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Åtgärder för en effektivare intern materialförsörjning : Genomlysning av förbättringsområden för lager till slutmontering av gruvmaskiner med fördjupning inom frekvensläggning – en studie vid Epiroc Rock Drills AB / Measures for a more efficient internal material supply : Review of improvement areas in warehouse for final assembly of mining machines with a focus in slotting optimization – a study at Epiroc Rock Drills ABEskilsson, Niklas, Magnuson, David January 2019 (has links)
Epiroc har åtnjutit en längre period av kraftig tillväxt samtidigt som flertalet effektiviseringsprojekt har genomförts med syfte att öka produktionsvolymen för att möta marknadens efterfrågan. Detta genom att bland annat implementera en variant av Lean production – The Way We Produce. En av dessa förändringar är en takad flödesorienterad montering med just-in-time sekvenserad materialförsörjning. Detta har i sin tur ökat kraven på materialförrådet Logistikcenter (LC) där ledningen nu börjat undersöka möjliga effektiviseringsåtgärder. Därav är studiens syfte till att ta fram realiserbara förbättringsförslag för logistikverksamheten vid Logistikcenter tillhörande Epiroc Rock Drills AB i Örebro för att öka effektiviteten och leveranssäkerheten. Studien har genomförts i två faser; identifieringsfasen och fördjupningsfasen. Under identifieringsfasen genomfördes en kartläggning av nuläget i LC, där underlaget för kartläggningen baserar sig på intervjuer, observationer och analyser. Genom en rotorsaksanalys, med målet att identifiera källor till ineffektivitet, kunde nio förbättringsområden identifieras varav en av dessa vidare skulle utredas i fördjupningsfasen. Dessa utvärderades utifrån en effekt-insats matris för att välja det förbättringsområde med störst effektiviseringspotential i förhållande till den förväntande insatsen. Analysmodellen för effekt-insats matrisen var de åtta slöserierna i Lean (Petersson, et al., 2015), dess förväntade påverkan på effektiviteten samt den förväntade komplexiteten av en implementation. Resultatet från rotorsaksanalysen gav artikelklassificering som det primära förbättringsområdet där den undersökta åtgärden var en alternativ tillämpning av frekvensläggning för att minimera rörelsetiden mellan lagerplatser vid plock. Under fördjupningsfasen undersöktes den nuvarande artikelklassificeringen och frekvensläggningen genom syntes av en alternativ modell för klassificeringen av artiklar och lagerplatser utifrån en fördjupad litteraturstudie. För att undersöka om en alternativ klassificering kan öka effektiviteten utvecklades en utvärderingsmodell som modellerar rörelsetiden för historiskt data från plocklistor. Den användes för att testa vilken kombination av storlekar på artikelklasserna som gav den minsta möjliga totala rörelsetiden. En kombination av 60/30/10 % (A/B/C) av det ackumulerade antalet plock gav den lägsta totala rörelsetiden för en plockhistorik på 15 månader med en reduktion av rörelsetiden motsvarande 33 % (1760 h) jämfört mot nuläget. Vidare undersöktes en alternativ sortering av plocklistorna för automathissar respektive pallställage med utvärderingsmodellen som gav en reduktion på 4 % respektive 11 % mot nuläget. Slutligen undersöktes olika former på zonerna för klassificering av lagerplatser i pallställage W3, där utlämningsplatsen är placerad halvvägs in i ställaget. En tyngdpunkt placerat centralt mellan ingången och utlämningsplatsen gav det bästa simuleringsresultatet. Sammanfattningsvis fastställdes artikelklassificering som det förbättringsområde med störst realiserbar effektiviseringspotential med lägst komplexitet utifrån en rotsorsaksanalys. Epiroc rekommenderas att implementera klassificeringsmodellen med tre klasser av storleken 60/30/10 % (A/B/C) av den ackumulerade antalet plockrader, samt att implementera att den alternativa sorteringen av plockrader på plocklistor för pallställage. / Epiroc has had a long period of strong growth, where several efficiency projects have been implemented to increase production volume in order to meet market demand. A variant of Lean production has been implemented over several years – called The Way We Produce by Epiroc. A large part of that change has been the implementation of sequenced flow-oriented assembly with just-in-time sequenced material deliveries to the assembly floor. This, in turn, has increased the requirements for the warehouse Logistic Center (LC) and management has now begun to investigate ways to improve efficiency at LC. Hence, the aim of the study is to develop realistic improvement proposals for the logistics operations at Logistics Center of Epiroc Rock Drills AB at Örebro to increase efficiency and delivery reliability. The study has been conducted in two phases; the identification phase and the in-depth phase. During the identification phase, a mapping of the current situation in LC was carried out, where the basis for the survey is based on interviews, observations and analyzes. Through a root cause analysis, with the goal of finding sources of inefficiency, nine areas of improvement could be identified, one of which would be chosen for further investigation in the in-depth phase. The areas of improvement were evaluated with an effect-input matrix to choose the area of improvement that provides the greatest efficiency potential in relation to the expected effort. The analysis model for the effect-input matrix was the eight wastes of Lean based on Petersson et al. (2015), its expected impact on efficiency and the expected complexity of an implementation. The result of the root cause analysis gave article classification as the primary area of improvement, where the measure is an alternative slotting strategy to minimize the movement time between storage locations. During the in-depth phase, the current article classification and frequency setting were examined by synthesis of an alternative model for the classification of articles and storage locations based on an in-depth literature study. To investigate whether an alternative classification can increase efficiency, an evaluation model was developed that models the movement time from historical data from pick lists. It was used to test which combination of sizes for the article classes gave the smallest possible total movement time. A combination of 60/30/10% (A/B/C) gave the lowest total movement time for a picking history of 15 months with a reduction corresponding to 33% (1760 h) compared to the current situation. Furthermore, an alternative sorting of the pick lists for the vertical lift modules and pallet racking was examined with the evaluation model, which gave a reduction of 4% and 11% respectively. Finally, various forms for the zones were examined for the classification of storage locations in pallet rack W3, where the delivery site is located three-quarter way into the pallet rack. A center of gravity for the for the A-class placed between the entrance and the delivery point gave the best simulation result. In summary, the article classification improvement area was established with the greatest realizable efficiency potential with the least effort based on the root cause analysis. Epiroc is recommended to implement the classification model with three classes of size 60/30/10 % (A/B/C) of the accumulated number of picking rows, and to implement that alternative sorting of pick rows on picklists for pallet racking.
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Cost engineering: Kostnadsidentifiering i en verksamhet : En fallstudie om identifiering av osynliga kostnaderSundberg, Frida, Viklund, Carina January 2018 (has links)
Det har alltid varit viktigt för företag i alla branscher att ständigt kunna fatta rätt beslut. För att kunna göra det måste företagen vara medvetna om vilka kostnader som finns och kan uppstå i deras verksamhet. För att kunna göra en rättvis kostnadsuppskattning bör företag även sträva efter att identifiera osynliga kostnader. Detta arbete baseras på en fallstudie som genomfördes på företaget LKAB som bedriver gruvverksamhet. Syftet med denna studie var att utreda huruvida osynliga kostnader och förlorade intäkter existerar och i så fall var och i vilken mån de bör beaktas. Studiens mål var att ta fram ett underlag som ska kunna tillämpas i effektiviserings syfte hos olika företag. Studiens frågeställningar kunde besvaras. Med hjälp av intervjuer utformades en värdeflödeskarta som påvisade var kostnader uppstår. En ABC-kalkyl användes för att kunna identifiera kopplingen mellan kostnader och deras aktiviteter och resurser. För att kunna avgöra vilka av kostnaderna och de förlorade intäkterna som bör påverkas användes Leans definition på slöseri. Resultatet som erhölls visade på att osynliga kostnader och förlorade intäkter existerar samt i vilken mån de bör beaktas och hanteras vid den taktiska planeringen. De kostnader som bör påverkas visade sig vara Demurrage-kostnaden, transportkostnaden och särhanteringskostnaden. De förlorade intäkterna som inte visade sig skapa något värde för kunden och då borde beaktas var dödfrakt, fraktkompensation, rabatterat pris och förlorade intäkter som uppstår i samband med produktionsstopp och produktkassering. Resultatet visade även på att det går att få ett användbart resultat utan att använda en ABC-kalkyl fullt ut. / Before making a decision based on a fair cost estimate, careful consideration should be given to ensure that invisible operational costs have been adequately identified and incorporated into the estimate. We present the results of a case study conducted at the Swedish mining company LKAB in which we investigated to what extent do invisible costs exist, their impact on lost revenue, and whether they can be adequately captured within the framework of a fair cost estimate. Using data obtained from a series of interviews conducted at LKAB, we built a value-flow chart to model costs and used an ABC calculation to determine the linkage between the costs and their activities and resources. We then applied Lean's definition of waste to identify which, and to what extent, these costs and lost revenue should be affected. Specifically, we determined that the costs which should be considered are: Demurrage, transport, and special handling and the sources of lost revenue are: dead freight, freight compensation, discounted prices, production stoppages, and discard a product. Finally, our analysis showed that fair cost estimates including invisible costs and lost revenue are useful in making decisions about the efficiency of a company and that a full ABC calculation is not required to get a useful result.
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Potencial de sequestro de carbono no solo e dinâmica da matéria orgânica em pastagens degradadas no Brasil / Potential of soil carbon sequestration and organic matter dynamics in degraded pastures in BrazilOliveira, Daniele Costa de 08 March 2018 (has links)
As pastagens são o principal uso da terra no mundo, ocupando dois terços de sua área agricultável e três quartos da área agricultável do Brasil. Quando bem manejadas, as pastagens possuem alta capacidade de estocar C no solo, porém podem perder até 50% do C do solo quando em avançado estágio de degradação. Os objetivos desse trabalho foram quantificar o potencial de sequestro de C e avaliar a dinâmica da matéria orgânica no solo em locais de conversão de pastagens degradadas para bem manejadas no Brasil. Para tanto, foram avaliadas as alterações dos estoques de C no solo nas pastagens do Brasil através de uma meta-análise, bem como os fatores de manejo e as taxas de variações do estoque de C no solo em diferentes status de pastagens. Em sete cronossequências situadas em Vila Bela da Santíssima Trindade (MT), Nova Xavantina (MT), Conquista D\'Oeste (MT), Dueré (TO), Carmolândia (TO) e Paraíso (TO), foram determinadas as alterações na quantidade e na qualidade da Matéria Orgânica do Solo (MOS). Foram determinados os estoques de C e N e as taxas de variação locais. A qualidade da MOS foi avaliada por meio de análise isotópica, fracionamento físico, Índice de Manejo do C (IMC), grau de humificação (HFIL) e teor de C na Biomassa Microbiana (C-BM). Através da meta-análise, estimou-se que no Brasil os solos sob pastagens degradadas apresentam redução dos estoques de C de 0,13 Mg ha-1 ano-1. As pastagens nominais foram capazes de aumentar o estoque de C no solo, enquanto as pastagens melhoradas nem sempre mantiveram os estoques semelhantes aos da vegetação nativa. A recuperação das pastagens promove acúmulo de C no solo na faixa de 0,40 Mg ha-1 ano-1. O cumprimento da meta de recuperação dos 30 milhões de hectares de pastagens resultará no acumúlo de 12 Tg ha-1 ano-1 de C. A degradação das pastagens nos estados do Mato Grosso e Tocantins reduziu os estoques de C no solo numa faixa de -0,06 Mg ha-1 ano-1, enquanto a recuperação das mesmas proporcionou aumento de 0,12 Mg ha-1 ano-1. Nas pastagens avaliadas, cerca de 54% do C é originado das gramíneas cultivadas. A fração orgânica foi a mais sensível às Mudanças de Uso da Terra (MUT), promovendo diminuição dos estoques de C nessa fração. A implantação de pastagens tem impacto negativo na qualidade da MOS, com diminuição do IMC de até 70%. Contudo, se bem manejadas, o IMC das pastagens pode ser superior ao da vegetação nativa, como acontece quando a pastagem é consorciada com Pueraria spp. O C-BM e o grau de humificação não foram alterados após a implantação ou a recuperação das pastagens, não sendo bons indicadores de qualidade da MOS. As alterações dos estoques de C nas frações da MOS e o IMC foram os indicadores mais eficientes de alterações da qualidade da MOS em pastagens. As recuperação de pastagens degradadas promove aumento no estoque de C no solo e melhoria da qualidade da MOS. / Pastures are the main land use in the world, occupying two-thirds of the world\'s arable land and three quarters of agricultural areas of Brazil. When well-managed, pastures have a high capacity to store SOC, but they may lose up to 50 % of stock SOC when in an advanced stage of degradation. The objective of this work was to quantify the potential of carbon sequestration and to evaluate soil organic matter dynamics in the conversion of degraded pastures to well-managed in Brazil. We evaluated changes in stocks SOC in pastures in Brazil through a meta-analysis, determining the management factors and the rates of stocks SOC changes in different pasture status. In seven cronossequences located in Vila Bela da Santíssima Trindade (MT), Nova Xavantina (MT), Conquista D\'Oeste (MT), Dueré (TO), Carmolândia (TO) and Paraíso (TO) were determined the changes in the quantity and quality of soil organic matter (SOM). C and N stocks and rates of local variations were determined. The quality of the SOM was evaluated through isotopic analysis, physical fractionation, C management index (CMI), degree of humification (HFIL) and C content in microbial biomass (MB-C). Through the meta-analysis it was estimated that in Brazil degraded pastures present a reduction of stocks SOC of 0.13 Mg ha-1 year -1. Nominal pastures were able to increase stock SOC, while improved pastures did not always maintain stocks similar to native vegetation. The recovery of pastures promotes the accumulation of C in the soil at the rate of 0.40 Mg ha-1 year -1. Meeting the recovery goal of 30 million ha of pasture will result in the accumulation of 12 Tg C ha-1year -1. The degradation of pastures in the states of Mato Grosso and Tocantins reduces C stocks in the soil at a rate of 0.06 Mg C ha-1 year-1. While the recovery of degraded pastures has the potential to increase C stocks in the soil with rate of 0.12 Mg C ha-1 year-1. In the pastures evaluated, about 54% of the C originates from the cultivated grasses. The organic fraction was the most sensitive to LUC, promoting the decrease of C stocks in this fraction. Pasture implantation has a negative impact on SOM quality, with a reduction in the CMI of up to 70%; however, if managed well, the CMI of pastures may be higher than that of native vegetation. The MB-C and degree of humification were not altered after the implantation or the recovery of the pastures. Changes in C stocks in SOM fractions and CMI were the most efficient indicators of changes in SOM quality in pastures. The recovery of degraded pastures promotes an increase in C stock in the soil and an improvement in the quality of the MOS.
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\"Estudo do desenvolvimento floral em espécies arbóreas da família Meliaceae\" / Floral development in woody species of the Meliaceae familyGouvêa, Cantidio Fernando 25 August 2005 (has links)
A família Meliaceae compreende cerca de 51 gêneros e 550 espécies distribuídas principalmente na região Neotropical. Incluídas nesta família, estão espécies de elevado interesse comercial para a produção de madeiras nobres. Há carência de informações quanto à biologia floral dessas espécies, que aliada a problemas silviculturas dificulta a elaboração de programas efetivos de melhoramento genético das espécies de interesse econômico da família. O presente trabalho visa contribuir para o entendimento do desenvolvimento floral na família Meliaceae, com destaque para sete espécies de interesse econômico e/ou ecológico: Cedrela fissilis L., Cedrela odorata L., Swietenia macrophylla R. A. King, Trichilia claussenii C. DC., Guarea guidonea (L.) Sleumer, Toona ciliata M. J. Roem e Melia azedarach L. Para os estudos morfo-anatômicos utilizaram-se técnicas de microscopia óptica e microscopia eletrônica de varredura. Analisou-se igualmente o padrão de expressão de genes do modelo ABC, relacionados ao desenvolvimento floral, via hibridização in situ. Os estudos morfo-anatômicos permitiram caracterizar o desenvolvimento dos primórdios dos órgãos florais e o estabelecimento de estágios arbitrários de desenvolvimento, auxiliando na caracterização da expressão gênica. Estes resultados permitiram ainda a identificação de flores funcionalmente femininas ou masculinas em S. macrophylla C. fissilis, C. odorata e T. ciliata, as quais apresentam dimorfismo sexual. O padrão de expressão dos homólogos dos genes: APETALA1 (AP1), APETALA3 (AP3) e AGAMOUS (AG) foram diferentes daqueles previstos pelo modelo ABC. A expressão dos homólogos de AP1 foi verificada em todo o meristema floral nos estágios iniciais de desenvolvimento, em todas as espécies estudadas. O sinal de expressão concentrou-se nos primórdios das sépalas e pétalas em estágios mais avançados do desenvolvimento, porém um fraco sinal de hibridização de AP1 foi verificado em todos os verticilos. A expressão dos homólogos de AP3 foi verificada nas regiões previstas pelo modelo ABC, correspondentes ao segundo e terceiro verticilos. Porém um fraco sinal de AP3 foi também observado no quarto verticilo, o que não é previsto pelo modelo teórico. A expressão dos homólogos de AG foi restrita à região central do meristema floral, correspondente ao terceiro e quarto verticilos, em todos os estágios do desenvolvimento floral de todas as espécies estudadas. Entretanto, sinais adicionais de hibridização dos homólogos de AG foram visualizados na região abaxial dos primórdios de sépalas e pétalas em C. fissilis, C. odorata e T. ciliata. Os estudos da expressão dos genes do modelo ABC em Meliaceae revelaram padrões de expressão que não concordam com o modelo teórico vigente do controle molecular da determinação da identidade dos órgãos florais. / The Meliaceae family comprises approximately 51 genus and 550 species mainly distributed in the Neotropical region. In this family there are many species of commercial interest for the production of noble wood. The lack of information on the floral biology associated with silvicultural problems limits the elaboration of effective breeding programs with species of economic interest. The present work aims to contribute with the understanding of floral development in Meliaceae, focusing in seven species of economical or ecological interest: Cedrela fissilis L., Cedrela odorata L., Swietenia macrophylla R. A. King, Trichilia claussenii C. DC., Guarea guidonea (L.) Sleumer, Toona ciliata M. J. Roem e Melia azedarach L. Morpho-anatomical analyses were done by light and scannning electron microscopy. The expression pattern of the ABC model genes, which are related to floral development, was analyzed by in situ hybridization. The characterization of the development of floral organ primordia and the establishment of arbitrary stages of floral development was done and was important for the characterization of gene expression. These results allowed for the identification of functionally female and male flowers in Swietenia macrophylla, Cedrela fissilis, Cedrela odorata and Toona ciliata, characterizing sexual dimorphism. The patterns of gene expression of the homologous of APETALA 1, APETALA 3 and AGAMOUS were different from those predicted by the ABC model. AP1 expression was observed in the entire floral meristem in the initial stages of development, in all seven species. The expression signal was more concentrated in sepal and petal primordia in further stages of development, although a weak hybridization signal of AP1 was verified in all four whorls. The expression of AP3 homologs was observed in the second and third whorls, as predicted by the ABC model. However, a weak signal of AP3 was also observed in the forth whorl, which was not predicted by the ABC model. AG homolog expression was restricted to the central region of the floral meristem, corresponding to the third and forth whorls, in all stages of development of all seven species. However, additional hybridization signal of AG homologs were seen in the abaxial region of sepal and petal primordia in Cedrela fissilis, Cedrela odorata and Toona ciliata. The analyses of the ABC model genes in Meliaceae revealed that the expression patterns do not agree with the theoretical currently accepted model that determines the molecular control of the identity of the floral organs.
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A interdisciplinaridade nas licenciaturas das áreas constituintes das ciências naturais: um estudo de caso na Universidade Federal do ABC / Interdisciplinary degrees of constituents in the natural sciences: a case study at the Federal University of ABCSaraiva, Karla Beatriz Gomes 06 October 2014 (has links)
O presente trabalho objetivou compreender a importância da interdisciplinaridade na formação de professores das áreas constituintes das ciências naturais de acordo com a Universidade Federal do ABC. Para isso, fez-se uma pesquisa qualitativa, de caráter exploratório e descritivo, na forma de estudo de caso, que realizou uma análise documental do Projeto Político Pedagógico Acadêmico de cada uma das licenciaturas constituintes da área das ciências naturais, Ciências Biológicas, Física e Química, da UFABC. A partir dela, analisamos mais profundamente esses documentos e as características contidas neles, procurando entender a importância de uma formação interdisciplinar para a atuação de professores das áreas constituintes das ciências naturais de acordo com essa instituição de ensino superior. Para compreendê-los melhor, entrevistou-se três dos professores que o elaboraram, com o auxílio de um roteiro de entrevista semi-dirigido. Através da realização desta pesquisa, com base nos documentos analisados e nas entrevistas feitas, conclui-se que a formação interdisciplinar de professores é fundamental para que o futuro docente perceba a complexidade dos conhecimentos globais, que envolvem as esferas social, ambiental e tecnológica. Um curso de licenciatura interdisciplinar é essencial para que o professor entenda como acontece a interação entre todos os campos científicos e saiba se posicionar perante todos eles, capacitando o seu aluno a essa competência. A abordagem interdisciplinar fundamentada na discussão e, principalmente no diálogo entre pessoas provenientes de diferentes campos científicos e com distintas visões habilita o professor a enfrentar as demandas educacionais de um mundo em vertiginosas mudanças. Essa formação, com certeza, reflete na atuação didático-pedagógica do docente, que forma, assim, um cidadão apto a se posicionar diante das questões do cotidiano e opinar, conscientemente, sobre problemas cada vez mais atuais. / This study aimed to understand the importance of interdisciplinarity in training teachers of the constituent areas of the natural sciences according to the Federal University of ABC. For this, there was a qualitative research, exploratory and descriptive, in the form of case study, we conducted a documentary analysis of the Project Political Scholar of each constituent degrees of natural science, Biological Sciences, Physics and chemistry, UFABC. From there, we analyze further these documents and features contained in them, trying to understand the importance of interdisciplinary training for practicing teachers of the constituent areas of the natural sciences according to this HEI. To understand them better, if interviewed three of the teachers who prepared with the aid of a script semi-directed interview. Through this research, based on the documents reviewed and the interviews, it is concluded that the interdisciplinary training of teachers is essential to the future teacher realizes the complexity of global knowledge, involving social, environmental and technological spheres. An interdisciplinary undergraduate course is essential for the teacher to understand how the interaction happens between all scientific fields and learn to stand before them all, enabling your student to that jurisdiction. The interdisciplinary approach based on the discussion and especially the dialogue between people from different scientific fields and with different views enables the teacher to meet the educational demands of a world in vertinosas changes. This training surely reflects the didactic-pedagogic activities of teachers, which thus forms a fit to stand on everyday questions and comment knowingly on increasing current problems citizen.
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Aufreinigung und funktionelle Charakterisierung der peroxisomalen ABC-Transporter Pxa1p-Pxa2p aus Saccharomyces cerevisiaeSchreiber, Gabriele 19 December 2007 (has links)
Die peroxisomalen ABC-Transporter Pxa1p und Pxa2p sind Halbtransporter. Genetische Studien ergaben Hinweise, dass sie zur Bildung aktiver Transporter heterodimerisieren und am Import von langkettigen Fettsäuren in die Peroxisomen von S. cerevisiae beteiligt sind. Es wurden epitopmarkierte Varianten der Proteine als Komplex isoliert. Damit wurde gezeigt, dass Pxa1p und Pxa2p ein stabiles Heterodimer bilden. Zur Charakterisierung der ATP Bindeeigenschaften wurden die Transporter mit 8-azido-[alpha-32P]-ATP inkubiert und kovalent verknüpft. Dabei konnte gezeigt werden, dass Pxa1p und Pxa2p eine unsymmetrische Bindung des ATP Analogons aufweisen. Pxa2p bindet deutlich mehr azido-ATP als Pxa1p, bei sehr ähnlichen Dissoziationskonstanten. Die reduzierte ATP Bindung von Pxa1p spiegelt sich durch degenerierte Sequenzmotive der an der ATP Bindung beteiligten Sequenzen wieder. Die isolierten ABC-Transporter wurden für ATPase Messungen eingesetzt. Sie zeigten eine basale ATPase Aktivität, die durch Zugabe langkettiger Coenzym A aktivierter Fettsäuren, wie Oleoyl-CoA und Palmitoyl-CoA stimulierbar war. Eine Lysin Mutation im Walker A Motiv von Pxa1p hatte keine Funktionalitätseinbuße zur Folge. Dieselbe Mutation bei Pxa2p führte im Wachstumstest auf Festmedium mit Ölsäure als Kohlenstoffquelle zu einem deutlich verlangsamten Wachstum. Diese Ergebnisse korrespondieren mit der beobachteten unsymmetrischen ATP Bindung von Pxa1p und Pxa2p, da bei dem schwächer bindenden Pxa1p die Mutation wirkungslos blieb. Keine Übereinstimmung war bei den ATPase Aktivitätsmessungen der aufgereinigten Mutanten zu verzeichnen. Beide Mutanten zeigten eine unbeeinträchtigte ATPase Aktivität. Die ABC-Transporter wurden in Proteoliposomen eingebaut und für Transportmessungen mit einem Spin-Label markierten Oleoyl-CoA verwendet. Die Transportmessungen zeigten einen ATP abhängigen Transport, woraus geschlossen wurde, dass Pxa1p-Pxa2p tatsächlich Coenzym A Ester langkettiger Fettsäuren transportiert. / The peroxisomal ABC-transporters Pxa1p and Pxa2p are half transporters. Previous genetic investigations have demonstrated that Pxa1p and Pxa2p have to dimerise in order to build a functional transporter, which is very likely involved in the import of long chain fatty acids into peroxisomes of S. cerevisiae. In this work, tagged versions of the proteins were purified as a complex. This proved for the building of a stable hetero dimer. For characterisation of the ATP binding properties, the transporters were incubated and cross linked with 8-azido-[alpha-32P]-ATP. This revealed an asymmetric binding of the ATP analogue. Pxa2p binds much more azido-ATP, than Pxa1p, while the dissociation constants are rather similar. The poorer ATP binding of Pxa1p is reflected by degenerated sequence motifs in the nucleotide binding fold. The purified ABC-transporters have been used for ATPase assays. They showed a basal ATPase activity, which could be stimulated by addition of long chain fatty acid CoAs, like oleoyl-CoA and palmitoyl-CoA. Mutants with a lysine mutation in the walker A motive of Pxa1p led to no functional impairment, while the corresponding lysine mutation in Pxa2p led to reduced growth on agar plates with oleic acid as sole carbon source. The result corresponds with the ATP binding properties of Pxa1p. Because of the poorer ATP binding, even in the wild type protein, the mutation was not supposed to have a big influence. No accordance was found in respect to the ATPase measurements of the isolated mutant proteins. Both mutants revealed unaffected ATPase activity. The purified ABC-transporters were reconstituted in proteoliposomes and used for translocation assays of a spin-labelled oleoyl-CoA derivative. The measurements revealed an ATP dependent transport of the oleoyl-CoA analogue. This led to the conclusion, that Pxa1p-Pxa2p is indeed the transporter of long chain acetyl CoA esters, which were transported in an ATP dependent manner.
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Substratbindung und -freigabe während des Katalysezyklus eines biotinspezifischen ECF-TransportersFinkenwirth, Friedrich 10 April 2017 (has links)
ECF (Energy-Coupling Factor)-Transporter sind prokaryotische Aufnahmesysteme für Mikronährstoffe, die eine spezielle Gruppe von Transportern mit ATP-Bindekassette (ABC) darstellen. Sie beinhalten zwei asymmetrische Membranproteine, von denen eins (S) für die spezifische Bindung und Translokation des Substrates und das andere (T) für die Kopplung mit den ATPasen (A1,A2) zuständig ist. Bei ECF-Transportern der Subklasse I bilden diese Komponenten eine Einheit, während bei Vertretern der Subklasse II ein AAT-Modul mit wechselnden S-Einheiten interagiert. In der vorliegenden Arbeit wurde der Transportmechanismus, der eine Drehung der kompletten S-Einheit in der Membran beinhaltet, anhand des Biotintransporters BioMNY erstmals experimentell validiert. Durch Rekonstitution in Lipid-Nanodiscs, chemische Quervernetzung, fluoreszenz- und ESR-spektroskopische Techniken sowie einen Bindungstest mit radioaktivem Biotin wurde gezeigt, dass (i) die ATP-Bindung an die ATPasen zu einer Aufrichtung der S-Einheit (BioY) führt, (ii) diese Bewegung die Substratbeladung ermöglicht und (iii) BioY dabei ununterbrochen mit der T-Einheit (BioN) interagiert. Dies stellt einen Gegensatz zu Systemen der Subklasse II dar, für die ein ATP-abhängiger Austausch von S-Einheiten im Transportzyklus gezeigt worden war. Darüber hinaus wurde ein Escherichia coli-Stamm konstruiert, der durch Blockierung seines hochaffinen Biotintransporters und des -synthesewegs auf Spuren von Biotin nicht wachsen kann. Dieser Stamm ermöglichte einen eindeutigen Nachweis der Transportaktivität einiger solitärer BioY-Proteine. Aufgrund der einheitlichen Topologie von S-Einheiten ist ein Kippen auch für solitäre BioY-Varianten wahrscheinlich. Auch die metallspezifischen S-Einheiten CbiM und NikM besitzen ohne AAT-Modul eine basale Co2+- bzw. Ni2+-Transportaktivität. Ein ESR-spektroskopischer Kobaltnachweises zeigte, dass die aus nur zwei Membranhelices bestehende CbiN-Einheit für die Metallbeladung von CbiM essentiell ist. / ECF (Energy-Coupling Factor) transporters are a subgroup of ABC transporters that mediate uptake of micronutrients into prokaryotic cells. In contrast to canonical ABC importers, ECF transporters comprise two unrelated membrane proteins, one of which is responsible for specific and high affinity substrate binding (S) and the other one constitutes the coupling component (T) between S and the cytosolic ABC-ATPases (A1,A2). Subclass I transporters consist of four dedicated components whereas in subclass II transporters, a central AAT-module may interact with various S units. The biotin specific subclass I ECF transporter BioMNY was used to experimentally verify the hitherto hypothetic transport mechanism, which involves a rotation of the S unit within the membrane. With a series of experiments including reconstitution of BioMNY into lipid nanodiscs, site-specific cross-linking, a substrate binding assay with radioactive biotin and both fluorescence and EPR spectroscopic techniques, the ATP-dependent rotation of BioY (S) as a prerequisite for substrate binding and release was shown for the first time for an ECF transporter. Unlike subclass II transporters, for which an ATP-dependent release of the S unit was proposed, BioY interacts continuously with BioN (T) during the transport cycle. In a second focus of the work, an Escherichia coli reporter strain for biotin transporters was constructed. Due to inactivation of both biotin synthesis and the intrinsic high affinity biotin transporter, this strain was not capable of growing on trace amounts of biotin. With the use of this strain, transport activity of recombinantly produced solitary BioY proteins that naturally lack other ECF components was evidenced. Transport activity in the absence of AAT modules is also a feature of the Co2+ and Ni2+ specific S components CbiM and NikM. An EPR spectroscopic Co2+ detection assay helped underscoring the essential role of the small membrane protein CbiN for Co2+ loading of CbiM.
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Modulation unterschiedlicher Formen der Multidrug-Resistenz mittels eines MultitargetmultiribozymesKowalski, Petra 27 July 2006 (has links)
Tumorzellen entwickeln häufig Resistenzen gegen verschiedene strukturell und funktionell unabhängige Zytostatika, was als Multidrug-Resistenz (MDR) bezeichnet wird und die Hauptursache für das Scheitern einer Chemotherapie ist. Mit Hilfe von in vitro-Untersuchungen wurden erhöhte Genexpressionen der ABC-Transporter MDR1, MRP2 und BCRP als maßgebliche Resistenzfaktoren identifiziert, wie z.B. in den Magenkarzinomzellinien EPG85-257RDB (MDR1) und EPG85-257RNOV (BCRP) sowie in der Ovarialkarzinomzellinie A2780RCIS (MRP2). Ziel der Arbeit war die Entwicklung eines auf Ribozym-Technologie basierenden Therapieansatzes, welcher die Expressionen der oben genannten ABC-Transporter simultan inhibiert und dessen Anwendung zur Reversion der zellulären MDR führt. Dazu wurde ein Multitargetmultiribozym (MTMR) entwickelt, das aus in trans-aktiven Ribozymen besteht, die gegen die ABC-Transporter mRNAs gerichtet sind sowie aus in cis-spaltenden Ribozymen und aus Spacer-RNA-Sequenzen. Durch autokatalytische Spaltung in cis konnten die in trans-aktiven Ribozyme aus dem Gesamt-MTMR freigesetzt werden. Die Analyse der kinetischen Parameter des MTMRs ergab, daß die autokatalytisch entstandenen MTMR-Fragmente ihre Substrat-RNAs im Vergleich zu den korrespondierenden Monoribozymen ohne Einbuße an Effizienz spalten können. Darüber hinaus wurde die MTMR-Sequenz stabil in den drei eingangs genannten MDR-Zellinien exprimiert, was in einer signifikanten Reduktion der jeweiligen Ziel-mRNAs (97 % MDR1-, 80 % BCRP-, 96 % MRP2-mRNA) und der entsprechenden Proteine resultierte. Die Multidrug-Resistenz konnte aufgrund der MTMR-Expression um 70% (A2780RCIS), 95% (257RNOV), 100% (257RDB) und die Zytostatikumsakkumulation um 90% (257RNOV-Zellen) sowie 100% (257RDB-Zellen) revertiert werden. Das MTMR stellt erstmalig ein RNA-Konstrukt dar, welches in der Lage ist, simultan mehrere unabhängige Gene funktionell auszuschalten. Es besitzt daher ein großes Potential für zukünftige gentherapeutische Ansätze. / Cancer cells are often insensible against structurally and functionally unrelated drugs that is known as multiple drug resistance (MDR) and the main cause for treatment failure. Overexpression of the ABC-transporters P-gp (ABCB1), MRP2 (ABCC2), and BCRP (ABCG2) is associated with MDR in several cancer cell lines, e.g. in the stomach carcinoma cell lines EPG85-257RDB (P-gp), EPG85-257RNOV (BCRP), and in the ovarian carcinoma cell line A2780RCIS (MRP2). We aimed the development of a novel hammerhead ribozyme-based therapeutic approach capable of simultaneous silencing of the prementioned ABC-transporters, and consequently of reversing MDR phenotypes. We designed a so-called multitarget multiribozyme (MTMR) consisting of trans-acting hammerhead ribozymes directed against the MDR1, MRP2, and BCRP transcripts, of MDR1 homologous spacer sequences, and of cis-acting ribozymes against the spacer sequences. Autocatalytic cleavage in cis excised the full-length MTMR, and released trans-acting hammerhead ribozymes. We also evaluated the catalytic features of the MTMR using large RNA target molecules. Comparison of the kinetic values of the autocatalytically derived MTMR fragments with those of corresponding mono-ribozymes demonstrated an MTMR-mediated substrate cleavage without distinct loss in catalytic efficiency. Moreover, the MTMR was stably expressed in the prementioned multidrug-resistant cancer cell lines, and decreased the targeted transcripts about 97% (MDR1), 80% (MRP2), and 96% (BCRP) as well as the corresponding protein levels, respectively. Cellular MDR could be reverted about 70% (A2780RCIS), 95% (257RNOV), and 100% (257RDB). Additionally, the MTMR reversed mitoxantrone accumulation entirely, and daunorubicin accumulation about 90% in stomach carcinoma cells, respectively. Taken together, the MTMRs capability of simultaneous silencing of multiple genes provides an effective instrument to knockdown genes of interest.
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Integridade biótica dos ecossistemas na região do Arquipélago dos Alcatrazes, São Sebastião - SP / Biotic integrity from the ecosystems of the Alcatrazes archipelago surroundings, São Sebastião - SPHoff, Natasha Travenisk 23 June 2015 (has links)
A Estação Ecológica (ESEC) Tupinambás, uma UC marinha de proteção integral, está localizada no litoral norte do estado de São Paulo (SP). Foi criada em 1987, sendo utilizada pela Marinha do Brasil para exercícios de tiro até recentemente no arquipélago dos Alcatrazes (São Sebastião, SP). Isto justifica a baixa quantidade de informações disponíveis sobre essa região. Através do levantamento de informações sobre a composição biológica, aspectos socioeconômicos e oceanográficos da área do arquipélago, da utilização de índices ecológicos (dominância, diversidade, riqueza e equabilidade) e multimétricos (Índice ABC e Índice de Integridade Biótica), e da elaboração da Carta de Sensibilidade Ambiental ao Derramamento de Óleo, verificou-se (1) a semelhança da composição ictiofaunística do arquipélago dos Alcatrazes em relação à Santos e São Sebastião, (2) se a região mantém a integridade biótica da ictiofauna mesmo localizando-se entre regiões impactadas, e (3) se o ponto mais sensível ecologicamente detectado pela carta SAO pode ser a área de maior suscetibilidade de ser atingida por um derramamento de óleo. Os dados analisados são provenientes de trabalhos pretéritos e coletas realizadas em setembro de 2011 e janeiro de 2014. A composição ictíica variou ao longo do tempo e períodos de coleta, respondendo à composição sedimentar e à variabilidade das propriedades hidrográficas. Foi constatada alta similaridade da ictiofauna do arquipélago com aquelas encontradas na plataforma continental de Santos e São Sebastião. Em relação à integridade biótica local, a partir da ictiofauna, as duas técnicas aplicadas refletiram o histórico no estado de conservação da ESEC, com a melhora dos índices ao longo do período estudado. A produção da carta SAO resultou em um produto único, que poderá auxiliar a gestão da UC e em planos de contenção, e em um compêndio das espécies encontradas na região, dentre as quais se encontram algumas endêmicas e muitas ameaçadas, segundo critérios nacionais e internacionais. Finalmente, espera-se que a alta biodiversidade do entorno do arquipélago, que representa uma vasta fonte de conhecimentos para o futuro, se encontre protegida pelo respeito à legislação e fiscalização da ESEC em que se encontra, pela Área Delta da Marinha do Brasil, e pela distância da costa e de fontes de contaminação. / The Tupinambás Ecological Station (TES), a Marine Protected Area (MPA) of integral protection, is located on the northern coast of São Paulo state (SP). It was established in 1987 and it has been used by the Brazilian Navy for shooting practice until recently in the Alcatrazes archipelago (São Sebastião, SP). That accounts for the low amount of the available data in this region. Using information on the biological composition, socioeconomic and oceanographic aspects variables, combined with ecological (dominance, diversity, richness and evenness) and multimetric (ABC method and Biotic Integrity Index) indexes and the preparation of the Environmental Sensitivity Index (ESI) Map, we (1) assessed the similarity of the ichthyofauna of the Alcatrazes archipelago regarding compared to Santos and São Sebastião, (2) verified whether the region retains the ichthyofauna biotic integrity despite being located between affected regions and (3) verified whether the most ecologically sensitive region detected by the ESI map is the area most likely to be hit by an oil spill. The analyzed data come from previous works and samplings carried out in September 2011 and January 2014. The ichthyofauna varied over time and sampling points. Its distribution also reflected the sedimentary composition and the variability of hydrographic properties. High similarity between the ichthyofauna of the archipelago and those found on the continental shelf off Santos and São Sebastião. Regarding the biotic integrity, the two techniques applied reflected the conservation history of the TES, with an improvement in the indexes during the study period. The production of the ESI map resulted in an unique product that can assist in the management of the MPA and containment plans, and a species compendium found in the region, among which many of them are endemic and endangered, according to national and international classifications. Finally, it is expected that the high biodiversity of the archipelago surroundings, which still represents a vast source of knowledge for the future, is protected by the Delta Area of the Brazilian Navy, by the law that ensure the TES and by the distance from the coast.
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Développement d’un modèle in vitro de Barrière Hémato-Encéphalique humaine pour des études pharmacologiques : Interactions avec les anticoagulants oraux directs / Development of an in vitro model of a human Blood Brain Barrier for pharmacological studies : Interactions with directs oral anticoagulantsPuech, Clémentine 13 December 2018 (has links)
La barrière hémato-encéphalique (BHE) contrôle le passage des médicaments, en partie par la présence d’ATP Binding Cassette (ABC) transporteurs. Dans de nombreuses pathologies cérébrales, la BHE est altérée. Parmi elles, les hémorragies intracérébrales (HIC), qui sont un effet iatrogène des anticoagulants. Des analyses cliniques montrent que les patients sous Anticoagulants Oraux Directs (AODs) présentent moins d’HIC que les patients traités avec les anticoagulants de référence, les anti-vitamine K (AVK), sans que les mécanismes cellulaires soient élucidés. Une des différences entre les AODs et les AVK résident dans leur profil pharmacocinétique, effectivement, les AODs sont des substrats des ABC transporteurs contrairement aux AVKs. Au cours des HIC, la thrombine est activée et entraine une altération de la BHE par clivage et des récepteurs protease activated receptor (PAR). Les objectifs de ce travail de thèse ont été de mettre en place un modèle in vitro de BHE afin d’étudier les interactions des médicaments avec les ABC transporteurs. Ensuite, le modèle est utilisé pour étudier les interactions des AODs en condition pathologique. Le modèle développé est basé sur la lignée HBEC-5i, peu décrite dans la littérature. Les cellules ont été cultivées en monocouche sur insert avec milieu conditionné issu d’astrocytes humains. Le modèle permet l’étude de l’interaction de thérapeutiques avec des ABC transporteurs par des mesures d’efflux ratios. Le modèle a été validé par des études de transport de molécules pharmacologiques. Ensuite, nous avons comparé, sur notre modèle, les effets de l’exposition à la thrombine avec ou sans prétraitement d’anticoagulants (rivaroxaban, dabigatran, apixaban, warfarine et héparine). Les AODs limitent l’ouverture de la BHE induite par la thrombine contrairement aux autres anticoagulants. Nos résultats ont montré que l’altération de la BHE est médiée par le clivage du récepteur PAR-1 par la thrombine. Ce clivage n’est pas le même en fonction de la classe d’anticoagulants utilisée, les AODs minimisant ce clivage. L’ensemble de ce travail de thèse a permis de donner des premières explications cellulaires quant aux mécanismes d’ouverture de la BHE consécutifs aux HIC sous AODs. / The blood-brain barrier (BBB) controls the passage of drugs, in part through the expression of the ATP Binding Cassette (ABC) transporters. In many brain diseases, the BBB is altered. Among them, intracerebral haemorrhages (ICH), which are an iatrogenic effect of anticoagulants. Clinical analyses show that patients with Direct Oral Anticoagulants (DOACs) treatment have less HIC than patients treated with the reference anticoagulants, Vitamin K Antagonist (VKA), without understanding the cellular mechanisms. One of the differences between DOACs and VKA lies in their pharmacokinetic profile, indeed, DOACs are substrates of ABC transporters unlike VKA. During HIC, thrombin, is activated and causes alterations in the BBB by the cleavage of the protease activated receptor (PAR). The objectives of this thesis work were first to set up an in vitro model of the BBB in order to study the passage of drugs and their interactions with ABC transporters. In a second step, the model is used to study the interactions of DOACs in pathological conditions. The model developed is based on the HBEC-5i cell line seldom described in the literature. The cells were cultured in monolayer on insert with conditioned medium from human astrocytes. It allows the study of the interaction of therapeutics with ABC transporters by measuring efflux ratios. The model has been validated by transport studies of pharmacological molecules. In order to meet our second objective, we compared the effect of thrombin exposure with or without pretreatment with anticoagulants (rivaroxaban, dabigatran, apixaban, warfarin and heparin sodium) on our model. DOACs limit the BBB damage induced by the thrombin unlike other anticoagulants. Our results showed that alteration of the BBB is mediated by the cleavage of the PAR-1 receptor by thrombin. This cleavage is not the same depending on the class of anticoagulants used, DOACs minimizing this cleavage. All this thesis work made it possible to provide the first cellular explanations of the opening mechanisms of the BBB following HIC under DOACs.
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