The presented work deals with the linking of software product EPLAN Electric P8 with production. The work briefly introduces the problems of computer-aided in manufacturing and control of software EPLAN. The work analyzed the equipment in production and created a solution for linking with output data of the program.

Microparticles produced from kafirin, the sorghum grain prolamin protein, by molecular selfassembly using coacervation with acetic acid solvent are vacuolated. They have shown considerable potential for encapsulation of antioxidants and for preparation of high quality free-standing bioplastic films. However, the functional quality of these kafirin microstructures needs to be improved to exploit their potential application, particularly as biomaterials. Wet heat, transglutaminase and glutaraldehyde treatments were used to modify the physical structure and chemical properties of the kafirin microstructures. Heat treatment (50–96°C) increased microparticle average size by up to four-fold to ≈20 μm, probably due to disulphide cross-linking of kafirin proteins. The vacuoles within these microparticles enlarged up to >10-fold, probably due to greater expansion of air within the microparticles with higher temperature, as the vacuoles are probably footprints of air bubbles. As with heat treatment, glutaraldehyde (10–30%) treatment resulted in oval microparticles, up to about four-fold larger than the control, probably due to covalent glutaraldehyde-polypeptide linkage. Transglutaminase (0.1–0.6%) treatment had only slight effect on the size and shape of microparticles, probably because kafirin has very low lysine content, inhibiting transglutaminase-catalysed cross-linking through ε-(-glutamyl)-lysine bonding. Surface morphology using atomic force microscopy indicated that the microparticles apparently comprised coalesced nanostructures. With heat and transglutaminase treatments, the microparticles seemed to be composed of round nanostructures that coalesced into random irregular shapes, indicative of non-linear protein aggregation. In contrast, with glutaraldehyde treatment, the nanostructures were spindle-shaped and had a unidirectional orientation, probably due to linear alignment of the nanostructures controlled by glutaraldehyde-polypeptide linkage. Thin (<50 μm) films prepared from kafirin microparticles and conventional cast kafirin films were compared in terms of their water stability and other related properties. Films cast from microparticles were more water-stable compared to conventional kafirin films, probably because the large vacuoles within the kafirin microparticles may have enhanced protein solubility in the casting solution, thereby improving the film matrix cohesion. The films prepared from microparticles treated with glutaraldehyde were more water-stable compared to the control, despite the loss of plasticizer, probably due to formation of the covalent glutaraldehyde-polypeptide linkages. The potential of modified kafirin microparticles to bind bone morphogenetic protein-2 (BMP- 2) was investigated. Compared to a collagen standard, the BMP-2 binding capacity of control, heat-treated, transglutaminase-treated and glutaraldehyde-treated kafirin microparticles were 7%, 18%, 34% and 22% higher, respectively, probably mainly due to the vacuoles within the microparticles creating greater binding surface area. The safety, biodegradability and effectiveness of kafirin microparticle film and kafirin microparticle film-BMP-2 system in inducing bone growth were determined by a subcutaneous bioassay using a rat model. Kafirin microparticle film and kafirin microparticle film-BMP-2 system was non-irritant to the animals, probably because kafirin is non-allergenic. The kafirin microparticle film implants showed signs of some degradation but a large proportion of these implants was still intact by Day 28 post implantation, probably because of the low susceptibility of kafirin to mammalian proteolytic enzymes. Kafirin microparticle film-BMP-2 system did not induce bone growth, probably mainly due to low BMP-2 dosage and short study duration. Modification of kafirin microparticles by wet heat or glutaraldehyde treatment both result in increased size of the microparticles with similar gross structure. However, it is apparent that with both treatments the proteins within the pre-formed kafirin microparticles undergo some form of further assisted-assembly through different mechanisms. It seems that heat-induced disulphide cross-linking reinforces a layer around the nanostructures, probably rich in γ- kafirin polypeptides, that stabilizes the structure of the nanostructures. In contrast, glutaraldehyde-treatment appears to destabilize this structure-stabilizing layer through formation of γ-kafirin polypeptide-glutaraldehyde covalent bonding. This probably offsets the balance of attractive and repulsive forces between the different kafirin subclasses within the nanostructures, thereby resulting in collapsed nanostructures and linear realignment. A deeper understanding of the mechanism of kafirin self-assembly will be important for further development of kafirin microstructures for different applications. / Thesis (PhD)--University of Pretoria, 2012. / Food Science / unrestricted

NK cells are a component of innate immunity system, which is derived from lymphoid progenitor. By a sophisticated receptor repertoire, which is expressed on their surface, they provide a surveillance against pathogenic, virus infected or tumour cells. Simultaneously they produce cytokines, thereby are involved in adaptive immune response. This work is focused on the study of structure of mice soluble mNkr-p1a isoform. Recently this short isoform was identified at the transcriptional level by a member of our laboratory and it is designated as isoform 2. The aim was to produce mNkr-p1a iso2 protein in the prokaryotic expression system and to perform its renaturation and purification in vitro. In the next phase of work, the obtained product was analyzed by the mass spectrometry methods. Recieved results made us think about that our protein is in unfolded state. This assumption was refuted by following biophysical methods, nuclear magnetic resonance, circular dichroism and dynamic light scattering measurement. Keywords: NK cells Receptor mNkr - p1a Short isoform mNkr - p1a iso2 Alternative splicing Protein biosynthesis Recombinant protein production Protein purification Mass spectrometry Disulfide bond Chemical cross-linking NMR, CD, DLS 5

Apoptosis signal-regulating kinase 1 (ASK1) is an apical kinase of the mitogen-activated protein kinase cascade. Its activity is triggered by various stress stimuli such as reactive oxygen species (ROS), cytokines, endoplasmic reticulum (ER) stress or osmotic stress resulting in the activation of p38 and c-Jun N-terminal kinase metabolic pathways and leading to inflammation or cell death. Dysregulation of ASK1 is linked to several pathologies such as neurodegenerative and cardiovascular diseases and cancer, which makes this protein a potential target of therapeutic intervention. The activity of ASK1 is regulated through protein-protein interactions with 14-3-3 proteins and thioredoxin1 being among the most important negative regulators and tumour necrosis factor receptor-associated factors being an example of positive regulators. Apart from that, ASK1 is also tightly regulated via oligomerization. Despite continual progress being made, the precise molecular mechanism of ASK1 regulation and the role of ASK1 oligomerization in this process still remains unclear to this day owing to the lack of structural data. Interaction of the N-terminal parts of two protomers of ASK1 dimer is one of the key steps in ASK1 activation. It was shown, that the isolated ASK1 catalytic domain (ASK1-CD) forms stable...

This dissertation is focused on developing cross-linking and mass spectrometry methodologies to study protein-protein interactions. Top-down cross-linking, in combination with mass spectrometry, provides advantages over bottom-up approaches, such as retaining posttranslational modification. Intermolecular cross-linking studies focus on defining protein complex topology and protein-protein interactions. We first developed the top-down MS approach to analyze intermolecular cross-linking in human hemoglobin. Both α-α and β-β intermolecular cross-linking were found and the cross-linking sites on the protein were identified, obtaining distance constraints between subunits of the human hemoglobin protein complex. This methodology would be a promising approach to characterize protein complexes and protein-protein interactions with high throughput and automation. This dissertation also focuses on development of cross-linking mass spectrometry to study synphilin-1 interactors and aggresome formation. Synphilin-1 is a protein that promotes the formation of protein aggregates and aggresome formation upon proteasome inhibition, and is implicated in Parkinson disease. Synphilin-1 contains several protein binding motifs. The biological functions of synphilin-1 and its role in aggresome formation and the pathogenesis of Parkinson disease remain to be elucidated. We utilized tandem affinity purification and label-free mass spectrometry to explore the patterns of cellular proteins associated with synphilin-1. We identified 57 synphilin-1 interacting proteins, and functional enrichment and pathway analysis showed that many of the associated proteins are involved in chromatin modulation, RNA and protein metabolism. Furthermore, we developed a proteomic strategy to identify synphilin-1 binary interacting partners as well as interacting domains using affinity purification followed by isotopically tagged cross-linking in combination with mass spectrometry. We found 24 newly discovered proteins that directly bind to synphilin-1. The proteins were mainly involved in RNA metabolism. The coiled-coil domain (CC), ankyrin-like repeat domain 2 (ANK2), and the protein aggregate promoting domain, appeared to the main regions that bound proteins. The functions of synphilin-1 interacting proteins, such as CK2, in aggresome formation were studied. The results show that CK2 is an important regulator of aggresome formation, but not through its kinase activities. Involvement of synphilin-1 in autophagy was also investigated. Knockdown of synphilin-1 shows that synphilin-1 impacts autophagy.

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El bajo rendimiento de las selecciones de la FDPN y la falta de infraestructura deportiva adecuada, vinculado con el mal diseño urbano ha suscitado que existe una necesidad en potenciar estos puntos y un nuevo lugar que funcione como hito tanto urbano como deportivo. Dentro de este fenómeno, el IPD, la FDPN y Municipalidad del Callao se han visto afectadas directamente debido al déficit de espacios tanto públicos como deportivos y vinculados entre sí. Sumando este problema a la necesidad general de espacio público de la ciudad, se decidió plantear crear un centro deportivo que funcionará como un equipamiento para el distrito y además abastecerá con crear deportistas adecuados para competencias y resultados. El proyecto enfatiza la idea del vínculo e integración. La idea es generar una atmosfera de confraternización, inspirando y concientizando a los deportistas de todos los niveles a través de una comunicación mutua, vinculándose unos con otros. Esto se logra a través de una red de espacios vinculadores, donde se fomenta la congregación y encuentro de deportistas experimentados y los amateurs experimentas sensaciones producidas por la tectónica y contacto con los elementos naturales. Además, el proyecto trabaja una innovación con la materialidad. Se usa en gran parte madera laminada como coberturas. La utilización de la madera laminada es algo nuevo en este tipo de proyectos para el país. La idea nace de tener una cobertura con la cual sea menos impactante en el medio ambiente, así como una más resistente en caso de sismo y al fuego. / The poor performance of the FDPN teams and the lack of adequate sports infrastructure, linked to poor urban design has raised the need to strengthen these points and a new place that functions as both urban and sports landmarks. Within this phenomenon, the IPD, the FDPN and the Municipality of Callao have been directly affected due to the deficit of both public and sports and interlinked spaces. Adding this problem to the general need for public space in the city, it was decided to create a sports center that will function as an equipment for the district and provide adequate sports for competitions and results. The project emphasizes the idea of the link and integration. The idea is to generate an atmosphere of fraternization, inspiring and raising awareness among athletes of all levels through mutual communication, linking with each other. This is achieved through a network of binding spaces, where the congregation and meeting of experienced athletes is encouraged, and amateurs experience sensations produced by tectonics and contact with natural elements. In addition, the project works an innovation with materiality. Laminated wood is largely used as toppings. The use of laminated wood is something new in this type of projects for the country. The idea stems from having a coverage with which it is less impactful on the environment as well as a more resistant in case of earthquake and fire. / Tesis

The molecular weight of the vasoactive intestinal peptide (VIP) receptor was assessed in bovine aorta, and rat liver, lung, and brain by covalent cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The receptor in all four tissues was found to be a single polypeptide of approximate Mr 54,000, contradicting previous claims for substantial heterogeneity in the molecular weight of this receptor. Guanine nucleotides inhibit cross-linking of 125I-VIP to its receptor, and cross-linking with ethylene glycolbis(succinimidylsuccinate) provides further evidence for complex formation between VIP, its receptor and a guanine nucleotide-binding regulatory protein (G-protein). The precise mechanism of receptor-G-protein coupling may differ between the aorta and other tissues.

In dieser Arbeit wird ein neuer Ansatz beschrieben, elektroaktive Verbindungen auf mesoporösen Elektroden zu fixieren. Dies wurde durch die Bildung eines sich selbst organisierenden Monolayers auf der Innenseite eines mesoporösen Trägers (ITO, FTO, ATO, TiO2) erreicht. Dieser Layer wurde dann vernetzt und in Richtung des Porenzentrums weiter aufgebaut durch Substitutions- Kondensations- oder Elektropolymerisations- Reaktionen. Es wurde ein Vernetzungsverfahren entwickelt, welches die Herstellung stabiler elektrochromer Bilder mit verbessertem Kontrast und einer Haltbarkeit von mehr als 18 Monaten erlaubt. Es beinhaltet die Synthesen von molekularen Einheiten mit latent vorhandenen oder voll entwickelten elektrochromen Eigenschaften. Diese Einheiten waren 4,4´- Bipyridine, die entweder mit optionalen N-Alkyl, N-Benzyl oder N-Phenyl Gruppen mit nukleophilem oder elektrophilen Eigenschaften oder mit TiO2 Ankergruppen versehen waren. Die Kaskadenreaktion ergab Elektroden mit unterschiedlichen Oberflächenkonzentrationen und unterschiedlichen Pimerisationsgraden. Darüber hinaus gelang es, die Haltbarkeit und den Kontrast so weit zu steigern, dass sie kommerziellen Ansprüchen genügen. Die Optimierung der Gegenelektroden wurde durch ein ähnliches Verfahren unter Verwendung von Ferrocen- Derivaten erreicht. Die Ladungskapazitäten, die durch Multilayer Vernetzung auf ATO-Ferrocen Elektroden erhalten wurden, waren hervorragend mit Ausnahme der Tatsache dass ein schwach grüner Farbton, verursacht durch oxidiertes Ferrocen, vorhanden war. Schließlich wurde die Kaskadenreaktion auf B12 Derivate zur Herstellung von katalytisch aktiven TiO2 Elektroden angewandt. Die mit B12 modifizierten Elektroden zeigten verbesserte Stabilität, höhere turn over - Zahlen und größere turn over -Raten im Vergleich zu unvernetzten B12 modifizierten Elektroden.