Les systèmes de génétique inverse permettent de manipuler les génomes viraux et se sont révélés essentiels pour étudier les virus à ARN. Récemment, une méthode basée sur la PCR, la méthode ISA (Infectious Subgenomic Amplicons), a été développée. La première partie de cette thèse se concentre sur la simplification de la méthode ISA. La principale contrainte d'ISA est l'exigence de produire des fragments génomiques modifiés qui nécessite un promoteur de transcription à l’extrémité 5’ du premier fragment et un ribozyme du virus de l'hépatite delta, suivi du signal de polyadénylation du virus simien 40 (HDR / SV40pA) à l’extrémité 3’ du dernier fragment. Ici, nous proposons une nouvelle méthode simplifiée "Haiku", dans laquelle sont fournis ces deux séquences en tant qu'amplicons séparés. Cette technique améliorée a été appliquée avec succès à une large gamme de virus dans des cellules de moustiques et de mammifères. La deuxième partie de cette thèse est axée sur la caractérisation de la population virale issue de divers systèmes de génétique inverse en utilisant le HRV-B14 comme modèle.Nos résultats montrent que le choix de la méthode a influencé la diversité génétique des populations virales mais quelle que soit la méthode utilisée, la fitness réplicative était similaire. En outre, nos données ont révélé que le poly(A)25 est la longueur optimale pour récupérer le HRV-B14 avec une efficacité élevée. La dernière partie du présent travail a examiné le potentiel de la méthode «ISA» pour conserver le spectre mutant présent dans l'échantillon viral d'origine. Nous avons montré que cette méthode récapitule au moins partiellement les quasi-espèces de la population virale native. / Reverse genetics systems allow manipulating viral genomes and have proved to be essential for studying RNA viruses. Recently, a PCR-based method, named ISA (Infectious Subgenomic Amplicons), was developed to facilitate the study of single-stranded positive-sense RNA viruses. The first part of the present work focused on simplifying the ISA method. The main constraint of the canonic protocol of the ISA method is the requirement to produce modified genomic fragments encompassing the transcription promoter and the terminator. Here, we propose the ultimately simplified "Haiku" design in which the promoter and the terminator are provided as additional separate DNA amplicons. This improved procedure was successfully applied to the rescue of a wide range of viruses in mosquito and mammalian cells. The second part of this work assessed the viral population issued from different reverse genetics systems. Using HRV B-14 as a model, we compared the genetic diversity and the replicative fitness of viruses generated using the most commonly used reverse genetics methods. Our results showed that the choice of the method influenced the genetic diversity of viral populations but whatever the method used, the replicative fitness was similar. In addition, Our data revealed that poly(A)25 is the optimal length to recover HRV-B14 with high efficiency and could be used to recover polyadenylated RNA viruses other than HRV-B14. The last part of the present work investigated the potential of the “ISA” method to conserve the mutant spectrum present in the original viral sample. We have showed that this method recapitulate at least partially the quasispecies of the native viral population.

Le rationnel pour l’utilisation des agonistes des Toll-Like Récepteurs (TLR) dans le traitement du cancer repose sur leurs effets bénéfiques au niveau des cellules du système immunitaire permettant une stimulation de la réponse immunitaire innée et adaptative. Cependant, certains types de cellules tumorales expriment les TLRs qui, une fois activés, peuvent déclencher des effets “délétères” comme la tumorigénèse.Pour mieux disséquer les effets biologiques directs et indirects d’un agoniste de TLR3, l’acide polyadenylique-polyurydilique (poly(A:U)), nous avons utilisé deux modèles tumoraux murins exprimant TLR3 et ne répondant pas à la chimiothérapie, mais capables de produire des grandes quantités de CCL5 et CXCL10 en réponse au poly(A:U) et à l’IFN de type I. In vivo, la combinaison chimiothérapie – poly(A:U) n’a permis d’obtenir qu’une faible activité anti tumorale sauf lorsqu’une vaccination contre les antigènes tumoraux est inclue dans le protocole de traitement et le récepteur CCR5 est bloqué (souris Ccr5-/- ou souris sauvages traitées avec MetRANTES). L’efficacité anti tumorale du traitement combiné est associée à l’induction de cellules T CD8+CXCR3+IFNγ+ et est perdue chez les souris nu/nu, Trif-/- et Cxcr3-/-. La source du ligand de CCR5 est constituée par les cellules tumorales dont la voie TLR3 est activée. L’efficacité du traitement combiné sur ces cellules a été améliorée en inhibant la production de CCL5 à l’aide d’un shARN spécifique pour CCL5. Ces résultats soutiennent la notion que le poly(A:U) peut directement agir sur l’épithélium tumoral pour promouvoir la libération de CXCL10 qui a un effet bénéfique (recrutement des LTCs), mais aussi la sécrétion de CCL5 qui a un rôle délétère (action sur des immunosuppresseurs au niveau de l’hôte). Le découplage des effets opposés des chimiokines et la vaccination anti tumorale préalable peuvent permettre aux LTCs dépendant des ligands de CXCR3 d’infirmer l’action d’immunosuppression CCR5 dépendante et devraient être intégrés dans les essais cliniques à venir utilisant les agonistes de TLR3. / The rationale for the use of Toll –Like Receptor (TLR) agonists in cancer therapy relies upon their “beneficial” effects on immune cells leading to enhanced innate and adaptive immune responses. However, a variety of cancer epithelia express TLRs which, upon triggering, may mediate “deleterious” effects such as tumorigenesis. To further dissect the direct versus indirect biological effects of the TLR3 agonist polyadenylic-polyuridylic acid (poly(A:U)), we took advantage of two murine tumor models expressing TLR3 that failed to respond to chemotherapy but did produce large amounts of CCL5 and CXCL10 in response to the poly(A:U) and type I IFN. In vivo, the combination of chemotherapy and poly(A:U) mediated low tumoricidal activity unless a vaccination against tumor antigens was included in the regimen and the CCR5 receptor was blocked (CCR5 loss-of-function mice or WT animals treated with MetRANTES). The antitumor efficacy of the combination therapy was associated with the elicitation of CD8+CXCR3+IFN+ T cells and abrogated in nu/nu, Trif-/- and Cxcr3-/- mice. The source of CCR5L is the TLR3-activated tumor cells in that stable inhibition of the chemokine production by specific shRNA CCL5 ameliorated the efficacy of the combination therapy. These results support the notion that poly(A:U) can directly act on tumor epithelia to promote the release of beneficial CXCL10 for the recruitment of intratumoral CTLs but also the release of deleterious CCL5 acting on host immunosuppressors. Uncoupling chemokine release and prior vaccination may enable the CXCR3L-dependent CTLs to overrule the CCR5-dependent suppression and may be integrated in future trials using TLR3 agonists.

In this modern age, society has become much more aware of the danger bacteria can have on people's health. Personal and household antimicrobial formulations are commonly used within the home to lower the levels of harmful bacteria such as E. Coli, Salmonella and Pseudomonas. The active which kills the bacteria within the formulation is described as a biocide. This research looks at the often neglected potential of cationic polyelectrolyte as a biocide, firstly within solution and secondly in creating an antimicrobial surface. The solution properties and antimicrobial activity for a range of commercially available cationic polyelectrolytes (polymeric quaternary ammonium compounds (QAC) and biguanides) of differing molecular weights were investigated. All polyelectrolytes were observed to have some level of antimicrobial activity. The second phase of this research investigated polyelectrolyte/surfactant/water mixture of similar charge (cationic). Two QAC surfactants were investigated: Alkyl (C12 70%; C14 30%) dimethyl benzyl ammonium chloride (BAC) and Didecyldimethylammonium chloride (DDQ). At a critical concentration, these mixtures segregatively phase separate into a surfactant rich upper phase and polyelectrolyte rich lower phase. This phase separation phenomenon was investigated in respect of surfactant and polyelectrolyte type as well as polyelectrolyte molecular weight. Surfactant type was observed to be the dominant factor in determining the onset of phase separation and by mixing different ratios of surfactants the ability to tune this phase separation concentration was shown. Dilute solutions of these mixtures well below their respective phase separation concentration were then deposited onto glass substrates via a drop cast or inkjet printer method. The surfactant/polyelectrolyte film composites left after drop evaporation ranged from an amorphous film to nodular like structures. The ability to order/structure actives onto a surface could alter active adhesion and surface roughness properties of the film. This change in surface property could consequently affect antimicrobial performance.

Le développement important de la chimie haut-débit, rendu possible par l'avènement de la microfluidique, apporte des besoins croissants en méthodes de caractérisation rapides et ne nécessitant que des volumes d'échantillons réduits. Ces travaux de thèse ont permis de développer un dispositif à ondes acoustiques de surface associé à une puce microfluidique dans le but de caractériser les propriétés rhéologiques de fluides complexes. À partir de volumes de l'ordre du microlitre, des phénomènes tels que l'apparition d'enchevêtrements dans des polymères en solution ont pu être observés. Une méthode de caractérisation du module de rigidité transverse complexe a également été développée, permettant d'envisager la conception d'un micro-rhéomètre à ondes acoustiques.

<p>A novel strategy for synthesising segmented poly(urethane urea) (PUU) without using a chain extender but nevertheless with the opportunity to vary the hard segment content has been developed. The strategy is based on amine formation from isocyanate upon reaction with water. By adding a dissolved soft segment to an excess of diisocyanate followed by the addition of water in the gas phase, amines are formed <i>in situ</i>. Urea linkages are then formed when these amines react with the excess of isocyanate groups. The gas phase addition facilitates addition in a slow and continuous manner. The hard segment content can easily altered by varying the diisocyanate/soft segment ratio. Even though the strategy is shown to be applicable to different diisocyanates, the focus has been on the potentially biodegradable methyl-2,6-diisocyanatehexanoate (LDI) and 1.4-butanediisocyanate (BDI) and various well known biodegradable polyesters and polycarbonates. </p><p>All the synthesised materials exhibited pronounced phase separation and hydrogen bonding within the hard domains. However, a major increase in hydrogen bonding strength was seen when a symmetric diisocyanate was used instead of an asymmetric. Based on FTIR measurements, PUUs with BDI and a polydisperse hard segment can exhibit the same degree of phase separation and hydrogen bonding as the monodisperse product.</p><p>The elastic properties of this new group of PUUs were exceptional with an elongation at break from 1600% to almost 5000% and the elastic modulus could be varied from a few MPa up to a couple of hundreds. </p><p>Hydrolytic degradation was greater in the polyester-based than in the polycarbonate-based PUUs due to the more reactive ester bonds. Low mass loss but a considerable loss in molecular weight was seen in the polyester PUUs. The tensile strength decreased dramatically due to the loss of strain hardening.</p><p>An MTT seeding assay using human fibroblasts and an in vivo biocompatibility study were performed and no signs of cytotoxicity were seen and the inflammatory response was comparable to other inert polymers.</p><p>A biodegradable PUU with properties that can be tailored through an easy synthesis is here presented. </p>

Scaling down of the CMOS technology requires thinner gate dielectric to maintain high performance. However, due to the depletion of poly-Si gate, it is difficult to reduce the gate thickness further especially for sub-65 nm CMOS generation. Fully silicidation metal gate (FUSI) is one of the most promising solutions. Furthermore, FUSI metal gate reduces gate-line sheet resistance, prevents boron penetration to channels, and has good process compatibility with high-k gate dielectric. Poly-SiGe gate technology is another solution because of its enhancement of boron activation and compatibility with the conventional CMOS process. Combination of these two technologies for the formation of fully germanosilicided metal gate makes the approach very attractive. In this paper, the deposition of undoped Poly-Si₁₋xGex (0 < x < 30% ) films onto SiO₂ in a low pressure chemical vapor deposition (LPCVD) system is described. Detailed growth conditions and the characterization of the grown films are presented. / Singapore-MIT Alliance (SMA)

Ziel dieser Arbeit ist die phänomenologische Untersuchung der Feuchteempfindlichkeit der elektrischen Eigenschaften dünner Polymerschichten. Diese Untersuchungen stellen gleichzeitig Vorarbeiten zur Entwicklung von Prototypen von zwei polymeren Dünnschicht-Feuchtesensoren dar, die sich durch die spezielle Auswahl der feuchtesensitiven Materialien jeweils durch eine besondere Eigenschaft gegenüber kommerziellen Massenprodukten auszeichnen. Ziel der Entwicklungsarbeiten für den ersten Prototypen war die Konstruktion eines schnellen Feuchtesensors, der plötzliche und sprunghafte Feuchteänderungen in der umgebenden Atmosphäre möglichst rasch detektieren kann. Dafür wurden dünne Schichten von Poly-DADMAC auf Interdigitalstrukturen aufgebracht, die einen möglichst direkten Kontakt zwischen feuchtesensitiver Schicht und umgebender, feuchter Atmosphäre gewährleisten. Als Messgrößen dienten die Wechselstromgrößen Widerstand und Kapazität der Schichten. Die Feuchtekennlinien der Schichten zeigen gute Konstanz und hohe Reproduzierbarkeit. Der Widerstand der Schichten ändert sich durch den Einfluss von Feuchte je nach Schichtdicke um 3 bis 5 Größenordnungen und eignet sich als Messgröße für die Feuchtigkeit im gesamten Feuchtebereich. Die Hysterese der Filme konnte auf kleiner als 2,5% r.F. bestimmt werden, die Reproduzierbarkeit auf besser als 1% r.F. Die Ansprechzeit der Schichten lässt sich schichtdickenabhängig zu 1 bis 10 Sekunden bestimmen. Hierbei zeigen besonders die dünnen Schichten kurze Ansprechzeiten. Zielstellung für den zweiten Feuchtesensor war die Entwicklung eines Prototypen, dessen sensitive Schicht sich biostatisch und biozid verhält, so dass er in biotischen Umgebungen eingesetzt werden kann. Es wurden fünf Polysulfobetaine synthetisiert, deren Biozidität und Biostatik mit dem Kontakttest nach Rönnpagel, dem ISO846-Test und Abbautests bestimmt wurde. Zwei Polymere – Poly-DMMAAPS (BT2) und Poly-[MSA-Styren-Sulfobetain] (BT5) – erwiesen sich als ausreichend biozid und biostatisch. Schichten dieser Polymere wurden auf Interdigitalstrukturen aufgezogen, anschließend wurden die Kennlinien dieser Proben aufgenommen. Die Messwerte zeigen für beide Polymere gute Konstanz und eine hohe Reproduzierbarkeit. BT2-Proben sind zwischen 20% und 80% r.F. besonders empfindlich und zeigen über einen Monat keine Langzeitdrift. Vernetzte Proben zeigen bis 50°C keinen temperaturbedingten Abfall der Feuchteempfindlichkeit. Der Einsatz vernetzter BT5-Schichten als kapazitiver Feuchtesensor ist bis etwa 70°C möglich, die Schichten sind selbst nach Lagerung im Hochvakuum und mehrfacher Betauung stabil. Damit liegen zwei funktionsfähige Prototypen von Feuchtesensoren vor, für die die meisten Kennwerte denen von vergleichbaren kommerziellen Feuchtesensoren entsprechen. Gleichzeitig zeichnen sie sich aber durch eine sehr niedrige Ansprechzeit bzw. eine ausreichende Lebensdauer unter biotischen Bedingungen aus. / The topic of this work is the phenomenological investigation of the humidity sensitivity of the electrical properties of thin polymer films. These investigations also act as preparatory work for the development of prototypes of two polymer thin film humidity sensors, which by the choice of the humidity sensitive materials stand out by one special property each compared with commercial mass products. The aim of the development work for the first prototype was the construction of a fast humidity sensor, which can rapidly detect sudden and erratic humidity changes in the surrounding atmosphere. Thin films of poly-DADMAC were prepared on interdigitated structures that allow for undisturbed contact between the sensitive layer and the surrounding humid atmosphere. Measured variables were the ac resistance and capacity of the films. The humidity characteristics of the films show good stability and high reproducibility. Under the influence of humidity the resistance of the films changes by 3 to 5 orders of magnitude, depending on the film thickness, and is suitable to measure the humidity in the entire humidity range. The hysteresis of the films is lower than 2.5% r.h., the reproducibility better than 1% r.h. The response time of the layers can be determined to 1 to 10 seconds, depending on their thickness. The thin films, specifically, show very short response times. The aim for the second humidity sensor was the development of a prototype, where the sensitive film is biostatic and biocide, so that it can be used to measure the humidity in biotic environments. Five polysulfobetaines were synthesized and their biocidity and biostatics were tested with the contact test according to Rönnpagel, the ISO846 test and degradation tests. Two of the polymers - poly-DMMAAPS (BT2) and poly-[MSA-styrene-sulfobetaine] (BT5) - showed sufficient biocidity and biostatics. Films of these polymers were prepared on interdigitated structures, then their humidity characteristics were recorded. The measured values show good stability and high reproducibility for both polymers. Samples of BT2 are highly sensitive between 20% and 80% r.h. and show no long-term drift after one month. Cross-linked samples show no decrease of the humidity sensitivity in temperatures up to 50 degrees Celsius. Cross-linked films of BT5 can be used as a capacitive humidity sensor up to 70 degrees Celsius, and the samples are stable even after storage in high vacuum and multiple dew cycles. These two functional prototypes of humidity sensors show characteristic data comparable to those of commercial sensors. However, they stand out, respectively, with a very short response time and a sufficient life time in biotic environments.

Die vorliegende Arbeit beschäftigt sich mit der Synthese und den Eigenschaften von doppelthydrophilen Blockcopolymeren und ihrer Anwendung in einem biomimetischen Mineralisationsprozeß von Calciumcarbonat und Bariumsulfat. Doppelthydrophile Blockcopolymere bestehen aus einem hydrophilen Block, der nicht mit Mineralien wechselwirkt und einem zweiten Polyelektrolyt-Block, der stark mit Mineraloberflächen wechselwirkt. Diese Blockcopolymere wurden durch ringöffnende Polymerisation von N-carboxyanhydriden (NCA′s) und a-methoxy-ω-amino[poly(ethylene glycol)] PEG-NH2 als Initiator hergestellt.<br /> Die hergestellten Blockcopolymere wurden als effektive Wachstumsmodifikatoren für die Kristallisation von Calciumcarbonat und Bariumsulfat Mineralien eingesetzt. Die so erhaltenen Mineralpartikel (Kugeln, Hantel, eiförmige Partikel) wurden durch Lichtmikroskopie in Lösung, SEM und TEM charakterisiert. Röntgenweitwinkelstreuung (WAXS) wurde verwendet, um die Modifikation von Calciumcarbonat zu ermitteln und die Größe der Calciumcarbonat- und Bariumsulfat-Nanopartikel zu ermitteln. / This work describes the synthesis and characterization of double hydrophilic block copolymers and their use in a biomimetic mineralization process of Calcium Carbonate and Barium Sulfate.<br /> Double hydrophilic block copolymers consist of a hydrophilic block that does not interact with minerals and another hydrophilic polyelectrolyte block that strongly interacts with mineral surfaces. These polymers were synthesised via ring opening polymerisation of N-carboxyanhydride (NCA), and the first hydrophilic block a-methoxy-ω-amino[poly(ethylene glycol)] PEG-NH2 was used as an initiator.<br /> The prepared block copolymers were used as effective crystal growth modifiers to control the crystallization of Calcium Carbonate and Barium Sulfate minerals. The resulting mineral particles (spheres, dumbbells, egg-like particles) were characterised by light microscopy in solution, by SEM, and by TEM. X-Ray scattering measurements (WAXS) were used to prove the modification of Calcium Carbonate particles and to calculate the size of Calcium Carbonate and Barium Sulfate nanoparticles.

A novel strategy for synthesising segmented poly(urethane urea) (PUU) without using a chain extender but nevertheless with the opportunity to vary the hard segment content has been developed. The strategy is based on amine formation from isocyanate upon reaction with water. By adding a dissolved soft segment to an excess of diisocyanate followed by the addition of water in the gas phase, amines are formed in situ. Urea linkages are then formed when these amines react with the excess of isocyanate groups. The gas phase addition facilitates addition in a slow and continuous manner. The hard segment content can easily altered by varying the diisocyanate/soft segment ratio. Even though the strategy is shown to be applicable to different diisocyanates, the focus has been on the potentially biodegradable methyl-2,6-diisocyanatehexanoate (LDI) and 1.4-butanediisocyanate (BDI) and various well known biodegradable polyesters and polycarbonates. All the synthesised materials exhibited pronounced phase separation and hydrogen bonding within the hard domains. However, a major increase in hydrogen bonding strength was seen when a symmetric diisocyanate was used instead of an asymmetric. Based on FTIR measurements, PUUs with BDI and a polydisperse hard segment can exhibit the same degree of phase separation and hydrogen bonding as the monodisperse product. The elastic properties of this new group of PUUs were exceptional with an elongation at break from 1600% to almost 5000% and the elastic modulus could be varied from a few MPa up to a couple of hundreds. Hydrolytic degradation was greater in the polyester-based than in the polycarbonate-based PUUs due to the more reactive ester bonds. Low mass loss but a considerable loss in molecular weight was seen in the polyester PUUs. The tensile strength decreased dramatically due to the loss of strain hardening. An MTT seeding assay using human fibroblasts and an in vivo biocompatibility study were performed and no signs of cytotoxicity were seen and the inflammatory response was comparable to other inert polymers. A biodegradable PUU with properties that can be tailored through an easy synthesis is here presented.

The knowledge regarding certain issues in polymer crystallization e.g. the possible existence of short–lived mesophases remains inconclusive due to experimental limitations. Polymers undergo chain folding upon crystallization, which introduces some complications that are not found in crystallization of low molar mass materials. Chain–folded crystals are far from their equilibrium shape and they rearrange rapidly at the crystallization temperature. This, together with the slow experimental techniques traditionally used, impedes the observation of the originally formed structures. To approach this problem, molecularly constrained polymer structures (in which the crystallizing chains are fixed at one end whereas the other end is free to move) have been studied by X–ray diffraction, differential scanning calorimetry, polarized optical microscopy, transmission electron microscopy and atomic force microscopy. The crystallization studies performed in star–branched polyesters showed that the dendritic cores have a pronounced effect on the crystallization of the linear poly(ε–caprolactone) (PCL) arms attached to them. The star–branched polymers showed slower crystal rearrangement, higher equilibrium melting point, higher fold surface free energy, moderately lower crystallinity, and a greater tendency to form spherulites in comparison with linear PCL. The crystal unit cell was the same in both linear and star–branched PCL. Single crystals of the star–branched polymers were more irregular and showed smoother fold surfaces than linear PCL crystals. No sectorial preference was observed in the crystals of the star–branched polymers upon melting while the single crystals of linear PCL showed earlier melting in the {100} sectors than in the {110} sectors. Some of the differences observed can be attributed to the dendritic cores, which must be placed in the vicinity of the fold surface and thus influence the fold surface structure, the possibility of major crystal rearrangement and the presence of a significant cilia phase during crystal growth causing diverging crystal lamellae and consequent spherulite formation. The attachment of the many crystallizable chains to a single core reduces the melt entropy, which explains the higher equilibrium melting point of star–branched PCL. The crystallization behavior of a series of poly(ethylene oxybenzoate)s was also studied. The polymers showed a profound tendency for crystal rearrangement during melting even at high heating rates. The Hoffman–Weeks extrapolation method was found to be unsuitable to calculate the equilibrium melting point of the samples studied because the melting point vs. crystallization temperature data were sensitive to the variations in crystallisation time, which led to significant variations in the equilibrium melting points obtained. / QC 20100914