An essential ingredient in the generation of environmentally compatible pulp bleaching chemicals is sodium chlorate. Chlorate is produced in electrochemical cells, where the electrodes are the key components. In Sweden the so-called DSA !R electrodes with catalytic coatings have been produced for more than 35 years. The production of chlorate uses a large amount of electric energy, and a decrease of just five percent of this consumption would, globally, decrease the consumption of electrical energy corresponding to half a nuclear power reactor. The aim of this project is to improve the electrode design on the nanoscale to decrease the energy consumption. The success of the DSA!R depends on the large catalytic area of the coating, however, little is known about the actual structure at the nanometer level. To increase the understanding of the nanostructure of these coatings, we used a number of methods, including atomic force microscopy, transmission electron microscopy, X-ray diffraction, porosimetry, and voltammetric charge. We found that the entire coating is built up of loosely packed rutile mono-crystalline 20 − 30 nm sized grains. The small grain sizes give a the large area, and consequently, lower cell-voltage and reduced energy consumption. A method to control the grain size would thus be a way to control the electrode efficiency. To alter the catalytically active area, we made changes in the coating process parameters. We found a dependency of the crystal-grain sizes on the choice of ruthenium precursor and processing temperature. The use of ruthenium nitrosyl nitrate resulted in smaller grains than ruthenium chloride and lowering the temperature tended to favour smaller grains. A more radical way would be to create a totally different type of electrode, manufactured in another way than using the 1965 DSA !R recipe. Such new types of electrodes based on, for example, nanowires or nanoimprint lithography, are discussed as future directions.

Alternative bone repair strategies are frequently sought after in orthopaedic surgery to address the growing need for improved morbidity and healing rates. This thesis sought to initiate and validate such an alternative, harnessing the flexible nature of a biomaterial substrate and the unique potential of glycosaminoglycan sugars. A novel, biodegradable biomaterial polymer, PHBV, has previously been identified to have the potential to mimic the characteristics of bone necessary for tissue repair and in this study, it was hypothesized that PHBV would be able to support bone formation. When tested in vitro, PHBV was found to support osteoblast cell attachment, proliferation and differentiation, despite its rougher, more hydrophobic surface characteristics compared to tissue culture plastic (TCP). However, unlike the progression of cells on TCP, PHBV caused a developmental delay at each stage of osteogenesis, suggesting a sub-optimal cell-substrate interaction. The expression profiles of genes involved in the maintenance of the extracellular matrix were monitored to investigate this phenomenon further. The results suggested that cells cultured on PHBV appeared to preference 7 against a collagen-based ECM and, instead, trigger an increase in the expression of other factors, such as osteopontin, presumably to modify the biomaterial microenvironment to optimise continued growth and differentiation. This finding led to the next hypothesis that functionalisation of PHBV with suitable compounds could optimise and enhance the osteogenic development at the implant site by facilitating the desired and appropriate cell-substrate interactions. Non-protein factors are often preferred for functionalisation to material scaffolds over proteins, as they are relatively robust and can survive many of the processes used in the manufacture of biomaterials. Glycosaminoglycan (GAG) sugars were appropriate candidates for this purpose, as they are not only abundantly expressed in bone, but more importantly, they are capable of binding and facilitating the activity of growth factors. Furthermore, they are resistant to several environmental influences including changes in pH, heat and desiccation. To identify a GAG that could be integrated with PHBV or any other biomaterial substrate, GAGs were extracted from phenotypically-distinct stages of MG-63 osteosarcoma cells. These GAGs were identified to display gross structural differences, as well as differences in the enzymes synthesising them, between immature and mature osteoblastic cells, with the increased production of a larger GAG species observed as the cells differentiated. Unexpectedly, however, when these GAGs were subsequently dosed back into the media of growing MG-63 cells, their bioactivity did not match the stage at which they had been harvested: all GAG species were able to influence cell survival and growth to varying degrees but were not capable of affecting cell differentiation. However, if these same GAGs were exposed to cells by first being attached to the growth substrate, they induced varying degrees of aggregation in human mesenchymal stem cells (hMSCs), with more mature GAGs producing the most profound effects. Interestingly, a similar phenomenon was not observed when MG-63 cells where cultured in a similar manner. A direct correlation between the GAGs expressed by osteoblasts and the specific cellular processes they functionally influence has yet to be identified. While the experiments presented here demonstrate an effect of GAGs in osteoblastic cell survival, a role for GAGs in the progression of bone formation was not revealed. Loss-of-function studies were therefore necessary to determine the role of GAGs in bone, but this was hampered by the limited availability of procedures that allow the alteration of GAGs and the subsequent detection of these effects. Therefore, a tool to screen the efficacy of a loss of GAG function was developed. TAT-EGFP, a purpose-designed fluorescent GAG-binding peptide, was able to confirm that treatment with sodium chlorate was an effective 8 strategy to hinder GAG expression in MG-63 cells with minimal cytotoxicity to the cells. Following more extensive studies with chlorate treatment, it was found that a recoverable disruption to both proliferation and mineralisation could be induced in MG-63 cells. This suggested a role for GAGs in osteogenesis. A series of experiments then carried out following gene expression microarray analysis indicated that GAG de-sulfation by chlorate gives rise to an S-phase block in the cell cycle and a disruption to the actin cytoskeleton, which appeared to be associated with a change in the activity of cell-surface proteoglycans, most likely syndecan 4. It was also found that cells up-regulated plasma membrane ALP activity and cholesterol synthesis, presumably in an attempt to recover from a chlorate-induced loss in GAG function. Cholesterol is known to be important in establishing connections between membrane elements and the actin cytoskeleton, and its up-regulation here may reflect dysfunctions in these units and a dysfunction in syndecan 4 activity. With further confirmation, this would suggest that syndecan 4 plays a pivotal role in maintaining osteogenesis, in at least MG-63 cells, and that sulfated GAGs function principally to facilitate this role. The effective use of GAGs in bone repair strategies will require further understanding of GAG/syndecan 4/osteogenesis relationship.

<p>Arbetet beskriver en analys av innehållet av membranbundna och periplasmiska c-cytokromer hos perkloratodlade GR-1 och jämförelser med c-cytokrominnehållet hos Ideonella dechloratans och andra kända c-cytokromer, samt med genomet för Dechloromonas aromatica. Den jämförande studien av c-cytokromer gjordes med syftet att undersöka en hypotes om att bakterierna använder olika vägar för elektronöverföring till det periplasmiska enzymet (per)kloratreduktas. Cellmembran från GR-1 renframställdes genom ultracentrifugering och periplasma preparerades fram med hjälp av osmotisk chock. Fraktionerna analyserades sedan med SDS-PAGE och peptider med kovalent bundet hem (c-cytokromer) detekterades med hjälp av en specifik färgreaktion. Även Touchdown PCR med degenererade primrar genomfördes på isolerat DNA från GR-1 i ett försök att finna en gen kodande för ett NapC/NirT-liknande protein. Slutligen kolmonoxidbubblades reducerade membran för att undersöka förekomsten av cbb3-typ oxidas.</p><p>Separation och infärgning av periplasmiska och membranbundna proteiner från GR-1 resulterade i sju respektive åtta peptidband med molekylvikter mellan 8-60 kDa. Inget som framkommit under arbetet talar emot hypotesen om att GR-1 och D. aromatica skulle använda ett NapC/NirT-liknande protein som elektronöverförare till det periplasmiska (per)kloratreduktaset. PCR-analysen resulterade i en produkt som troligtvis är en sekvens från en gen som kodar för ett NapC/NirT-liknande protein och två, eventuellt tre, kandidater för ett NapC/NirT-liknande protein hittades i membranet hos GR-1. Dessutom framkom att GR-1 troligtvis använder cbb3-typ oxidas som terminalt oxidas vid reduktion av syrgas under mikroaerofila förhållanden.</p><p>Vad gäller I. dechloratans och hypotesen om att denna bakterie använder ett lösligt cytokrom c för elektronöverföring till sitt kloratreduktas så har inget framkommit under arbetet som talar emot detta. Tre kandidater för lösliga cytokrom c-proteiner hittades. För teorin talar även att de försök som tidigare gjorts med att påvisa genen för ett NapC/NirT-liknande protein hos denna bakterie gett negativt resultat.</p> / <p>This work describes an analysis of membrane-anchored and periplasmic c-type cytochromes of perchlorate grown GR-1, and a comparison with the c-type cytochrome content of Ideonella dechloratans, other known c-type cytochromes and the genome of Dechloromonas aromatica. The aim of the comparison was to investigate a hypothesis that the bacteria use different routes for electron transfer to the periplasmic enzyme (per)chlorate reductase. Cell membrane from GR-1 was prepared through ultracentrifugation and periplasm was prepared through osmotic chock. The fractions were separated by SDS-PAGE and peptides containing covalently bound heme (c-type cytochromes) were detected by a specific staining reaction. In an attempt to probe a gene coding for a NapC/NirT-like protein Touchdown PCR was performed on isolated DNA from GR-1, using degenerate primers. Finally, reduced membranes were treated with carbon monoxide to investigate the presence of cbb3-type oxidase.</p><p>Separation and detection resulted in seven periplasmic peptides and eight membrane anchored peptides, all with molecular weights in a range of 8-60 kDa. Nothing has been revealed during this work that opposes the hypothesis of GR-1 and D. aromatica using a NapC/NirT-like protein as an electron carrier to their periplasmic (per)chlorate reductase. The PCR resulted in a product that most likely is a sequence from a gene coding for a NapC/NirT-like protein and two, maybe three, candidates for a NapC/NirT-like protein were also found in the membrane of GR-1. Analysis also revealed that GR-1 most likely makes use of a cbb3-type oxidase for reduction of oxygen during microaerofilic conditions.</p><p>Concerning I. dechloratans, nothing has been revealed during this work that opposes the hypothesis of this bacterium using a soluble cytochrome c as an electron carrier to its chlorate reductase. Three candidates for a soluble cytochrome c protein were found. The theory is also supported by the negative result from earlier attempts to probe a gene coding for a NapC/NirT-like protein in this bacterium.</p>

Arbetet beskriver en analys av innehållet av membranbundna och periplasmiska c-cytokromer hos perkloratodlade GR-1 och jämförelser med c-cytokrominnehållet hos Ideonella dechloratans och andra kända c-cytokromer, samt med genomet för Dechloromonas aromatica. Den jämförande studien av c-cytokromer gjordes med syftet att undersöka en hypotes om att bakterierna använder olika vägar för elektronöverföring till det periplasmiska enzymet (per)kloratreduktas. Cellmembran från GR-1 renframställdes genom ultracentrifugering och periplasma preparerades fram med hjälp av osmotisk chock. Fraktionerna analyserades sedan med SDS-PAGE och peptider med kovalent bundet hem (c-cytokromer) detekterades med hjälp av en specifik färgreaktion. Även Touchdown PCR med degenererade primrar genomfördes på isolerat DNA från GR-1 i ett försök att finna en gen kodande för ett NapC/NirT-liknande protein. Slutligen kolmonoxidbubblades reducerade membran för att undersöka förekomsten av cbb3-typ oxidas. Separation och infärgning av periplasmiska och membranbundna proteiner från GR-1 resulterade i sju respektive åtta peptidband med molekylvikter mellan 8-60 kDa. Inget som framkommit under arbetet talar emot hypotesen om att GR-1 och D. aromatica skulle använda ett NapC/NirT-liknande protein som elektronöverförare till det periplasmiska (per)kloratreduktaset. PCR-analysen resulterade i en produkt som troligtvis är en sekvens från en gen som kodar för ett NapC/NirT-liknande protein och två, eventuellt tre, kandidater för ett NapC/NirT-liknande protein hittades i membranet hos GR-1. Dessutom framkom att GR-1 troligtvis använder cbb3-typ oxidas som terminalt oxidas vid reduktion av syrgas under mikroaerofila förhållanden. Vad gäller I. dechloratans och hypotesen om att denna bakterie använder ett lösligt cytokrom c för elektronöverföring till sitt kloratreduktas så har inget framkommit under arbetet som talar emot detta. Tre kandidater för lösliga cytokrom c-proteiner hittades. För teorin talar även att de försök som tidigare gjorts med att påvisa genen för ett NapC/NirT-liknande protein hos denna bakterie gett negativt resultat. / This work describes an analysis of membrane-anchored and periplasmic c-type cytochromes of perchlorate grown GR-1, and a comparison with the c-type cytochrome content of Ideonella dechloratans, other known c-type cytochromes and the genome of Dechloromonas aromatica. The aim of the comparison was to investigate a hypothesis that the bacteria use different routes for electron transfer to the periplasmic enzyme (per)chlorate reductase. Cell membrane from GR-1 was prepared through ultracentrifugation and periplasm was prepared through osmotic chock. The fractions were separated by SDS-PAGE and peptides containing covalently bound heme (c-type cytochromes) were detected by a specific staining reaction. In an attempt to probe a gene coding for a NapC/NirT-like protein Touchdown PCR was performed on isolated DNA from GR-1, using degenerate primers. Finally, reduced membranes were treated with carbon monoxide to investigate the presence of cbb3-type oxidase. Separation and detection resulted in seven periplasmic peptides and eight membrane anchored peptides, all with molecular weights in a range of 8-60 kDa. Nothing has been revealed during this work that opposes the hypothesis of GR-1 and D. aromatica using a NapC/NirT-like protein as an electron carrier to their periplasmic (per)chlorate reductase. The PCR resulted in a product that most likely is a sequence from a gene coding for a NapC/NirT-like protein and two, maybe three, candidates for a NapC/NirT-like protein were also found in the membrane of GR-1. Analysis also revealed that GR-1 most likely makes use of a cbb3-type oxidase for reduction of oxygen during microaerofilic conditions. Concerning I. dechloratans, nothing has been revealed during this work that opposes the hypothesis of this bacterium using a soluble cytochrome c as an electron carrier to its chlorate reductase. Three candidates for a soluble cytochrome c protein were found. The theory is also supported by the negative result from earlier attempts to probe a gene coding for a NapC/NirT-like protein in this bacterium.

In this work, the formation and effects of chlorate ion were studied over hydrogen evolution reaction (HER), focusing for the chlorine and alkalis production by diaphragm process, using a pilot and bench cells, respectively. Studies in pilot cell revealed that chlorate formation is dependent of NaOH concentration in liquor and brine dwelling time. It was also observed that the NaOH concentration increase in the cathodic side provoked decreasing of diaphragm structure lifetime. This phenomenon reduced the blockage efficiency to back migration and consequently, increasing NaOH concentration in the anodic chamber, further increase in the undesirable parallels reaction rate, for example, the chlorate formation. After formation of chlorate in the anodic chamber the migration to the cathodic side is inevitable. Therefore to complete the studies we decided to verify the influence the chlorate, in bench cell, in the cathodic side, specifically for HER process. Potential polarization experiments in presence of chlorate, evidenced effects of HER wave displacement over SAE1020 mild steel carbon steel plate employed in industrial cells for chlorine and alkalis production using diaphragm process. Alternatively to SAE1020, other duplex alloys like SAF2205, SAF2304 and SAF2507 were also investigated. SAF2205 alloy presented a very interesting electrocatalytic response for HER. Pilot cell results pointed for the problem detected with the NaOH migration to the anodic side of cell, and invoked the single possibility to solve this problem - the changing of diaphragm cell separator. Bench experiments revealed chlorate influence on HER over SAE1020 carbon steel, claiming for urgent developing of alternative materials/cathodes with chlorate-immune character. / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Esse trabalho estuda a formação e os efeitos de íon clorato sobre a reação de desprendimento de hidrogênio (RDH) com ênfase para a produção de cloro-soda por processo de diafragma, com a utilização de célula piloto e de bancada, respectivamente. Os estudos em célula piloto revelaram que a formação de clorato é dependente da concentração de hidróxido de sódio no licor de célula e do tempo de residência da salmoura. Observou-se também que o aumento da concentração de hidróxido de sódio no lado catódico provoca envelhecimento prematuro da estrutura do diafragma, e este fenômeno reduz a eficiência do bloqueio à back migration de forma mais intensa. Esta perda de eficiência promove uma maior alcalinização do anólito, e consequentemente, um aumento nas velocidades das reações paralelas indesejáveis, como por exemplo, a formação de clorato. Uma vez formado clorato no compartimento anódico, sua percolação para o lado catódico é inevitável. Por essa razão, optou-se por fazer estudos de bancada do processo catódico, particularmente na região de RDH. Os dados de polarização revelaram a dependência da RDH em presença de clorato sobre o aço carbono SAE1020, atualmente empregado nas células industriais no processo de diafragma para produção de cloro-soda. Alternativamente ao SAE1020, foram estudadas as ligas duplex SAF2205, SAF2304 e SAF2507. Os estudos de polarização mostraram a dependência da RDH para as ligas SAF2304 e SAF2507, por outro lado, a liga SAF2205 apresentou resposta eletrocatalítica para o desprendimento de hidrogênio. Os estudos em célula piloto mostram que a queda de rendimento de produção é observada de forma mais imediata na parte anódica do processo, e fica evidenciada a importância de otimização do separador de diafragma. Nos experimentos de bancada foi constatada a influência do clorato sobre a RDH para o SAE1020, mostrando a necessidade de materiais alternativos que não apresentem susceptibilidade em presença de clorato.

Die vorliegende Arbeit befasst sich mit Untersuchungen zur Herstellung der Erdalkalimetallchlorate und zu den Lösungsgleichgewichten in den reziproken Salzpaaren MCl2 + 2NaClO3 = M(ClO3)2 + 2NaCl/H2O (M = Mg2+, Ca2+, Sr2+, Ba2+) bei 25°C, 50°C und 75°C. Neben den Löslichkeiten und Bodenkörperparagenesen sind die Dichten und Viskositäten bestimmt worden. Auf der Basis der erarbeiten Daten erfolgte die Bilanzierung der Herstellungsprozesse der Erdalkalimetallchlorate durch reziproke Umsetzung. Zusammenfassend kann eingeschätzt werden, dass mit etwa 0,2 bis 0,5 kg wasserfreies Chlorat pro kg Umlauflösung durchaus effektive Prozesse der polythermen reziproken Umsetzung vorliegen. Über die IR- und Raman-Spektroskopie und die Thermoanalyse wurden die Erdalkalimetallchlorate bzw. Erdalkalimetallchlorat-Hydrate näher charakterisiert, u.a. bezüglich ihres Zersetzungsverhaltens bis zum Erdalkalimetallchlorid.

Actuellement le polyéthylènimine (PEI) est l’agent de transfection transitoire le plus utilisé par l’industrie pharmaceutique pour la production de protéines recombinantes à grande échelle par les cellules de mammifères. Il permet la condensation de l’ADN plasmidique (ADNp) en formant spontanément des nanoparticules positives appelées polyplexes, lui procurant la possibilité de s’attacher sur la membrane cellulaire afin d’être internalisé, ainsi qu’une protection face aux nucléases intracellulaires. Cependant, alors que les polyplexes s’attachent sur la quasi-totalité des cellules seulement 5 à 10 % de l’ADNp internalisé atteint leur noyau, ce qui indique que la majorité des polyplexes ne participent pas à l’expression du transgène. Ceci contraste avec l’efficacité des vecteurs viraux où une seule particule virale par cellule peut être suffisante. Les virus ont évolués afin d’exploiter les voies d’internalisation et de routage cellulaire pour exprimer efficacement leur matériel génétique. Nous avons donc supposé que l’exploitation des voies d’internalisation et de routage cellulaire d’un récepteur pourrait, de façon similaire à plusieurs virus, permettre d’optimiser le processus de transfection en réduisant les quantités d’ADNp et d’agent de transfection nécessaires. Une alternative au PEI pour transfecter les cellules de mammifèreest l’utilisation de protéines possédant un domaine de liaison à l’ADNp. Toutefois, leur utilisation reste marginale à cause de la grande quantité requise pour atteindre l’expression du transgène. Dans cette étude, nous avons utilisé le système E/Kcoil afin de cibler un récepteur membranaire dans le but de délivrer l’ADNp dans des cellules de mammifères. Le Ecoil et le Kcoil sont des heptapeptides répétés qui peuvent interagir ensemble avec une grande affinité et spécificité afin de former des structures coiled-coil. Nous avons fusionné le Ecoil avec des protéines capables d’interagir avec l’ADNp et le Kcoil avec un récepteur membranaire que nous avons surexprimé dans les cellules HEK293 de manière stable. Nous avons découvert que la réduction de la sulfatation de la surface cellulaire permettait l’attachement ciblé sur les cellules par l’intermédiaire du système E/Kcoil. Nous démontrons dans cette étude comment utiliser le système E/Kcoil et une protéine interagissant avec l’ADNp pour délivrer un transgène de manière ciblée. Cette nouvelle méthode de transfection permet de réduire les quantités de protéines nécessaires pour l’expression du transgène. / Pharmaceutical industry often employs polyethylenimine (PEI) for large scale protein production processes by transient transfection of mammalian cells. PEI condenses plasmid DNA (pDNA) by spontaneously forming positive nanoparticles known as polyplexes. Condensed pDNA is favoured for cell surface binding, internalization and protection from intracellular nucleases. While most of the cells efficiently uptake polyplexes, only 5 to 10% of captured pDNA reaches the nucleus for transgene expression. This suggests that polyplexes are hampered in their ability to route and to translocate to the nucleus necessitating large amounts of polyplexes to achieve high expression levels. By contrast, many viruses can efficiently transduce cells with only one or a few viral genome copies. Viruses have evolved to exploit cellular internalization and routing properties to express their own genetic material. We hypothesized that less pDNA would be used in an optimized transfection process if we exploited the internalization and routing properties that viruses use. DNA binding proteins could be used as an alternative to PEI to transfect mammalian cells. However, their usage is marginal due to the large protein quantities required to bind pDNA for transgene expression. If less pDNA is used less binding protein is needed. In this study, we used the E/Kcoil system to target a membrane receptor to deliver pDNA in mammalian cells. The Ecoil and Kcoil are two repeated heptapeptides which interact with a high affinity and specificity to form coiled-coil structures. We fused the Ecoil with a recombinant pDNA-binding protein. The Kcoil was fused to a stably-expressed membrane receptor in HEK293 cells. We discovered that low sulfation of the cell surface reduced non-specific binding of the pDNA:protein complex and permitted targeted binding via the E/Kcoil interaction. We demonstrate how to use recombinant pDNA-binding protein and the E/Kcoil system for targeted transgene delivery. This newly developed system provides a new transfection method, with reduced pDNA-binding protein quantities needed to achieve transgene expression.

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Title from second page of PDF document. Includes bibliographical references (p. 144-152).