the assumption that all drug added to an incubation mixture is available for metabolism or inhibition. Many drugs bind non-specifically to the membrane of the in vitro enzyme source. The aims of this thesis were to: 1) investigate the comparative importance of lipophilicity (as log P), and pKa as determinants of the non-specific binding of drugs to human liver microsomes; 2) develop and validate an ANS fluorescence technique for measuring the non-specific binding of drugs to human liver microsomes; 3) characterise the non-specific binding of a large dataset of physicochemically diverse drugs using the ANS fluorescence procedure; 4) evaluate relationships between selected physicochemical characteristics and the extent of non-specific binding of drugs to human liver microsomes and; 5) computationally model the non-specific binding of drugs to discriminate between high binding (fu(mic) less than 0.5) and low binding (fu(mic) greater than 0.5) drugs. The comparative binding of the basic drugs atenolol (log P = 0.1; fu(mic) = 1.00), of propranolol (log P = 3.1; fu(mic) = 0.36 - 0.84), and imipramine (log P = 4.8; fu(mic) = 0.42 - 0.82) suggested that lipophilicity is a major determinant of non-specific binding. In contrast, the comparative binding of diazepam (pKa = 3.3; fu(mic) = 0.69 - 0.80), a neutral compound; and the bases propranolol (pKa = 9.5; fu(mic) = 0.36 - 0.84) and lignocaine (pKa = 9.5; fu(mic) = 0.98), indicated that pKa was not a determinant of the extent of non-specific binding. The non-binding of lignocaine, a relatively lipophilic base, was unexpected and confirmed by the non-binding of the structurally related compounds bupivacaine and ropivacaine. These results implicated physicochemical characteristics other than lipophilicity and charge as important for the non-specific binding of drugs to human liver microsomes. An assay based on 1-anilinonaphthalene-8-sulfonate (ANS) fluorescence was developed using the seven drugs employed in the initial study. Non-specific binding data from equilibrium dialysis and the ANS fluorescence methods were compared and a linear correlation (r2 = 0.92, p less than 0.01) was observed at drug concentrations of 100 and 200 micromolar. The approach was further validated by characterising the microsomal binding of nine compounds (bupropion, chloroquine, chlorpromazine, diflunisal, flufenamic acid, meclofenamic acid, mianserine, triflupromazine, and verapamil) using both binding methods (i.e. equilibrium dialysis and ANS fluorescence). A significant logarithmic relationship (r2 greater than or equal to 0.90) was demonstrated between fu(mic) and the modulus of ANS fluorescence for all drugs and for basic drugs alone at concentrations of 100 and 200 micromolar, while the acidic/neutral drugs showed a significant linear relationship (r2 greater than or equal to 0.84) at these two concentrations (p less than 0.01). The non binding of bupropion provided further evidence that physicochemical properties other than log P and charge were important for non-specific binding of drugs to human liver microsomes. The ANS fluorescence technique was then used to characterise the non-specific binding of 88 physicochemically diverse compounds. In general, acids and neutrals bound to a ‘low’ extent (fu(mic) greater than 0.5) whereas bases bound the full fu(mic) range (0.0001 to 1). Statistically significant relationships were observed between the non-specific binding of bases and log P, the number of hydrogen bond donors and hydrogen bond acceptors per molecule, and molecular mass. Preliminary in silico modeling of the dataset generated by the ANS fluorescence technique, using the program ROCS, provided discrimination of all but one (itraconazole) of the ‘high’ binding bases. However, there were 14 false positives, resulting in low overall prediction accuracy. Taken together, the studies conducted in this thesis provide important insights into the physicochemical factors that determine the non-specific binding of drugs to human liver microsomes.

The objective of this dissertation is to improve the performance of surface acoustic wave (SAW) biosensors for use in point-of-care-testing (POCT) applications. SAW biosensors have the ability to perform fast, accurate detection of an analyte in real time without the use of labels. However, the technology suffers from the inability to differentiate between specific and non-specific binding. Due to this limitation, direct testing of bodily fluids using SAW sensors to accurately determine an analyte's concentration is difficult. In addition, these sensors are challenged by the need to detect small concentrations of a biomarker that are typically required to give a clinical diagnosis. Sensitivity, selectivity and reliability are three critical aspects for any sensing platform. To improve sensitivity the delay path of a SAW sensor has been modified with microcavities filled with various materials. These filled cavities increased sensitivity by confining wave energy to the surface by way of constructive interference and waveguiding. Thus, the improved sensitivity will result in a lower limit of detection. In addition, insertion loss is decreased as a consequence of increased wave confinement to the surface. Sensor selectivity and reliability are adversely affected by non-specific binding of unwanted species present in a sample. To address this issue a multifunctional SAW sensor is presented. The sensor consists of two SAW delay lines oriented orthogonal to each on ST-quartz in order to generate two distinct wave modes. One wave mode is used for sensing while the other is used to remove loosely bound material. By using the same transduction mechanism for both removal and sensing, the sensor chip is simplified and complex electronics are avoided. The findings of this research involve the technological advances for SAW biosensors that make their use in POCT possible.

The purpose of this investigation was to create and optimise a capture assay for the detectionof anti-drug antibodies (ADA) in human plasma, using Biacore. We also dealt with the nonspecificplasma binding to mouse-derived anti-biotin which may occur in the capture assay.By paying attention to these things we aimed at reaching as high sensitivity as possible for theADA detection. The capture assay also benefited and gained flexibility from using the same regenerationsolution irrespective of drug and from having a composition that minimises the risk ofdamaging drug epitopes.

Development of repellent surfaces which can supress bacteria adhesion, blood contamination and thrombosis, and non-specific adhesion on diagnostic devices has been a topic of intense research as these characteristics are in high demand. This thesis focused on design and development of omniphobic surfaces based on hierarchical structures and their application for preventing pathogenic contamination and biosensing. First, a flexible hierarchical heat-shrinkable wrap featuring micro and nanostructures, was developed with straightforward scalable methods which can be applied to existing surfaces. These surfaces reduced biofilm formation of World Health Organization-designated priority pathogens as well as minimized risk of spreading contamination from intermediate surfaces. This is due to the broad liquid repellency and the presence of reduced anchor points for bacterial adhesion on the hierarchical surfaces. Next, the developed surfaces were applied to minimize blood contamination and clot formation as well as facile integration of hydrophilic patterns. This led to droplet compartmentalization and was utilized for detection of Interleukin 6 in a rapid dip-based assay. Furthermore, in a review article the need for anti-viral or virus repellent surfaces and future perspectives were discussed as the global COVID-19 pandemic surged and attracted interest toward innovative technologies for suppressing the spread of pathogens. To address the pressing issue of non-specific adhesion in diagnostics devices, an omniphobic liquid infused electrochemical biosensor was developed. This was achieved by electroplating gold nanostructures on fluorosilanized gold electrodes. These electrodes demonstrated rapid and specific detection of Escherichia coli within an hour in complex biological liquids (blood, urine, etc.) without dilutions or amplification steps from clinical patient samples which are major bottle necks when rapid detection systems are sought for at the point of care. / Thesis / Doctor of Philosophy (PhD) / Repellent surfaces have a variety of applications in healthcare, for coating medical devices (e.g. indwelling implants, stethoscopes, and other external devices.), coating hospital surfaces for blood and pathogen repellency, and for developing anti-fouling diagnostic devices. Furthermore, they can be applied in the food sector for limiting contaminations, and in public areas on high-touch surfaces to eliminate the spread of infection. Therefore, there is a need for repellent surface which can be easily applied to surfaces with various form factors while having an easy fabrication method. Featuring hierarchical structures on a heat-shrinkable material, a repellent wrap was designed to be integrated on existing surfaces and repel pathogens and suppress the spread of infection as an intermediate surface. Similar concept was used for designing blood repellent surfaces which were patterned with hydrophilic regions for a rapid dip-based biosensing platform. Finally, surface textures on conductive materials with liquid infused repellent coatings were investigated for electrochemical biosensing in complex biological liquids.

<p>Gene expression is regulated in response to metabolic necessities and environmental changes throughout the life of a cell.</p><p>A major part of this regulation is governed at the level of transcription, deciding whether messengers to specific genes are produced or not.</p><p>This decision is triggered by the action of transcription factors, proteins which interact with specific sites on DNA and thus influence the rate of transcription of proximal genes.</p><p>Mapping the organisation of these transcription factor binding sites sheds light on potential causal relations between genes and is the key to establishing networks of genetic interactions, which determine how the cell adapts to external changes.</p><p>In this work I review briefly the basics of genetics and summarise popular approaches to describe transcription factor binding sites, from the most straight forward to finally discuss a biophysically motivated representation based on the estimation of free energies of molecular interactions.</p><p>Two articles on transcription factors are contained in this thesis, one published (Aurell, Fouquier d'Hérouël, Malmnäs and Vergassola, 2007) and one submitted (Fouquier d'Hérouël, 2008).</p><p>Both rely strongly on the representation of binding sites by matrices accounting for the affinity of the proteins to specific nucleotides at the different positions of the binding sites.</p><p>The importance of non-specific binding of transcription factors to DNA is briefly addressed in the text and extensively discussed in the first appended article:</p><p>In a study on the affinity of yeast transcription factors for their binding sites, we conclude that measured in vivo protein concentrations are marginally sufficient to guarantee the occupation of functional sites, as opposed to unspecific emplacements on the genomic sequence.</p><p>A common task being the inference of binding site motifs, the most common statistical method is reviewed in detail, upon which I constructed an alternative biophysically motivated approach, exemplified in the second appended article.</p>

Pregnancy tests and related immunoassays are heavily dependent on specific and non-specific protein adsorption. These interfacial processes are affected by many factors that influence the in situ conformations of interfacially immobilised antibodies. This thesis examines a number of representative features with dual polarisation interferometry (DPI) and neutron reflection (NR), thus combining real-time dynamic monitoring with high interfacial structural resolution. Bovine serum albumin (BSA) was initially used as a model system to compare the surface coverage and thickness measurements of DPI and NR. The results show that DPI and NR provided similar surface coverage data but the measured thicknesses differed at BSA concentrations above 0.1 mg/ml. This discrepancy arose from the adoption of the uniform-layer model used by DPI for data analysis and the greater thickness sensitivity of NR. A model pregnancy immunoassay was built in steps on a silica surface so that the adsorption of each protein could be accurately monitored. Both DPI and NR provided evidence of BSA insertion into the gaps on the surface between the antibody molecules. This suggests that BSA adsorption is an excellent method to block the non-specific adsorption of target antigens to the immunoassay test surface. A magnetic tweezer system was designed and built in order to measure the specific antibody/antigen binding force. The antibodies and antigens were used to immuno-link magnetic beads to the experimental surface before the immuno-links were broken by increasing the attractive force between the magnetic tweezers and beads. The force per antibody/antigen immuno-link was estimated to lie between the values of 13.6 pN and 43.8 pN.Immuno-link detachment as a function of time was investigated. It was found that the immuno-link comprised both a strong and a weak interaction. The dissociation constant of the strong antibody/antigen interaction was found to equal 3E-4 /s and had an interaction length of 0.06 nm. The low population of beads bound by the second, weaker interaction meant that it was not possible to obtain accurate values of the dissociation constant and bond length of the second weaker interaction.

The aim of this study was to investigate the molecular interactions of the anti-cancer drug Doxorubicin (DOX) with poly(methacrylic acid) grafted starch nanoparticles (PMAA-g-St). In order to fully understand the DOX/PMAA-g-St system, we conducted in-depth studies on DOX dimer dissociation and DOX/PMAA-g-St binding interactions using various techniques such as isothermal titration calorimetry (ITC), dynamic light scattering (DLS), and fluorescence and absorption spectroscopy. Based on our experimental results, we developed a quantitative thermodynamic model with relevant parameters such as dissociation constant, Kd, as well as enthalpy of binding, ΔH, in order to explain DOX/PMAA-g-St interactions. In addition, we also studied the effect of environmental factors such as pH and NaCl on DOX self-association and DOX/PMAA-g-St complex formation. In conclusion, the combination of results obtained from various techniques as well as the multispecies equilibrium model, enables us to interpret quantitatively the data of drug loading onto and release from polymeric nanoparticles.

In this thesis we have attempted to study four basic aspects of DNA-protein interactions: Affinity, specificity, accessibility and kinetics. With NF-kB as our model transcription factor, we wanted to investigate how a particular dimer recognizes a specific binding sequence? How fast are these interactions? And finally, how does the NF-kB interact with it binding site in the chromatin context? Specificity of NF-kB-DNA interactions has recently come into focus after it was shown that these dimers can bind to the sequences which do not fall into the NF-kB general consensus motif. We studied seven such sequences for their specificity for four NF-kB dimers. Our results show that p50 homodimers are least discriminative and can bind specifically to all these sequences. While as, RelA homodimers were highly discriminative and did not bind to most of these nontraditional sequences. We used two different methods to measure binding affinities: traditional gel mobility shift assay (EMSA) and a novel technique called as UV laser footprinting. Our results show that UV laser footprinting is the better method to determine the binding constants.For studying the dynamics of NF-kB-DNA binding, we combined UV laser footprinting with stopped flow device. This combination, not only give us one base pair resolution but also milli-second time resolution. Using p50 homodimers as a model transcription factor, we showed that the binding of this factor follows a two-step mechanism. First step involves the fast recognition of the sequence and second step follows a slower kinetics most likely for the stabilization of the complex. Our experiments suggest that flanking sequences play a role in the recognition and stabilization process of the complex formation.Finally, we also studied the accessibility of nucleosomes to NF-kB. Our in vitro data sheds light on the in vivo requirements for the alterations in chromatin structure necessary for the productive binding of NF-kB. These include either a removal of H2A-H2B dimers from the nucleosome and/or chromatin remodeler induced relocation of the histone octamer.Our data sheds light on the in vivo requirements for the alterations in chromatin structure necessary for the productive binding of NF-kB. We hypothesize that some factors like PU.1 might be able to target the chromatin remodeling/dimer eviction machinery to particular nucleosomes and lead to productive binding of NF-kB.

The aim of this study was to investigate the molecular interactions of the anti-cancer drug Doxorubicin (DOX) with poly(methacrylic acid) grafted starch nanoparticles (PMAA-g-St). In order to fully understand the DOX/PMAA-g-St system, we conducted in-depth studies on DOX dimer dissociation and DOX/PMAA-g-St binding interactions using various techniques such as isothermal titration calorimetry (ITC), dynamic light scattering (DLS), and fluorescence and absorption spectroscopy. Based on our experimental results, we developed a quantitative thermodynamic model with relevant parameters such as dissociation constant, Kd, as well as enthalpy of binding, ΔH, in order to explain DOX/PMAA-g-St interactions. In addition, we also studied the effect of environmental factors such as pH and NaCl on DOX self-association and DOX/PMAA-g-St complex formation. In conclusion, the combination of results obtained from various techniques as well as the multispecies equilibrium model, enables us to interpret quantitatively the data of drug loading onto and release from polymeric nanoparticles.

Gene expression is regulated in response to metabolic necessities and environmental changes throughout the life of a cell. A major part of this regulation is governed at the level of transcription, deciding whether messengers to specific genes are produced or not. This decision is triggered by the action of transcription factors, proteins which interact with specific sites on DNA and thus influence the rate of transcription of proximal genes. Mapping the organisation of these transcription factor binding sites sheds light on potential causal relations between genes and is the key to establishing networks of genetic interactions, which determine how the cell adapts to external changes. In this work I review briefly the basics of genetics and summarise popular approaches to describe transcription factor binding sites, from the most straight forward to finally discuss a biophysically motivated representation based on the estimation of free energies of molecular interactions. Two articles on transcription factors are contained in this thesis, one published (Aurell, Fouquier d'Hérouël, Malmnäs and Vergassola, 2007) and one submitted (Fouquier d'Hérouël, 2008). Both rely strongly on the representation of binding sites by matrices accounting for the affinity of the proteins to specific nucleotides at the different positions of the binding sites. The importance of non-specific binding of transcription factors to DNA is briefly addressed in the text and extensively discussed in the first appended article: In a study on the affinity of yeast transcription factors for their binding sites, we conclude that measured in vivo protein concentrations are marginally sufficient to guarantee the occupation of functional sites, as opposed to unspecific emplacements on the genomic sequence. A common task being the inference of binding site motifs, the most common statistical method is reviewed in detail, upon which I constructed an alternative biophysically motivated approach, exemplified in the second appended article. / QC 20101110