Modification of Natural Rubber using Thermoset Resins

This thesis presents the findings of a study on incorporating vanous thermoset resins into natural rubber for property improvement. Natural rubber is an important elastomer with the unique attribute of being a renewable agricultural product. The study was undertaken to investigate the extent to which the drawbacks of natural rubber, especially its poor thermal and oil resistance propel1ies could be nullified by blending with common thermoset resins. A thorough and comparative understanding of the perfonnance of different resins from this viewpoint will be beneficial for both natural IUbber processors and consumers. In this study the thennoset resins used were epoxy resin, phenolics, epoxidised phenolics and unsaturated polyester resin.The resins were incorporated into NR during compounding and their effects on the properties of NR were studied after vulcanization. Properties were studied for both gum and filled N R compounds. The important properties studied are cure characteristics, mechanical properties, ageing propel1ies, thermal propel1ies, crosslink density and extractability. Characterization studies were also conducted using FTIR, TGA and DSC.Improvement in mechanical properties was noticed in many cases. The results show that most resins lead to a reduction in the cure time of NR. The perfonnance of epoxy resin is most noticeable in this respect. Mechanical properties of the modified IUbber show maximum improvement in the case of epoxidised novolacs. Most resins are seen to improve the thermal and oil resistance propel1ies of NR. Epoxy novolacs show maximum effect in this respect also. However the presence of tillers is found to moderate the positive effects of the thermoset resins considerably.

Applications of cardanol in rubber processing

This research project explores the utilization of cardanol in various capacities for rubber processing. Cardanol is a phenol with a long side chain in the meta position of the benzene ring. It is obtained by the vacuum distillation of cashew Hut shell liquid (CNSL) which is a cheap agro-byproduct. In this study, the plasticizer property of cardanol was investigated in silica filled and HAF black filled NR, NBR, EPDM and CR by comparing cure characteristics and mechanical properties of vulcanizates containing conventional plasticizer with those containing cardanol as plasticizer. The co-activator, antioxidant and accelerator properties were investigated in gum samples of NR, NBR, EPDM and CR by comparing the properties of vulcanizates which contain conventional co-activator, antioxidant and accelerator with those in which each of them was replaced by cardanol. The general effectiveness of cardanol was investigated by determination of cure time , measurement of physical and mechanical properties, ageing studies, crosslink density, extractability, FTIR spectra, TGA etc.The results show that cardanol can be a substitute for aromatic oil in both silica filled and HAF black filled NR. Again, it can replace dioctyl phthalate in both silica filled and HAF black filled NBR. Similarly, cardanol Can replace naphthenic oil in silica filled as well as HAF black filled EPDM and CR. The cure characteristics and mechanical properties are comparable in all the eight cases. The co-activator property of cardanol is comparable to stearic acid in all the four rubbers. The cure characteristics and mechanical properties in this case are also comparable. The antioxidant ,property of cardanol is comparable to TQ in all the four rubbers. The antioxidant property of cardanol is comparable to TQ in all the four case of NBR and EPDM.The accelerator property of cardarlol is comparable with CBS in the case of NBR and EPDM. No accelerator property is observed in the case of NR. The accelerator property of cardanol in CR is not negligible when compared with TMTD.

Covulcanization of elastomer blends with special reference to NR/EPDM blends

Attempts have been made to attain satisfactory network structures in each of the phases of a rubber blend by minimising the cure rate imbalance by employing methods such as grafting of accelerators to the slow curing rubber, chemically bonding the crosslinking agents to the rubber in which it has lower solubility, functionalisation of the slow curing rubber, masterbatching of the curing agents to the slow curing rubber etc. Functionalisation of the slow curing constituents of NR/IIR and NR/EIPDM blends is tried using novel reagents as the first part of this study. However, the crux of the present study is a more direct approach to attaining a covulcanized state in NR/IIR and NR/EPDM blends: Precuring the slow curing rubber (IIR or EPDM) to a low level when it can still blend with NR and then to ck) the final curing after blending with NR. TNM3 precuring is also likely to minimise the viscosity mismatch. Since a low level of resmmal crosslink density is likely to be present lJ1 reclaimed rubbers, blending heat resistant reclaimed rubber such as butyl reclaim with NR may also have the same effect of precuring IIR, and then blending with NR. Hence use of IIR reclaim for developing blends with NR is also proposed to be investigated in this study

Biologische Funktionsanalyse und Identifizierung neuer Substrate der Methyltransferase Dnmt2

Obwohl die DNA Methyltransferase 2 (Dnmt2) hoch konserviert ist und zu der am weitesten verbreiteten eukaryotischen MTase-Familie gehört, ist ihre biologische Funktion nach wie vor unklar. Nachdem lange Zeit keine DNA Methylierungsaktivität nachgewiesen werden konnte, wurde vor einigen Jahren über geringe Mengen an 5-Methylcytosin (5mC) in Retroelementen der “Dnmt2-only”-Organismen D. melanogaster, D. discoideum und E. histolytica berichtet (Kunert et al. 2003; Fisher et al. 2004; Kuhlmann et al. 2005; Phalke et al. 2009). Als kurze Zeit später robuste Methylierung der tRNAAsp durch humane Dnmt2 gezeigt wurde (Goll et al. 2006), wurde zunächst eine Dualspezifität des Enzyms vorgeschlagen (Jeltsch et al. 2006). Neuere Daten zum 5mC-Status verschiedener „Dnmt2-only“-Organismen bilden Anlass für kontroverse Diskussionen über Ausmaß und Bedeutung der DNA Methyltransferaseaktivität von Dnmt2 (Schaefer et al. 2010a; Krauss et al. 2011). Die vorliegende Arbeit konzentriert sich auf die Identifizierung neuer RNA Substrate des Dnmt2-Homologs DnmA aus D. discoideum sowie die biologische Bedeutung der tRNA-Methylierung durch Dnmt2. Wie in anderen Organismen beschrieben, fungiert auch DnmA als tRNAAsp(GUC) MTase in vitro und in vivo. Zusätzlich konnte in vitro tRNAGlu(UUC) als neues Substrat der Dnmt2-Homologe aus D. discoideum und dem Menschen identifiziert werden. In einem Kooperationsprojekt wurde außerdem auch tRNAAsp-Methylierungsaktivität für das Dnmt2-Homolog aus S. pombe (Pmt1) nachgewiesen. Crosslink-RNA-Immunopräzipitationen (RNA-CLIP) mit anschließender Next-Generation-Sequenzierung der mit DnmA assoziierten RNAs zeigen, dass DnmA mit tRNA Fragmenten interagiert, die sich vom Anticodonloop bis in den T-loop erstrecken. Neben der tRNAAsp(GUC) und tRNAGlu(UUC/CUC) sind Fragmente der tRNAGly(GCC) verstärkt angereichert. Inwiefern diese Fragmente eine biologische Funktion haben oder spezifische Degradationsprodukte darstellen, ist noch ungeklärt. Interessanterweise sind von einigen tRNAs wenige Sequenzen von antisense-Fragmenten in den RNA-CLIP Daten zu finden, die etwas kürzer, jedoch exakt komplementär zu den genannten sense-Fragmenten sind. Besonders stark sind diese Fragmente der tRNAGlu(UUC) vertreten. In einem weiteren RNA-CLIP Experiment wurden U-snRNAs, snoRNA und intergenische Sequenzen mit DnmA angereichert. Bei nachfolgenden in vitro Methylierungsstudien konnte ausschließlich die U2-snRNA als potentielles Nicht-tRNA-Substrat der hDnmt2 und DnmA identifiziert werden. Da tRNA Modifikationen im Anticodonloop die Codonerkennung beeinflussen können, wurde ein System etabliert um die Translationseffizienz eines GFP-Reportergens in Wildtyp- und dnmAKO-Zellen zu messen. In D. discoideum wird das Aspartat-Codon GAU ca. zehnmal häufiger genutzt als das GAC Codon, allerdings ist nur eine tRNAAsp(GUC) im Genom der Amöbe kodiert. Aus diesem Grund wurde zusätzlich die Frage adressiert, inwiefern die DnmA-abhängige Methylierung dieser tRNA das „Wobbling“ beeinflusst. Dazu wurde dem Reportergen jeweils eine (GAU)5- und (GAC)5-Leadersequenz vorgeschaltet. Entgegen der Annahme wurde der (GAC)5-Leader in beiden Stämmen etwas effizienter translatiert. Insgesamt zeigte der dnmAKO-Stamm eine leicht erhöhte Translationseffizienz der Reportergene. Vergleichende Analysen zur Aufnahme von Fremd-DNA zeigten signifikant reduzierte Transformationseffizienzen mit einem integrierenden Plasmid in dnmAKO-Zellen. Ein weiterer dnmAKO-Stamm zeigte diesen Effekt jedoch nicht, wobei bei derselben Mutante eine deutlich reduzierte Aufnahme eines extrachromosomalen Plasmids zu verzeichnen war. Untersuchungen zum Einfluss von DnmA auf die Regulation des Retroelements skipper ergaben keinen Zusammenhang zwischen der Generierung kleiner RNAs und der erhöhten Transkription des Retrotransposons in dnmAKO-Zellen (Kuhlmann et al. 2005). Durch Kompensationsversuche sowie Experimente mit einer weiteren dnmAKO-Mutante konnte die Mobilisierung des Retrotransposons nicht eindeutig als DnmA-Funktion eingeordnet werden. In einem weiteren Projekt wurden die Bindung des m5C-bindenden Proteins EhMLBP aus E. histolytica an DNA mittels Rasterkraftmikroskopie abgebildet (Lavi et al. 2006). Neben vermutlich unspezifischen Endbindungsereignissen konnte eine bevorzugte Bindungsstelle des Proteins an LINE DNA (long intersperesed nuclear element) identifiziert werden. Möglicherweise fällt diese mit einem von zwei A/T-reichen Bereichen der LINE DNA zusammen, von denen vermutet wird, dass diese für die Bindung von EhMLBP an DNA von Bedeutung sind. Insgesamt bestätigen die Ergebnisse dieser Arbeit die tRNAAsp Methylierungsaktivität als konservierte Dnmt2-Funktion. Darüber hinaus erweitern sie das Substratspektrum der Dnmt2-Methyltransferasen im Bereich der tRNA. Außerdem wird erstmals ein potentielles Nicht-tRNA Substrat vorgeschlagen. Zusätzlich geben neu entdeckte Phänotypen Hinweise auf vielfältige zelluläre Dnmt2-Funktionen.

Electronic couplings and on-site energies for hole transfer in DNA: systematic quantum mechanical/molecular dynamic study

The electron hole transfer (HT) properties of DNA are substantially affected by thermal fluctuations of the π stack structure. Depending on the mutual position of neighboring nucleobases, electronic coupling V may change by several orders of magnitude. In the present paper, we report the results of systematic QM/molecular dynamic (MD) calculations of the electronic couplings and on-site energies for the hole transfer. Based on 15 ns MD trajectories for several DNA oligomers, we calculate the average coupling squares 〈 V2 〉 and the energies of basepair triplets X G+ Y and X A+ Y, where X, Y=G, A, T, and C. For each of the 32 systems, 15 000 conformations separated by 1 ps are considered. The three-state generalized Mulliken-Hush method is used to derive electronic couplings for HT between neighboring basepairs. The adiabatic energies and dipole moment matrix elements are computed within the INDO/S method. We compare the rms values of V with the couplings estimated for the idealized B -DNA structure and show that in several important cases the couplings calculated for the idealized B -DNA structure are considerably underestimated. The rms values for intrastrand couplings G-G, A-A, G-A, and A-G are found to be similar, ∼0.07 eV, while the interstrand couplings are quite different. The energies of hole states G+ and A+ in the stack depend on the nature of the neighboring pairs. The X G+ Y are by 0.5 eV more stable than X A+ Y. The thermal fluctuations of the DNA structure facilitate the HT process from guanine to adenine. The tabulated couplings and on-site energies can be used as reference parameters in theoretical and computational studies of HT processes in DNA

Estimates of electronic coupling for excess electron transfer in DNA

Electronic coupling Vda is one of the key parameters that determine the rate of charge transfer through DNA. While there have been several computational studies of Vda for hole transfer, estimates of electronic couplings for excess electron transfer (ET) in DNA remain unavailable. In the paper, an efficient strategy is established for calculating the ET matrix elements between base pairs in a π stack. Two approaches are considered. First, we employ the diabatic-state (DS) method in which donor and acceptor are represented with radical anions of the canonical base pairs adenine-thymine (AT) and guanine-cytosine (GC). In this approach, similar values of Vda are obtained with the standard 6-31 G* and extended 6-31++ G* basis sets. Second, the electronic couplings are derived from lowest unoccupied molecular orbitals (LUMOs) of neutral systems by using the generalized Mulliken-Hush or fragment charge methods. Because the radical-anion states of AT and GC are well reproduced by LUMOs of the neutral base pairs calculated without diffuse functions, the estimated values of Vda are in good agreement with the couplings obtained for radical-anion states using the DS method. However, when the calculation of a neutral stack is carried out with diffuse functions, LUMOs of the system exhibit the dipole-bound character and cannot be used for estimating electronic couplings. Our calculations suggest that the ET matrix elements Vda for models containing intrastrand thymine and cytosine bases are essentially larger than the couplings in complexes with interstrand pyrimidine bases. The matrix elements for excess electron transfer are found to be considerably smaller than the corresponding values for hole transfer and to be very responsive to structural changes in a DNA stack

Effect of the anti-neoplastic drug doxorubicin on XPD-mutated DNA repair-deficient human cells

Doxorubicin (DOX), a member of the anthracycline group, is a widely used drug in cancer therapy. The mechanisms of DOX action include topoisomerase II-poisoning, free radical release, DNA adducts and interstrand cross-link (ICL) formation. Nucleotide excision repair(NER) is involved in the removal of helix-distorting lesions and chemical adducts, however, little is known about the response of NER-deficient cell lines to anti-tumoral drugs like DOX. Wild type and XPD-mutated cells, harbouring mutations in different regions of this gene and leading to XP-D, XP/CS or TTD diseases, were treated with this drug and analyzed for cell cycle arrest and DNA damage by comet assay. The formation of DSBs was also investigated by determination of gamma H2AX foci. Our results indicate that all three NER-deficient cell lines tested are more sensitive to DOX treatment, when compared to wild type cells or XP cells complemented by the wild type XPD cDNA, suggesting that NER is involved in the removal of DOX-induced lesions. The cell cycle analysis showed the characteristic G2 arrest in repair-proficient MRC5 cell line after DOX treatment, whereas the repair-deficient cell lines presented significant increase in sub-G1 fraction. The NER-deficient cell lines do not show different patterns of DNA damage formation as assayed by comet assay and phosphorylated H2AX foci formation. Knock-down of topoisomerase II alpha with siRNA leads to increased survival in both MRC5 and XP cells, however, XP cell line still remained significantly more sensitive to the treatment by DOX. Our study suggests that the enhanced sensitivity is due to DOX-induced DNA damage that is subject to NER, as we observed decreased unscheduled DNA synthesis in XP-deficient cells upon DOX treatment. Furthermore, the complementation of the XPD-function abolished the observed sensitivity at lower DOX concentrations, suggesting that the XPD helicase activity is involved in the repair of DOX-induced lesions. (C) 2009 Elsevier B.V. All rights reserved.

Thermal and spectrophotometric studies of new crosslinking method for collagen matrix with glutaraldehyde acetals

Despite the many existing crosslinking procedures, glutaraldehyde (GA) is still the method of choice used in the manufacture of bioprosthesis. The major problems with GA are: (a) uncontrolled reactivity due to the chemical complexity or GA solutions; (b) toxicity due to the release of GA from polymeric crosslinks; and (c) tissue impermeabilization due to polymeric and heterogeneous crosslinks formation, partially responsible for the undesirable calcification of the bioprosthesis. A new method of crosslinking glutaraldehyde acetals has been developed with GA in acid ethanolic solution, and after the distribution inside de matrix, GA is released to crosslinking. Concentrations of hydrochloride acid in ethanolic solutions between 0.1 and 0.001 mol/L with GA concentration between 0.1 and 1.0% were measured in an ultraviolet spectrophotometer to verify the presence of free aldehyde groups and polymeric compounds of GA. After these measurements, the solutions were used to crosslink bovine pericardium. The spectrophotometric results showed that GA was better protected in acetal forms for acid ethanolic solution with HCl at 0.003 mol/L and GA 1.0%(v/v). The shrinkage temperature results of bovine pericardium crosslinked with acetal solutions showed values near 85 C after the exposure to triethylamine vapors.

Effects of MWNT nanofillers on structures and properties of PVA electrospun nanofibres

In this study, we have electrospun poly(vinyl alcohol)(PVA) nanofibres and PVA composite nanofibres containing multi-wall carbon nanotubes (MWNTs) (4.5 wt%), and examined the effect of the carbon nanotubes and the PVA morphology change induced by post-spinning treatments on the tensile properties, surface hydrophilicity and thermal stability of the nanofibres. Through differential scanning calorimetry (DSC) and wide-angle x-ray diffraction (WAXD) characterizations, we have observed that the presence of the carbon nanotubes nucleated crystallization of PVA in the MWNTs/PVA composite nanofibres, and hence considerably improved the fibre tensile strength. Also, the presence of carbon nanotubes in PVA reduced the fibre diameter and the surface hydrophilicity of the nanofibre mat. The MWNTs/PVA composite nanofibres and the neat PVA nanofibres responded differently to post-spinning treatments, such as soaking in methanol and crosslinking with glutaric dialdehyde, with the purpose of increasing PVA crystallinity and establishing a crosslinked PVA network, respectively. The presence of carbon nanotubes reduced the PVA crystallization rate during the methanol treatment, but prevented the decrease of crystallinity induced by the crosslinking reaction. In comparison with the crosslinking reaction, the methanol treatment resulted in better improvement in the fibre tensile strength and less reduction in the tensile strain. In addition, the presence of carbon nanotubes reduced the onset decomposition temperature of the composite nanofibres, but stabilized the thermal degradation for the post-spinning treated nanofibres. The MWNTs/PVA composite nanofibres treated by both methanol and crosslinking reaction gave the largest improvement in the fibre tensile strength, water contact angle and thermal stability.

Toughened electrospun nanofibers from crosslinked elastomer-thermoplastic blends

A crosslink-able elastomeric polyester urethane (PEU) was blended with a thermoplastic, polyacrylonitrile (PAN), and electrospun into nanofibres. The effects of the PEU/PAN ratio and the crosslinking reaction on the morphology and the tensile properties of the as-spun fibre mats were investigated. With the same overall polymer concentration (9 wt %), the nanofibre containing higher composition of PEU shows a slight decrease in the average fibre diameter, but the tensile strength, the elongation at break and tensile modulus of the nanofibre mats are all improved. These tensile properties are further enhanced by slight crosslinking of the PEU component within the nanofibres.

Cytotoxicity of Diepoxybutane and Epichlorohydrin in Relation to Stages of the Cell Cycle

Work conducted in the Millard Biochemistry Research Laboratory examines the dual nature of molecules as carcinogens and anti-tumor agents through the molecular mechanisms of duplex DNA damage by bifunctional alkylating agents. Diepoxybutane (DEB) and epichlorohydrin (ECH) are polar molecules that form covalent DNA interstrand lesions by cross-linking the N7 position of deoxyguanosine residues. A recent experiment indicated that ECH preferentially targets nuclear DNA over mitochondrial DNA, whereas DEB shows similar rates of lesion formation for both loci. It was concluded that preferential targeting of nuclear DNA results from relatively poor uptake of ECH across the mitochondrial membrane. The objective of my honors research was to determine if the cytotoxicities of DEB and ECH vary according to the presence of the nuclear envelope in 6C2 chicken erythro-progenitor cells. The cytotoxicity of DEB and ECH was compared between cells randomly distributed throughout the cell cycle (Go/G, and S » G2/M) and cells enriched in G2/M stages. Results indicated that ECH is more cytotoxic than DEB in both unsynchronized control 6C2 cells and synchronized 6C2 cells enriched in G2/M stages of the cell cycle. Treatment with either bifunctional alkylating agent induced greater cytotoxicity in 6C2 cells enriched in G2/M stages than in unsynchronized control 6C2 cells, suggesting that the presence of the nuclear envelope-or any plasma membrane-may inhibit the reactivity of DEB and ECH.

Epichlorohydrin Cross-Linking of Synthetic DNA Oligomers

Epichlorohydrin (ECH), an important chemical in the synthetic polymer industry, is a bifunctional alkylating agent with the potential to form DNA interstrand crosslinks. Occupational exposure to this suspect carcinogen leads to chromosomal aberrations, and ECH has been shown to undergo reaction with DNA in vivo and in vitro. We are using denaturing polyacrylamide gel electrophoresis to assess cross-linking of synthetic DNA oligomers by both ECH and the related compound, epibromohydrin (EBH). Both epihalohydrins produce a low-mobility band on denaturing gels consistent with an interstrand cross-link. Moreover, the efficiencies, sequence preferences, reaction kinetics, and pH dependence differ for the two compounds, suggesting different mechanisms of reaction. Understanding these alkylation reactions may help explain the role of the epihalohydrins in cancer development.

Quantitative Analysis of Diepoxybutane Damage within the Chicken β-globin Domain

In industrial polymer and synthetic rubber production facilities, workers are exposed to 1,3-butadiene. This compound is converted in vivo to 1,2,3,4-diepoxybutane (DEB) and has been linked to increased incidences of cancer in these individuals. Carcinogenesis has been attributed to formation of DEB induced DNA interstrand cross-links. Previous studies have demonstrated that DEB cross-links deoxyguanosine residues within 5'-GNC sequences in synthetic DNA, in restriction fragments, and in defined sequence nucleosomes. The current study utilized the polymerase chain reaction (PCR) to examine DEB damage frequencies within nuclear genes, found within "open" regions of chromatin, as compared to regions of unexpressed sequence that reside in tightly packed, "closed" chromatin, to more closely model DEB reactivity in vivo. These initial studies have been performed in chicken liver homogenates. Preliminarily, we have found a dose-dependent DEB lesion-forming response within "open" chromatin. DEB appears to have little-to-no effect upon regions of "closed" chromatin.

Mapping of Diepoxybutane Damage in the Cytochrome b Domain of Mitochondrial DNA and the β-globin Domain of Nuclear Chicken DNA Using Quantitative PCR

Diepoxybutane (DEB), a known industrial carcinogen, reacts with DNA primarily at the N7 position of deoxyguanosine residues and creates interstrand cross-links at the sequence 5'-GNC. Since N7-N7 cross-links cause DNA to fragment upon heating, quantative polymerase chain reaction (QPCR) is being used in this experiment to measure the amount of DEB damage (lesion frequency) with three different targets-mitochondrial (unpackaged), open chromatin region, and closed chromatin region. Initial measurements of DEB damage within these three targets were not consistent because the template DNA was not the limiting reagent in the PCR. Follow-up PCR trials using a limiting amount of DNA are still in progress although initial experimentation looks promising. Sequencing of these three targets to confirm the primer targets has only been successfully performed for the closed chromatin target and does not match the sequence from NIH used to design that primer pair. Further sequencing trials need to be conducted on all three targets to assure that a mitochondrial, open chromatin, and closed chromatin region are actually being amplified in this experimental series.

Replacement of RNA hairpins by in vitro selected tetranucleotides

An in vitro selection method based on the autolytic cleavage of yeast tRNAPhe by Pb2+ was applied to obtain tRNA derivatives with the anticodon hairpin replaced by four single-stranded nucleotides. Based on the rates of the site-specific cleavage by Pb2+ and the presence of a specific UV-induced crosslink, certain tetranucieotide sequences allow proper folding of the rest of the tRNA molecule, wheras others do not. One such successful tetramer sequence was also used to replace the acceptor stem of yeast tRNAPhe and the anticodon hairpin of E.coli tRNAPhe without disrupting folding. These experiments suggest that certain tetramers may be able to replace structurally non essential hairpins in any RNA.