Au cours de la dernière décennie, quelques (rares) exemples de copolymères à blocs présentant des liens supramoléculaires entre les blocs constitutifs ont été décrits. En raison du caractère réversible de l’association des blocs macromoléculaires, de tels polymères sont d’un grand intérêt pour le développement de matériaux nanostructurés, ayant des propriétés auto-cicatrisantes ou à processabilité améliorée. L’objectif principal de cette thèse est d’élaborer de nouvelles architectures supramoléculaires, en combinant la polymérisation RAFT et l’association spécifique par liaisons H des groupements thymine et diaminopyridine. La stratégie employée a consisté dans un premier temps en la synthèse d’agents de transfert et d’un amorceur radicalaire fonctionnalisés par des unités complémentaires thymine et diaminopyridine. Ces précurseurs ont permis de générer, par polymérisation RAFT, des polymères α- ou α,ω-fonctionnalisés de manière quantitative par ces motifs à liaisons H. L’auto-assemblage des blocs polymères ainsi obtenus a été mis en évidence par RMN 1H, AFM et par des mesures rhéologiques. Cette démarche a été adaptée dans un second temps à l’élaboration de copolymères greffés supramoléculaires hydrophiles, basés sur le complexe d’inclusion β-cyclodextrine/adamantane. / Over the past decade, some (rare) examples of block copolymers with supramolecular links between the building blocks have been described. Because the association between macromolecular blocks is a reversible process, such polymers are of great interest in the field of nanostructured materials, self-healing materials, or processing aid. The main goal of this work is to develop new supramolecular architectures, by a combination of RAFT polymerization and H-bonding. In a first step, several chain transfer agents and a radical initiator possessing complementary thymine or diaminopyridine H-bonding moeties were synthesized. These precursors were used to generate a panel of polymers α- or α,ω-functionalized with these H-bonding stickers in a quantitative manner. The self-assembly of the resulting polymer blocks was highlighted by 1H NMR, AFM and rheological measurements. This approach was subsequently adapted to the development of hydrophilic supramolecular comb-shaped polymers, based on the β-cyclodextrin/adamantane host/guest complexation.

La première partie de ce travail traite des propriétés mécaniques de matériaux silicones de type HCR réalisés à partir de macromolécules dont la structure a dû être déterminée. Les relations existantes entre macromolécules initiales et matériaux finaux sont discutées. Cela nous a permis d’isoler un seul paramètre à ajuster pour développer un procédé permettant l’architecturation des propriétés mécaniques d’une membrane en silicone : la densité de réticulation. Ensuite, nous avons comparé trois classes d’élastomères silicones en tentant de comprendre comment les différences de réseau, comprenant une matrice commune mais des moyens de réticulation et renforcements différents, influaient sur les propriétés mécaniques simples (par exemple de traction et de dureté) et plus avancées (comme les propriétés dynamiques et élastiques). L’influence du réseau, des charges, du mécanisme de réticulation et les différentes interactions existantes entre eux, ont orienté notre choix sur la formulation dont la réticulation est la plus contrôlée, et les propriétés élastiques les meilleures, c’est-à-dire les LSR. Nous avons alors développé un procédé permettant de contrôler localement la cinétique de réticulation d’une formulation silicone par dégradation du catalyseur aux UV. Ce procédé très simple nous a permis d’obtenir une large gamme de propriétés mécaniques avec des conditions de réticulations semblables. Les deux premières études ont permis d’expliquer plus facilement le comportement mécanique des matériaux-sous-réticulés. Des membranes anisotropes ont pu être réalisées à l’aide de masques à motifs. Les membranes obtenues ont même ouvert la porte à des perspectives très intéressantes, comme l’architecturation de surface par lavage au solvant, la post-fonctionnalisation chimique via les fonctions non réagies, et le collage cohésif silicone-silicone. / The first part of this work deals with the mechanical properties of high consistency silicone rubbers crafted from macromolecules whose structure had to be determined, and the existing links between initial macromolecules and final material behavior. It allowed us to choose only one parameter to adjust in order to manufacture mechanically architectured silicone membranes: the crosslinking density. Then, three types of silicone elastomers were compared in an attempt to understand how network differences, comprising the same matrix but different crosslinking chemistry and reinforcement, influenced the mechanical properties, both simple (such as hardness and modulus), and more complex (dynamic, and elastic). The influence of the network, fillers, matrix and the different interactions between them oriented our choice towards the silicone ty with the best control of the crosslinking and best elasticity, namely LSR. Then, we developed a process allowing the local control of the crosslinking kinetics of a silicone formulation by UV degradation of the crosslinking catalyst prior to regular heat crosslinking. From this simple and versatile process, materials with a wide range of mechanical properties were manufactured and characterized. The first two studies helped us to understand the mechanical properties of under and regularly crosslinked materials. Anisotropic membranes were also manufactured thanks to the use of patterned masks during irradiation. The obtained materials opened up new promising perspectives, such as surface architecturation through solvent wash of under crosslinked zone, chemical post functionalization of unreacted moieties, or even cohesive silicone-silicone gluing.

L’objectif de cette thèse était d’allier, dans un même polymère, le contrôle de l’architecture macromoléculaire, la fonctionnalité et la possibilité de sa mise en oeuvre en phase fondue, tout en préservant l’aspect environnemental. Les structures polymères synthétisées sont basées sur des chaînes biodégradables et/ou biosourcées d’acide polylactic (PLA), de polyhydroxybutyrate (PHB) et de polycaprolactone (PCL). Ces dernières ont été assemblées dans des structures macromoléculaires branchées à design contrôlé et portant des fonctions thiols, ces fonctions ont permis le greffage de monomères dotés de groupements ammoniums quaternaires, sur les structures obtenues, via une addition radicalaire thiol-ène.Les produits obtenus ont été mélangés en phase fondue, par extrusion, avec des matrices de PLA et de PCL, pour préparer des films. Ces derniers ont fait l’objet d’une étude d’activité antibactérienne qui a montré une grande efficacité envers différents types de bactéries / The aim of this work was to develop polymers that combine controlled macromolecular architectures, functionality, melt processing and an environmentally friendly aspect. The obtained polymeric structures were based on biodegradable and/or biosourced chains of polylactic acid (PLA), polycaprolactone (PCL) and polyhydroxybutyrate (PHB). The lasts were assembled in branched macromolecular structures with controlled design and bearing thiol functions, these functions allowed the grafting of quaternary ammoniumcontaining monomers on the branched structures according to a thiol-ene radical addition mechanism. The final products were blended with neat matrices of PLA and PCL in the melt state, by extrusion process, to make polymeric films. The obtained film-shaped blends were subjected to antibacterial activity study showing there high efficiency against different types of bacteria

<p dir="ltr">The recent COVID-19 pandemic has emphasized the risk of respiratory infections leading to acute respiratory distress syndrome (ARDS). A significant factor contributing to poor ARDS outcomes is the impairment of lung surfactant due to infiltrating surface-active proteins and phospholipases during lung inflammation. Lung surfactant's vital role in stabilizing alveoli by reducing air-water interfacial tension becomes evident as its dysfunction severely compromises respiratory function. Although lung surfactant (LS) replacement therapy effectively addresses neonatal LS deficiencies, its efficacy in ARDS treatment for adults remains limited. The challenge lies in the chemical similarity between current animal-extracted surfactants and human lung surfactant which are both phospholipid-based. To address this issue, this dissertation outlines a transformative "polymer lung surfactant (PLS)" designed to overcome the limitations of conventional exogenous surfactants in treating ARDS.</p><p dir="ltr">Firstly, a formulation method, referred to as equilibration-nanoprecipitation (ENP), is established which achieves reproducibility, controls sizing, and limits dispersity of the PLS formulation consisting of block copolymer (BCP) kinetically "frozen" micelles/nanoparticles suspended in water. The method uses a two-step approach of 1) equilibrating the BCP nanoparticles in a water/co-solvent mixture and 2) removing co-solvent using dialysis against a large water reservoir. Comparison of ENP with a conventional solvent-exchange technique through experimental and computational analysis yields further insights into ENP's advantages.</p><p dir="ltr">Next, various studies are highlighted which provide fundamental characterizations of the air-water surface behavior and physical properties of BCP nanoparticles in water. The air-water surface properties of block copolymers have been studied extensively when spread as free chains in organic solvent; however, little was previously known about air-water interfacial behavior of water-spread polymer nanoparticles. The studies address such topics as the effect of nanoparticle size, effect of nanoparticle core chemistry, and the effect of temperature on surface-mechanical behavior. Insights into nanoparticle molecular structure at the interface are provided through X-ray reflectivity and grazing incidence X-ray diffraction. The effect of temperature is further characterized by developing novel NMR and Langmuir trough methods to determine the physical state (glassy vs rubbery) of the core domain in the nanoconfined state at temperatures above and below physiologic temperature.</p><p dir="ltr">Lastly, <i>in vivo </i>studies are presented which demonstrate the detailed and promising proof-of-concept results on the efficacy of the PLS technology in mouse models of lung injury. The PLS therapy not only improves biomechanical function of the lung, but it also significantly lowers the extent of lung injury as shown by histological analysis and inflammatory marker measurements. An additional <i>in vivo </i>study is presented which highlights challenges in the delivery of the liquid PLS suspension to the lungs. The <i>in vivo </i>studies ultimately provide solid motivation for continued research into the development of the PLS therapy.</p><p dir="ltr">Given the promising potential of the PLS technology shown in the <i>in vivo</i> studies, the materials characterizations shared in this presentation offer valuable insights into the design of a novel PLS therapy. From these insights, key design parameters such as nanoparticle size characteristics, core chemistry, and core molecular weight can be chosen to produce the most desirable material properties. Overall, this dissertation furthers the progress of PLS therapeutic development and will hopefully ultimately contribute to improved health outcomes in patients suffering from ARDS.</p>

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<p dir="ltr">DNA sequence design requires the ability to identify possible tertiary structural defects, secondary structure disruptions, and self-complimentary stretches that will disallow your complimentary strands to come together to form the desired duplex design. However, there is a need for those self-complimentary stretches, especially when designed with the intent for this to homo-oligomerize into the desired building block. With the programmability of nucleic acid hybridization, there is an expanding field wherein this specific, self-complimentary design feature can give new possibility of fine-tuning DNA self-assembly (Chapter 1) or overcome a previously thought limit of DNA ligation (Chapter 2).</p><p dir="ltr">The first chapter will closely look at the branched kissing loop interaction. This interaction was studied as a homo-polymerizable DNA building block that is topologically closed. As such, this paranemic motif has increased stability due to the Watson-Crick base pairing being “protected” by a 3-base adenine branch which close the loop of the sticky-end, meaning no free ends in the binding region. With this, herein we report that the intended higher-level structure could influence the lower-level building block formation. In DNA nanotechnology, this could mean the final higher-level structure would allow for fine-tuning as this would dictate the building blocks that fill in the defected parts of the higher-level structure.</p><p dir="ltr">The second chapter looks at the more finite than broad picture. Whilst the first chapter focusses on the impact the microscale has on the nanoscale through a homo-polymerizable design, the second chapter focusses on the ability to break barriers with homo-polymerizable design. In this chapter, we prove that with our splint strand design, when improved with a hairpin loop on the terminal ends, we can ligate DNA strands enzymatically as short as 16 nucleotides with an efficiency of 97% at high concentrations (100 uM). These hairpins allow for a stable, robust splint strand as they are a self-complimentary region which will maintain its shape throughout the process of joining together the 5’ and 3’ ends of the target strand.</p><p dir="ltr">Overall, this dissertation hopes to prove that homo-polymerizable DNA sequence designs are helping expand upon the DNA nanotechnology toolbox by introducing new possibilities for nanoscale design, as well as push past previously held boundaries through necessary added stability afforded by the self-complimentary strands.</p>

<p dir="ltr">Ebola virus is an enveloped filamentous shaped RNA virus which causes severe hemorrhagic fever in humans. Multiple outbreaks of different strains of ebolavirus have been reported in the past years with limited therapeutics available for treatment. Despite some advances in treatment, there remains a lack of knowledge about the mechanisms of ebolavirus replication in host cells.</p><p dir="ltr">Ebolavirus encodes for seven structural proteins with matrix protein (VP40) being the most abundantly expressed viral protein. VP40 is essential for viral assembly and budding as expression of VP40 alone is sufficient for formation of virus-like particles (VLPs). VP40 also disassembles during viral entry to help in viral and host cell membrane fusion. Oligomerization of VP40 has been reported to decrease viral replication and transcription. VP40 can perform these diverse functions by virtue of changes in conformation and oligomerization state. VP40 predominantly exists as a dimer through hydrophobic interactions between the alpha helices of the two protomers. Furthermore, VP40 oligomerizes into a hexamer which serves as the structural unit for cylindrical matrix layer formation. VP40 also forms a ring-shaped octamer for regulation of viral transcription. The different oligomeric forms of VP40 exist in an equilibrium for successful viral infection. However, the exact mechanism of formation, stability, and energetics of conversion between these oligomeric forms is unknown.</p><p dir="ltr">In this study, we performed biophysical analysis on the dimerization interface and identified keystone interactions which when abrogated lead to complete disruption of dimer interface. In addition, peptidomimetics approach was used to design and synthesize a library of compounds to probe the dimerization interface. The compounds were screened using thermal shift assay and then compared using MST and ITC studies. We identified that a peptide mimicking the alpha helical region stabilized by a p-xylene di-cysteine staple was able to bind to VP40 dimer. We also determined that this peptide binds near the dimer interface and was able to slightly shift equilibrium of VP40 dimer towards monomer formation.</p><p dir="ltr">Overall, this report sheds light on critical interactions required for VP40 dimer formation and stability and introduces use of peptidomimetics to probe for VP40 dimerization interface to understand energetics of oligomerization equilibrium, thereby increasing our knowledge about disease mechanism and paving way for development of therapeutics.</p>

<p dir="ltr">CaaX proteins, comprising approximately 300 members in the human protein database, represent a diverse group implicated in fundamental cellular processes, including proliferation, differentiation, trafficking, and gene expression. To carry out such vital cellular functions, CaaX proteins need to undergo three sequential post-translational modifications (PTM) through the CaaX pathway, which consists of isoprenylation (farnesylated or geranylgeranylated), endoproteolysis, and methylation. Among the CaaX family of protein, the Ras superfamily, plays a pivotal role in cell growth and survival. Mutations in <i>Ras proteins</i> are associated with a spectrum of cancers, presenting a significant challenge for therapeutic intervention. This thesis explores the intricate landscape of PTMs of CaaX proteins, with a focus on methylation, which is carried out by membrane protein isoprenylcysteine carboxyl methyltransferase (Icmt), and its potential as a therapeutic target, particularly for Ras-driven cancers.</p><p><br></p><p dir="ltr">Icmt is unique as it is the sole methyltransferase which carries out the third PTM of methyl esterification of CaaX proteins with the aid of co-substrate SAM, which serves as the methyl donor. Additionally, how Icmt, a membrane protein localized in the endoplasmic reticulum (ER), brings these two chemically diverse molecules in close enough proximity to promote catalysis, is very intriguing and is not yet fully understood. This thesis focuses on studying the structural and functional properties of Ste14, the yeast homolog of Icmt, in order to better understand the Icmt family of proteins. Ste14 is a functional homolog of human Icmt, sharing 41% sequence identity and 62% sequence similarity. Furthermore, Ste14 can be functionally purified unlike human Icmt. Together, these attributes make Ste14 an ideal system to study.</p><p dir="ltr"><br>The first project explores Ste14 and substrate binding, focusing on residues that determine farnesylated vs geranylgeranylated substrate specificity. It is essential to note that wild-type Ste14 recognizes farnesylated and geranylgeranylated substrate equally, with no preference to one over the other. Conserved residues found in Loop 2 and Transmembrane 3 of Ste14 were mutated to alanine and assessed for their activity with AGGC, the minimal geranylgeranylated CaaX substrate. Mutants which showed nearly zero percent activity with AGGC in comparison to wild type were further analyzed to understand if this loss of mutant activity with AGGC was potentially due to the mutant's inability to bind with AGGC. A photoreactive AGGC analog was used to carry out the photolabeling experiments and residues were analyzed for their binding ability with geranylgeranylated substrate. Mutants were further analyzed to understand the effect of mutation on structural integrity, to gauge which residues are essential for catalysis and for maintaining structural integrity of Ste14. Results demonstrated that residues F80 and E98 are essential for structural stability while L81 and L82 are essential for catalysis. This project would overall help better understand the lesser studied Ste14-substrate binding.</p><p><br></p><p dir="ltr">In the second project, the focus shifts to study Ste14 and co-substrate SAM binding by using electron paramagnetic resonance spectroscopy (EPR) and site directed spin labeling (SDSL). The biophysical technique of EPR requires much less protein and serves as great tool to study conformational change Ste14 undergoes on SAM binding, 3 non conserved residues found in the SAM binding region of Ste14, were individually mutated to cysteine, and had a spin label MTSL attached to their purified active mutant forms. Through EPR the conformational changes of Ste14 during methylation specifically during SAM binding was analyzed by visualizing the movement of MTSL attached residue. Results showed of the three non-conserved residues, A223 and E227 were immobile during SAM binding while T164 residue displayed flexibility during SAM binding during SAM binding and release process. This study would help understand the protein dynamics that Icmt undergoes upon SAM binding.</p><p><br></p><p dir="ltr">The final section centers on inhibiting the third step of the CaaX pathway, which is methyl esterification, by targeting Icmt. The project involved testing a library of Icmt inhibitors and evaluating their ability to inhibit Icmt activity. Of this library of bi-substrate analog inhibitors, compounds YD 1-66, YD 1-67 and YD 1-77 emerge as promising inhibitors against human Icmt, laying the foundation for further studies to develop more potent inhibitors. This section accentuates the strategies employed to target Icmt and the potential of these inhibitors in combating Ras-driven cancers.</p><p><br></p><p dir="ltr">This thesis provides an extensive analysis of the structure and function of Ste14. The varied studies and their insights contribute to a comprehensive understanding of Icmt and pave the way for the rational design of potent chemotherapeutic inhibitors for Ras-driven cancers. The multifaceted research presented in this thesis reveals several new possibilities for targeted therapies in the field of oncology.</p>