Microbial nitrile hydratases are important industrial enzymes that catalyse the conversion of nitriles to the corresponding amides. A thermostable, cobalt-type Bacillus sp. RAPc8 microbial nitrile hydratase was cloned and expressed in E.coli. In this study the primary aim was to determine the molecular structure of Bacillus sp. RAPc8 microbial nitrile hydratase.

<p>The nitrilases are an important class of industrial enzymes that are found in all phyla. These enzymes are expressed widely in prokaryotes and eukaryotes. Nitrilases convert nitriles to corresponding acids and ammonia. They are used in industry as biocatalysts because of their specificity and enantioselectivity. These enzymes belong to the nitrilase superfamily in which members share a common &alpha / &beta / &beta / &alpha / structural fold and a unique cys, glu,lys catalytic triad with divergent N- and C-terminals.</p> <p>There are four atomic structures of distant homologues in the superfamily, namely 1ems, 1erz, 1f89 and 1j31. All structures have two-fold symmetry which conserves the &alpha / &beta / &beta / &alpha / -&alpha / &beta / &beta / &alpha / fold across the dimer interface known as the A surface. The construction of a 3D model based on the solved structures revealed the enzyme has two significant insertions in its sequence relative to the solved structures, which possibly correspond to the C surface. In addition there are intermolecular interactions in a region of a conserved helix, called the D surface. These surfaces contribute additional interactions responsible for spiral formation and are absent in the atomic resolution homologues.</p> <p>The recombinant enzyme from R.rhodochrous J1 was expressed in E. coli BL21 cells and eluted by gel filtration chromatography as an active 480 kDa oligomer and an inactive 80 kDa dimer in the absence of benzonitrile. This contradicts previous observations, which reported the native enzyme exists as an inactive dimer and elutes as a decamer in the presence benzonitrile. Reducing SDS-PAGE showed a subunit atomic mass of ~40 kDa. EM and image analysis revealed single particles of various shapes and sizes, including c-shaped particles, which could not form spirals due to steric hindrances in its C terminal.</p> <p>Chromatographic re-elution of an active fraction of 1-month old J1 nitrilase enabled us to identify an active form with a mass greater than 1.5 MDa. Reducing SDS-PAGE, N-terminal sequencing and mass spectroscopy showed the molecular weight was ~36.5 kDa as result of specific proteolysis in its C terminal. EM revealed the enzyme forms regular long fibres. Micrographs (109) were recorded on film using a JEOL 1200EXII operating at 120 kV at 50K magnification. Two independent 3D reconstructions were generated using the IHRSR algorithm executed in SPIDER. These converged to the same structure and the resolution using the FSC 0.5 criterion was 1.7 nm.</p> <p>The helix structure has a diameter of 13nm with ~5 dimers per turn in a pitch of 77.23 &Aring / . Homology modeling and subsequent fitting into the EM map has revealed the helix is built primarily from dimers, which interact via the C and D surfaces. The residues, which potentially interact across the D surface, have been identified and these confer stability to the helix. The conservation of the insertions and the possibility of salt bridge formation on the D surface suggest that spiral formation is common among microbial nitrilases. Furthermore, the presence of the C terminal domain in J1 nitrilase creates a steric hindrance that prevents spiral formation. When this is lost &ndash / either by specific proteolysis or autolysis - an active helix is formed.</p>

<p>The nitrilases are an important class of industrial enzymes that are found in all phyla. These enzymes are expressed widely in prokaryotes and eukaryotes. Nitrilases convert nitriles to corresponding acids and ammonia. They are used in industry as biocatalysts because of their specificity and enantioselectivity. These enzymes belong to the nitrilase superfamily in which members share a common &alpha / &beta / &beta / &alpha / structural fold and a unique cys, glu,lys catalytic triad with divergent N- and C-terminals.<br /> <br /> There are four atomic structures of distant homologues in the superfamily, namely 1ems, 1erz, 1f89 and 1j31. All structures have two-fold symmetry which conserves the &alpha / &beta / &beta / &alpha / -&alpha / &beta / &beta / &alpha / fold across the dimer interface known as the A surface. The construction of a 3D model based on the solved structures revealed the enzyme has two significant insertions in its sequence relative to the solved structures, which possibly correspond to the C surface. In addition there are intermolecular interactions in a region of a conserved helix, called the D surface. These surfaces contribute additional interactions responsible for spiral formation and are absent in the atomic resolution homologues.<br /> <br /> The recombinant enzyme from R.rhodochrous J1 was expressed in E. coli BL21 cells and eluted by gel filtration chromatography as an active 480 kDa oligomer and an inactive 80 kDa dimer in the absence of benzonitrile. This contradicts previous observations, which reported the native enzyme exists as an inactive dimer and elutes as a decamer in the presence benzonitrile. Reducing SDS-PAGE showed a subunit atomic mass of ~40 kDa. EM and image analysis revealed single particles of various shapes and sizes, including c-shaped particles, which could not form spirals due to steric hindrances in its C terminal.</p> <p>Chromatographic re-elution of an active fraction of 1-month old J1 nitrilase enabled us to identify an active form with a mass greater than 1.5 MDa. Reducing SDS-PAGE, N-terminal sequencing and mass spectroscopy showed the molecular weight was ~36.5 kDa as result of specific proteolysis in its C terminal. EM revealed the enzyme forms regular long fibres. Micrographs (109) were recorded on film using a JEOL 1200EXII operating at 120 kV at 50K magnification. Two independent 3D reconstructions were generated using the IHRSR algorithm executed in SPIDER. These converged to the same structure and the resolution using the FSC 0.5 criterion was 1.7 nm.<br /> <br /> The helix structure has a diameter of 13nm with ~5 dimers per turn in a pitch of 77.23 &Aring / . Homology modeling and subsequent fitting into the EM map has revealed the helix is built primarily from dimers, which interact via the C and D surfaces. The residues, which potentially interact across the D surface, have been identified and these confer stability to the helix. The conservation of the insertions and the possibility of salt bridge formation on the D surface suggest that spiral formation is common among microbial nitrilases. Furthermore, the presence of the C terminal domain in J1 nitrilase creates a steric hindrance that prevents spiral formation. When this is lost &ndash / either by specific proteolysis or autolysis - an active helix is formed.</p>

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, fulfilment of the requirements for the degree of Master of Science. Johannesburg 2017. / Pichia pastoris is an established platform for the production of industrial enzymes. This nonfermentative methylotrophic yeast has many attractive features for the production of heterologous protein both in the laboratory and in industry. The PichiaPinkTM multi-copy secreted expression system was selected for the heterologous production of the fluorinase from Streptomyces cattleya. Fluorinase enzymes are useful for the production of fluorinated compounds which are applied in the agrochemical and pharmaceutical industries. The gene was cloned into the pPinkα-HC vector and used to transform four host srains by electroporation. Protein production was induced with 0.5% methanol and expression and activity was analysed by SDS-PAGE and a HPLC activity assay. Construction of the pPinkαHC-fLA expression plasmid and transformation of the host strains proved succesful. The PichiaPinkTM integrants showed genetic instability as the expression cassette showed signs of gene excision, thereby reducing the gene copy number. The wild-type strain1 efficiently secreted the foreign protein into the culture media, but the α-MF secretion signal was not processed correctly and secretion failed for the three protease knockout strains. However, the enzyme in both the secreted and intracellular protein fraction showed activity. Secretion methods need to be optimised and intracellular expression should be explored. The fluorinase enzyme was successfully cloned and expressed in four PichiaPinkTM strains and a biologically active protein was produced. / XL2017

Background: Gay men have adopted the use of people-nearby applications (PNA) to connect with members of the LGBT+ community. PNA uses global positioning system (GPS) data to locate other users in the area and facilitates communication between users through online profiles and instant messaging services. Objectives: This thesis explored gay men’s process of using PNA to connect with other users. Methods: The thesis work was conducted in two phases. The first phase was a review of the existing literature with literature synthesised into major themes. The second phase was an original qualitative study that used group sessions within a qualitative descriptive method and used thematic analysis to explore experiences of PNA use. Findings: The reviewed articles (n = 40) evolved into four major themes: risk, stigma, sexuality, and community. The theme of risk was overrepresented in the literature and comprised research that reviewed the sexual health risks of using PNA to meet partners. The review themes aligned closely with the four themes that emerged from participants’ (n = 6) experiences that were revealed in the original qualitative study: community, hope, stigma, and doubt. The themes of hope and doubt were found to be driving forces in a cyclical pattern of use reported by the participants, wherein users will repeatedly experience cycling phases of app use and disuse. Conclusion: Phase one of this thesis work exposed a gap in the knowledge related to the process of gay men using PNA. Phase two began to fill that gap by exploring the process of using PNA and furthering academic knowledge of how gay men interact and experience PNA use. The knowledge created in this thesis may assist nurses by providing them with improved cultural understanding of gay men and facilitate open communication between nurses and gay clients.

The diffusion rate of a large range of solvents into carbon fibre reinforced PEEK (APC-2) has been measured to discover the effect of the physical characteristics of the solvent. Three dimensional graphs have been plotted which correlate four parameters (solvent size, shape, hydrogen bonding capacity and solubility parameter) to solvent uptake. In the composite the effects of: thickness, lay-up, background water content and strain level on solvent diffusion have been assessed. The effect of composite thickness can be predicted using the film thickness scaling law provided the diffusion is Fickian. The effect of background water content is small, tending to make the diffusion profile two-stage. The effect of lay-up has been shown to have a major Affect on diffusion rate, unidirectional lay-ups having a much slower diffusion rate. Several theories have been postulated to explain this behaviour. The effects of stress on diffusion rate can be predicted by free volume models, provided that the stress/strain is kept below a certain critical level. It has been shown that the damage caused by a solvent, provided the stress does not exceed a critical value, is dependent on the amount of solvent in the matrix. This is due to plasticisation effects. Attempts to model this behaviour using free volume models have proved successful. Stress has been shown to enhance environmental attack. With certain solvents, above a critical stress or strain, environmental stress cracking occurs, leading to a considerable reduction in mechanical properties. Photographic evidence shows that cracking is initiated at stress concentrators within the matrix. Crack propagation is entirely matrix related and independent of spherulite boundaries. Overall, APC-2 has been shown to possess excellent environmental resistance when used in aerospace applications.

by Nancy Wang. / Bibliography: leaves 126-144 / Thesis (M.Ph.)--Chinese University of Hong Kong, 1984

This project was focused on the synthesis of novel Polymers of Intrinsic Microporosity (PIMs) that are soluble in common low boiling point solvents so that self-standing films can be prepared for gas permeability measurements. The common building unit of these novel PIMs was triptycene and its derivatives. Modification of these triptycene compounds enabled the alteration of the polymeric backbone, so that we could tune the gas permeability properties. Modifications included the substitution of different functional groups (e.g. addition of methyl groups) and also the extension via benzoannulation of the triptycene structure. The synthesis of the PIMs was based around three different polymerisation techniques. The first one involved the formation of triptycene-based polyimides (PIs) using a triptycene based dianhydride, prepared in a multistep synthesis. Shorter and cheaper synthetic routes were attempted, but all to no avail. The resulting triptycene monomer was reacted with a variety of commercial and non-commercial bisanilines for the formation of several PIM-PIs, all exhibiting different performances. Robust self-standing films were obtained for two of these PIM polyimides. In addition to the formation of polyimides, the synthesis of Tröger’s Base (TB) polymers, also based on triptycene components, were achieved. This type of polymerisation involves the reaction between a “bisaniline” monomer and a source of “formaldehyde”, such as dimethoxymethane (DMM), in a strong acid media, typically trifluoroacetic acid (TFA). Modification of these triptycene-based bisanilines has led to the formation of TB-PIMs, all with distinctive gas permeation properties. TB-PIM copolymers (reaction between two different bisaniline monomers with DMM and TFA) were synthesised in an attempt to further tune the performance of the polymers. Finally, the preparation of polybenzodioxan polymers based around extended triptycene monomers (i.e. benzotriptycenes) was studied. By using a variety of substituted benzotriptycene biscatechol monomers and performing the polymerisation using tetrafluoroterephthalonitrile, in the presence of K2CO3, the synthesis of a series of substituted benzotriptycene polybenzodioxane polymers was successfully achieved and the polymers showed enhanced gas permeation properties.

<p>Streaming applications like multimedia and radar signal processing applications are becoming </p><p>increasingly compute-intensive. To overcome the computational demands new parallel </p><p>architectures are emerging. </p><p> </p><p>The programming tools provided with these architectures require low-level programming, which </p><p>creates a need for a common high-level architecture independent language that can exploit </p><p>parallelism efficiently. One such language is StreamIt, designed around the notions of streams </p><p>and stream transformers, which allows efficient mapping to a variety of architectures. </p><p> </p><p>The overall goal of this master’s thesis is to evaluate the StreamIt language from a </p><p>programmability and portability point of view. An MPD-application has been developed in </p><p>StreamIt, which is executed on the RAW simulator. Furthermore, a code generator is designed to </p><p>compile and execute the application on the XPP simulator. </p><p> </p><p>The conclusions drawn are that StreamIt is easy to learn, but hard to use because of its </p><p>programming paradigm as compared to conventional languages. StreamIt programming involves </p><p>thinking in terms of streams instead of globally accessed memory. The structure of StreamIt </p><p>makes it easy to reuse components and modify the application. The construction of the compiler </p><p>makes it possible to port StreamIt application to various architectures.</p>

<p>Transportation has expanded the scope of human mobility, increasing the distances we cover on a</p><p>regular basis. The large benefits of transportation have resulted in a huge recent increase in the</p><p>number of vehicles. This, however, implies an increased number of traffic accidents that cause</p><p>many fatalities and injuries every year. It also leads to problems like increased delay for</p><p>commuters, and negative effects on the environment. Not to mention, the money spent in the</p><p>wasted fuel, as well as the costs of fixing damaged equipment and property.</p><p>To help in mitigating these problems the vehicles and the road infrastructure should be equipped</p><p>with intelligent devices that allow them to communicate and collaborate with each other and</p><p>exchange safety information concerning accidents, road traffic conditions, and weather conditions</p><p>as well as non-safety information. Recently, this topic termed telematics has gathered</p><p>considerable interest constituting a lot of work and research all included under the title of</p><p>Intelligent Transportation System (ITS).</p><p>The thesis work defines general communication requirements of future telematics applications</p><p>and investigates various wireless carriers that are important to achieve communication inbetween</p><p>vehicles and between vehicles and nearby infrastructure. We analyse several future</p><p>applications related to the ITS field and describe their communication requirements. Based on the</p><p>communication requirements the applications are grouped into different profiles in order to</p><p>determine the most suitable carrier for each profile.</p><p>Disclaimer: This paper reflects only the authors’ views and the European Community (as cofounders</p><p>of the CVIS, SAFESPOT and PReVENT Sixth Framework Programme projects,</p><p>through the European Commission DG Information Society and Media) is not liable for any use</p><p>that may be made of the information contained within.</p>