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The focus of this research was to investigate the effect of thermal degradation upon the mechanical properties of natural rubber compounds and apply those effects to the life prediction of off axis 2-ply cord rubber laminates. The work examined both the quasi-static and dynamic mechanical properties of two natural rubber vulcanizates, which had been subjected to isothermal anaerobic aging. Thermal aging was performed on two different natural rubber vulcanizates. The thermal aging was conducted between the temperatures of 80 and 120°C for times ranging from 3 to 24 days. The effect of thermal degradation was measured from the changes in the chemical composition of the vulcanizates as functions of time at temperature. A master curve relationship between the changes in the chemical composition of the vulcanizates due to thermal degradation and their static and dynamic mechanical properties has been developed. This relationship allowed for the prediction of the vulcanizate mechanical properties after thermal aging. It was found that the mechanical properties correlated with the percentage of poly and monosulfidic crosslinks, where in general higher levels of polysulfidic crosslink gave rise to the highest mechanical properties. Crack propagation in an aged and unaged natural rubber vulcanizate was measured using a double cantilever beam, DCB. This type of testing arrangement exhibits a plane strain condition and resulted in crack growth rates two orders of magnitude faster than traditional plane stress testing geometries. To validate the DCB specimens, an investigation into the potential cavitation inside the rubber of the DCB specimens was performed. It was found that no cavitation occurred due to the high speed of the fracture. DCB samples were thermally aged to determine the effect of thermal aging upon the crack growth rate. It was found that crack growth rates increase with thermal aging. Life prediction of the aged 2-ply laminates was performed using a finite element analysis. In order to verify the finite element models used in the life prediction, the fatigue failure and crack growth characteristics of off axis 2-ply cord-rubber laminates were examined with a delamination analysis. This analysis allowed for the determination of the modulus of off axis 2-ply laminates in the presence of damage as well as the calculation of the crack growth rates of the laminate. The failure of unaged and thermally aged 2-ply laminates was evaluated and compared to the crack growth rates of thermally aged DCB specimens. The trend due to thermal aging between the two types of testing specimens was consistent. The finite element analysis was sectioned into two approaches: crack initiation and crack propagation. The former utilized a residual strength approach, while the latter applied a fracture mechanics approach. The predicted stress versus cycles, S-N, curves were not in complete agreement with the experimental data. The error between the predicted and the experimental is discussed and future work to correct that error is suggested. While there was not complete agreement between the predicted and the experimental data, this dissertation outlines a comprehensive approach to track the effects of thermal degradation and apply those effects to a real world application. / Ph. D.

Diese Arbeit beschreibt die Realisierung mehrerer theoretischer Ansätze zur Synthese von Oligonucleotiden, die an gezielter Position mit der trans-{PtII(NH3)2}2+ - Spezies modifiziert sind. Es wurde ein Synthesebaustein "Pt-T" synthetisiert, der die direkte Einführung eines N3-platinierten Thymidins während der Oligonucleotidsynthese ermöglicht. Unter den Bedingungen der Standard - H-Phosphonat - Synthese werden bei Verwendung des synthetisierten platinierten Synthesebaustein "Pt-T" platinierte Oligonucleotide erhalten, die eine trans-[PtII(NH3)2Py(N3-T)]+ - Modifizierung enthalten. Mit Hilfe der Sanger - Sequenzierung konnte gezeigt werden, daß die an T angebundene trans-{PtII(NH3)2Py}2+ - Platinspezies T7 DNA Polymerase blockiert. Weiterhin konnte gezeigt werden, daß die Spaltung der 5'-Phosphorsäurediesterbindung durch Schlangengift - Phosphodiesterase infolge dieser Platinmodifizierung deutlich langsamer abläuft. Durch Ersatz von Pyridin durch 1,6-Lutidin bleibt die Reaktionsfähigkeit der trans-Position am Platin erhalten. Durch geeignete Reaktionsbedingungen wurde das nach erfolgter Oligonucleotidsynthese an einem 4-mer gebundene Platin bifunktional an den selben Strang gebunden und so trans-[PtII(NH3)2{d(TTTG)-N3-T(2),N7-G(4)}]+, ein kurzes Oligonucleotid mit intrastrand-crosslink, synthetisiert. Die durchgeführte enzymatische Hydrolyse zeigt eine hohe Beständigkeit gegenüber dem Abbau mit Schlangengift - Phosphodiesterase und alkalischer Phosphatase. / This work describes the synthesis of oligodeoxynucleotides, which are modified at a specific position with the trans-{PtII(NH3)2}2+ - species. A platinated monomer building block "Pt-T" has been synthesized separately prior to automated synthesis. Platin modified oligonucleotides were elongated by use of standard H-phosphonate chemistry. The use of the synthesized platinated building block "Pt-T" leads to platinated oligonucleotides with a trans-[PtII(NH3)2Py(N3-T)]+ - modification. The synthesized oligonucleotides have been subjected to sequencing by the Sanger - method and it could be shown, that this modification blocks T7 DNA polymerase. Furthermore it could be shown, that the cleavage of the 5'-phosphodiester bond by snake venom phosphodiesterase due to these modification runs down clearly more slowly. In consequence of substitution of pyridine by 2,6-lutidine the reactivity of the trans - position of the platinum remains received. After oligonucleotide synthesis the platinum became crosslinked, thus trans-[PtII(NH3)2{d(TTTG)-N3-T(2),N7-G(4)}]+, a short oligonucleotide with intrastrand-crosslink, was synthesized. The enzymatic hydrolysis showed a high constancy facing the degradation with snake venom phosphodiesterase and alkaline phosphatase.

<p>Translesion synthesis is a fundamental biological process that enables DNA replication across lesion sites to ensure timely duplication of genetic information at the cost of replication fidelity, and it is implicated in development of cancer drug resistance after chemotherapy. The eukaryotic Y-family polymerase Rev1 is an essential scaffolding protein in translesion synthesis. Its C-terminal domain (CTD), which interacts with translesion polymerase &#950; through the Rev7 subunit and with polymerases &#954;, &#953; and &#951; in vertebrates through the Rev1-interacting region (RIR), is absolutely required for function. </p><p>In chapter 1, the solution structures of the mouse Rev1 CTD and its complex with the Pol &#954; RIR are reported, revealing an atypical four-helix bundle. Yeast two-hybrid assays were used to identify a Rev7-binding surface centered at the &#945;2-&#945;3 loop and N-terminal half of &#945;3 of the Rev1 CTD. Binding of the mouse Pol &#954; RIR to the Rev1 CTD induces folding of the disordered RIR peptide into a three-turn &#945;-helix, with the helix stabilized by an N-terminal cap. RIR-binding also induces folding of a disordered N-terminal loop of the Rev1 CTD into a &#946;-hairpin that projects over the shallow &#945;1-&#945;2 surface and creates a deep hydrophobic cavity to interact with the essential FF residues juxtaposed on the same side of the RIR helix. The combined structural and biochemical studies reveal two distinct surfaces of the Rev1 CTD that separately mediate the assembly of extension and insertion translesion polymerase complexes.</p><p>The multifaceted abilities of the Rev1 CTD are further explicated in chapter 2 where the purification and structure determination of a quaternary translesion polymerase complex consisting of the Rev1 CTD, the heterodimeric Pol &#950; complex, and the Pol &#954; RIR is reported. Yeast two-hybrid assays were employed to identify important interface residues of the translesion polymerase complex. The structural elucidation of such a quaternary translesion polymerase complex encompassing both insertion and extension polymerases bridged by the Rev1 CTD provides the first molecular explanation of the essential scaffolding function of Rev1 and highlights the Rev1 CTD as a promising target for developing novel cancer therapeutics to suppress translesion synthesis. Our studies support the notion that vertebrate insertion and extension polymerases could structurally cooperate within a mega translesion polymerase complex (translesionsome) nucleated by Rev1 to achieve efficient lesion bypass without incurring an additional switching mechanism.</p><p>Chapter 3 explores the ubiquitin-binding capacity of the FAAP20 UBZ in an effort to begin understanding its requirement for recruitment of the Fanconi anemia complex to interstrand DNA crosslink sites and for interaction with the translesion synthesis machinery through recognition of monoubiquitinated Rev1. FAAP20 is an integral component of the Fanconi anemia core complex that mediates the repair of DNA interstrand crosslinks. Although the UBZ-ubiquitin interaction is thought to be exclusively encapsulated within the &#946;&#946;&#945; module of UBZ, it is revealed that the FAAP20-ubiquitin interaction extends beyond such a canonical zinc-finger motif. Instead, ubiquitin-binding by FAAP20 is accompanied by transforming a disordered tail C-terminal to the UBZ of FAAP20 into a rigid, extended &#946;-loop that latches onto the complex interface of the FAAP20 UBZ and ubiquitin, with the invariant C-terminal tryptophan emanating toward I44Ub for enhanced binding specificity and affinity. Substitution of the C-terminal tryptophan with alanine in FAAP20 not only abolishes FAAP20-ubiquitin binding in vitro, but also causes profound cellular hypersensitivity to DNA interstrand crosslink lesions in vivo, highlighting the indispensable role of the C-terminal tail of FAAP20, beyond the compact zinc finger module, toward ubiquitin recognition and Fanconi anemia complex-mediated DNA interstrand crosslink repair.</p><p>Having structurally elucidated the molecular basis of the essential scaffolding function of the Rev1 CTD, the search for small molecule inhibitors of the Rev1-Rev7 interaction has been initiated toward the goal of developing novel adjuvants to DNA targeting chemotherapeutics. Screening efforts have led to the discovery of a lead compound, JH-RE-06*NaOH, that specifically targets the Rev7-binding hydrophobic pocket of the Rev1 CTD with low micromolar affinity, effectively inhibiting the Rev1-Rev7 interaction in an in vitro ELISA assay developed for high-throughput screening of small molecule libraries. With the potential for positive outcomes in future in vivo assays, we hope to develop JH-RE-06*NaOH into the first potent inhibitor of translesion synthesis in cancer patients being treated with DNA-targetng chemotherapeutics to aid in sensitization and prevention of chemoresistance development in malignancies.</p> / Dissertation

L’embolisation thérapeutique est devenu le traitement de choix pour l’hémorragie, les malformations artériovéneuses ou certains types de cancer. Parmi différents agents d’embolisation,les microsphères non dégradables (Embozene®, Bead BlockTM,…) sont les plus utilisées. Leur forme bien sphérique et leur taille calibrée permettent un meilleur ciblage dans les vaisseaux et une bonne qualité de l’occlusion. Dans certains cas cliniques, l’embolisation temporaire, envisageable avec l’utilisation des microsphères résorbables peut être bénéfique pour les patients. Le but du travail réalisé au cours de cette thèse a été le développement de microsphères résorbables satisfaisant les différents critères pour être employées comme matériaux d’embolisation (taille calibrée,biocompatibles, élastique pour être injectée au travers des cathéters mais suffisamment rigide pour résister à la pression sanguine). Dans cet objectif, nous avons développé une méthode de synthèse de microsphères constituées d’hydrogels hydrolysables par polymérisation en suspension. Une large gamme de microsphères ont été synthétisées en modulant la nature du réticulant et/ou la composition des milieux de polymérisation. Les expériences in vitro ont démontré que les microsphères obtenues sont satisfaisantes pour permettre leur injection au travers des cathéters. La dégradation rapide des ponts de réticulation a été confirmée à travers la diminution du module élastique G’ et du pH du surnageant, accompagnée d’une augmentation du taux de gonflement.Malgré une dégradation partielle des microsphères (due à une réaction secondaire formant des liaisons de réticulation non dégradables), le temps de l’hydrolyse a répondu parfaitement au cahier de charges (entre 7 et 49 jours). Des études complémentaires pour optimiser la réaction de polymérisation vont permettre le développement de microsphères totalement dégradables. / Therapeutic embolization is nowadays a first line treatment for haemorrhage, arteriovenous malformation or tumors. Among different embolization agents, non degradable microspheres(Embozene®, Bead BlockTM,…) are the most employed thanks to their well calibrated spherical shape which allows good occlusion. In some cases including treatment of uterine fibroids or chemo-sensitive tumors, it may be interesting to achieve a temporary embolization to avoid definitive destruction of the tissue. Temporary embolization would be possible using biodegradable microspheres. The aim of our work was to develop degradable microspheres having all requiredcharacteristics to be used as embolization material (well calibrated in size, biocompatible, rigide enough to resist blood pressure but elastic enough to remain intact during injection through catheter). To this purpose, we have developed hydrolysable hydrogel based microspheres by suspension polymerization. A wide range of microspheres was synthesized by varying the type of crosslinker and composition of the polymerization medium. In vitro test showed that the microspheres have suitable characteristics to pass through catheter. Degradation studies revealed a rapid diminution of G’ modulus and the pH of the supernatants, accompanied by an increase of swelling ratio due to the hydrolysis of the crosslinkings. Although microspheres were not totally degradable as expected (since a side reaction had created non degradable crosslinking during the polymerisation), characterisations showed promising results that the degradation did occur within a suitable time scale requirements for temporal embolization.

<p>Validerat; 20130918 (global_studentproject_submitter)</p>

<p>5-azacytidine and its derivatives are cytidine analogs used for leukemia chemotherapy. The primary effect of 5-azacytidine is the prohibition of cytosine methylation, which results in covalent DNA-methyltransferase crosslinks at cytosine methylation sites. These DNA-protein crosslinks have been suggested to cause chromosomal rearrangements and contribute to cytotoxicity, but the detailed mechanisms of DNA damage and the repair pathways of DNA-protein crosslinks have not been elucidated. </p><p>We used 2-dimensional agarose gel electrophoresis and electron microscopy to analyze plasmid pBR322 replication dynamics in Escherichia coli cells grown in the presence of 5-azacytidine. 2-dimensional gel analysis revealed the accumulation of specific bubble- and Y-molecules, dependent on overproduction of the cytosine methyltransferase EcoRII and treatment with 5-azacytidine. Furthermore, a point mutation that eliminates a particular EcoRII methylation site resulted in disappearance of the corresponding bubble- and Y-molecules. These results imply that 5-azacytidine-induced DNA-protein crosslinks block DNA replication in vivo. RecA-dependent X-structures were also observed after 5-azacytidine treatment. These molecules may be generated from blocked forks by recombinational repair and/or replication fork regression. In addition, electron microscopy analysis revealed both bubbles and rolling circles after 5-azacytidine treatment. These results suggest that replication can switch from theta to rolling circle mode after a replication fork is stalled by a DNA-methyltransferase crosslink. The simplest model for the conversion of theta to rolling-circle mode is that the blocked replication fork is cleaved by a branch-specific endonuclease. Such replication-dependent DNA breaks may represent an important pathway that contributes to genome rearrangement and/or cytotoxicity. </p><p>In addition, we performed a transposon mutagenesis screen and found that mutants defective in the tmRNA translational quality control system are hypersensitive to 5-azacytidine. The hypersensitivity of these mutants requires expression of active methyltransferase, indicating that hypersensitivity is dependent on DNA-methyltransferase crosslink formation. Furthermore, the tmRNA pathway is activated upon 5-azacytidine treatment in cells expressing methyltransferase, resulting in increased SsrA tagging of cellular proteins. These results support a "chain-reaction" model, in which transcription complexes blocked by 5-azacytidine-induced DNA-protein crosslinks result in ribosomes stalling on the attached nascent transcripts, and the tmRNA pathway is invoked for cleaning up the resulting pile-ups. In support of this model, an ssrA mutant is also hypersensitive to antibiotic streptolydigin, which blocks RNA polymerase elongation. These results reveal a novel role for the tmRNA system in clearance of coupled transcription/translation complexes in which RNA polymerase has become blocked.</p> / Dissertation

New polymer cure chemistry is described, wherein the onset of free radical cross-linking is delayed without compromising cure yields. The addition of an acrylate-functionalized nitroxyl, 4-acryloyloxy-2,2,6,6-tetramethylpiperidine-N-oxyl (AOTEMPO), to a peroxide-cure formulation quenches free radical activity during the initial stages of the cross-linking process, trapping alkyl radicals as alkoxyamines that bear acrylate functionality. Polymer cross-linking by macro-radical combination is suppressed until all nitroxyl is consumed, at which point radical oligomerization of polymer-bound acrylate groups generates the desired covalent network. As a result, cross-link density losses incurred during radical trapping are recovered during the oligomerization phase of the process. The effectiveness of this approach is demonstrated for a range of polymers, peroxide initiators, reaction temperatures and reagent loadings. Furthermore, AOTEMPO formulations are compared directly to other delayed-onset additives that are used in commercial practice. / Thesis (Master, Chemical Engineering) -- Queen's University, 2012-06-11 10:09:20.848

Thin films with the ability to impart oxygen and other types of gas barrier are crucial to commercial packaging applications. Commodity polymers, such as polyethylene (PE), polycarbonate (PC) and polyethylene terephthalate (PET), have insufficient barrier for goods requiring long shelf life. Current gas barrier technologies like plasma-enhanced vapor deposition (PECVD) often create high barrier metal oxide films, which are prone to cracking when flexed. Bulk composites composed of polymer and impermeable nanoparticles show improved barrier, but particle aggregation limits their practical utility for applications requiring high barrier and transparency. Layer-by-layer (LbL) assemblies allow polymers and nanoparticles to be mixed with high particle loadings, creating super gas barrier thin films on substrates normally exhibiting high gas permeability. Branched polyethylenimine (PEI) and poly (acrylic acid) (PAA) were deposited using LbL to create gas barrier films with varying pH combinations. Film thickness and mass fraction of each component was controlled by their combined charge. With lower charge density (PEI at pH 10 and PAA at pH 4), PEI/PAA assemblies exhibit the best oxygen barrier relative to other pH combinations. An 8 BL PEI/PAA film, with a thickness of 451 nm, has an oxygen permeability lower than 4.8 x 10^-21 cm^3 * cm/cm^2 * s * Pa, which is comparable to a 100 nm SiOx nanocoating. Crosslinking these films with glutaraldehyde (GA), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide methiodide (EDC) or heating forms covalent bonds between PEI and/or PAA. Oxygen transmission rates (OTR) of 8 BL films crosslinked with 0.1M GA or 0.01M EDC show the best oxygen barrier at 100% RH. Graphene oxide (GO) sheets and PEI were deposited via LbL with varying GO concentration. The resulting thin films have an average bilayer thickness from 4.3 to 5.0 nm and a GO mass fraction from 88 to 91wt%. Transmission electron microscopy and atomic force microscopy images reveal a highly-oriented nanobrick wall structure. A 10 BL PEI/GO film that is 91 nm thick, made with a 0.2 wt% GO suspension, exhibits an oxygen permeability of 2.5 x 10^-20 cm^3 * cm/cm^2 * s * Pa. Finally, the influence of deposition time on thin film assembly was examined by depositing montmorillonite (MMT) or laponite (LAP) clays paired with PEI. Film growth and microstructure suggests that smaller aspect ratio LAP clay is more dip-time dependent than MMT and larger aspect ratio MMT has better oxygen barrier. A 30 BL PEI/MMT film made with 10 second dips in PEI has the same undetectable OTR as a film with 5 minute dips (with dips in MMT held at 5 minutes in both cases), indicating LbL gas barrier can be made more quickly than initially thought. These high barrier recipes, with simple and efficient processing conditions, are good candidates for a variety of packaging applications.

Orientador: Marcelo Ganzarolli de Oliveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-25T12:07:45Z (GMT). No. of bitstreams: 1 Marcilli_RaphaelHenriqueMarques_M.pdf: 7897278 bytes, checksum: 7868f663066dcea221688ac8dc901af2 (MD5) Previous issue date: 2013 / Resumo: Condições fisiológicas associadas à diminuição do nível de óxido nítrico (NO) na vasculatura, como na síndrome de Raynaud e na angiopatia diabética, têm estimulado o desenvolvimento de novos biomateriais capazes de liberar NO topicamente. Neste trabalho efetuamos a modificação do poli(álcool vinílico), PVA, pela sua reticulação química através da esterificação com o ácido mercaptosuccínico. Esta reação permitiu a moldagem de filmes de PVA sulfidrilados (PVA-SH). A calorimetria diferencial de varredura e a difratometria de raios X mostraram que a reação de reticulação suprimiu a cristalização do PVA, levando a um material sem poros, com uma distribuição homogênea de grupos sulfidrila(-SH). Os grupos hidroxila remanescentes na rede de PVA-SH conferiram hidrofilicidade parcial ao material, associada a um grau de intumescimento de 80 a 120 % em meio aquoso. Os filmes de PVA-SH, foram submetidos a uma reação de S-nitrosação dos grupos ¿SH produzindo um PVA contendo grupos S-nitrosotióis (PVA-SNO). Medidas amperométricas e por quimiluminescência mostraram que os filmes de PVA-SNO contém ca. 6 nmols de NO/mg de filme e são capazes de liberar NO espontaneamente após imersão em meio fisiológico. A fluxometria por laser Doppler, usada para medir o fluxo sanguíneo na microcirculação dérmica, permite verificar que a aplicação tópica dos filmes de PVA-SNO hidratados sobre a pele saudável leva a um aumento dependente da dose e do tempo de mais de 5 vezes no fluxo sanguíneo basal em menos de 10 min de aplicação com uma ação prolongada de mais de 4 h durante a aplicação contínua. Estes resultados mostram que os filmes de PVA-SNO podem representar um novo material com potencial para o tratamento tópico no tratamento de desordens microvasculares da pele / Abstract: Pathological conditions associated with the impairment of nitric oxide (NO) production in the vasculature, like Raynaud¿s syndrome and diabetic angiopathy, have stimulated the development of new biomaterials capable of delivering NO topically. In this work, we have modified poly(vinyl-alcohol) (PVA) by chemically crosslinking it via esterification with mercaptosuccinic acid. This reaction allowed the casting of sulfhydrylated PVA (PVA-SH) films. Differential scanning calorimetry and X-ray diffractometry showed that the crosslinking reaction suppressed the crystallization of PVA, leading to a non-porous material with a homogeneous distribution of -SH groups. The remaining hydroxyl groups in the PVA-SH network confered partial hydrophylicity to the material, associated with a swelling degree of 80 to 120 % in aqueous medium. The PVA-SH films were subjected to an S-nitrosation reaction of the SH groups yielding a PVA containing S-nitrosothiol groups (PVA-SNO). Amperometric and chemiluminescence measurements showed that the PVA-SNO films contain ca. 6 nmol NO/mg of film and are capable of releasing NO spontaneously after immersion in physiological medium. Laser Doppler-flowmetry, used to assess the blood flow in the dermal microcirculation, showed that the topical application of hydrated PVA-SNO films on the health skin leads to a dose and time dependent increase of more than 5-fold in the dermal baseline blood flow in less than 10 min with a prolonged action of more than 4 h during continuous application. These results show that the PVA-SNO films may represent a new material with potential for the topical treatment of microvascular skin disorders / Mestrado / Físico-Química / Mestre em Química