<p dir="ltr">This dissertation investigates novel approaches for modifying cellulose nanofibers (CNFs) to develop sustainable alternatives to petroleum-based plastics. As plastic production continues to rise dramatically – from 2 million tons in 1950 to a projected 1231 million tons by 2060 – the need for renewable, biodegradable alternatives has become increasingly urgent. This work presents three interconnected studies exploring different aspects of CNF modification and application.</p><p dir="ltr">The first study introduces an innovative method for developing superhydrophobic (SHP) coatings using CNFs lyophilized from a 10 wt% <i>tert</i>-butyl alcohol slurry. Through solvent-free mechanochemical modification, we successfully produced oleic acid-modified CNFs (OL-CNFs), which exhibited exceptional SHP properties, demonstrating high contact angles, low hysteresis, and remarkable durability. Suspensions of OL-CNF were utilized in various spray coating applications, including moisture barriers and atmospheric water harvesting systems.</p><p dir="ltr">The second study explores the trifluoroacetylation of CNFs using trifluoroacetic anhydride (TFAA) without additional base. By employing glucopyranosides as small molecule models to guide reaction optimization, we developed a method for controlled trifluoroacetylation while preserving CNF crystallinity. Notably, we introduce a novel approach for quantifying the degree of substitution using ¹⁹F NMR spectroscopy of saponified trifluoroacetylated CNFs (TFA-CNFs) in methanol-<i>d4</i>, offering improved accuracy over traditional methods.</p><p dir="ltr">The third study examines the potential of TFA-CNFs as reinforcing materials in biodegradable polymer composites, particularly with poly(butylene adipate-co-terephthalate) (PBAT). We investigate the dispersibility of TFA-CNFs in various organic solvents and explore methods for creating PBAT/TFA-CNF blends. The research reveals promising aspects of TFA-CNFs, including their compatibility with biodegradable polymers and rapid ester hydrolysis in soil, while also identifying key challenges and opportunities for future development.</p><p dir="ltr">Together, these studies advance our understanding of sustainable CNF modification strategies and their applications in developing eco-friendly materials. This work contributes to ongoing efforts to address environmental challenges posed by conventional plastics while maintaining high performance standards for material applications.</p>

<p dir="ltr">Phospholipase Cε (PLCε) is required for normal cardiovascular function, and dysregulation of its expression or activity has been shown to cause cardiac hypertrophy and heart failure. However, regulation of PLCε by the RhoA small GTPase protects the heart against ischemia-reperfusion injury, particularly downstream of G_12/13-coupled receptors. Despite the role of RhoA and PLCε in driving the cardioprotective response, little is known about how these proteins interact to increase lipase activity.<b> </b>RhoA was initially thought to bind to PLCε through one of its C-terminal Ras association (RA) domains, which are essential for its regulation by other GTPases. However, the RA domains are dispensable for both RhoA binding and activation, and further truncations of PLCε narrowed its binding site to the highly conserved PLC catalytic core. Functional studies implicated an insertion within the catalytic TIM barrel domain, known as the Y-box, as a requirement for RhoA-dependent activation of PLCε. However, the Y-box does not bind the GTPase. The goal of this dissertation is to identify the molecular mechanism by which RhoA binds to PLCε and increases its activity using structural and functional studies. The successful completion of these studies will map the interaction between these two critical signaling proteins, as well as identify elements in PLCε required for activation at the membrane. Ultimately, this knowledge can be exploited to develop lead therapeutic compounds that modulate this interaction to improve cardiovascular health.</p>

<p dir="ltr">The Centers for Disease Control and Prevention’s 2019 Antibiotic Resistance Threats Report highlights more than 2.8 million antibiotic infections each year, with at least 35,000 deaths per annum attributed to antibiotic resistance. The CDC’s 2022 COVID-19 Impact Report emphasizes a 15% increase in hospital-acquired resistant infections between 2019 and 2020, many which are caused by Gram-negative bacteria, bacteria characterized by two encapsulating membranes. The limited treatment options for Gram-negative bacterial infections underscore the critical need for new strategies to combat these pathogens. The β-barrel assembly machinery complex (BAM) is a protein complex located in the outer membrane (OM) of Gram-negative bacteria, facilitating the folding and insertion of β-barrel outer membrane proteins (OMPs) into the OM. Inhibiting the function of this complex is lethal for Gram-negative bacteria, making BAM a significant and promising drug target.</p><p dir="ltr"><i>Fusobacterium nucleatum</i> is a Gram-negative pathogen that functions in the oral microbiome, interacting with multiple levels of biofilm colonizers. <i>F. nucleatum</i> causes oral infections and is linked to colorectal cancer, impacting treatment response and disease recurrence. The pathogenicity of <i>F. nucleatum</i> in both biofilm formation and in cancer involves OMPs whose biogenesis relies on BAM; however, BAM has not been characterized in this organism. The goal of our study here is to better understand the composition, structure, and function of BAM and its potential as a drug target for <i>F. nucleatum</i>. We first used bioinformatics analysis and proteomics to investigate the putative composition of the BAM complex in <i>F. nucleatum</i>. While the core component BamA was identified, there was a notable absence of other typical accessory proteins in this organism's genome. Therefore, we postulate that unlike other bacteria such as <i>E. coli</i> and <i>A. baumannii</i>, the biogenesis of OMPs in <i>F. nucleatum</i> is mediated solely by BamA without the need of accessory components.</p><p dir="ltr">To investigate how BamA can accomplish OMP biogenesis itself, we employed biophysical techniques to analyze the structure of <i>Fn</i>BamA. We resolved the cryo-EM structure of <i>Fn</i>BamA in complex with several Fabs which showed novel structural features not previously observed in bacteria. In these structures, <i>Fn</i>BamA was found to contain four N-terminal POTRA domains arranged in a J-shaped conformation, rather than elongated. The Fab was found to bind primarily along POTRA 3 which likely stabilizes the unique conformation of the POTRA domains. The C-terminal 16-stranded b-barrel domain was observed as an inverted dimer, with the dimer interface mediated by direct interaction of the b1 strands along the lateral seam of both barrel domains. Additionally, we determined the X-ray crystal structure of the barrel domain alone which was found as a monomer. Measurements of the barrel domain of <i>Fn</i>BamA reveal it has a different shape and size than is found in other BamA structures such as in <i>E. coli</i>. Together, these structural differences provide clues to how <i>Fn</i>BamA alone may accomplish OMP biogenesis when additional components are required in other bacteria. Our ongoing studies aim to further characterize the molecular structure and function of <i>Fn</i>BamA in conjunction with promising antibiotics and other putative BAM components if discovered.</p>

<p dir="ltr">Anti-CRISPR (Acr) proteins are produced by phages to deactivate CRISPR–Cas systems in bacteria and archaea, thus expanding the CRISPR toolbox for genome editing. In this study, we present the structure and function of AcrIF24, an Acr protein that inhibits the type I-F CRISPR–Cas system in Pseudomonas aeruginosa. AcrIF24 forms a homodimer that binds to two surveillance complexes (Csy), preventing CRISPR RNA from hybridizing with target DNA. Additionally, AcrIF24 acts as an anti-CRISPR-associated (Aca) protein, suppressing the transcription of the acrIF23-acrIF24 operon. Whether alone or in complex with Csy, AcrIF24 binds to the acrIF23-acrIF24 promoter DNA with nanomolar affinity. The 2.7 Å structure of the Csy–AcrIF24–promoter DNA complex reveals how transcriptional suppression occurs. Our findings demonstrate that AcrIF24 functions as an Acr-Aca fusion protein and enhance our understanding of the varied mechanisms employed by Acr proteins.</p><p dir="ltr">In the ongoing evolutionary struggle between bacteria and bacteriophages, the emergence of CRISPR and anti-CRISPR systems has shaped host-pathogen interactions significantly. Bacteriophages exert intense selective pressure on bacteria, driving the evolution of defense mechanisms such as restriction enzymes and the CRISPR-Cas system. Conversely, bacteriophages have evolved anti-CRISPR proteins (Acrs) to counteract CRISPR-Cas-mediated targeting. Here, we investigate the interactions and regulatory mechanisms within co-encoded Acrs, focusing on AcrVA1-5 from a prophage within Moraxella bovoculi. Our findings reveal that AcrVA1 and AcrVA2 form a stable complex capable of inhibiting Cas12a-mediated DNA cleavage, with AcrVA1 regulating the activity of AcrVA2. Additionally, AcrVA4 and AcrVA5 form a complex that modulates Cas12a activity by inhibiting DNA binding and lysin acetylation, respectively. Structural and biochemical analyses uncover a complex regulatory network governing the function of co-encoded Acrs, highlighting their role in downregulating DNA targeting in response to Cas12a presence and aiding the survival of both phage and host bacteria during infection.</p>

Mathematical models are important tools in systems biology, since the regulatory networks in biological cells are too complicated to understand by biological experiments alone. Analytical solutions can be derived only for the simplest models and numerical simulations are necessary in most cases to evaluate the models and their properties and to compare them with measured data. This thesis focuses on the mesoscopic simulation level, which captures both, space dependent behavior by diffusion and the inherent stochasticity of cellular systems. Space is partitioned into compartments by a mesh and the number of molecules of each species in each compartment gives the state of the system. We first examine how to compute the jump coefficients for a discrete stochastic jump process on unstructured meshes from a first exit time approach guaranteeing the correct speed of diffusion. Furthermore, we analyze different methods leading to non-negative coefficients by backward analysis and derive a new method, minimizing both the error in the diffusion coefficient and in the particle distribution. The second part of this thesis investigates macromolecular crowding effects. A high percentage of the cytosol and membranes of cells are occupied by molecules. This impedes the diffusive motion and also affects the reaction rates. Most algorithms for cell simulations are either derived for a dilute medium or become computationally very expensive when applied to a crowded environment. Therefore, we develop a multiscale approach, which takes the microscopic positions of the molecules into account, while still allowing for efficient stochastic simulations on the mesoscopic level. Finally, we compare on- and off-lattice models on the microscopic level when applied to a crowded environment.

This thesis covers a wide variety of projects within the domain of computational structural biology. Structural biology is concerned with the molecular structure of proteins and nucleic acids, and the relationship between structure and biological function. We used molecular modeling and simulation, a purely computational approach, to study DNA-linked molecular nanowires. We developed a computational tool that allows potential designs to be screened for viability, and then we used molecular dynamics (MD) simulations to test their stability. As an example of using molecular modeling to create experimentally testable hypotheses, we were able to suggest a new design based on pyrrylene vinylene monomers. In another project, we combined experiments and molecular modeling to gain insight into factors that influence the kinetic binding dynamics of fibrin "knob" peptides and complementary "holes." Molecular dynamics simulations provided helpful information about potential peptide structural conformations and intrachain interactions that may influence binding properties. The remaining projects discussed in this thesis all deal with RNA structure. The underlying approach for these studies is a recently developed chemical probing technology called 2'-hydroxyl acylation analyzed by primer extension (SHAPE). One study focuses on ribosomal RNA, specifically the 23S rRNA from T. thermophilus. We used SHAPE experiments to show that Domain III of the T. thermophilus 23S rRNA is an independently folding domain. This first required the development of our own data processing program for generating quantitative and interpretable data from our SHAPE experiments, due to limitations of existing programs and modifications to the experimental protocol. In another study, we used SHAPE chemistry to study the in vitro transcript of the RNA genome of satellite tobacco mosaic virus (STMV). This involved incorporating the SHAPE data into a secondary structure prediction program. The SHAPE-directed secondary structure of the STMV RNA was highly extended and considerably different from that proposed for the RNA in the intact virion. Finally, analyzing SHAPE data requires navigating a complex data processing pipeline. We review some of the various ways of running a SHAPE experiment, and how this affects the approach to data analysis.

Xu, Xiao-ping. / "March 1999." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 152-166). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

L’objectif de cette thèse est d’étudier la synthèse et les propriétés d’auto-assemblage en milieu aqueux de copolymères « intelligents » capables de former des agrégats supramoléculaires en réponse à deux stimuli : la température du milieu et la présence de dioxyde de carbone (CO2). Pour cela, une gamme de copolymères diblocs amphiphiles composés d’un bloc hydrophile d’acrylate de polyethylèneglycol méthyléther) (PEGA) et d’un bloc stimulable contenant une distribution statistique d’unités PEGA et acrylate de diéthlèneglycol éthyléther (DEGA) (thermosensibles) et acrylate de diéthylaminoéthyle DEAEA (CO2-sensible), a été préparée par polymérisation radicalaire contrôlée par les nitroxydes (NMP). Dans un second temps, il a été mis en évidence que la température ainsi que la présence de CO2 dans la solution influencent le comportement auto-associatif des copolymères dans l’eau. Par la suite, le bloc hydrophile a été remplacé par une séquence de dextrane, ce qui a permis de préparer de nouveaux copolymères diblocs fonctionnels, stimulables par la température et le CO2. Dans ce cas, deux techniques de polymérisation radicalaire contrôlée (NMP et ATRP) ont été testées afin d’obtenir les copolymères possédant l’architecture la mieux définie possible. / This project aims to investigate the synthesis and properties of dual stimuli-responsive block copolymers able to self-assemble into supramolecular aggregates in response to two stimuli: the temperature and the presence of carbon dioxide in the aqueous solution. Therefore, a range of amphiphilic diblock copolymers composed of a hydrophilic block of polyethylene glycol methylether acrylate (PEGA) and a statistical block of PEGA, diethylene glycol ethyl ether acrylate (DEGA) and diethylaminoethyl acrylate (DEAEA) was prepared via nitroxide-mediated polymerization (NMP) and the level of control of each synthesis was studied. We evidenced that temperature and CO2 play a different role in the self-assembly of such block copolymers. Finally, the introduction of dextrane as hydrophilic block coming from renewable resources allows for the preparation of novel “smart” amphiphilic diblock copolymers. In order to synthesize these block copolymers with well-defined structure, both NMP and atom transfer radical polymerization (ATRP) were investigated in parallel.

Le polyéthylène à ultra haute masse molaire (UHMWPE) présente une viscosité si forte à l’état fondu que seuls des procédés de mise en forme de type frittage peuvent être employé. Ce procédé rarement utilisé pour les polymères reste peu étudié. En particulier les deux principaux mécanismes généralement mentionnés que sont le réenchevêtrement et la cocristallisation aux interfaces sont difficilement observables séparément. Le UHMWPE, grâce à sa très haute viscosité à l’état fondu et grâce à son plateau caoutchoutique extrêmement étendue en température, peut faire l’objet d’essais mécaniques à la fois à l’état semi-cristallin et à l’état fondu. Des poudres natives de UHMWPE de masses molaires comprises entre 0,6 et 10,5 Mg.mol-1 sont utilisées comme matériau de départ pour leur mise en oeuvre par frittage. La consolidation des interfaces par soudage des particules a été effectuée sous pression à différentes températures supérieures au point de fusion et pour différentes durées. Des expériences de traction effectuées soit à température ambiante soit au-dessus du point de fusion ont permis de distinguer le rôle de l'interdiffusion des chaînes au travers des interfaces de celle de la cocristallisation dans les mécanismes de soudage de particules. Il s'est avéré qu’un soudage efficace se produit dans une échelle de temps très courte. La très faible influence de la durée de frittage par rapport à celle de la température de frittage a prouvé que l'interdiffusion des chaînes n'est pas régie par un mécanisme de reptation. L'explosion à la fusion des cristaux « hors-équilibre » de la poudre native est suggérée être le mécanisme principal permettant un réenchevêtrement dans un laps de temps beaucoup plus court que celui de la reptation. La cocristallisation est un phénomène si efficace dans la consolidation de l'interface à l'état solide qu'elle masque significativement la cinétique de réenchevêtrement gouverné par la température, visible dans les tests mécaniques à l’état fondu. / One of the main issues of ultra-high-molecular-weight polyethylene (UHMWPE) is to overcome its very high viscosity. Powder sintering is then often required instead of injection or extrusion. However, sintering mechanisms remain partially understood. Indeed, the two main mechanisms generally mentioned for interparticle welding, i.e. re-entanglement and cocrystallization, can hardly be observed separately. Fortunately, due to its very high molecular weight, UHMWPE exhibits an exceptionally broad rubbery plateau so that mechanical tensile tests can be easily performed both below and above the melting point. Four UHMWPE of molecular weight in the range of 0.6.106 g.mol-1 to 10.5.106 g.mol-1 have been processed by means of sintering of nascent powders. The interface consolidation or particle welding was carried out under pressure at various temperatures above the melting point and for various durations. Tensile drawing experiments performed either at room temperature or above the melting point enabled to discriminate the role of chain interdiffusion through the particle interface from that of cocrystallization in the mechanism of particle welding. It turned out that an efficient welding occurred within a very short time scale. The very weak influence of sintering time compared to that of sintering temperature gave evidence that chain interdiffusion was not governed by a reptation mechanism. The entropy-driven melting explosion of the “non-equilibrium” crystals in the nascent powder is suggested to be the main mechanism of the fast chain reentanglement and subsequent particle welding within a time scale much shorter than the reptation time. Cocrystallization is so much efficient in the interface consolidation in the solid state that it significantly hides the temperature-governed kinetics.

Particles and macromolecular components, including biopolymers (protein and carbohydrate), are viewed as the main foulants in the complex feed submerged membrane filtration systems such as membrane bioreactor (MBR). This work focused on two aspects of fouling in complex fluids: 1- Assessing fouling propensity and mechanisms for various model solutions. 2- Using of two specific solutions modelling biomass found in MBR for a better understanding of the fouling mechanisms in submerged MBR processes. Filtrations were carried out with 0.22 ??m PVDF hollow fibre membrane. Alginate was used as a model for polysaccharide, bovine serum albumin (BSA) as a model for protein, (un)washed yeast and bentonite were representing suspended solid contents. According to the data obtained during this study the fouling propensity of each model solution was classified as follow in a decreasing order: Alginate &gt unwashed yeast &gt washed yeast &gt BSA &gt bentonite for one-component solutions; and Alginate-washed yeast &gt Alginate-BSA &gt Alginate-bentonite &gt Alginate-unwashed yeast for two-component solutions. Introducing the alginate increased the reversible fouling (except BSA). Passive adsorption had a significant effect on fouling of alginate even before the beginning of the filtration. Washed yeast and a mixture of washed yeast + BSA were then used as model solutions to simulate the activated sludge found in MBR. The concentration of washed yeast and BSA used in this study were calculated in order for the characterisations of the two model solution to match (in terms of biopolymer contents) those of MBR biomasses reported in the literature. By rinsing, backwashing and chemical cleaning of the membrane, three fouling layers of upper, intermediate and lower were defined respectively. Results obtained from the analysis of the biopolymers found in the cleaning solutions allow a better understanding of the fouling mechanisms occurring for the two model solutions used in this study: for washed yeast, the lower layer and for washed yeast + BSA , the upper and intermediate layers were found to have relatively high biopolymeric composition. This was explained by higher concentration of solids on the membrane surface and by higher biopolymer interactions when washed yeast was mixed with BSA.