The dynamic rearrangements of RNA and protein complexes and the fidelity of pre-mRNA splicing are governed by DExD/H-box ATPases. One of the spliceosomal ATPases, Brr2, is believed to facilitate conformational rearrangements during spliceosome activation and disassembly. It features an unusual architecture, with two consecutive helicase-cassettes, each comprising a helicase and a Sec63 domain. Only the N-terminal cassette exhibits catalytic activity. By contrast, the C-terminal half of Brr2 engages in protein interactions. Amongst interacting proteins are the Prp2 and Prp16 helicases. The work presented in this thesis aimed at studying and assigning functional relevance to the bipartite architecture of Brr2 and addressed the following questions: (1) What role does the catalytically inert C-terminal half play in Brr2 function, and why does it interact with other RNA helicases? (2) Which RNAs interact with the different parts of Brr2? (1) In a yeast two-hybrid screen novel brr2 mutant alleles were identified by virtue of abnormal protein interactions with Prp2 and Prp16. Phenotypic characterization showed that brr2 C-terminus mutants exhibit a splicing defect, demonstrating that an intact C-terminus is required for Brr2 function. ATPase/helicase deficient prp16 mutants suppress the interaction defect of brr2 alleles, possibly indicating an involvement of the Brr2 C-terminus in the regulation of interacting helicases. (2) Brr2-RNA interactions were identified by the CRAC approach (in vivo Crosslinking and analysis of cDNA). Physical separation of the N-terminal and C-terminal portions and their individual analyses indicate that only the N-terminus of Brr2 interacts with RNA. Brr2 cross-links in the U4 and U6 snRNAs suggest a step-wise dissociation of the U4/U6 duplex during catalytic activation of the spliceosome. Newly identified Brr2 cross-links in the U5 snRNA and in pre-mRNAs close to 3’ splice sites are supported by genetic analyses. A reduction of second step efficiency upon combining brr2 and U5 mutations suggests an involvement of Brr2 in the second step of splicing. An approach now described as CLASH (Cross-linking, Ligation and Sequencing of Hybrids) identified Brr2 associated chimeric sequencing reads. The inspection of chimeric U2-U2 sequences suggests a revised secondary structure for the U2 snRNA, which was confirmed by phylogenentic and mutational analyses. Taken together these findings underscore the functional distinction of the N- and C-terminal portions of Brr2 and add mechanistic relevance to its bipartite architecture. The catalytically active N-terminal helicase-cassette is required to establish RNA interactions and to provide helicase activity. Conversely, the C-terminal helicase-cassette functions solely as protein interaction domain, possibly exerting regulation on the activities of interacting helicases and Brr2 itself.

A wide variety of techniques have been explored to synthesize small diameter tissue engineered blood vessels. Toward this end, we are exploring direct cell seeding and culture on tubular mandrels to create engineered vascular tissues. In the present study, v-shaped channels cast from polydimethyl siloxane (PDMS) were used as cell seeding wells. Fibrin microthreads placed in the chamber were used as model tubular seeding mandrels. Human mesenchymal stem cells (hMSCs) were seeded onto fibrin microthreads in v-shaped channels for 4 hours. Cell attachment to the microthreads was confirmed visually by Hoechst nuclear staining and a cell quantification assay showed that 5,114 ± 339 cells attached per 1 cm fibrin microthread sample (n = 6). Fibrin microthreads were completely degraded by hMSCs within 5 days of culture, therefore UV crosslinking was used to increase their mechanical strength and prolong the amount of time cells could be cultured on fibrin microthreads and generate tubular tissue constructs. Cell attachment was unaffected on UV-crosslinked microthreads compared to uncrosslinked microthreads, resulting in a count of 4,944 ± 210 cells per 1 cm of fibrin microthread sample (n = 3). Long term culture of the hMSCs on the UV-crosslinked fibrin microthreads showed an increase in cell number over time to 11,198 ± 582 cells per cm of microthread after 7 days with 92% cell viability (CYQUANT NF/DEAD staining) and evidence of cell proliferation. The results show that the v-well cell seeding technique was effective in promoting rapid hMSC attachment on UV-crosslinked fibrin microthreads and encouraged their growth, maintained viability and also promoted their proliferation over the culture period. In conclusion, the technique could serve as an efficient model system for rapid formation of tissue engineered vascular grafts.

Polymeric isoporous membranes have many interesting properties leading to various specific applications in different fields. However, such structures also have one main drawback, namely their poor solvent stability, which should be improved to extend the range of their possible applications. Therefore, this project will focus on the enhancement of solvent stability of polymeric isoporous membranes by UV cross-linking. Stable isoporous films were obtained by creating honeycomb membranes from star polystyrene (PS) and its derivatives. The star PS was synthesized by Atom Transfer Radical Polymerization (ATRP) method and was then functionalized with methacrylate groups. The isoporous films made from these materials maintained the honeycomb structures after curing by UV light and immersion in chloroform. The crosslinking of PS under UV light exposure rather than the cross-linking of the methacrylates groups was responsible for the solvent stability of these membranes. To further investigate the effect of specific end-groups on the film stability, PEG2k-G3-PCL30 linear-dendritic-linear hybrid polymers and its derivatives with allyl, acrylate, methacrylate end-groups were employed to cast films. Functionalized PEG2k-G3-PCL30 linear-dendritic-linear hybrid isoporous films were cross-linked by UV-induced thiol-ene reactions and allyl reactions. However, no significant increase in the solvent stability of these kinds of films was observed. When mixing PEG2k-G3-PCL30 linear-dendritic-linear hybrids with star PS, stable isoporous films could be obtained. The pores became smaller but the isoporous structures were still kept.

Thermally rearranged (TR) polymers have outstanding gas separation properties, but are limited in their industrial application due to being mechanically brittle. A series of low volume fraction of a poly(arylene ether sulfone) (PAES) block was introduced into the TR precursor polyhydroxyimide (PI) chain to improve mechanical properties without compromising gas transport properties. The multiblock copolyhydroxyimide incorporated the PAES in systematically varied amounts and copolymerized it with 4,4'-(hexafluoroisopropylidene)diphthalic anhydride and 3,3’-dihydroxy-4,4’-diaminobiphenyl. Before thermal rearrangement, the PI-co-PAES precursors exhibited much more improved mechanical properties (tensile stress and strain at break) than those of homo polyimide precursor. After thermal rearrangement, tensile stress and strain at break of all TR copolymers decreased comparing to their corresponding precursors, but improved comparing to the homo TR polymer. Poly(phenylene oxide) (PPO) based copolymers (Chapter 4) and polymer blends (Chapter 5) were also studied for use as gas separation membranes. The polymer materials were cast into films, then crosslinked in the solid state with UV light. The ketone and benzylic methyl groups crosslinked upon exposure to UV light. For the study of PPO copolymers, copolymers were prepared by polycondensation of a difunctional PPO oligomer with 4,4’-difluorobenzophenone or 1,3-bis(4-fluorobenzoyl)benzene respectively. This study offers a means for fabrication of membrane films, fibers or composites, as well as tuning of gas transport properties through crosslinking in the solid state. While for the study of PPO polymer blends, PPO polymers with Mn’s from 2000-22,000 g/mole were synthesized and blended with a poly(arylene ether ketone) derived from bisphenol A and difluorobenzophenone (BPA-PAEK). The crosslinked blends had improved gas selectivities over their linear counterparts. The 90/10 wt/wt 22k PPO/BPA PAEK crosslinked blends gained the most O2/N2 selectivity and maintained a high permeability. / Ph. D. / Membrane gas separation has become a fast-growing industrial technology because of its many advantages over traditional separation technologies including low capital cost and energy consumption relative to thermal distillation methods, and higher operational flexibility. Currently, membrane gas separation is widely used in processing raw natural gas to meet certain specifications before delivery to pipelines. Researches in this dissertation mainly focus on synthesis and characterization of membrane gas separation materials. In chapter 3, one type of thermally rearranged copolymer membrane was obtained, which could be potentially used in industrial field due to its improved mechanical property. In chapter 4 and 5, a series of poly(phenylene oxide) based copolymers and blends were studied. After UV-crosslinking reaction, poly(phenylene oxide) membranes showed improved gas selectivities over their linear counterparts.

Pre-mRNA (precursor messenger RNA) splicing is a fundamental process in eukaryotic gene expression. In order to catalyse the excision of the intervening intronic sequence between two exons, the spliceosome is assembled stepwise on the pre-mRNA substrate. This ribonucleoprotein machine is extremely dynamic: both its activation and the progression through the catalytic stages require extensive compositional and structural remodelling. The first part of this thesis aims at understanding how the spliceosome is activated after assembly. When this work was started, the GTPase Snu114 was thought to activate the helicase Brr2 to unwind the U4/U6 snRNA duplex, which ultimately leads to the formation of the spliceosome active site. To explore the role of Snu114, a complex built from Snu114 and a part of Prp8 was expressed and analysed in its natural context, bound to U5 snRNA. However, before I was able to obtain highly diffracting crystals, the structure of Snu114 was determined in the context of a larger spliceosomal complex by electron cryo-microscopy by competitors. Regardless, the role of Snu114 in spliceosome activation remains elusive. In a short section of this thesis, genetic and biochemical analysis suggest Snu114 to be a pseudo-GTPase, precluding a role for Snu114-catalyzed GTP hydrolysis in activation. The second and larger part of the thesis describes the development of a novel, biochemical method to analyse spliceosome remodelling events that are caused by the eight spliceosomal helicases. Purified spliceosomes assembled on a defined RNA substrate are analysed by UV crosslinking and next-generation sequencing, which allows for the determination of the RNA helicase binding profile at nucleotide resolution. In vitro spliceosome iCLIP (individual-nucleotide resolution UV crosslinking and immunoprecipitation) was initially developed targeting the helicase Prp16 bound to spliceosomal complex C. The obtained binding profile shows that Prp16 contacts the intron, about 15 nucleotides downstream of the branch in the intron-lariat intermediate. Our finding supports the model of Prp16 acting at a distance to remodel the RNA and protein interactions in the catalytic core and thereby it promotes the transition towards a conformation of the spliceosome competent for second step catalysis. Control experiments, which locate SmB protein binding to known Sm sites in the spliceosomal snRNAs, validated the method. Preliminary results show that in vitro spliceosome iCLIP can be adapted to analyse additional spliceosomal helicases such as Prp22. Finally, I performed initial experiments that give promising directions towards time-resolved translocation profiles of helicases Brr2 and Prp16.

Small proteins are difficult to study at high resolution with single-particle cryo-electron microscopy (cryo-EM). In general, sample properties such as large size (> 80 kDa), symmetry and rigidity are key to utilize this technology. To facilitate structural studies of small proteins as well, using cryo-EM, this project aims to incorporate a photo-inducible cross-link in a large and symmetric scaffold that is amenable for study, and covalently bind small proteins of interest to this scaffold. The scaffold in this project consists of rabbit muscle aldolase (157 kDa in tetrameric state) with an engineered affibody affinity protein (7 kDa) attached to the N-terminus of each aldolase monomer via a rigid helix fusion. The affibody-domain of the scaffold will be cross-linked to small proteins of structural interest, with a focus on a model target consisting of a second affibody with affinity for the affibody displayed on the aldolase scaffold. Photoconjugation of the affibody Zwt was performed to crosslink both the Fc of IgG and the anti-idiotypic affibody Z963, revealing that a methionine acceptor in the target is preferable but not necessary for UV crosslinking using BPA. Binding of affibodies rigidly displayed on of the scaffold to targets such as affibodies and antibody fragments was demonstrated , using surface plasmon resonance (SPR). / Att studera små protein vid hög upplösning med enpartikelsrekonstruktion i kryo-elektronmikroskopi (kryo-EM) är utmanande. Generellt så krävs stora (> 80 kDa), symmetriska och stabila protein för att använda sig av kryo-EM. Med målet att möjliggöra strukturbestämning och strukturella studier av små protein, så ska detta projekt föra in en foto-aktiverad korslänk i ett stort och symmetriskt bärarprotein. Bäraren består av aldolas från kaninmuskel (157 kDa som tetramer) med en affibody (7 kDa) kopplad till N-terminalen av varje aldolas-monomer via en rigidt fuserad helix. Affibody-domänen av bärarproteinet kan bilda korslänkar till små protein vars struktur sedan kan studeras. Fokus i projektet är ett modellprotein som består av en annan affibody som binder den affibody i bäraren. Fotokonjugering av affibodyn Zwt utfördes för att skapa korslänkar till både Fc av IgG, samt den anti-idiotypiska affibodyn Z963, vilket påvisade att en metionin-mottagare i målproteinet är fördelaktigt för UV korslänkning med BPA, men inte ett krav. Affinitet av affibodies i bärarproteinet till målprotein såsom andra affibodies och antikroppsfragment påvisades.

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This thesis focuses on the synthesis and characterization of various poly(arylene ether)s to improve the efficiency of gas separation and water desalination membranes. This class of polymers includes polymers such as poly(arylene ether sulfone), poly(arylene ether ketone) and poly(phenylene oxide) which offer excellent thermal and mechanical stability and usually have high enough rigidity to support gas separation and water desalination operations. Besides the plethora of properties offered by the homopolymers, these polymers can also be post-modified to cater to specific needs. For example, the polyphenylene oxides have been brominated to increase the permeability for gas separation applications. Blending is another viable method to impart desirable properties to polymers. Bisphenol A based poly(arylene ether ketone) (BPAPAEK) has been blended with commercially available poly(2,6-dimethylphenylene oxide)s (PPO) of different molecular weights in a fixed ratio (66/34 wt/wt) and in various ratios of a 22000 g/mol PPO. All the blends were UV crosslinked to minimize plasticization by condensable gases and analyzed for gel fractions, whereas, only the 22,000 g/mol blends were tested for transport properties since they yielded the highest gel fractions and exhibited the best mechanical properties. The crosslinking reduced the free volume and improved the selectivity with some drop in permeability. The blends with 90% of the 22000 g/mol PPO by weight was plotted closest to the upperbound. A phosphine oxide based poly(arylene ether ketone) (POPAEK) was blended with the various PPOs in a similar manner. The results were compared to the BPAPAEK based blends in terms of miscibility behavior and transport properties. It was found that the POPAEK based blends had higher permeability due to the higher fractional free volumes of the POPAEK. The POPAEK was more compatible with the PPOs than BPAPAEK as seen by analyzing various blend permeability models, mechanical properties and scanning electron microscope images. Moreover, blends with both the PAEKs displayed only a small drop in mechanical properties, such as the Young's modulus and the yield strength in comparison to the parent polymers. Hydroquinone based poly(arylene ether sulfone) oligomers were synthesized, post-sulfonated and chemically crosslinked to determine the effect of water uptake, fixed charge concentration and block length of oligomers on the salt permeability and the hydrated mechanical properties of the networks. The sulfonic acid groups were placed strategically and quantitatively on the hydroquinone units. The strategic placement of the acid groups may help in maintaining high rejection of monovalent ions in the presence of divalent ions, as shown in unpublished work by our group. It was found that the water uptake and fixed charge density had the opposite effects on the salt permeability. Also, the salt permeability varied differently for 5000g/mol and 10000g/mol block based networks. Another polymer that was investigated in this thesis was poly(2-ethyl-2-oxazoline) (PEtOx). An elaborate account of synthesis of monofunctional, heterobifunctional and telechelic poly(2-ethyl-2-oxazoline)s using different initiators including methyl triflate, activated alkyl halides (e.g., benzyl halides), and non-activated alkyl halides has been presented in this thesis. Endgroup functionalities and molecular weight distributions were studied by SEC, 1H NMR and titrations. The oligomers initiated with the benzyl or xylyl chloride had a PDI of 1.3-1.4 which is broader than expected for a living cationic ring opened polymer. This was attributed to the participation of covalent species which propagated slowly in the activated halide reactions. These oligomers were quantitatively terminated as proven by NMR and titrations. Due to the molecular weight distributions and quantitative termination these oligomers were deemed to be desirable for drug delivery applications. / PHD / This work pivots around the synthesis and characterization of different classes of polymers which are long molecules made by joining small molecules. The structure-property relationships of different polymers with respect to applications such as gas separation, water desalination and drug delivery were examined. The first two projects were focused of gas separation applications. Gas separation is an essential process used to recover the required gas from a mixture of gases. This process is used in a number of industries such as natural gas, hydrogen recovery and air dehumidification. In these projects, gas separation membranes were used to remove non combustible components of natural gas such as carbon dioxide and hydrogen sulfide. Two different types of poly(arylene ether ketone)s (PAEKs) (a kind of polymer) were blended with a commercial polymer called poly(phenylene oxide) (PPO) and crosslinked at the surfaces to improve the gas transport properties of the commercial polymer. PPOs have high gas permeability and a low selectivity. In other words even though the PPO membranes would alow the gasses to pass through easily, the efficiency of gas separation would be low. The blending with the PAEKs improved the selectivity of the PPOs without much loss in throughput. These blends of the two different PAEKs were compared for transport and other relavent properties. It was found that the transport properties of the commercial polymer were improved markedly without much loss in mechanical properties which are usually sacrificed upon blending of two uncomaptible polymers. Water desalination applications were looked into for a polymer class called polysulfones. About 40% of the world’s population lives in water stressed areas. In order to address the water crisis, there is a need to look beyond primitive methods such as distillation which are inefficient. Hence, the polymeric membrane separations which do not involve phase change (eg liquid to gas and then back to liquid in distillation) were examined. The currently used polyamide membranes have a rough surface because of the way they are made, making them prone to deposition of salt and organic matter. This deposition makes them inefficient. They are also prone to degradation by chlorine. The polysulfones membranes have a smoother surface less prone to these depositions. Their resistance to chlorine makes them more viable for water desalination applications. The polysulfones were post modified to introduce charges to make them more suitable for water desalination purposes. The charges repelled the ions of same polarity and made the polymer more hydrophilic. However, as the number of charges increased, the water uptake of the polymer increased which resulted in a decrease in the effectiveness of salt /ion rejection. To increase the charge density of the polymers by (the effectiveness of ion rejection), the polymer chains were crosslinked at the ends. For deleniating the structure property relationships, the amount of charges were varied and two sets of chain lengths were studied. The salt permeability decreased with increase in fixed charge concentration and decrease in water uptake. Poly(2-Oxazolines), were investigated as potential drug delivery vehicles. Polymeric drug delivery vehicles have been used to control the rate of release of drugs in the body to avoid side effects. Another advantage of polymeric drug delivery systems is making the water insoluble drugs more compatible with the fluids in the body. Currently, polyethylene oxides are being used as drug delivery vehicles. However, these polymers have been known to produce antibodies in some people. In this work, poly(2-oxazolines) which are known to be more compatible with human body than PEOs were prepared using different initiators and end cappers to prepare an elaborate repertoire of controlled molecular weight and controlled functionality oligomers for further modification.

L’engouement mondial pour les appareils nomades et la course à la sobriété énergétique font de la diminution de la taille des systèmes microélectroniques (MEMS) un enjeu majeur pour les prochaines années. Les micro batteries au lithium sont aujourd'hui le moyen le plus efficace pour stocker et alimenter des dispositifs avec une très forte densité énergétique. Les incorporer dans des cartes de crédit comportant un écran et des touches intégrés est l’un des défis que relèvent les multinationales comme ST Micro Electronics. Ces micro batteries contiennent cependant du lithium métallique qui peut s'avérer très dangereux quand il est en contact avec de l’eau ou de l’air humide. Ainsi, afin de protéger les composants à une exposition à l’humidité, une encapsulation de l’ensemble de la batterie est nécessaire. L'encapsulation polymère a l’avantage, comparativement à d’autres matériaux, de présenter un faible coût de mise en forme et un faible poids. Cependant, de tels systèmes d'encapsulation sont aujourd'hui insuffisants pour garantir une durée de vie de plusieurs années des composants car en présence d’humidité ou d’une variation de température importante la tenue mécanique des assemblages peut être fragilisée. L'objectif de la thèse est donc de réaliser et d'étudier le comportement mécanique et structural d’assemblage de couches minces de polymères et de métaux en température et en humidité. Deux types de polymères ont été choisis pour ce projet :1. Le chlorure de polyvinylidène (PVDC), un polymère commercial très utilisé pour ses bonnes propriétés barrières à l'eau 2. Un oligomère acrylate reticulable par voie thermique et UV synthétisé au sein de l'IMMM. / The increasing number of mobile devices and the race to energy sobriety make the decrease of the size of microelectronic systems (MEMS) a major challenge. Today, Lithium micro batteries are currently the best solution for high-power-and-energy applications. Incorporate them into credit cards containing a screen or associate them to electronic sensors for the supervision is the challenge which raises international companies such as ST Microelectronics. However, these micro batteries contain some lithium metal which can be dangerous if the metallic lithium is in contact with water or humid air. In addition, the substance can spontaneously ignite in the contact of the humidity. So, in order to avoid the problems of safety, we absolutely have to protect the lithium contained in our micro batteries using an encapsulation layer. Polymeric encapsulation has the advantage, compared with other materials (ceramic, metal), to present a moderate cost of shaping and a low weight. However, such systems of encapsulation are today insufficient to guarantee a satisfactory life cycle of components. Indeed, in the presence of humidity or of a too important temperature variation, the mechanical assemblies can be weakened and engender an irreparable break. The objective of the thesis is therefore to realize and study the mechanical and structural behavior of assembly of thin layers of polymers and metals in temperature and humidity.Two types of polymers were selected for this project:1. Polyvinylidene chloride (PVDC), a commercial polymer widely used for its good barrier properties to water.2. A thermally and UV-crosslinkable acrylate oligomer synthesized in the IMMM.

Hemocompatibility tuning was adopted to explore and refine an innovative, GA-free preparation strategy combining decellularization, riboflavin/UV crosslinking, and low-energy electron irradiation (SULEEI) procedure. A SULEEI-protocol was established to avoid GA-dependent deterioration that results in insufficient long-term aortic valve bioprosthesis durability. Final SULEEI-pericardium, intermediate steps and GA-fixed reference pericardium were exposed in vitro to fresh human whole blood to elucidate effects of preparation parameters on coagulation and inflammation activation and tissue histology. The riboflavin/UV crosslinking step showed to be less efficient in inactivating extracellular matrix (ECM) protein activity than the GA fixation, leading to tissue-factor mediated blood clotting. Intensifying the riboflavin/UV crosslinking with elevated riboflavin concentration and dextran caused an enhanced activation of the complement system. Yet activation processes induced by the previous protocol steps were quenched with the final electron beam treatment step. An optimized SULEEI protocol was developed using an intense and extended, trypsin-containing decellularization step to inactivate tissue factor and a dextran-free, low riboflavin, high UV crosslinking step. The innovative and improved GA-free SULEEI-preparation protocol results in low coagulant and low inflammatory bovine pericardium for surgical application.