Surface plasmon microscopes are mostly built around the prism based Kretschmann configuration. In these systems, an image of a sample can be obtained in terms of an intensity map, where the intensity of the image is dependent on the coupling of the light into the surface plasmons. Unfortunately the lateral resolution of these systems relies on the ability of plasmons to propagate along the metallised layer and is usually limited to a few microns unless special measures are taken. The widefield surface plasmon microscope (WSPR), used here enables surface plasmon imaging at significantly higher lateral resolutions than prism based systems. In this study we demonstrate the functionality of the WSPR by imaging a sequence of binding events between micro-patterned extracellular matrix proteins and their specific antibodies. Using the WSPR system a change in contrast was observed with each binding event. Images produced via the WSPR system were analyzed and compared qualitatively and quantitatively. Consequently, we confirm that the WSPR microscope described here can be used to study sequential monomolecular layer binding events on a micron scale. These results have significant implications in the development of new micron scale bioassays.

Yes / The Sustainable Development Goal (SDG) 6.1, established by the United Nations General Assembly in 2015, targets universal and equitable access to safe and affordable drinking water for all by 2030. An essential factor in achieving this goal is the harnessing of “green” coagulants – naturally occurring, environmentally friendly materials which are effective coagulants for use in water treatment, with good availability in developing countries, inherent renewable properties and ease of biodegradation. In order to gain from these benefits, it is essential to fully understand how such coagulants may best be utilised, particularly concerning their practical application in developing countries. In this study, three different plant-based coagulation products (PCPs), namely Hexane (HxKP), saline (StKP) and crude (CrKP) extracts of Kenaf plant seed (Hibiscus cannabinus, a species of the Hibiscus plant), were applied to high (HTW), medium (MTW) and low (LTW) turbidity water in order to determine their performance and coagulation ability. The ability of the three Kenaf coagulant products (KCPs) to remove hydrophobic fractions of natural organic matter (NOM) was measured. The impact of KCPs on the treated water organic matter content (a known disinfection by-product (DBP) precursor) was examined using known surrogates of natural organic matter (NOM) i.e. the dissolved organic carbon (DOC), ultraviolet absorbance at 254 (UV254) and specific ultraviolet absorbance (SUVA254). Results obtained quantify the implications of using these coagulants during the water disinfection process. A parametric study, measuring the effect of different operating parameters, such as untreated water turbidity, pH, dosages, retention time, and KCP storage time, was completed. Turbidity removal performance for HxKP and StKP was very good with > 90% removal recorded for HTW and MTW, respectively, at pH seven within 2 hours retention time. Images obtained from scanning electron microscopy (SEM) analysis revealed a high likelihood of the coagulation mechanism of KCPs to be adsorption-interparticle bridging brought about by their flake-like structures and surfaces charges. Varying pH had no measurable influence on the coagulation performance of the KCPs. Comparing their efficiency with Moringa Oleifera (MO, a previously researched PCP) and alum showed that HxKP had a negligibly different particle removal as MO. StKP turbidity removal performance was below HxKP by 1% for HTW and LTW and 2% for MTW but performed higher than the CrKP by 5% and 7% in HTW and MTW, respectively. The optimum dosage of HxKP and StKP reduced DBP surrogate values, indicating that its precursor is also minimized, although a slight shift from this optimum dosage showed a significant rise in their concentration thus signifying a potential increase in DBPs during disinfection.

Inter-molecular interaction is at the heart of biological function. Proteins can interact with ligands, peptides, small molecules, and other proteins to serve their structural or functional purpose. With advances in combinatorial chemistry and the development of high throughput binding assays, the available inter-molecular interaction data is increasing exponentially. As the space of testable compounds increases, the complexity and cost of finding a suitable inhibitor for a protein interaction increases. Computational drug discovery plays an important role in minimizing the time and cost needed to study the space of testable compounds. This work focuses on the usage of various computational methods in identifying protein interaction inhibitors and demonstrates the ability of computational drug discovery to contribute to the ever growing field of molecular interaction. A program to predict the location of binding surfaces on proteins, STP (Mehio et al., Bioinformatics, 2010, in press), has been created based on calculating the propensity of triplet-patterns of surface protein atoms that occur in binding sites. The use of STP in predicting ligand binding sites, allosteric binding sites, enzyme classification numbers, and binding details in multi-unit complexes is demonstrated. STP has been integrated into the in-house high throughput drug discovery pipeline, allowing the identification of inhibitors for proteins whose binding sites are unknown. Another computational paradigm is introduced, creating a virtual library of -turn peptidomimetics, designed to mimic the interaction of the Baff-Receptor (Baff-R) with the B-Lymphocyte Stimulator (Blys). LIDAEUS (Taylor, et al., Br J Pharmacol, 2008; 153, p. S55-S67) is used to identify chemical groups with favorable binding to Blys. Natural and non-natural sidechains are then used to create a library of synthesizable cyclic hexapeptides that would mimic the Blys:Baff-R interaction. Finally, this work demonstrates the usage and synergy of various in-house computational resources in drug discovery. The ProPep database is a repository used to study trends, motifs, residue pairing frequencies, and aminoacid enrichment propensities in protein-peptide interaction. The LHRLL protein-peptide interaction motif is identified and used with UFSRAT (S. Shave, PhD Thesis, University of Edinburgh, 2010) to conduct ligand-based virtual screening and generate a list of possible antagonists from the EDULISS (K. Hsin, PhD Thesis, University of Edinburgh, 2010) compound repository. A high throughput version of AutoDock (Morris, et al., J Comput Chem, 1998; 19, p. 1639-62) was adapted and used for precision virtual screening of these molecules, resulting in a list of compounds that are likely to inhibit the binding of this motif to several Nuclear Receptors.

Borrelia hermsii, the primary etiological agent of tick-borne relapsing fever in North America, binds the complement regulatory protein factor H (FH) as a means of evading opsonophagocytosis and the alternative pathway of complement. The sequence of the gene encoding the FH-binding protein has been determined. The protein is unique to B. hermsii and has been designated FhbA. Analyses of B. hermsii isolates revealed that FhbA is expressed by 24 of the 25 isolates tested. fhbA was demonstrated to be a single genetic locus through pulsed-field gel electrophoresis, restriction digest, and hybridization analyses, and all isolates possessing fhbA carry it on a 200 kb linear plasmid, whereas isolates that lack fhbA instead carry a 170 kb linear plasmid.To investigate the molecular basis of the interaction between FhbA and FH, truncated FhbA proteins were generated and tested for FH binding. Binding required both N- and C-terminal domains indicating conformational determinants are needed for FH binding. Further mutagenesis established that two C-terminal coiled-coil domains and a loop are involved in the interaction with FH. Potential variation in FhbA among isolates was analyzed by DNA sequence analysis. Two FhbA types, FhbAl and FhbA2, were delineated. Both FhbA types share a conserved C terminus containing the coiled-coil structures involved in binding FH. Additionally, it was demonstrated through whole-cell adsorption and ALBI assays that B. hermsii also binds FHL-1. To assess the specificity of the immune response to FhbA, recombinant FhbAl and FhbA2 were screened with serum from infected mice and humans. FhbA was found to be expressed and antigenic during infection. To localize the epitopes of FhbAl and FhbA2, truncations were screened with infection serum. The epitopes were determined to be conformational. Lastly, type-specific PCR primers were generated to implement rapid differentiation of strains bearing fhbA1 versus fhbA2.Together, these analyses indicate that FH/FHL-1 binding is a prevalent virulence mechanism allowing complement evasion by B. hermsii and provide insight into the antigenic structure of FhbA. Additionally, the data can be applied to the future development of species-specific diagnostic tools and will advance the studies on the epidemiology of relapsing fever in North America.

Etant donné leur implication dans de nombreux processus biochimiques, les ARN sont maintenant considérés comme des cibles thérapeutiques très prometteuses. Cependant, nos connaissances limitées concernant les phénomènes d'interaction entre les ARN et de petites molécules, compliquent l'élaboration de nouveaux ligands (ou médicaments), capables de reconnaître sélectivement une structure complexe d'ARN. En absence de toute approche rationnelle, une stratégie de criblage pourrait permettre de mieux comprendre ces phénomènes de reconnaissance. Cette thèse porte sur la mise au point d'une méthode électrochimique, simple, adaptée pour du criblage haut-débit et permettant de détecter et de quantifier les interactions ARN/ligands. Le principe de la méthode repose sur la différence de coefficient de diffusion qui existe entre la forme libre d'un ligand possédant des propriétés redox et sa forme complexée à l'ARN. Cette stratégie de détection par voie électrochimique présente comme avantages d'être peu coûteuse, rapide, simple d'utilisation, adaptée pour du criblage haut-débit de molécules et utilisable dans de faibles volumes. Cette méthodologie a été utilisée pour caractériser la formation d'un complexe entre un analogue d'aminoglycoside porteur d'un groupe ferocene et une séquence d'ARNr 16S23. De plus, des expériences de compétition entre le complexe ARN/ligand redox et des aminoglycosides non modifiées permettent d'étendre la méthode à la détermination de constantes de dissociation (KD) pour des molécules non marquées en phase homogène. Ces expériences de compétition pourront être généralisées pour mesurer le KD de librairies de molécules, permettant ainsi de trouver de meilleurs ligands d'ARN. / RNA molecules play a major role in various biochemical processes and they are now considered as an important drug target. However, our limited understanding of the interactions occurring between small molecules and RNA complicate the search for new ligands (or drugs) with improved specific interaction and binding to elaborated RNA structures. In the absence of any rational approach, a screening strategy could shed light on the ligand/RNA interactions. In this thesis, we describe a simple electrochemical approach allowing for high-throughput detection and quantification of small molecule/RNA interactions. The principle of the method relies on the difference of diffusion rates between a redoxmolecular probe free or bound to its RNA target and thus to the ability to more easily electrochemically detect the forme rover the latter in a homogenous solution. This electrochemical detection strategy has the advantages of being affordable,fast, easy to use, sensitive and well-adapted to a high-throughput screening strategy in small volume samples. This methodology was used to characterize the binding of an aminoglycoside analog bearing a ferocenyl group to the ribosomal RNA fragment (rRNA 16S23). Furthermore, competitive binding of unlabelled aminoglycosides on theRNA/electrochemical probe complex allowed us to evaluate their dissociation constants (KD). These competitive experiments could further be generalized to measure KD values for libraries of molecules, which could help to find better RNA ligands.

In this thesis, we focused on local structural features of the DNA backbone in protein-complexed DNA and non-complexed (naked) DNA, and its dependence on types of a base pairing in DNA, and on the base sequence. To reach this goal we analyzed about 1,400 crystal structures of DNA in complexes with proteins and more than 400 crystal structures of naked DNA. DNA local conformations were structurally classified into 38 dinucleotide conformers ntCs, which were described previously (Svozil et al. Nucleic Acids Res. 2008). The ntC were further clustered into 16 structural alphabet classes ntA to reduce the number of analyzed variables. We assembled base-paired dinucleotides from double helical DNA structures accord- ing to their assigned structural alphabet classes into so called Association matrices. Three basic Association matrices were analyzed; two compare ntA/ntA associations between dinucleotides forming only Watson-Crick base pairs in protein/DNA com- plexes and in naked DNA, respectively; the third one ntA/ntA associations between dinucleotides base-paired also by non-Watson-Crick pairs. We also analyzed As- sociation matrices of dinucleotides as a function of their sequences. The analyzes revealed differences in structural behavior of various ntA and their dependence on dinucleotide sequences.

Surface plasmon microscopes are mostly built around the prism based Kretschmann configuration. In these systems, an image of a sample can be obtained in terms of an intensity map, where the intensity of the image is dependent on the coupling of the light into the surface plasmons. Unfortunately the lateral resolution of these systems relies on the ability of plasmons to propagate along the metallised layer and is usually limited to a few microns unless special measures are taken. The widefield surface plasmon microscope (WSPR), used here enables surface plasmon imaging at significantly higher lateral resolutions than prism based systems. In this study we demonstrate the functionality of the WSPR by imaging a sequence of binding events between micro-patterned extracellular matrix proteins and their specific antibodies. Using the WSPR system a change in contrast was observed with each binding event. Images produced via the WSPR system were analyzed and compared qualitatively and quantitatively. Consequently, we confirm that the WSPR microscope described here can be used to study sequential monomolecular layer binding events on a micron scale. These results have significant implications in the development of new micron scale bioassays.

High-throughput whole-genome biological assays are highly intricate and difficult to interpret. The molecular interaction networks generated from evaluation of those experiments suggest that cellular functions are carried out by modules of interacting molecules. Reverse-engineering the modular structure of cellular interaction networks has the promise of significantly easing their analysis. We hypothesize that: • cellular wiring diagrams can be decomposed into overlapping modules, where each module is a set of coherently-interacting molecules and • a cell responds to a stress or a stimulus by appropriately modulating the activities of a subset of these modules. Motivated by these hypotheses, we develop models and algorithms that can reverse-engineer molecular modules from large-scale functional genomic data. We address two major problems: 1. Given a wiring diagram and genome-wide gene expression data measured after the application of a stress or in a disease state, compute the active network of molecular interactions perturbed by the stress or the disease. 2. Given the active networks for multiple stresses, stimuli, or diseases, compute a set of network legos, which are molecular modules with the property that each active network can be expressed as an appropriate combination of a subset of modules. To address the first problem, we propose an approach that computes the most-perturbed subgraph of a curated pathway of molecular interactions in a disease state. Our method is based on a novel score for pathway perturbation that incorporates both differential gene expression and the interaction structure of the pathway. We apply our method to a compendium of cancer types. We show that the significance of the most perturbed sub-pathway is frequently larger than that of the entire pathway. We identify an association that suggests that IL-2 infusion may have a similar therapeutic effect in bladder cancer as it does in melanoma. We propose two models to address the second problem. First, we formulate a Boolean model for constructing network legos from a set of active networks. We reduce the problem of computing network legos to that of constructing closed biclusters in a binary matrix. Applying this method to a compendium of 13 stresses on human cells, we automatically detect that about four to six hours after treatment with chemicals cause endoplasmic reticulum stress, fibroblasts shut down the cell cycle far more aggressively than fibroblasts or HeLa cells do in response to other treatments. Our second model represents each active network as an additive combination of network legos. We formulate the problem as one of computing network legos that can be used to recover active networks in an optimal manner. We use existing methods for non-negative matrix approximation to solve this problem. We apply our method to a human cancer dataset including 190 samples from 18 cancers. We identify a network lego that associates integrins and matrix metalloproteinases in ovarian adenoma and other cancers and a network lego including the retinoblastoma pathway associated with multiple leukemias. / Ph. D.

L’ARN non-codant 7SK forme la charpente d’un complexe, 7SK snRNP, qui régule l’activité du facteur d’élongation de la transcription P-TEFb, intervenant dans la levée des pauses transcriptionelles chez les métazoaires. Le 7SK snRNP comprend les protéines LARP7, essentielle pour la stabilité de l’ARN 7SK et MePCE, participant à sa coiffe. Dans le cadre d’une investigation du rapport entre structure et fonction de l’ARN7SK, le projet était de comprendre commen la protéine LARP7 reconnait et assemble l’ARN dans le 7SK snRNP. La protéine LARP7, membre d’une famille reliée à laprotéine La, est spécifique de 7SK. Les éléments responsables de l’interaction ont été analysés par des méthodes biochimiques dans des complexes reconstitués à partir d’ARN synthétique et de protéines recombinantes. Le module La, dans la région N-terminale, reconnaît et lie les trois uridines à l’extrémité 3’ de l’ARN et, additionellement,une séquence conservée au pied de la tigeboucle en 3’, induisant une conformation fermée de l’ARN. L’autre extrémité de la protéine comprend un domain RRM de reconnaissance de l’ARN, qui se lie à la boucle apicale de la tige-boucle 3’. La protéine LARP7 reconnaît également une région conservée au centre de l’ARN. Dans l’ensemble, LARP7 utiliserait ses domaines terminaux et central pour envelopper l’ARN et le stabiliser. Au cours de ces travaux, une interaction directe du domaine C-terminal avec la tige-boucle 5’ a également été mise en évidence. Celle-ci comprend le site de liaison à la HEXIM, la protéine qui déclenche l’interaction avec P-TEFb et un rôle fonctionnel de LARP7 est envisagé. / The non-coding RNA 7SK is the scaffold for the 7SK snRNP complex that regulates PTEFb, the positive transcription elongation factor, which relieves transcription pauses in metazoans. The 7SK snRNP comprises the proteins LARP7, essential for 7SK stability and MePCE, involved in capping. In the frame of an investigation of how the structure of the7SK RNA sustains its function, the project was to understand how is the RNA recognized and assembled in the 7SK snRNPby the associated protein LARP7. LARP7, a La-related protein is specific for 7SK. The elements responsible for the interaction were investigated by biochemical approaches in vitro with complexes reconstituted from purified recombinant proteins and transcribed RNA. The La-module of LARP7 recognizes and binds the triplet of uridines at the 3’-end of the 7SK RNA and additionally binds to a conserved region at the foot of the 3’-hairpin.This may stabilize a closed conformation of the 7SK. On the other end of the LARP7molecule, the C-terminal domain comprising a RRM (RNA Recognition Motif) binds to the apical loop of the 3’hairpin. Further investigations showed that a conserved region in the core of the RNA is also involved. On the whole, this strongly suggests thatLARP7 wraps around 7SK using its N terminal, C-terminal and linker domains to ensure the RNA stabilization into a functional core. In the course of the investigation, was revealed a direct interaction of the C-terminal domain of LARP7 with the 5’-hairpin of the RNA, which is responsible for 7SK function as it contains the binding site of HEXIM, the protein which bridges 7SK and P-TEFb. A possible functional role of LARP7 is envisioned.

abstract: Measuring molecular interaction with membrane proteins is critical for understanding cellular functions, validating biomarkers and screening drugs. Despite the importance, developing such a capability has been a difficult challenge, especially for small molecules binding to membrane proteins in their native cellular environment. The current mainstream practice is to isolate membrane proteins from the cell membranes, which is difficult and often lead to the loss of their native structures and functions. In this thesis, novel detection methods for in situ quantification of molecular interactions with membrane proteins are described. First, a label-free surface plasmon resonance imaging (SPRi) platform is developed for the in situ detection of the molecular interactions between membrane protein drug target and its specific antibody drug molecule on cell surface. With this method, the binding kinetics of the drug-target interaction is quantified for drug evaluation and the receptor density on the cell surface is also determined. Second, a label-free mechanically amplification detection method coupled with a microfluidic device is developed for the detection of both large and small molecules on single cells. Using this method, four major types of transmembrane proteins, including glycoproteins, ion channels, G-protein coupled receptors (GPCRs) and tyrosine kinase receptors on single whole cells are studied with their specific drug molecules. The basic principle of this method is established by developing a thermodynamic model to express the binding-induced nanometer-scale cellular deformation in terms of membrane protein density and cellular mechanical properties. Experiments are carried out to validate the model. Last, by tracking the cell membrane edge deformation, molecular binding induced downstream event – granule exocytosis is measured with a dual-optical imaging system. Using this method, the single granule exocytosis events in single cells are monitored and the temporal-spatial distribution of the granule fusion-induced cell membrane deformation are mapped. Different patterns of granule release are resolved, including multiple release events occurring close in time and position. The label-free cell membrane deformation tracking method was validated with the simultaneous fluorescence recording. And the simultaneous cell membrane deformation detection and fluorescence recording allow the study of the propagation of the granule release-induced membrane deformation along cell surfaces. / Dissertation/Thesis / Doctoral Dissertation Electrical Engineering 2018