The spliceosomal U5 snRNP contains several proteins with well characterised functions in splicing, including: Brr2, an ATPase/RNA helicase that disrupts U4/U6 and U2/U6 snRNA base pairing during activation of the spliceosome; Snu114, a GTPase that controls the action of Brr2; and Prp8, the largest and most conserved protein considered to have a central role in the spliceosome, which interacts directly with Snu114 and Brr2. Yeast Cwc21 is one of twelve Bact complex proteins that associate with spliceosomes shortly before the first step of splicing catalysis. Cwc21 interacts directly with Prp8 and Snu114, as does its human orthologue, the SR protein SRm300/SRRM2. Although, Cwc21 is not essential for yeast cell viability, it is required for sporulation. This work aims to identify the function of Cwc21 during meiosis. PP1 is a protein phosphatase required for both steps of splicing. Multiple sequence alignments of Snu114 and Prp8 revealed the presence of putative PP1 binding motifs that are well conserved among different species. This led me to hypothesize that PP1 may interact with Snu114 and/or Prp8 to regulate these or other interacting proteins. By screening intron-containing genes that are expressed in meiosis, I found that Cwc21 is required for splicing HRB1 transcripts. In addition, I show that HRB1 is also required during meiosis. The HRB1 intron contains an unusual branchsite sequence, TACTAATG, which when changed to the consensus branchsite sequence restores sporulation in the absence of Cwc21. Therefore, it is likely that Cwc21 promotes the expression of HRB1 during an early stage of meiosis by stabilising its pre-mRNA in the catalytic centre of the spliceosome. This study demonstrates a novel function for Cwc21 during meiosis. Using yeast two hybrid assay I have identified the interacting regions of Cwc21, PP1 and Brr2 in Snu114. Through biochemical studies I provide evidence for mutually exclusive interaction of Cwc21 and PP1 in the putative PP1 binding motif situated in Snu114 domain ‘IVa’. In the case of yeast Snu114, the PP1 binding motif has a novel sequence ‘YGVQYK’. I also show that the affinity of Cwc21 and PP1 for Snu114 is influenced by the different nucleotide-bound states of Snu114. Furthermore, I show that mutations in Snu114 domain ‘IVa’ restrict Snu114 function during meiosis and affect the MER1 splicing regulatory network. Therefore, Snu114 may play a role in modulating the conformational state of the catalytic spliceosome through its interactions with Cwc21/PP1 in regulating subsets of genes during meiosis. Finally, I show that PP1 is a putative regulator of Prp8.

Octa-heme cytochrome c tetrathionate reductase (OTR) from Shewanella oneidensis MR-1 is a periplasmic protein and shows several extraordinary structural features around its active-site heme. OTR has been found able to catalyse the in vitro reduction of tetrathionate, nitrite, hydroxylamine and hydrogen peroxide. However the physiological function of this novel protein remains unknown. The subject of this thesis is the in vitro catalytic mechanism and the in vivo function of OTR. As OTR displays great similarity with bacterial penta-heme cytochrome c nitrite reductase (NrfA) in several aspects, it has been proposed that OTR might be physiologically involved in the metabolism of nitrite or other nitrogenous compounds. However kinetics assays and phenotypes studies carried out in this project suggest this is not the case. In vitro kinetic assays of the reduction of nitrite and hydroxylamine catalysed by OTR showed no significant difference in enzyme activities among the wild-type OTR and its mutant forms which have one active site residue replaced by alanine, namely OTR K153A, C64A, N61A and D150A. And the nitrite reductase activity of OTR (kcat/Km = 1.0×105 M-1•s-1) are much lower than that of NrfA (kcat/Km = ~108 M-1•s-1). These results indicate that OTR is not specifically adapted to reduce nitrite and it cannot compete for nitrite against NrfA in vivo. No phenotype difference was identified between the wild-type and the Δotr strain of Shewanella oneidensis MR-1 when nitrite or nitrate served as the sole electron acceptor. OTR appears not to be involved in the respiration or detoxification of nitrite, which is consistent with previous transcriptional and phenotype reports that involve OTR or its homologues. The in vitro tetrathionate reduction activity of OTR was unable to be reproduced in this project for unknown reasons. Although transcriptomic data from the literature suggest that OTR may be related to the metabolism of sulphur-containing compounds, kinetic and phenotype studies reveal that OTR does not directly participate in the respiration of thiosulfate, sulfite, tetrathionate, polysulfide or elemental sulphur. Cysteine 64 is a highly-conserved amino acid residue of OTR close to the active site and its side-chain sulphur atom is covalently bonded by either an oxygen or a sulphur atom as observed in the crystal structure. Such a modification is potentially important to the function of OTR. ESI mass spectroscopy results show that in native OTR the modified form is around 48 Da heavier than the unmodified form, and the MALDITOF peptide mass spectra show that the modified form could be converted into the unmodified form by reducing agent DTT. These results suggest that the modification could be a cysteine persulfide attaching an extra oxygen atom in the form of water or hydroxide anion.

The RNA World hypothesis proposes a period of time during the origins of life in which RNA molecules were the only source of both genotypes and phenotypes. Although a vast amount of evidence has been obtained in support of this hypothesis, a few critical demonstrations are lacking. A most crucial one is a demonstration of self-replication of RNA molecule from prebiotic soup. Previously in the Lehman laboratory, it has been demonstrated that a 198-nucleotide molecule derived from the Azoarcus group I intron can self-assemble from up to four fragments of RNA via recombination. Furthermore, the covalent full-length molecules are catalytically active and can make copies of themselves from the remaining pieces in the solution leading to their autocatalytic growth. I was able to demonstrate how this recombination system can overcome different obstacles and evolve to be an efficient replicating system. I discovered the ability of a single RNA fragment to be multifunctional in a single reaction pathway during RNA recombination events that avoids the necessity of multiple genotypes. I also confirmed the capacity of self-replicating ribozymes to form cooperative catalytic cycles and networks that would potentially prevent informational decay. Finally, I have discovered a recycling phenomenon in the RNA recombination system that exploits dynamic covalent chemistry. Recycling provides the earliest replicating system with adequate concentrations of reagents and ability to explore sequence space. Together these findings have improved our understanding of RNA recombination and bolstered the plausibility of the RNA World.

Dynamic Kinetic Selection (DKS) suggests that kinetic, rather than thermodynamic, stability will dictate the composition of a replicating population of biomolecules. Here, the results obtained from a series of five related reactions involving gradually increasing percentages of randomly-mutated substrate fragments to generate variants of full-length Azoarcus group I intron through an autocatalytic self-assembly reaction involving a series of recombination events, showed DKS as a driving factor in dictating the population composition of full-length product assembled from substrates that had fewer positions available to randomization. In trying to elucidate a plausible scheme for the origins of complex biomolecules on the prebiotic Earth, the suggestion that networks comprised of interacting molecules were more likely to evolve into biomolecules capable of obtaining and sustaining characteristics attributed to living molecules has gained traction within the past few years. Of specific interest is the catalytic efficacy of ribozymes whose genotypes require that they interact with molecules of the same genotype (selfish systems) to be effective catalysts versus those that are more effective when accomplishing catalysis by cooperating with ribozymes of a different genotype (cooperative systems). Here, the Azoarcus I ribozyme was used to compare these two types of system. Both systems were shown to robustly produce full-length product. Two different methods of introducing random mutations into substrate fragments for the reactions described in this thesis were employed. The differences in the preparation methods for the substrates was not expected to have an impact on the nature of the full-length product. However, there was no correlation between the positions that tended to be more tolerant of accepting random mutations between the products arising from the two preparation methods. One preparation method yielded full-length ribozymes more consistent with the secondary structure of the wild-type ribozyme and followed substitution patterns found in in vivo nucleic acid substitutions, whereas the other method provided full-length ribozymes that tolerated mutations that would be expected to greatly affect the secondary structure of the ribozyme and those positions tended to mutate evenly to any of the three possible alternative nucleobases. Point mutations introduced into ribozyme substrate fragments may have a deleterious, neutral, or beneficial effect, depending on their impact on the catalytic capability of the molecule vis-á-vis the effect, if any, the change has to the secondary and tertiary structure of the ribozyme. In this dissertation, the results of two series of point mutation reactions are addressed. The first set showed a point mutation to have a deleterious effect, whereas concerted mutations did not significantly affect activity of the ribozyme. The second series of reactions involved point mutations at a position that had previously been determined to be highly tolerant of random mutations. Results suggested that substitutions at this position had a minimal impact on ribozyme activity.

Reports indicate that the worldwide energy consumption and fossil fuel energy production level will have an opposite trend in the coming two decades. The former will continue to increase while the later will decrease. Therefore, additional sources of energy need to be developed. Field pennycress (Thlaspi, arvense L.) has been found to be an ideal source of energy because it has prolific yield and has no value as food. We demonstrated conventional and catalytic fast pyrolysis of whole pennycress biomass in a fluidized bed reactor. Characterization studies on field pennycress showed that the biomass had a potential to be converted to energy-rich bio-fuel. Thermogravimetric and kinetic study on field pennycress provided vital information on the degradation behavior of the feedstock. A parametric study was conducted on conventional rapid pyrolysis by using the effects model. The optimum experimental condition that gave maximum liquid yield was found to be at a temperature of 500 °C and a gas flow rate of 24 l/min. The catalysts used for catalytic fast pyrolysis were HZSM-5, a commercial catalyst, and red mud, an alumina industry waste material. The liquid products obtained from pennycress were found to have better qualities compared to a typical lignocellulosic feedstocks pyrolysis bio-oil. The bio-oil from the red mud catalyzed experiment had almost neutral pH of 6.5 and the pH in the case of HZSM-5 was 5.7. In comparison to bio-oil from conventional rapid pyrolysis, HZSM-5 and red mud reduced the viscosity of the bio-oil by 3 and 5 times, respectively. However, red mud was only found to be effective in improving the higher heating value (HHV) of the bio-oil from 33.18 MJ/kg (dry basis) in conventional pyrolysis to 35.7 MJ/Kg (dry basis). The HHV of HZSM-5 catalyzed bio-oil was 33.63 MJ/kg. The composition of non-condensable gases and the chemical makeup of the bio-oil from the two catalysts were different, suggesting that the reaction pathways could be different. HZSM-5 had higher selectivity for aromatics whereas red mud produced longer aliphatic chains. The bio-oil obtained from red mud catalytic pyrolysis of field pennycress is a promising alternative energy source that could replace petroleum fuels after some upgrading.

Fluidization characteristics and co-gasification of pulverized sub-bituminous coal, hybrid poplar wood, corn stover, switchgrass, and their mixtures were investigated. Co-gasification studies were performed over temperature range from 700°C to 900°C in different media (N2, CO2, steam) using a bubbling fluidized bed reactor. In fluidization experiments, pressure drop (ΔP) observed for coal-biomass mixtures was higher than those of single coal and biomass bed materials in the complete fluidization regime. There was no systematic trend observed for minimum fluidization velocity (Umf) with increasing biomass content. However, porosity at minimum fluidization (εmf) increased with increasing biomass content. Channeling effects were observed in biomass bed materials and coal bed with 40 wt.% and 50 wt.% biomass content at low gas flowrates. The effect of coal pressure overshoot reduced with increasing biomass content. Co-gasification of coal and corn stover mixtures showed minor interactions. Synergetic effects were observed with 10 wt.% corn stover. Coal mixed with corn stover formed agglomerates during co-gasification experiments and the effect was severe with increase in corn stover content and at 900°C. Syngas (H2 + CO) concentrations obtained using CO2 as cogasification medium were higher (~78 vol.% at 700°C, ~87 vol.% at 800°C, ~93 vol.% at 900°C) than those obtained with N2 medium (~60 vol.% at 700°C, ~65 vol.% at 800°C, ~75 vol.% at 900°C). Experiments involving co-gasification of coal with poplar showed no synergetic effects. Experimental yields were identical to predicted yield. However, synergetic effects were observed on H2 production when steam was used as the co-gasification medium. Additionally, the presence of steam increased H2/CO ratio up to 2.5 with 10 wt.% hybrid poplar content. Overall, char and tar yields decreased with increasing temperature and increasing biomass content, which led to increase in product gas.

"Catalytic promiscuous" enzymes, which possess additional activities besides their "native" activity, albeit with a lower efficiency than the main reaction, have become a new frontier for biochemistry and have received considerable attention. Catalytic promiscuity has been suggested to contribute to enzyme evolution through the mechanism of gene duplication followed by specialization of one of the two copies for the new function. Mimicking this evolutionary shortcut could also provide a more efficient route to changing the function of proteins by directed evolution. The promiscuous phosphatase PP1 is a member of the phosphoprotein phosphatase (PPP) gene family, which is critical for the control of many cellular pathways by antagonizing the effects of protein phosphorylation mediated by kinases. The catalytic promiscuity of PP1&gamma WT and two mutants has been investigated with a set of monoanionic and dianioic phosphester substrates. PP1&gamma is an effective catalyst for the hydrolysis of both monoanionic and dianionic phosphate-ester based substrates 1-5, with second-order rate accelerations that fall within the narrow range of 1011 to 1013. While the transition states of the uncatalyzed hydrolysis reactions of these substrates differ, those for the PP1&gamma-catalyzed reactions are similar. Thus, the enzyme catalyzes the hydrolysis of these substrates by transition states that are controlled by the active site environment more than by the intrinsic nature of the substrates. The reason for the inability of PP1&gamma to catalyze the hydrolysis of a sulfate ester is unclear, and unexpected, since the charge and transition state of this substrate are well within the range of those of the phosphorus-based substrates that are effectively catalyzed. Inhibition experiments suggest that the PP1&gamma active site is tolerant of variations in the geometry of bound ligands. This characteristic may permit the effective catalysis even of substrates whose steric requirements may result in perturbations to the positioning of the transferring group, both in the initial enzyme-substrate complex and in the transition state. The conservative mutation of arginine 221 to lysine results in a mutant that more effectively catalyzes monoanionic substrates than the native enzyme. The surprising result in substrate preference from a single, conservative mutation lends support to the notion that mutations following gene duplication can result in an altered enzyme with different catalytic capabilities and preferences, and may, following subsequent mutations, provide a pathway for the evolution of new enzymes.

Nanoscale patterning of organic molecules has received considerable attention in current nanoscience for a broad range of technological applications. In order to provide a viable approach, this thesis describes catalytic stamp lithography, a novel soft-lithographic process that can easily produce sub-100 nm patterns of organic monolayers on surfaces. Catalytic stamps were fabricated through a two-step procedure in which the nanoscale patterns of transition metal catalysts are first produced on SiOx/Si surfaces via the use of self-assembled block-copolymers, followed by the production of the poly(dimethylsiloxane) (PDMS) stamps on top of the as-patterned metals. Simply peeling off the as-formed PDMS stamps removes the metallic nanostructures, leading to the functional stamps. A number of different patterns with various metals were produced from a commercially available family of block copolymers, polystyrene-block-poly-2-vinylpyridine, by controlling the morphology of thin-film templates through the modulation of molecular weights of polymer blocks or solvent vapor annealing. Using these catalytic stamps, hydrosilylation-based catalytic stamp lithography was first demonstrated. When terminal alkenes, alkynes, or aldehydes were utilized as molecular inks, the metallic (Pt or Pd) nanopatterns on catalytic stamps were translated into corresponding molecular arrays on H-terminated Si(111) or Si(100) surfaces. Since localized catalytic hydrosilylations took place exclusively underneath the patterned metallic nanostructures, the pattern formations were not affected by ink diffusion and stamp deformation even at the sub-20 nm scale, while maintaining the advantages of the stamp-based patterning (i.e., large-area, high-throughput capabilities, and low-cost). The concept of catalytic stamp lithography was further extended with other catalytic reactions, and successful nanoscale patterning was performed using hydrogenation (on azide-terminated SiOx surfaces) and the Heck reaction (on alkene- or bromphenyl-terminated SiOx surfaces). A range of nanopatterned surfaces with different chemical functionalities, including thiol, amine, and acid, were created, and they were further modified through appropriate chemical reactions. The potential utility of this simple approach for the construction of a higher degree of nanoarchitectures was suggested.

Nanostrukturierte Materialien zeichnen sich dadurch aus, dass sie aus sehr kleinen Baueinheiten zusammengesetzt sind. Typischerweise liegt die Grössenordnung dieser Bausteine im Bereich von einigen Nanometern. Ein Nanometer entspricht 10-9 Meter. Dadurch bekommen nanostrukturierte Materialien oft verbesserte, vielfach sogar ganz neue Eigenschaften, die für viele heutige wie auch zukünftige Anwendungen von Vorteil sind.<br /> <br /> Ein Weg, um solche nanostrukturierte Materialien herzustellen, ist die sogenannte &bdquo;Templatierungsmethode&ldquo;. Das Templat besteht aus einem einzelnen Molekül, einer Ansammlung von Molekülen oder aus einem festen Objekt. Beim Aufbau des nanostrukturierten Materials wirkt das Templat als Schablone oder als Gussform und beeinflusst damit die Struktur des Endproduktes. Normalerweise besteht dieser Prozess aus mehreren Schritten. Zuerst wird der Raum um das Templat mit dem Ausgangsstoff umhüllt oder ausgefüllt, dann wird der Ausgangsstoff chemisch in das gewünschte Endprodukt umgewandelt, wobei das Templat die Endform kontrolliert und am Schluss wird das Templat entfernt. Das geschieht meistens durch Erhitzen. Als Ausgangsstoff können dabei einzelne Moleküle verwendet werden, die sich leicht in das Endprodukt umwandeln lassen, oder aber vorgeformte Partikelchen, die nur noch zur entsprechenden Form angeordnet werden müssen.<br /> <br /> In dieser Arbeit wurden poröse Metalloxid-Kügelchen hergestellt, die aus einem Gemisch aus Titanoxid und entweder Aluminium-, Gallium- oder Indiumoxid bestehen. Als Template wurden poröse Kunststoffkügelchen eingesetzt, die man sonst für Chromatographiezwecke braucht. Bei der Synthese wurden die Poren der Kunststoffkügelchen mit dem Ausgangsmaterial gefüllt und mit Wasser in ein amorphes Netzwerk umgewandelt. Danach werden die Kügelchen erhitzt, wobei das Kunststofftemplat zersetzt wird. Gleichzeitig wird das amorphe Gerüst in stabile, kristalline Wände umgewandelt, die die Form der Kügelchen auch dann noch behalten, wenn das Templat verschwunden ist. Mit einem ähnlichen Prozess wurden auch Kügelchen aus Cer-Zirkonoxid erhalten. Als Ausgangsstoff wurden dabei aber vorgeformte Cer-Zirkonoxid-Nanopartikel eingesetzt, die in die Poren der Kunststofftemplatkügelchen hinein diffundieren. Diese Cer-Zirkonoxid-Nanopartikel lassen sich auch für die Herstellung von porösen Pulvern verwenden, wobei dann nicht Polymerkügelchen, sondern hochgeordnete Ansammlungen von Block Copolymeren als Template verwendet werden.<br /> <br /> Form, Struktur und Eigenschaften all dieser Materialien wurden systematisch unter Anwendung verschiedenster Analysemethoden untersucht. Die auf Titanoxid-basierten Kügelchen wurden auch auf ihre photokatalytische Verwendung zum Abbau von umweltschädlichem 2-Chlorophenol untersucht. Die Cer-Zirkonoxid-Kügelchen wurden für die Herstellung von Wasserstoff aus Methanol getestet. Wasserstoff gilt als hoffungsvoller, sauberer Energieträger der Zukunft und kommt in Brennstoffzellen zum Einsatz. / Nanostructured materials are the materials having structural features on the scale of nanometers i.e. 10-9 m. the structural features can enhance the natural properties of the materials or induce additional properties, which are useful for day to technology as well as the future technologies<br /> <br /> One way to synthesize nanostructured materials is using templating techniques. The templating process involves use of a certain &ldquo;mould&rdquo; or &ldquo;scaffold&rdquo; to generate the structure. The mould is called as the template, can be a single molecule or assembly of molecule or a larger object, which has its own structure. The product material can be obtained by filling the space around the template with a &ldquo;precursor&rdquo;, transformation of precursor into the desired material and then removal of template to get product. The precursor can be any chemical moiety that can be easily transformed in to the desired material. Alternatively the desired material is processed into very tiny bricks or &ldquo;nano building blocks (NBB)&rdquo; and the product is obtained by arrangement of the NBB by using a scaffold. <br /> <br /> We synthesized porous metal oxide spheres of namely TiO2-M2O3: titanium dioxide- M-oxide (M = aluminum, gallium and indium) TiO2-M2O3 and cerium oxide-zirconium oxide solid solution. We used porous polymeric beads as templates. These beads used for chromatographic purposes. For the synthesis of TiO2-M2O3 we used metal- alkoxides as precursor. The pore of beads were filled with precursor and then reacted with water to give transformation of the precursor to amorphous oxide network. The network is crystallized and template is removed by heat treatment at high temperatures. In a similar way we obtained porous spheres of CexZr1-xO2. For this we synthesized nanoparticle of CexZr1-xO2 and used then for the templating process to obtain porous CexZr1-xO2 spheres. <br /> <br /> Additionally, using the same nanoparticles we synthesized nano-porous powder using self-assembly process between a block-copolymers scaffold and nanoparticles. <br /> <br /> Morphological and physico-chemical properties of these materials were studies systematically by using various analytical techniques <br /> <br /> TiO2-M2O3 material were tested for photocatalytic degradation of 2-Chlorophenol a poisonous pollutant. While CexZr1-xO2 spheres were tested for methanol steam reforming reaction to generate hydrogen, which is a fuel for future generation power sources like fuel cells. All the materials showed good catalytic performance.

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