Background: The company has had a low outgoing delivery precision for a prolonged period. This creates problems as orders must be prioritized in order to be produced on time and partial deliveries are used to satisfy customers. The origin of the problem is unknown to the company, but there are suspicions that the problems arise in production. Purpose: The purpose of the study is to contribute to an improved outgoing delivery precision for Företag X. A current state analysis from the inbound to the outbound flow is conducted to find causes of delays. The study creates a prioritization basis for the company to identify the most critical article groups. This creates a tool for decision support, linked to delivery precision, for manufacturing companies. Methods: This study is a case study at Företag X. The study uses secondary data in the form of quantitative data from the company's business system, but primary data in the form of interviews and the author’s own observations. The data is used to create a cause and effect diagram and to perform an ABC/XYZ-analysis on the company's inventory that forms the basis for segmentation of the articles. Conclusions: The study's conclusion is that Företag X's low outbound delivery precision is due to several reasons such as communication gaps, insufficient production planning and measurement & variation in load. The most important cause is production planning. In order to improve this, other causes must also be solved first and Företag X must follow the priority order that the study has developed. Through this, Företag X can get a better outbound delivery precision and a more efficient flow. Keywords: Delivery precision, production planning, ABC/XYZ-analysis, classification, cause and effect diagram

På grund av rådande livsmedelstrender har efterfrågan på premiumvaror, som ekologiska och kolhydratfattiga livsmedel, ökat. Trenderna sätter tryck på livsmedelsföretag att erbjuda ett brett utbud av produkter. Detta får till följd att behovet av effektiva försörjningskedjor ökar samtidigt som det bundna kapitalet behöver minskas för att få ökad lönsamhet. För att identifiera var slöserier som driver upp ledtiden finns i ett produktionsflöde, kan värdeflödesanalys användas. Genom användning av värdeflödesanalys kan värdeskapande tid ställas mot icke värdeskapande tid för att upptäcka moment som inte tillför värde för kunden utan endast driver upp kostnader. Analys av dessa moment kan leda till identifiering av möjligheter för reduktion av kapitalbindning i form av minskade nivåer av produkter i arbete och lager. Kapitalbindning kan även reduceras genom ABC-klassificering av artiklar. Syftet med detta kandidatexamensarbete är att undersöka reduktion av ledtider i Lantmännen Cerealias pastafabrik med hjälp av värdeflödesanalys. Utöver detta undersöks hur kapitalbindningen kan reduceras samtidigt som ledtidsreduktionen genomförs. Slutligen ABCklassificeras produkterna. Dels för att minska kapitalbindningen, dels för att underlätta vid fluktuationer i efterfrågan hos fabriken. Slutsatsen svarar på om det är möjligt att reducera ledtiden i fabriken samtidigt som en reduktion av kapitalbindning görs. Värdeflödesanalys visade sig vara ett bra verktyg för att identifiera eventuella slöserier och förbättringsmöjligheter i produktionen. Svårigheten med analysen i detta fall låg i att finna korrekta siffror till analysen eftersom data för detta mottogs i sin helhet och ej mättes upp i fabriken. ABC-klassificeringens valda parametrar visades lämpliga då uppdelningen blev tydlig. Denna tog hänsyn till problem både rörande efterfrågefluktuationer och kapitalbindning. Slutsatsen drogs att om lagerhållningen styrs enligt denna uppdelning kan kapitalbindningen minskas samtidigt som företaget enklare kan möta fluktuationer i efterfrågan. Den reducerade ledtiden som värdeflödesanalysen resulterade i, gav effekten att medel-PIAnivån i produktionen kunde minskas. Detta skulle kunna ske genom att minska mellanlagringstiden i silos. Möjlighet saknas att på kort sikt reducera medel-PIA-nivån till den kritiska volymen av PIA, dock leder den möjliga minskningen till en reduktion av kapitalbindning. Den viktigaste slutsatsen som dras är att reduktion av ledtid ofta har en positiv inverkan även på kapitalbindningen. I detta fall visade sig lagerhållning ha stor betydelse för reduktion av både ledtid och kapitalbindning. Att reducera lagringstiden i färdigvarulagret ger större effekt på kapitalbindningen än en reduktion av mellanlagringstiden i silos då produktens värde är större i detta skede / Due to the prevailing food trends, customers demand premium goods such as organic and low carb products in addition to simpler alternatives. The trends puts pressure on food businesses to offer a wide selection of products, which leads to an increasing need for efficient supply chains as well as decreases in capitalization. Value stream mapping is a tool used to identify wastes. These wastes increases lead times in a production flow. The results gathered from the analysis can be used to compare value adding time with non-value adding time, in order to discover processes that drives up expenses without adding value for customers. Detailed analysis of these processes can identify possible reductions in capitalization, through reduced levels of work in process and stock. Capitalization can also be reduced by ABC classification of products. The purpose of this thesis is to examine how value stream mapping can help reduce lead times at Lantmännen Cerealias pasta production plant in Järna. Reduction of capitalization through reducing the amount of work in progress and stock is also studied. Furthermore, the effects of ABC classification are examined in order to ease fluctuations in demand and reduce capitalization at the plant. The conclusion answers whether or not it is possible to reduce the lead time in the production and simultaneously reduce capitalization. Value stream mapping proved to be an efficient tool to identify possible sources of waste and improvements in the production. In this case, the absence of accurate numbers made the analysis challenging and led to inaccurate results. The parameters used for the ABC classification led to a proper partition of products since it took both fluctuations in demand and problems regarding capitalization into account. This partition of products is assumed to reduce both capitalization and ease the effects of fluctuations in demand. Reducing the storage time of WIP using value stream mapping can result in reduced levels of WIP. Reducing WIP to the lowest possible level proved to be impossible in the short term. However, the possible reduction of WIP levels would result in reduced capitalization. The most important conclusion drawn from the analysis, is that a reduced lead time often has a positive impact on capitalization. Transformation of stockkeeping proved to be of great importance when reducing both lead times and capitalization. Since the value of finished goods are higher than the value of WIP, reducing storage time for inventory of finished goods proved to have a greater impact on capitalization.

Damasteel är ett företag som tillverkar damaskusstål genom en process av pulvermetallurgiståltillverkning som sker med hjälp av en gasatomiseringprocess. Då Damasteels verksamhet växer har företaget köpt en ny lokal nära produktionsanläggningen. Avsikten är att den nya lokalen ska ersätta det nuvarande mellanlagret. Därför har den byggts om och är nu utrustad med större lagringsutrymme och hanteringsutrustning än det befintliga mellanlagret. I det nuvarande mellanlagret finns detproblem med artikelplacering. Artiklarna blir placerade utan hänsyn till frekvensläggning. Det finns också ett problem med att de olika artiklarna placeras på fel platssom medför att arbetarna kan smida fel sorts stål till order. Det kan enbart upptäckas efter bearbetningen av materialet. Syftet med arbetet är således att undersökahur det nya mellanlagret kan möjliggöra effektivare materialhantering, materialflödeoch spårbarhet. Målet med arbetet är att utveckla och bereda artikelplacering utifrånfrekvensläggning och förbrukning. Ett märksystem ska implementeras för effektivbatchidentifiering, spårbarhet och kortbitshantering.För att uppnå arbetets mål och syfte genomfördes en litteraturstudie om materialflöde, arbetssättet i tillverkande företag och ABC-klassificering. Med hjälp av informationen från litteraturstudien kunde nulägeanalys utföras genom analys av nuvarande samt nya mellanlagret. Genom dubbel ABC-klassificering kunde de viktiga artiklarna uppmärksammas men även upptäckas. I enlighet med ABC-klassificeringenkunde ett kortsiktigt förbättringsförslag utvecklas, vilket består av strategisk artikelplacering och märkning på stängerna och grenställen. Detta för att möjliggöra effektivt materialflöde och mindre påfrestningar för arbetarna. Ett långsiktigt förbättringsförslag innefattar ett paternosterverk för långgods som skulle kunna medföraeffektivt utnyttjande av lagerutrymmet. Paternosterverk skulle sedan kunna kompletteras med ett insticksställage om behovet finns.Artikelklassificering genomfördes med förbruknings- och uttagsfrekvensdata frånåret 2021 eftersom datan från tidigare år inte kunde inhämtas. Resultaten från ABCanalysen kunde ha blivit annorlunda om data från flera år använts. Med hjälp av artikelklassificering kan storleken på säkerhetslagret och kapitalbindning minskas, vilketkan möjliggöra bättre utnyttjande av lagerutrymmet. Nulägeanalysen kan möjligtvisvara bristfällig då författaren saknar tidigare erfarenhet inom lagerstyrning. Artikelplaceringen kan utvärderas i ett senare skede när flera års data kan inhämtas. Livstidskostnaden för paternosterverk i förhållande till företagets behov och omsättningbör undersökas. / Damasteel manufactures damascus steel using a process of powder metallurgy steelmanufacturing, which is done through a gas atomization process. Due to the growthof Damasteel's business, the company has purchased a new premises next to its production facility. The intention with new premises is that it will become the new intermediate warehouse and replace the current one. Therefore, it has been rebuiltand is now equipped with a larger storage space and handling equipment. In the current intermediate warehouse, there are problems with item placement, due to thembeing placed without regard to the frequency of their consumption. There is also anissue with various items sometimes being misplaced which leads to workers pickingthe wrong material for a certain order. This can only be detected after processingthe material.The purpose of this thesis is to investigate how the new intermediate warehouse canenable more efficient material handling, material flow and traceability. The objective of this thesis is to develop and conduct item placement based on frequencyplacement and consumption. A labelling system will be implemented for efficientbatch identification, traceability and short-bit sorting. To achieve the objective andpurpose of this thesis, a literature review was conducted on material flow, workingmethods in manufacturing companies and ABC-classification. With the help of theinformation produced from the literature review, an observation was able to be conducted. Which was then followed by a current situation analysis of the workingmethods within the current intermediate warehouse and the new intermediatewarehouse. Through the method of double ABC-classification, the higher ranked articles that should be paid attention, could be sorted. In accordance with the classification, a short-term improvement proposal could be produced. It consists of strategic article placement and labelling on the bars and cantilever racks to allow efficientmaterial flow and less stress for the workers. Long-term improvement proposalsconsist of long goods paternoster that would entail efficient storage space utilizationand then can be supplemented with a roll rack if it is considered necessary.Item classification was carried out with consumption and withdrawal frequency datafrom the year 2021, only because previous years data could not be obtained. TheABC-analysis results can be different if data consisting of several years is used. Withthe help of item classification, the size of safety stock and the tied-up capital can bereduced, which can enable better inventory space utilization. The current situationanalysis can be insufficient since the executor lacks previous experience in inventorymanagement. The item placement can be evaluated in later stages when data consisting of several year’s usage can be obtained. Lifetime cost of paternoster in relationto the needs and revenue of the enterprise should be investigated.

[pt] O presente texto visa discutir as transformações no mundo do trabalho e na classe trabalhadora, em especial durante a crise mundial capitalista dos anos 70/80. Entendendo o movimento punk como um importante elemento que pode nos dar pistas desse processo no âmbito da cultura. Nossa hipótese é que este movimento foi ao mesmo tempo um sintoma e um agente. Um sintoma de uma classe operária em desagregação: da transição do trabalho operário chão de fábrica como força hegemônica, para a hegemonia do setor terciário: do setor de serviços, da informalidade, do subemprego, do que alguns vão chamar de precariado urbano. No entanto, esse movimento também se estabelece como um agente ativo, em especial no que tange o debate em torno de uma agência em relação à disciplina do trabalho capitalista e no caso brasileiro, uma agência dos de baixo (por meio da arte, música, cultura) em oposição à ditadura-empresarial-civil-militar em sua etapa final. Focaremos para tal, no movimento da cidade de São Paulo e do ABC paulista, bem como sua relação com a classe operária de São Paulo. Para tanto, utilizaremos fontes primárias, por meio de elementos da História oral, entrevistando participantes do movimento punk paulista e do ABC. Recorreremos também aos arquivos sobre movimento punk do CEDIC (Centro de Documentação Científica da PUC SP), contendo fanzines, discos, correspondências, etc. Utilizaremos também, como ferramenta, a análise de canções e poesias punks do período, tendo como importante elemento conceitual as concepções de agência e experiência presentes na obra do historiador inglês Edward Palmer Thompson. Ainda como aporte teórico utilizaremos algumas produções de livros e artigos a respeito do punk no Brasil e no mundo. / [en] This text aims to discuss transformations in the world of work and the working class, especially during the capitalist world crisis of the 1970s and 1980s. Understanding the punk movement as an important element that can give us clues of this process within the culture. Our hypothesis is that this movement was both a symptom and an agent. A symptom of a disintegrating working class to the work of an urban precarious. However, this movement also establishes itself as an active agent, especially in what concerns the debate around an agency in relation to the discipline of capitalist labor. We will focus on the movement of the city of São Paulo and the ABC of São Paulo, as well as its relationship with the working class of São Paulo. To do so, we will use primary sources, through elements of oral history, interviewing participants of the São Paulo punk movement and ABC. We will also visit the archives about punk movement of CEDIC (Center of Scientific Documentation of PUC SP), containing fanzines, discs, correspondences, etc. We will also use, as a tool, the analysis of punk songs and poems of the period, having as important conceptual element the conceptions of agency and experience present in the work of the English historian Edward Palmer Thompson. Still as a theoretical contribution, we will use some productions of books and articles about punk in Brazil and in the world.

Circular product flows are found as a business model in several companies and are characterized by the rental of various products. As a result of the products being returned, it creates changes in what can be regarded as a classic linear product flow. The study aims to evaluate the management of rental products at a specific case company and evaluate the advantages and disadvantages of selected methods when applied to a circular product flow. The case company with which the study was conducted is Hjälpmedelscentralen Region Västernorrland, which has the task of providing technical aids to individuals with special needs. The methods used to fulfill the purpose are Value Stream Mapping, ABC-XYZ classification and DEA. By applying the methods, it makes it possible to evaluate the efficiency of the case company and the advantages and disadvantages of the methods when applying to circular product flows. The Value Stream Mapping resulted in four potential future states and that a non-value-adding process could be identified. The ABC-XYZ classification shows that about 5 percent of the articles have a high demand and a low variation while about 58 percent have a low demand and a low variation. Based on the DEA analysis, Hjälpmedelscentralen is currently 75 percent efficient in relation to the potentially most efficient state. In evaluation of the studied models, it can be stated that the choice of parameters for ABC-XYZ classification on a circular product flow differs from the most common parameters used in ABC-XYZ. Furthermore, it appears that VSM is not adapted for circular product flows, which contributes to a source of uncertainty in the DEA. / Cirkulära produktflöden återfinns som affärsmodell hos flera företag och kännetecknas av uthyrning av diverse produkter. Till följd av att produkterna returneras skapar det förändringar i vad som kan anses som klassiska linjära produktflöden. Studien syftar till att utvärdera hanteringen av uthyrningsprodukter hos ett specifikt fallföretag samt utvärdera valda metoders för- och nackdelar vid applicering på ett cirkulärt produktflöde. Fallföretaget som studien är genomförd hos är Hjälpmedelscentralen Region Västernorrland, som har till uppdrag att tillhandahålla tekniska hjälpmedel till individer med speciella behov. Metoderna som används för att uppfylla syftet är värdeflödesanalys, ABC-XYZ klassificering samt DEA. Genom att tillämpa metoderna möjliggör det att utvärdera fallföretagets effektivitet och metodernas för- och nackdelar vid applicering på cirkulära produktflöden. Värdeflödesanalysens resultat är fyra potentiella framtida tillstånd och att en icke-värdeskapande process kan identifieras. ABC-XYZ klassificeringen påvisar att cirka 5 procent av artiklarna har en hög efterfrågan och en låg variation medan cirka 58 procent har en låg efterfrågan och en låg variation. Utifrån DEA-analysen är hjälpmedelscentralen i dagsläget cirka 75 procent effektiv i förhållande till det potentiellt effektivaste tillståndet. Vid en utvärdering av studiens modeller går det att konstatera att val av parametrar för ABC-XYZ klassificering på ett cirkulärt produktflöde skiljer sig från de vanligast förekommande parametrarna som används vid ABC-XYZ. Vidare framgår det att VSM inte är anpassad för cirkulära produktflöden, vilket bidrar till en osäkerhetskälla i DEA.

Multidrug Resistenz (MDR), die simultane Resistenz gegenüber strukturell und funktionell nicht-verwandten Zytostatika, stellt eine wesentliche Ursache für unzureichende Behandlungserfolge maligner Erkrankungen dar. Die inherente Resistenz bzw. Resistenzentwicklung gegenüber chemotherapeutischen Substanzen ist vor allem die Folge der Präsens und Regulation unterschiedlicher Transportproteine wie MDR1, MRP1, BCRP und MVP. In der Konsequenz kommt es zu alteriertem Influx und/oder Efflux von Zytostatika, verminderter Akkumulation und Effektivität von Chemotherapeutika. Sowohl Zytostatika als auch Zytokine zeigten modulierende Einflüsse auf die Expression der MDR-Gene MDR1, MRP1 und MVP (Kapitel 2-9). Zytostatika wie Adriamycin resultierten vorwiegend in induzierten MDR1-Expressionen, dem hauptsächlichen Interventionstarget zur Überwindung des klassischen MDR-Phänotyps. Zytokine wie TNFa führten, extern appliziert als auch durch Gentransfer, zur Chemosensitivierung der Tumorzellen, verbunden mit Down-Regulationen von MDR1 und MVP. Die Zytokin-vermittelte Überwindung des klassischen MDR-Phänotyps weist auf die Inklusion definierter Zytokine in etablierte Chemotherapieprotokolle hin, wie bereits angewendet bei der hyperthermen isolierten Extremitätenperfusion mit TNFa (Kapitel 13). Die Verwendung BCRP-spezifischer Ribozyme demonstrierte deren Potential zur Überwindung des BCRP-bedingten, atypischen MDR-Phänotyps. Darüber hinaus wurde gezeigt, dass die Expression der ABC-Transporter als auch des MVP durch Hyperthermie temperatur- und zeitabhängig induzierbar ist (Kapitel 10-13). Diese Hyperthermie-Induktion wird für MDR1 und MRP1 über den Transkriptionsfaktor YB-1 zeitnah zum Stressereignis vermittelt. In der klinischen Situation konnte anhand verfügbarer Biopsien von Kolonkarzinomen, Sarkomen und Melanomen, jeweils mittels Hyperthermie im Kontext multimodaler Behandlungsregime behandelt, kein direktes, generelles Risiko einer MDR1- oder MRP1-vermittelten, Hyperthermie-bedingten Induktion/Verstärkung einer MDR beobachtet werden. Die Analyse der Promotoren MDR-assoziierter Gene wie MDR1 und MVP zeigte deren Induzierbarkeit durch unterschiedliche Therapie-relevante Faktoren wie Zytostatika und Hyperthermie in verschiedenen in vitro- und in vivo-Modellen (Kapitel 10,14-20). Spezifische Sequenzmotive sind für die Stressfaktor-induzierte Bindung von Transkriptionsfaktoren wie YB-1 verantwortlich; Mutationen in diesen Sequenzbereichen modulierten die Induzierbarkeit (Kapitel 14,15,20). Der Einsatz Therapie-induzierbarer Promotoren unterschiedlicher MDR-Gene wie MDR1 (Kapitel 14-18) und MVP (Kapitel 19,20) erlaubt somit generell die Anpassung an etablierte Behandlungsprotokolle verschiedener Tumorentitäten. In fortführenden Arbeiten bleibt die erfolgreiche Anwendung von Therapie-induzierbaren MDR-Promotorsequenzen zur Expression therapeutisch relevanter Gene im Kontext einer Gentherapie maligner Erkrankungen zu prüfen. / Multidrug resistance, the simultaneous resistance towards structurally and functionally unrelated cytostatic drugs, still represents a major cause of cancer treatment failure. Inherent or acquired resistance against a wide variety of chemotherapeutic drugs depends mainly on the presence and regulation of different transporter proteins, such as MDR1, MRP1, BCRP, and MVP. Thus, decreased uptake and/or increased efflux, lowered net accumulation, and in consequence, less efficiency of anti-cancer drugs is the clinical hurdle to struggle with. Cytostatics as well as cytokines showed modulating effects on the expression of the MDR-associated genes MDR1, MRP1, and MVP (chapter 2-9). Cytostatics such as adriamycin resulted mainly in increased expression of the MDR1 gene, the most prominent intervention target for the reversal of the classical MDR phenotype. Cytokines such as TNFa, externally applied or by gene transfer, led to chemosensitization of tumor cells, and to down regulation of MDR1 and MVP. This cytokine-mediated reversal of the classical MDR phenoype refer to the inclusion of defined cytokines into established chemotherapy protocols, as already realized by the hyperthermic isolated limb perfusion with TNFa (chapter 13). The employment of BCRP-specific ribozymes demonstrated their potential to reverse the BCRP-mediated atypical MDR phenotype. Furthermore it was shown, that the expression of the ABC transporters as well as of MVP was inducible by hyperthermia in a temperature and time-dependet manner (chapter 10-13). This hyperthermia-caused induction of MDR1 and MRP1 is mediated by the transcription factor YB-1 timely close to the stress event. However, no direct, general risk of a MDR1- or MRP1-mediated hyperthermia-caused induction/enhancement of the MDR phenotype was observed in clinical settings, analyzed by using biopsies available from colon carcinomas, sarcomas, and melanomas, which were treated with hyperthermia in the context of multimodal regimes. The analyses of promoters of the MDR-associated genes MDR1 and MVP revealed their inducibility by different therapy-related factors such as cytostatics and hyperthermia in several in vitro- and in vivo models (chapter 10,14-20). Specific sequence motifs were found to be responsible for the stress-induced binding of transcription factors; mutations within these sequence regions modulated their inducibility (chapter 14,15,20). Thus, the employment of therapy-inducible promoters of different MDR genes such as MDR1 (chapter 14-18) and MVP (chapter 19,20) allows the improvement of established treatment protocols for different tumor localizations. Based on this, the succesful use of therapy-inducible MDR promoter sequences for the expression of therapeutically relevant genes in the context of a gene therapy of cancer represents an ambitious goal for the future.

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.

Energy-Coupling-Factor (ECF)-Transporter sind Aufnahmesysteme für Vitamine und Übergangsmetallkationen in Prokaryoten. Sie bestehen aus den zwei unverwandten Membranproteinen S und T sowie einem Paar ABC-ATPasen (A). Die S-Einheit vermittelt die Substratspezifität. Die Kombination aus der T- und den A-Einheiten wird als ECF bezeichnet. In dieser Arbeit wurden Fragen zur kontrovers diskutierten Stöchiometrie der Untereinheiten von ECF-Transportern sowie zur zuvor postulierten Substrattransport-Funktion einzelner S-Komponenten auch ohne ECF untersucht. Dazu wurden der ECF-Biotintransporter BioMNY, mehrere natürlicherweise in Organismen ohne ECF existierende biotinspezifische S Einheiten (BioY) sowie zwei Vertreter der metallspezifischen ECF-Systeme genutzt. Die S-Einheit BioY des dreiteiligen Biotinimporters lag in vitro als Monomer und Dimer vor. Oligomeres BioY wurde außerdem in lebenden Bakterienzellen beobachtet. „Pull-down“-Experimente zeigten, dass die T Komponente BioN im BioMNY-Komplex zum Teil als Dimer vorlag. Wachstumsuntersuchungen bestätigten die Transportfunktion von acht solitär vorkommenden BioY. Die in vitro auch für diese BioY-Proteine nachgewiesene Dimerisierung könnte die Transportfunktion von BioY ohne ECF erklären. Die metallspezifischen S Einheiten CbiM/NikM interagieren mit für die Transportfunktion essentiellen, zusätzlichen Transmembranproteinen (N) und zeichnen sich durch eine Topologie mit sieben Transmembranhelices und einem extrem konservierten, weit in das Proteininnere hineinragenden N-Terminus aus. Die Metallbindestelle besteht aus vier Stickstoffatomen von Met1, His2 und His67 und wird durch ein Netz aus Wasserstoffbrückenbindungen stabilisiert. Die Transport¬funktion von CbiMN bzw. Nik(MN) ohne ECF wurde in vivo mittels des nickelabhängigen Enzyms Urease als Indikator für die intrazelluläre Nickelkonzentration verifiziert. Zum gegenwärtigen Zeitpunkt ist die Funktion der für den Transport essentiellen N-Komponente jedoch noch unklar. / Energy-coupling factor (ECF) transporters are uptake systems for vitamins and transition metal cations in prokaryotes. They consist of the two unrelated membrane proteins S and T, and a pair of ABC ATPases (A). The S unit mediates substrate specificity. The combination of the T and the A units is called ECF. In this thesis the controversially discussed stoichiometry of the subunits of ECF transporters and the postulated substrate transport function of solitary S units without ECF were analysed. For this purpose, the biotin-specific ECF transporter BioMNY, several biotin-specific S units (BioY) encoded in organisms lacking any recognizable ECF and two metal-specific ECF transporters were used. The S unit BioY of the tripartite biotin importer existed in vitro as monomer and dimer. Furthermore, oligomeric BioY was observed in living bacterial cells. Oligomerisation of a part of the T unit BioN in the BioMNY complex was shown by “pull-down”- experiments. Growth analyses confirmed the transport function of eight solitary BioY proteins. The dimerisation, also proved for these solitary BioY proteins in vitro, could be an explanation for the transport function of BioY without ECF. The metal-specific S units CbiM/NikM interact with additional and for the transport function essential transmembrane proteins (N). The S units consist of seven transmembrane helices and an extremely conserved N-terminus, which extends deeply into the protein. The metal-binding site consists of four nitrogen atoms from Met1, His2 and His67 and is stabilised by a series of hydrogen bonds. The transport function of CbiMN and Nik(MN) without ECF was verified respectively in vivo using the nickel-depending enzyme urease as an indicator for intracellular nickel concentration, respectively. However, the role of the N component, which is essential for transport activity, is currently under investigation.

Acarbose hat als Inhibitor von Hydrolasen alpha-1,4-glykosidischer Bindungen medizinische Bedeutung. Das Acarbose-Biosynthese-Gencluster (acb) des grampositiven Produzenten Actinoplanes sp. wurde identifiziert und Genprodukte z. T. charakterisiert. Das Modell zum Acarbose-Metabolismus beschreibt einen Acarbosekreislauf, bei dem das Pseudotetrasaccharid als Carbophor fungiert. Das Molekül wird in das umgebende Medium abgegeben und durch das Zusammenwirken zweier extrazellulärer Enzyme nach Stärkehydrolyse mit einer unterschiedlichen Anzahl an Glukosemonomeren beladen. Nach dem vermuteten Re-Import über ein Bindeprotein-abhängiges ABC-Transportsystem AcbHFG stünde dem Organismus dann ein Gewinn an Glukosemolekülen zur Verfügung. Neben diesem Vorteil gegenüber Nahrungskonkurrenten im Habitat fungiert Acarbose ebenso als Hemmer der artfremden extrazellulären a-Amylasen. Die ökologische Funktion des Pseudotetrasaccharids wurde durch Untersuchungen zum Einfluss auf den Maltodextrin-Stoffwechsel von E. coli verifiziert und ausgeweitet. Es lässt sich ein ökonomisch sinnvolleres Konkurrenzverhalten von Actinoplanes sp. ableiten. Von den durch den Acarboseproduzenten selbst bereitgestellten Maltosacchariden aus Stärke profitieren artfremde Mikroorganismen nicht, da neben den Exoenzymen auch die Maltodextrin-Aufnahmesysteme in ihrer Funktion gehemmt sind. Außerdem wurde eine für Actinoplanes sp. geforderte Kapazität zur Aufnahme von Maltose und Maltodextrinen in vivo gefunden und in Transportexperimenten mit radioaktiv markierten Zuckern charakterisiert. Die Transportaktivität wird wahrscheinlich über zwei Bindeprotein-abhängige ABC-Importer mit multiplem Substratspektrum realisiert. Das ABC-Importsystem AcbHFG wurde heterolog in E. coli und S. lividans synthetisiert und z. T. erfolgreich gereinigt. In Substrat-Bindungsstudien konnte für das Bindeprotein AcbH eine Interaktion mit Acarbose und längerkettigen Derivaten, nicht jedoch mit Maltose/Maltodextrinen beobachtet werden. / Acarbose acts as an inhibitor of alpha-glucosidases and is therefore clinically used. The biosynthesis gene cluster (acb) was identified and partly characterized. The proposed model describes a pathway in which acarbose might function as a carbophor. The molecule is secreted into the medium where, after hydrolysis of starch, it is charged with additional glucose moieties. Re-uptake by a binding-protein dependent ABC importer AcbHFG would then result in a net gain of carbon and energy. Besides extracting glucose from the extracellular pool acarbose also acts as an inhibitor of alpha-amylases secreted by competitors in the natural environment. Prompted by the structural similarity between acarbose and maltotetraose, the effects of acarbose on the metabolism of maltose and maltodextrins in whole cells of E. coli and on individual components of the maltose / maltodextrin system were studied. The results demonstrate that acarbose is efficiently transported but not metabolized by E. coli due to its poor performance as a substrate of maltodextrin-degrading enzymes. Thus, besides acting as a carbophor acarbose also severely inhibits growth of competitors on maltodextrins. Actinoplanes using starch as carbon source should be able to import maltose and maltodextrins. Experiments with radioactive sugars indicate the action of two different binding-protein dependent ABC transport systems with a multiple substrate spectrum. Within the acb cluster a putative operon (acbHFG) encoding components of an ABC import system was found. To elucidate gene functions the products were overproduced in E. coli and S. lividans and some of the proteins were purified. Surface plasmon resonance analysis showed that the substrate binding protein AcbH binds acarbose and longer derivatives, but not maltose and maltodextrins.

ABC (ATP-binding-cassette)-Transporter katalysieren den ATP-abhängigen Transport diverser niedermolekularer Substanzen durch die biologische Zellmembran. Ihr Vorkommen erstreckt sich auf alle drei Domänen des Lebens. Der Maltose Transporter von E.coli gehört zu dieser Superfamilie der ABC-Transporter. Die Kristallstrukturen des Transporters MalFGK2 wurden kürzlich gelöst für dessen inaktiven Zustand als auch für dessen katalytischen Zwischenzustand. Um den Transportmechanismus besser verstehen zu können, müssen die Kristallstrukturen des Transporters und seiner Komponenten unter physiologischen Bedingungen genau geprüft werden, um den daraus katalytischen Mechanismus zu bewerten. Im rahmen der Dissertation konnte mittels Lösungs-NMR kann gezeigt werden, dass die periplasmatische Schleife P2 von MalF eine unabhängige Faltung aufweist und eine wohl definierte Tertiärstruktur einnimmt, die vergleichbar ist mit der im Kristall vorliegenden Konformation. MalF-P2 interagiert unabhängig von der Transmembranregion von MalF und MalG mit dem Maltose-Bindeprotein in An- und Abwesenheit des Substrats mit einem KD im mikromolaren Bereich. NMR Untersuchungen zu den an der Interaktion beteiligten Aminosäuren stehen in Einklang mit den Kristallstrukturdaten. Die Analyse residualer dipolarer Kopplungen (RDC) zeigt, dass die Konformation der zwei individuellen Domänen von MalF-P2 in Abwesenheit von MalE erhalten bleibt und der im Kristall ähnelt. Die Zugabe von MalE induziert eine Änderung der relativen Orientierung der zwei Domänen von MalF-P2 um so dem räumlichen Anspruch des Liganden gerecht zu werden. Besonders betroffen hiervon ist die Domäne 2 von MalF-P2, deren Konformation abweicht von der in der Kristallstruktur. Die Struktur der Domäne 1 dagegen bleibt konserviert, während sich lediglich ihre relative Orientierung zu Domäne 2 ändert. MD Simulationen des MalF-P2-MalE-Komplexes deuten auf eine stark dynamische Interaktion von MalF-P2 mit der MalE Bindungsregion hin. NMR CPMG Kinetikstudien weisen auf die Bildung eines ungewöhnlichen Knicks in alhpa-Helix alpha2 während der Assoziation hin. Diese konformelle Änderung der alpha-Helix findet auf einer Zeitskala von Millisekunden statt, was im Einklang mit der Austauschrate der Komplexbildung ist. / ABC (ATP-binding-cassette)-transporters catalyze the ATP-dependent transport of diverse solutes across the cellular membrane. They are present in all three kingdoms of life. The E.coli maltose transporter belongs to the ATP binding cassette (ABC) transporter superfamily. Recently, the crystal structures of the full transporter MalFGK2 in its resting and a catalytic intermediate state was solved. At the present state of research, it is of particular interest to scrutinize the X-ray structures of the transporter and its components under physiological conditions as well as to evaluate their implications for the catalytic mechanism. In the context of the PhD thesis, it could be shown using solution-state NMR that the periplasmic loop P2 of MalF folds independently in solution and adopts a well-defined tertiary structure, which is similar to the one found in the crystal structure. MalF-P2 interacts with the maltose binding protein, independent of the transmembrane region of MalF and MalG, with a KD in the µM range, in the presence and absence of substrate. NMR studies showed good agreement of the residues interacting in solution to those identified in the X-ray structure. Analysis of residual dipolar coupling (RDC) experiments shows that the conformation of the two individual domains of MalF-P2 is preserved in the absence of MalE, and resembles the conformation in the X-ray structure. Upon titration of MalE to MalF-P2, the two domains of MalF-P2 change their relative orientation in order to accommodate the ligand. In particular, a conformational change of domain 2 of MalF-P2 is induced, which is distinct to the conformation found in the X-ray structure. Domain 1 retains its structure but changes its relative orientation to domain 2. MD simulations of the MalF-P2 – MalE complex show a highly dynamic interaction of MalF-P2 to the MalE interface. From NMR CPMG kinetic studies, a peculiar kink of alpha-helix alpha2 can be seen introduced upon association. The transition time of this conformational change of the alpha-helix is on the ms timescale, which is matching the exchange rate of the complex formation.