<p>  </p> <p>The cGAS-STING axis represents a key pathway towards the activation of innate immunity against pathogens. However, persistent activation can lead to the development of autoimmune diseases, driving the need for the development of antagonists of cGAS-STING pathway. Herein, we describe the discovery of a small molecule STING binder HSD1077 through a STING based fluorescence polarization (FP) displacement assay. Initial SAR studies utilizing the FP displacement assay suggests the presence of pyrazole moieties critical for HSD1077 towards STING Binding. Additionally, we show that HSD1077 serves as an antagonist of the cGAS-STING pathway and effectively suppresses type-1 interferon expression upon 2’-3’cGAMP induction in both murine RAW macrophages and human THP-1 monocytes. HSD1077 in conclusion shows potential as a lead compound towards the further development of anti-inflammatory drugs. </p>

L’imagerie par Résonance des Plasmons de Surface (SPRI) est une technique d’analyse d’interactions moléculaires présentant de nombreux avantages. Elle peut être appliquée en temps réel et sans marquage, pour étudier un grand nombre d’interactions simultanément sur un même échantillon. La transduction d’un événement d‘interaction entre deux molécules complémentaires en un signal optique, repose sur la perturbation de l’onde plasmonique évanescente créée à la surface d’un film métallique mince.Toutefois, bien que la mesure SPR soit directe et sans marquage, sa spécificité repose entièrement sur celle des molécules sondes déposées à la surface de la puce et donc sur la chimie ayant servi à les immobiliser. Cette limitation devient problématique pour adresser les grands enjeux de santé actuels, liés à la détection de molécules à l’état de traces. En particulier, de nouveaux systèmes d’analyse plus sensibles sont requis pour pouvoir diagnostiquer le cancer au plus tôt, ou encore détecter la présence de contaminants agro-alimentaires en faible concentration.Dans cette perspective d’amélioration de la spécificité de détection, ce travail porte sur la mise au point d’un instrument bimodal couplant la SPRI, capable de quantifier la capture de molécules cibles, à la Spectrométrie Raman Exaltée de Surface (SERS), qui permet d’identifier la nature des molécules capturées en déterminant leur « empreinte » moléculaire. Cette thèse s’inscrit dans un projet ANR regroupant un consortium de partenaires académiques et un industriel.Ce document se concentre sur le développement de l’instrument optique combinant les deux systèmes de détection en un seul prototype. La mesure SPRI est réalisée en configuration Kretschmann, tandis que l’analyse SERS s’effectue par le dessus, en milieu liquide, à travers un hublot. Ces deux mesures simultanées sont rendues possibles grâce à la mise au point d’un substrat métallique nanostructuré. Une caractérisation détaillée du système optique est tout d’abord présentée, puis de premiers résultats de validation de la mesure bimodale sur un cas modèle d’interaction biomoléculaire ADN sont démontrés. Ces expériences prometteuses confirment le fonctionnement de l’instrument bimodal dans la perspective d’applications d’intérêt biologique. / Surface Plasmon Resonance Imaging (SPRI) is a powerful technique to study molecular interactions providing a real time, label free and high throughput analysis. The transduction of an interaction between complementary molecules into an optical signal is based on the perturbation of a plasmonic evanescent wave supported by a thin metallic film.However, despite its direct and label free assets, the specificity of SPR measurements is only guaranteed by the probe molecules grafted on the metallic surface and therefore by the quality of the surface chemistry. This limitation becomes an issue when addressing major health concerns relying on the detection of trace molecules. In particular, new systems are required to help early diagnosis and the control of food contaminants.In view of improving measurement’s specificity, this work reports the development of a bimodal instrument coupling SPRI, allowing the quantification of captured molecules, with Surface Enhanced Raman Spectroscopy (SERS), adding the precise identification of the molecules by measuring their spectroscopic fingerprint. This PhD is part of an ANR project bringing together academic and industrial partners.This manuscript focuses on the development of the optical instrument combining the two detection systems in a unique prototype. SPRI measurements are performed in the Kretschmann configuration while SERS analysis is implemented from the top, in solution, through a glass window. Nanostructured substrates have been designed and realized to allow the simultaneous experiment.The optical system is described, characterized and validated on the model case of a DNA hybridization. These first results prove the capabilities of the bimodal instrument in the perspective of more complex biological applications.

<p dir="ltr">Microfluidic techniques have been widely adopted in biomedical research due to the pre- cise control of fluids, small volume requirement, low cost and etc, and have boosted the development of biomolecular interaction analysis, point-of-care diagnostics, and biosensors.</p><p dir="ltr">Protein-protein interaction plays a key role in biological, biomedical and pharmaceutical research. The technical development of biosensors, new drugs and vaccines, and disease diagnostics heavily rely on the characterization of protein-protein interaction kinetics. The current gold standard assays for measuring protein-protein interaction are surface plasmon resonance (SPR), and bio-layer interferometry (BLI). These commercial devices are accurate but expensive, however.</p><p dir="ltr">Here, I have developed new microfluidic techniques and models in protein-protein in- teraction kinetics measurement, rotational diffusion coefficient modeling, electrochemical impedance spectroscopy-based biosensors, and two-phase porous media flow models. Firstly, I applied particle diffusometry (PD) in the streptavidin-biotin binding kinetics measurement, utilizing a Y-junction microchannel. Secondly, to reduce solution volumes used in an analysis experiment, I designed a low-volume chip and coupled it with PD to measure the binding kinetics of human immunodeficiency virus p24 antibody-antigen interactions. Thirdly, con- sidering the Brownian motion of the non-symmetric particles, I developed a new model to efficiently compute particles’ rotational diffusion coefficients. Fourthly, to make economic biosensors to detect multiple biomarkers, I created a new chip, enabling hundreds of tests in a single droplet (∼ 50 μL) on one chip. Finally, to understand the liquid flow in porous media, such as nitrocellulose in lateral flow assays, I built a new two-phase porous media flow model based on the Navier-Stokes equation and compared it with experiments. These techniques and models underwent rigorous experimental and computational validation, demonstrating their effectiveness and performance.</p>

Human schistosomiasis popularly known as bilharzias in many regions of Africa is a freshwater snail-transmitted disease caused by parasitic flatworms known as schistosomes. The growth and development of schistosomes typically requires developmental stages in multiple hosts and transmission stages in freshwater. These life cycle environments present a plethora of stressors. Certain gene families including heat shock proteins (HSPs/Hsps) and universal stress proteins (USPs) help schistosomes to respond to unfavourable conditions. The availability of genomes sequences information for Schistosoma japonicum, Schistosoma mansoni and Schistosoma haematobium provide unique research resources to apply bioinformatics analysis of its associated USPs to predict regulatory features from sequence analysis. The objectives of the research were to (i) Infer the biochemical and environmental regulation of universal stress proteins of Schistosoma species; (ii) Identify biological function relevant protein sequence and structure features for prioritized universal stress proteins from Schistosoma species; (iii) Determine the distinctive structural features of a predicted regulator of Schistosoma adenylate cyclase activity that has possible influence on the functioning of universal stress proteins. The findings revealed that (i) schistosomes USPs are hydrophilic and very reactive in the water environment or in aqueous phase, which seems adaptive with their immediate environment and developmental stages; (ii) The functions of Smp_076400 and Sjp_0058490 (Q86DW2) are regulated by conserved binding site residues and metallic ions ligands (Ca2+, Mg2+ and Zn2+), particularly Ca2+ predicted to bind to both USPs; (iii) The S. mansoni life cycle and stress resistance pathway protein (Smp_059340.1) is regulated by Ser53, Thr188, Gly210 and Asp207 residues. The overall scope has highlighted the role of bioinformatics in predicting exploitable regulatory features of schistosome universal stress proteins and biological pathways that might lead to identification of putative functional biomarkers of common environmental diseases. The findings of this research can be applicable to other areas of environmental health and environmental genomics. / Environmental Sciences / (D. Litt et Phil. (Environmental Sciences)

Ces dernières décennies, on a assisté à l’augmentation du nombre de technologies et de concepts permettant l’analyse des interactions intermoléculaires. Dans ce contexte, les puces à fluorescence restent les plus fréquemment utilisées. Cependant, cette technologie bien que très sensible et multiplexée, ne permet pas d’avoir accès aux paramètres cinétiques, indispensables au calcul des constantes d’affinité et la recherche de systèmes alternatifs s’impose. Dans cette optique, la résonance plasmonique de surface par imagerie (SPRi) est considérée comme une véritable option. Cette technologie se caractérise par l’absence de marquage et permet de suivre en temps réel d’infimes variations de masses consécutives à des interactions intermoléculaires sur la surface du prisme. L’obtention de constantes d’affinité est ainsi possible. En revanche, la SPRi présente un certain nombre de limites, principalement au niveau de la sensibilité et du multiplexage. Les objectifs de la thèse ont ainsi consisté à combler en partie ces différentes limites. La chimie de greffage basée sur l’utilisation d’oligonucléotides modifiés par un thiol a permis d’améliorer le multiplexage et de déposer plus de 1000 spots par cm² sur la surface d’or du prisme. Dans le même temps, la modification de la surface avec des colloïdes d’or et des dendrimères a permis pour des interactions ADN/ADN, d’atteindre une limite de détection de 2 nM (d’où un gain de 200%). En parallèle de ces travaux, diverses applications biologiques ont été effectuées. Une première étude a consisté à rechercher des ligands spécifiques des structures G-quadruplex des télomères. Une seconde étude s’est portée sur le complexe de partition bactérien. Par des études de criblage les bases impliquées dans l’interaction avec une protéine indispensable à la partition du plasmide F chez E.coli ont été identifiées. L’ensemble de ces travaux ont montré le fort potentiel de la SPRi et les applications potentielles qui en découlent sont nombreuses. / During the last decades a large number of technologies have been developed to analyze intermolecular interactions. In this context, the fluorescence biochips remain the most frequently used. Although this technology is very sensitive and multiplexed, it does not allow access to the kinetic parameters, essential to the calculation of the constants of affinity. Therefore, the research for alternative systems is essential. In this way, the Surface Plasmon Resonance imaging (SPRi) is considered as an opportunity. It is an optical detection process that can occur when a polarized light hits a prism covered by a thin metal layer. Under certain conditions free electrons at the surface of the biochip absorb incident light photons and convert them into surface plasmon waves. Perturbations at the surface of the biochip, such as an interaction between probes immobilized on the chip and targets, induce a modification of resonance conditions which can be measured. It is a label free technology which allows intermolecular interactions in real time and gives access to the kinetics parameters. However, SPRi is limited in sensitivity and multiplexing. The objectives of my PhD were to circumvent these various limits. Thus, we validated the immobilization of DNA probes on gold surface using thiol-modified oligonucleotide probes. Deposition carried out on non-modified gold surface, does not require electrical stimulation and expensive specific robotic devices. The thiol modification of the probes was shown to be very stable at room temperature, contrary to pyrrole and diazonium probes that need to be prepared just prior to their spotting. We demonstrate that thiol-modified oligonucleotide probes spotted on a gold surface of the SPRi-prisms are very robust and reproducible. We also demonstrated that this simple chemistry is compatible with high density arrays fabrication bearing more than 1000 spots using a classical spotter. Furthermore, the modification of the prism surface with gold colloids and dendrimers allowed for DNA/DNA interactions, to reach a detection limit of 2 nM. In parallel of this work, various biological applications were carried out and validate our previous developments. A first study was to screen G-quadruplex specific ligands to inhibit telomerase activity. We demonstrated that SPRi technology is particularly well adapted to the screening of interaction of small molecules with DNA probes and is sensitive enough to permit distinction between interactions with different DNA structures. The second study was on the bacterial partition complex. We study the DNA binding requirement involved in SopB-sopC specific interactions and analysed at the nucleotide level the bases involved in the binding efficiency and essential for the partition All this PhD work improved the SPRi technology and demonstrated its great potential in biological applications.

Lung cancer is one of the leading causes of cancer deaths in the United States. Radiation and chemotherapy are conventional treatments, but they result in serious side effects and the probability of tumor recurrence remains high. Therefore, there is an increasing need to enhance the efficacy of conventional treatments. Magnetic nanoparticles have been previously studied for a variety of applications such as magnetic resonance imaging contrast agents, anemia treatment, magnetic cell sorting and magnetically mediated hyperthermia (MMH). In this work, dextran coated iron oxide nanoparticles were developed and functionalized with peptides to target the nanoparticles to either the extracellular matrix (ECM) of tumor tissue or to localize the nanoparticles in subcellular regions after cell uptake. The magnetic nanoparticles were utilized for a variety of applications. First, heating properties of the nanoparticles were utilized to administer hyperthermia treatments combined with chemotherapy. The nanoparticles were functionalized with peptides to target fibrinogen in the ECM and extensively characterized for their physicochemical properties, and MMH combined with chemotherapy was able to enhance the toxicity of chemotherapy. The second application of the nanoparticles was magnetically mediated energy delivery. This treatment does not result in a bulk temperature rise upon actuation of the nanoparticles by an alternating magnetic field (AMF) but rather results in intracellular damage via friction from Brownian rotation or nanoscale heating effects from Neél relaxations. The nanoparticles were functionalized with a cell penetrating peptide to facilitate cell uptake and lysosomal escape. The intracellular effects of the internalized nanoparticles alone and with activation by an AMF were evaluated. Iron concentrations in vivo are highly regulated as excess iron can catalyze the formation of the hydroxyl radical through Fenton chemistry. Although often a concern of using iron oxide nanoparticles for therapeutic applications, these inherent toxicities were harnessed and utilized to enhance radiation therapy. Therefore, the third application of magnetic nanoparticles was their ability to catalyze reactive oxygen species formation and increase efficacy of radiation. Overall, iron oxide nanoparticles have a variety of cancer therapy applications and are a promising class of materials for increasing efficacy and reducing the side effects of conventional cancer treatments.

Head and neck cancer is a heterogeneous group of tumours, of which certain subgroups such as cancer of the mobile tongue frequently are associated with a relatively poor prognosis due to the high risk of regional failure and mortality rates that haven’t improved in a significant way over the last 3 decades, despite advancements in both diagnostics and treatment. Today we lack means to assess the biological aggressiveness of each individual tumour, which varies largely. Treatment comprises of surgery with additional radiotherapy and medical therapies in more advanced tumours. The focus in this thesis is on molecular biomarker expression in head and neck cancer and especially in association with radiotherapy. Increased knowledge paves the way to a more individualized cancer treatment aiming for better outcome and less overtreatment and sequelae. The aims of this thesis was: To map the effects of radiotherapy in both tumour and adjacent tissue for the possible markers hyaluronan, EGFR and mast cells. To investigate whether the expression of hyaluronan in the epithelium and connective tissue stroma and EGFR in the tumour correlates with the risk for developing cervical metastasis in N0 patients, and to find out whether the 3-year tumour-specific survival rates correlates with the expression of HA in the epithelium and EGFR in the tumour. To establish an animal model for radiation-induced mucositis and to use that model to examine the pattern of invading inflammatory cells. To investigate whether the expression of podoplanin in tongue cancer correlates with the risk for cervical metastasis and to determine whether the total amount of lymph vessels in the diagnostic biopsy has any impact on the clinical outcome. To investigate the differences in the metabolome of tongue cancer cell lines with different radiosensitivity. The most important findings of this thesis were: The expression of EGFR and hyaluronan hade the same pattern of expression in both tumour and adjacent tissues before radiotherapy. The expression of EGFR was increased in the epithelium of the adjacent tissue close to the tumour after radiotherapy. The intensity of the staining of hyaluronan was correlated to the 3-year survival rates in patients with tongue cancer. An experimental model for radiation-induced oral mucositis in rat was established and in this model a temporal pattern of macrophage invasion with two different subtypes of macrophages was found. There were no correlation between the expression of podoplanin in the tumour tissue and the cervical metastasis rate in patients with tongue cancer, but the younger patients were more likely to have a higher expression of podoplanin in their tumour than elder patients. Tongue cancer cell lines with different radiosensitivity respond to irradiation with different patterns of metabolic expressions.

The recreation of the complicated tissue microenvironment is essential to reduce the gap between in vitro and in vivo research. Polysaccharide-based hydrogels form excellent scaffolds to allow for three-dimensional cell culture owing to the favorable properties such as capability to absorb large amount of water when immersed in biological fluids, ability to form “smart hydrogels” by being shear-thinning and thixotropic, and eliciting minimum immunological response from the host. In this study, the biodegradable shear-thinning polysaccharide, gellan-gum based hydrogel was investigated for the conditions and concentrations in which it can be applied for the adhesion, propagation and assembly of different mammalian cell types in an unmodified state, at physiological conditions of temperature. Cell studies, to show successful propagation and assembly into three-dimensional structures, were performed in the range of hydrogels which were deemed to be optimum for cell culture and the cell types were chosen to represent each embryonic germ layer, i.e., human neural stem cells for ectoderm, human brain microvasculature cells for mesoderm, and murine β-cells for endoderm, along with a pluripotent cell line of human induced pluripotent stem cells, derived from human foreskin fibroblasts. Three-dimensional cell organoid models, to allow for gellan gum based bioprinting, were also developed using human induced pluripotent stem cells and human neural stem cells.

Energy functions are found to be a key of protein structure prediction. In this work, we propose a novel 3-dimensional energy function based on hydrophobic-hydrophilic properties of amino acid where we consider at least three different possible interaction of amino acid in a 3-dimensional sphere categorized as hydrophilic versus hydrophilic, hydrophobic versus hydrophobic and hydrophobic versus hydrophilic. Each of these interactions are governed by a 3-dimensional parameter alpha used to model the interaction and 3-dimensional parameter beta used to model weight of contribution. We use Genetic Algorithm (GA) to optimize the value of alpha, beta and Z-score. We obtain three energy scores libraries from a database of 4332 protein structures obtained from Protein Data Bank (PDB) server. Proposed energy function is found to outperform nearest competitor by 40.9% for the most challenging Rosetta decoy as well as better in terms of the Z-score based on Moulder and Rosetta decoy sets.

Biofuels produced from lignocellulosic biomass via the fermentation platform are sustainable energy alternatives to fossil fuels. Process Systems Engineering (PSE) uses computer-based tools and methods to design, simulate and optimize processes. Application of PSE tools to the design of economic biorefinery processes requires the development of simulation approaches that can be integrated with existing, mature PSE tools used to optimize traditional refineries, such as Aspen Plus. Current unit operation models lack the ability to describe unsteady state fermentation processes, link unsteady state fermentation with in situ separations, and optimize these processes for competing factors (e.g., yield and productivity). This work applies a novel architecture of commercial PSE tools, Aspen Plus and MATLAB, to develop techniques to simulate time-dependent fermentation without and with in situ separations for process design, analyses and optimization of the operating conditions. Traditional batch fermentation simulations with in situ separations decouple these interdependent steps in a separate “steady state” reactor followed by an equilibrium separation of the final fermentation broth. A typical mechanistic system of ordinary differential equations (ODEs) describing a batch fermentation does not fit the standard built-in power law reaction kinetics model in Aspen Plus. To circumvent this challenge, a novel platform that links the batch reactor to a FORTRAN user kinetics subroutine (incorporates the ODEs) combined with component substitution (to simulate non-databank components) is utilized to simulate an unsteady state batch and in situ gas stripping process. The resulting model system predicts the product profile to be sensitive to the gas flow rate unlike previous “steady state” simulations. This demonstrates the importance of linking a time-dependent fermentation model to the fermentation environment for the design and analyses of fermentation processes. A novel platform linking the genetic algorithm multi-objective and single-objective optimizations in MATLAB to the unsteady state batch fermentation simulation in Aspen Plus through a component object module communication platform is utilized to optimize the operating conditions of a typical batch fermentation process. Two major contributions are: prior concentration of sugars from a typical lignocellulosic hydrolysate may be needed and with a higher initial sugar concentration, the fermentation process must be integrated with an in situ separation process to optimize the performance of fermentation processes. With this framework, fermentation experimentalists can use the full suite of PSE tools and methods to integrate biorefineries and refineries and as a decision-support tool to guide the design, analyses and optimization of fermentation-based biorefineries.