På senare tid har forskning syftat till att främja miljövänligare teknik inom alla fält. Det visar sig inom materialframställningen som en önskan att använda material som inte är beroende av olja, inte tillverkade med tungmetaller samt nedbrytbara med en minimal miljöpåverkan. Detta har bland annat gett organiska katalysatorer en större plats inom forskningen. En av dessa är cyclodextrin, en cyklisk oligosackarid som har påvisats bilda makromolekylara komplex med andra molekyler. En aspekt av detta är att den kan hydrolysera polymerer då den bildar komplex med hydrofila molekyler och kan aktivera dessa genom vätebindning. Vilket Harada et. al. visade kunde utnyttjas for att polymerisera cycliska estrar. Detta examensarbete utforskar en del av det arbetet genom att dels verifiera polymerisationstekniken som ar en lösningsmedelsfri polymerisation dels försök till att framstalla en ny dimer av cyclodextrin som ska effektivt kunna polymerisera cycliska estrar mer effektivt an tidigare. Den nya dimern bygger på en thiourea-länk. Aven om polymerisation med vanlig cyclodextrin har visats fungera sa kan den föreslagna  dimern inte polymerisera lika effektivt. Syntesen ar problematisk och annars åtråvarda egenskaper hos cyclodextrin, som makromolekylär komplexbildning, kan inhibera polymerisationen och tidigare uppreningssteg.

<p>  </p> <p>Organic semiconductors have been considered a promising candidate to replace Silicon-based inorganic semiconductors in our electronics due to their lightweight, high flexibility, and solution processability. Recently, conjugated polymers were shown to be functional at up to 200°C, expanding organic semiconductors application territory into high-temperature electronics, which sorely depends on wide-bandgap semiconductors. To push the operational temperature boundary of polymer transistors even further than 200°C, our understanding of temperature impacts on the materials and charge transport mechanism in such harsh conditions needs to be improved. Here, we study the high temperature effect on polymer transistors from two main directions: via molecular design and via device engineering. First, via sidechain design, we explored the impact of close π-π packing on the thermal stability of semiconducting polymers. We discovered that maintaining close π-π packing can lead to lower chain distortion, thus improving the polymer transistors' operational stability at high temperatures. Then we study the impact from device factor, specifically contact resistance in device behavior at extreme conditions. We found that the contact area is more susceptible to high temperatures than other regions in the channels and is the main reason for the degraded performance. We then propose a facile method to minimize the contact problem, to achieve stable devices at above 200°C. And last, we proposed a simple method to attain quasi-temperature independent charge transport in polymer transistors from room temperature to 140°C by simply applying a prolonged bias gate voltage before heating. This research expands our knowledge on charge transport in conjugated polymers at high temperatures and provides a guide to make conjugated polymer transistors for extreme conditions in the future.</p>

<p> Cellulose-based materials have been attracting significant attention in recent years for their potential as renewable and biodegradable materials. Cellulose nanofibrils (CNFs) in particular are readily attainable from woody biomass in high purity and without harsh chemical processes. These CNFs can undergo chemical surface modifications after a simple workup, imbuing them with new attributes that differ from their naturally paper-like structure and properties. In this research, CNFs are modified with oleic acid—another common biomass found in high concentrations in some vegetable oils—which transforms the naturally hydrophilic cellulose into a superhydrophobic material. This transformation can be carried out using solventless mechanochemistry and worked up in ethanol, supporting a green process from start to finish.</p> <p><br></p> <p>Since cellulose contains many free, exposed hydroxyl groups, carboxylic acids can be condensed onto exposed hydroxyls to form esters. In this research, we focus specifically on the oleic acid moiety because its internal alkene has potential for further reactivity. Here we explore methods to introduce crosslinks into esterified CNF (eCNF) for structural and mechanical reinforcement between fibrils. Several methods are attempted, including methods involving thiolene chemistry and epoxide ring opening.</p> <p><br></p> <p>Additionally, efforts have been made to develop a method to disperse eCNF materials in ethyl acetate for deposition by spray coating. Dispersions of eCNF in ethyl acetate are sufficiently stable to enable deposition using simple airbrushing tools. The eCNF coatings are homogenous, superhydrophobic, and have good adhesion to a wide variety of surfaces. </p>

La régulation fine des mécanismes d’élimination des ARN messagers (ARNm) au sein des cellules contribue au contrôle de l’expression génétique ainsi qu’à l’adaptation rapide des niveaux de transcrits en réponse à divers événements cellulaires ou stimuli externes. Elle intervient ainsi dans différents aspects de la physiologie cellulaire : différentiation, prolifération, homéostasie, inflammation ou encore défense anti-parasitaire. Les ARNm eucaryotes matures sont protégés d’une dégradation incontrôlée par une coiffe et une queue poly(A), à chacune de leurs extrémités. Le premier événement amorçant la dégradation des ARNm est le raccourcissement de la queue poly(A) par le complexe CCR4/Not par un processus appelé déadénylation. Ensuite, la coiffe 5’ est éliminée pendant l’étape de « decapping » qui est considérée comme une étape cruciale, irréversible et extrêmement contrôlée, nécessaire à la dégradation rapide du corps du messager par Xrn1. L’étape de “decapping” est effectuée via le recrutement d’un complexe protéique formé de l’enzyme Dcp2 et de son co-activateur essentiel Dcp1. Cependant, ce complexe n’est que peu actif et nécessite de nombreux co-facteurs pour être pleinement efficace. Ces facteurs comprennent l’anneau LSm1-7 qui reconnaît l’extrémité 3’ des ARNm déadénylés et interagit avec Pat1, une protéine plateforme qui recrute l’hélicase Dhh1 et les protéines activatrices du decapping Edc1-2-3. Tous ces facteurs sont organisés au sein d’un réseau d’interaction complexe et dynamique qui, dans certaines conditions, colocalise dans les P-bodies, des foyers cytoplasmiques impliqués dans la dégradation des ARNm et dans la répression de la traduction.Même si de nombreuses études ont révélé l’importance des interactions protéine/protéine dans le processus de decapping, peu d’informations sont disponibles sur les mécanismes moléculaires du recrutement et d’activation de Dcp2 par ses différents co-facteurs. De même, en raison de l’absence de structure de Dcp2 en complexe avec un ARNm coiffé, les détails moléculaires de la reconnaissance et du clivage de la coiffe sont inconnus. Mon projet de thèse a pour but de répondre à ces questions par l’étude fonctionnelle et structurale des acteurs du decapping, en utilisant les protéines de la levure Saccharomyces cerevisiae comme système modèle, puisque la plupart des acteurs du decapping sont conservés au sein des eucaryotes. Dans ce but, j’ai exprimé par génie génétique et isolé la majorité des facteurs impliqués dans le “decapping” et reconstitué plusieurs sous complexes comprenant Dcp2 et ses différents cofacteurs. / MRNA decay is a highly regulated process allowing cells to rapidly adapt their abundance of transcripts to environmental conditions. Eukaryotic mRNAs are protected from uncontrolled decay by a cap structure (m7GpppX) and a poly(A) tail at their 5’ and 3’ ends, respectively. The first event initiating the 5’ to 3’ degradation pathway is the shortening of the poly(A) tail by the CCR4/Not complex through a process known as deadenylation. Then the 5’ cap is degraded during the decapping step, which is considered as a crucial and irreversible step before rapid degradation of RNAs. Decapping is accomplished by the recruitment of a protein complex formed by the Dcp2 catalytic subunit and its activator Dcp1. However, this complex has a low intrinsic decapping activity and requires several accessory factors to be fully efficient. These include the Lsm1-Lsm7 complex that binds to the 3’ end of deadenylated mRNAs and promotes decapping. This complex binds to Pat1, a scaffolding protein recruiting other accessory proteins such as Dhh1 and Edc1-3 proteins (Enhancer of Decapping), which favor decapping. After efficient removal of the cap, Xrn1 (the major cytoplasmic 5’-3’ exonuclease) is recruited and degrades the resulting uncapped RNAs. Interestingly, all these proteins are part of dynamic and multifunctional protein assemblies that, under conditions, localize into cytoplasmic foci known as P-bodies.Although many studies have revealed the importance of these protein/protein interactions, little is known concerning the mechanisms of recruitment and activation of the decapping enzyme by its numerous co-factors. Moreover, in the absence of Dcp2 in complex with a capped RNA, molecular details of cap recognition and cleavage are lacking. My thesis project aims at answering these open questions with the structural and functional studies of the decapping machinery, using yeast Saccharomyces cerevisiae as a model organism, as most of decapping actors are well conserved among eukaryotes. For this purpose, I expressed and purified the majority of the decapping factors and reconstituted several sub-complexes including Dcp2 and its cofactors.

<p> DNA origami is a powerful method to construct DNA nanostructures. It requires long, single-stranded DNAs. The preparation of such long DNA strands is often quite tedious and has a limited production yield. In contrast, duplex DNAs can be easily prepared via enzymatic reactions in large quantities. Thus, we ask a question: can we design DNA nanostructures in such a way that the two complementary strands can simultaneously fold into the designed structures in the same solution instead of hybridizing with each other to form a DNA duplex? By engineering DNA interaction kinetics, herein, we are able to provide multiple examples to concretely demonstrate a positive answer to this question. The resulting DNA nanostructures have been thoroughly characterized by electrophoresis and atomic force microscopy imaging. The reported strategy is compatible with the DNA cloning method; thus, would provide a convenient way for large-scale production of the designed DNA nanostructures. </p>

<p>Conserved across metazoans, cell division depends upon the synchronous assembly and disassembly of a robust, mitotic spindle for the congression and separation of duplicated chromosomes. Composed of mostly microtubules, mitotic spindle generation depends on three different microtubule nucleation mechanisms to build its distinctive bipolar assembly. These three mechanisms are centrosomal-based, kinetochore-based, and branching microtubule nucleation. Branching microtubule nucleation occurs when microtubules nucleate from the sides of pre-existing microtubules within the mitotic spindle. Without branching microtubules, a weaker spindle apparatus can result in mitotic delay, chromosomal misalignment, multi-polar spindles, and/or aneuploidy. </p> <p>Several important complexes and proteins mediate branching microtubule nucleation. These proteins are the γ-tubulin ring complex (γ–TuRC), the homologous to augmin subunits (HAUS) complex (or simply augmin), the targeting protein for Xklp2 (TPX2), colonic and hepatic tumor overexpressed gene (chTOG), and echinoderm microtubule-associated protein-like 3 (EML3) among others. This work focused on discerning the molecular architecture of the augmin complex while also endeavoring to establish heterologous expression and purification methodologies for the γ–TuRC and TPX2. </p> <p>Augmin consists of proteins HAUS1–8 (H1–8) which bind to the sides of pre-existing microtubules and orient the γ–TuRC, the template for making microtubules, via NEDD1 to create new microtubules at shallow angles (~<20°). Despite its importance in cell division, the structure of augmin has eluded determination. This work utilized a multi-pronged approach of the baculovirus insect cell protein complex expression, cryo-EM, new protein structure prediction methodologies, and crosslinking mass spectrometry (CLMS) to elucidate the molecular architecture of the augmin complex. Further work studying the isolation, structure prediction and comparison across model organisms, and phosphorylation studies was also conducted. The results will aid the structure-assisted development of novel chemotherapeutics that target the augmin complex as well as provide deeper insights into how this complex functions in cell division. </p> <p>To help better understand the molecular mechanisms, regulation, and interactions between the different machinery involved in branching microtubule nucleation, the γ–TuRC and TPX2 also became a focus of this work. My primary effort was to overexpress and purify from the heterologous baculovirus insect cell protein complex expression system sufficient quantities of γ–TuRC for biochemical and biophysical characterization. Thus, efforts shifted to establish an expression and purification methodology for this complex. Similarly, a methodology for purification of TPX2 were also initiated. The goal of these endeavors is to establish <em>in vitro</em> biochemical reconstitution of branching microtubule nucleation utilizing the augmin complex, γ–TuRC, and TPX2 utilizing total internal reflection fluorescence microscopy (TIRF-M). </p> <p>Lastly, in unrelated work, a section on other work focuses on the roles of anti-CRISPR proteins that inhibit the Csy surveillance complex from <em>Pseudomonas aeruginosa</em> can be found. Cryo-EM studies revealed the structures of AcrIF4, AcrIF7, and AcrIF14. These anti-CRISPR proteins inhibit the Csy complex by different mechanisms. AcrIF4 prevents conformational changes necessary to recruit a Cas2/3 nuclease for degradation of invading mobile genetic elements while AcrIF7 acts as a dsDNA mimic preventing invading phage DNA recognition. Lastly, AcrIF14 functions by binding in the grove where the crRNA of Csy is and prevents hybridization between target invading MGE DNA and the crRNA. These mechanisms exemplify convergent evolution among anti-CRISPR proteins while also showing the diversity of structures produced by phages in their ongoing molecular arms race with their hosts.</p>

<p dir="ltr">Ubiquitination and ADP-ribosylation are reversible post-translational modifications involved in various cellular activities. Pathogens like <i>Legionella pneumophila</i> and <i>Chromobacterium violaceum</i> target host ubiquitin system via modifications involving ADP-ribosylation. Specifically, <i>Legionella pneumophila</i> mediates atypical ubiquitination of host targets using the SidE effector family in a process that involves ubiquitin ADP-ribosylation on arginine 42 as an obligatory step. On the other hand, <i>Chromobacterium violaceum</i> effector CteC ADP-ribosylates threonine 66 of ubiquitin and causes overall blocking of host ubiquitin signaling. Removal of ADP-ribosylation requires (ADP-ribosyl)hydrolases, with macrodomain enzymes being a major family in this category. In the current study, a proteome-wide screening of ubiquitin interactors in the <i>Legionella</i> secreted proteome was performed, which led to the <i>Legionella</i> macrodomain effector MavL as a regulator of the SidE-mediated ubiquitination pathway by reversing the ubiquitin arginine ADP-ribosylation, likely to minimize potential detrimental effects caused by modified ubiquitin. Crystal structure of ADP-ribose-bound MavL was determined, providing structural insights into substrate recognition and catalytic mechanism. Further bioinformatical analyses reveal DUF4804 as a class of MavL-like macrodomain enzymes uniquely selective for mono-ADP-ribosylated arginine residue. The arginine-specific macrodomains are also present in eukaryotes, as exemplified by two previously uncharacterized (ADP-ribosyl)hydrolases in <i>Drosophila melanogaster</i>. Crystal structures of several proteins in this class provide insights into arginine specificity and a shared mode of ADP-ribose interaction distinct from previously characterized macrodomains. The crystal structure of NAD⁺-bound CteC was also determined, which provided insights into its ADP-ribosylation activity and its ubiquitin specificity. Collectively, the studies described here provide biochemical and structural characterizations and mechanistic insights into bacterial effectors associated with ubiquitin ADP-ribosylation.</p>

<p>    </p> <p>Protein kinases are a highly targeted class of enzymes for cancer therapeutics. All current FDA approved drugs bind within the ATP site and many demonstrate toxicity due to off-target effects. A new avenue is to design drugs that compete with substrate interactions. This requires the knowledge of how kinases interact with their substrates. Current structure information on kinase-substrate interactions is particularly deficient due to the transient nature of tyrosine kinase- substrate complexes. To overcome this deficiency, we used biophysical approaches in the native solution state to characterize structural patterns for substrate binding of Src tyrosine kinase, a well- known cancer-related drug target. We developed a new peptide substrate of Src kinase with SPOT peptide array screening. Binding kinetics and activity information was obtained with various biophysical and biochemical assays. NMR experiments, such as paramagnetic relaxation enhancement and chemical shift perturbation, were exploited to define the peptide pose for Src- peptide complexes. Our data suggests an alternative binding mode for Src substrate recognition. While peptide substrates inform on interactions near the active site, the native protein substrate involves additional contacts. We obtained high-quality NMR spectra of a Src-Csk protein-protein complex that probe for the interaction of the substrate near the kinase catalytic site. Given the strong correlation between protein tyrosine kinase dysfunction and cancer, substrate recognition patterns become critical for drug discovery. Finding of tight binders and clarifying substrate recognition modes would facilitate development of more specific drugs for future cancer treatments. </p>

<p>   Organic electronics has been a highly researched field owing to the low cost, biocompatibility, mechanical flexibility, and superior performance relative to their inorganic counterparts in some applications. Significant advancement has been achieved across various device platforms including organic light-emitting diodes (OLEDs), organic field effect transistors (OFETs), and organic solar cells, for instance. Recently, soft materials that can conduct both charge and ions simultaneously (i.e., organic mixed conductors) have been a major catalyst in the fields of biosensors and energy storage. Extensive research efforts in the organic electronics field are being invested to establish the relevant structure-property relationships to design and develop higher performing organic mixed conductors. Simultaneously, these materials are utilized in developing prototype biosensors with the aim of superior performance, lower cost, and better patient comfort and outcomes than currently available technologies. Following suit, this dissertation is dedicated to furthering organic electronics on both fundamental and applied fronts. Specifically, this work examines a novel class of redox-active macromolecules, radical polymers, as the organic electrochemical transistor (OECT) active layer. In addition, wearable ocular biosensors utilizing soft materials to realize design innovation are presented.</p> <p>   For the first part of the present dissertation, radical polymer-based blends are evaluated for mixed electron and ion conduction in OECTs. Traditional macromolecular design motifs for OECT active layer materials have been a closed-shell macromolecular backbone for electron conduction with charge-neutral hydrophilic side chains (e.g., triethylene glycol) for ion conduction. When poly(4-glycidyloxy-2,2,6,6-tetramethylpiperidine-1-oxyl) (PTEO) is blended with poly(3-hexylthiophene) (P3HT), 2,2,6,6-tetramethylpiperidin-N-oxy (TEMPO) radicals in PTEO act as an independent voltage regulator that modulates the ionic and hence electronic transport of the OECT devices. Electrochemical analysis of the blend films reveals that the ionic transport and hence electrochemical doping of the P3HT phase occur when the applied bias matches the onset oxidation potential of TEMPO radicals in PTEO even though that of P3HT is lower than that of TEMPO oxidation. By optimizing the blend ratio, figure-of-merit (i.e., μC*) values over 150 F V–1 cm–1 s–1 at loadings as low as 5% PTEO (by weight) are achieved, placing the performance on the same order as top-performing conjugated polymers despite the mediocre performance of pristine P3HT (<10 F V–1 cm–1 s–1). These findings suggest that introduction of open-shell moieties in the OECT active layer as a secondary redox-active species may significantly improve OECT performance metrics and offer a new paradigm for future macromolecular designs.</p> <p>   In the second part of the dissertation, novel design strategies for wearable ocular electroretinography (ERG) sensors are presented. Typically, wearable sensors are custom-made contact lenses fabricated in a bottom-up fashion where the pre-fabricated sensor component is either embedded in the contact lens body or sandwiched between two. The present work instead utilizes commercially available contact lenses, and the corneal electrode is integrated via electropolymerization of poly(3,4-ethylenedioxythiophene):iron(III) p-toluenesulfonate (PEDOT:Tos) on the lens surface. Electrochemical analysis of the PEDOT:Tos reveals that the measured impedance is several orders of magnitude lower than that of noble metals (e.g., Au) used as the working electrode in commercial electrodes. The mechanical and chemical stability along with the soft form factor of the present design strategy enables high-fidelity recording of ERG signals in human subjects without the need for topical anesthesia.</p> <p>   Following the similar strategy, a new seamless wearable ocular sensor integration strategy utilizing polydopamine (PDA) conformal coating is demonstrated. In this work, we utilize its strong adhesive property originating from the van der Waals interactions between catechol moieties of PDA and various hydrophilic functional groups (e.g., hydroxy, ether, etc.) already present in commercial contact lens materials. The facile integration demonstrates high peeling strength (> 55 J m-2), chemical and mechanical stability. A series of <em>in vivo</em> assessments demonstrates high accuracy, reliability, and user comfort of the fabricated wearable sensor in both animal and human subjects. The findings suggest that the PDA-assisted integration strategy may be applied in designing various future-generation wearable ocular electrophysiological sensors.</p>

For systems biology, it is important to describe the kinetic and thermodynamic properties of enzyme-catalyzed reactions and reaction cascades quantitatively under conditions prevailing in the cytoplasm. While in part I kinetic models based on irreversible thermodynamics were tested, here in part II, the influence of the presumably most important cytosolic factors was investigated using two glycolytic reactions (i.e., the phosphoglucose isomerase reaction (PGI) with a uni-uni-mechanism and the enolase reaction with an uni-bi-mechanism) as examples. Crowding by macromolecules was simulated using polyethylene glycol (PEG) and bovine serum albumin (BSA). The reactions were monitored calorimetrically and the equilibrium concentrations were evaluated using the equation of state ePC-SAFT. The pH and the crowding agents had the greatest influence on the reaction enthalpy change. Two kinetic models based on irreversible thermodynamics (i.e., single parameter flux-force and two-parameter Noor model) were applied to investigate the influence of cytosolic conditions. The flux-force model describes the influence of cytosolic conditions on reaction kinetics best. Concentrations of magnesium ions and crowding agents had the greatest influence, while temperature and pH-value had a medium influence on the kinetic parameters. With this contribution, we show that the interplay of thermodynamic modeling and calorimetric process monitoring allows a fast and reliable quantification of the influence of cytosolic conditions on kinetic and thermodynamic parameters.