Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Lenka Burgetová Supervisor: PharmDr. Martina Čečková PhD. Specialized supervisor: Dr. Kathryn Taylor PhD. Title of diploma thesis: Investigating the role of zinc transporter ZIP 6 and STAT3 in mitosis It has been shown that STAT3 (signal transducer and activator of transcription 3) plays a role in the development of cancer. ZIP6 is the downstream target of this transcription factor. Previous research has focused mainly on the activation of STAT3 by tyrosine phosphorylation, while the effect of phosphorylation at a second site, serine 727, remained relatively uninvestigated. In this study, it is proposed that serine-phosphorylated STAT3 is activated throughout mitosis in tamoxifen-resistant (TamR) breast cancer cells and that zinc transporter ZIP6 and serine-phosphorylated STAT3 are involved in a zinc-mediated mitotic mechanism. After using nocodazole to induce mitotic arrest, the expression of tyrosine- phosphorylated STAT3 protein was observed to be reduced while the expression of serine- phosphorylated STAT3 was increased. Zinc supplementation after nocodazole treatment appeared to push cells through mitotic-arrest and cause proteolytic cleavage of STAT3 suggesting a novel...

Within the world economy, the automobile industry is the largest industrial sector and has since a long time reached a level of overcapacity. The majority of components used in a vehicle is produced by subcontractors and the competition on the market is large. To survive on the market, the companies need to be competitive and often need to offer high quality products with low prices. There are a lot of methods to be more cost effective in an industrial environment and many companies’ choses to use the method Lean which focuses on minimizing waste. Internal transportation is one costs that does not increase the value of the product and is therefore seen as a waste. With this in mind it is important to optimize the internal transportations in order to lower the costs related to them. A case study has been performed at Gestamp Hardtech since they had identified shortcomings within their internal transportation. These are shortcomings such as low utilization of the forklifts and that the production is limited due to the fact that the drivers does not have time to perform their tasks on time. Further, Gestamp Hardtech also has a focus on reducing costs within their manufacturing which leads to their need to increase the utilization on the transportation and shortening the lead times. Having that in mind, the purpose of this study is to see how the utilization of the internal transportations can be increased and lead times can be reduced by an improved logistical organisation. The study is limited to studying the current logistical organisation at Gestamp Hardtech. Further, the study is also limited to current machine and stock positions. The study was started by illustrating the entire logistical organisation. That is, five different forklift areas in the factory. During the study the utilization of the frequently used forklifts were established. The utilization of the mainly used forklifts in the organization were around 50 percent of the total work-time and the utilization of the lesser used forklifts were lower then 10 percent. Further, the total time machines are forced to wait due to a forklift being late and how frequently tasks are performed together with how long time it takes to perform a task is established. To complement the data gathered from the case company two benchmarking studies were performed at two other manufacturing companies in the same branch. Analysis of the data collection showed that the forklift positions used ad the case company is based on geographical areas which are compressed. However, all of the forklift positions also includes trips which forces the driver to leave the main geographical area. Machines are forces to stand in order to wait for the forklifts but the utilization of the transportation is low. The fact that the drivers are leaving their main area does not conform with how the competitive companies organizes their internal transportation. These deviant routes also create a problem is one of the machines were to stop producing whilst the driver performed a task outside of the main area. Because of these two areas are combined and later divided into two new forklift positions. One of the forklifts are to handle the material which is connected to the machines and one forklift are to handle the material which is carried of the main area. In Gestamp Hardtechs case this is emptying scrap tubs, stock and package handling. In theory the forklift that are to handle the machines were to be utilized up to maximum 90 percent whilst the forklift handling deviating routes would be utilized to less than 50 percent. In reality, both of the forklifts would be utilized less than this which will enable for adding more tasks onto the deviating are. A suggestion is to ass other deviating routes from the other forklift positions or other types of scrap handling. / Inom världsekonomin är fordonsindustrin den största industriella sektorn och har sedan länge nått en nivå av överkapacitet. Majoriteten av de komponenter som används i ett fordon härstammar från underleverantörer och konkurrensen på marknaden är hög. För att överleva på marknaden krävs konkurrenskraft, ofta i form av låga priser och god kvalitet på produkten. Det finns många metoder för att kostnadsbespara i en industriell miljö och många företag väljer att använda sig av metoden Lean som fokuserar på att minimera slöseri i verksamheten. Interna transporter är en kostnad som inte ökar produktens värde och ses därför som ett slöseri. Av den anledningen är det viktigt att optimera och effektivisera för att kunna minska kostnader relaterade till interna transporter. En fallstudie har utförts vid Gestamp Hardtech eftersom de har identifierat brister inom deras interna transport. Detta är brister såsom låg utnyttjandegrad av truckarna samt att produktionen begränsas då truckförarna inte hinner utföra sina arbetsuppgifter i tid. Vidare har även Gestamp Hardtech stort fokus på kostnadsreduceringar i tillverkningen vilket leder till att de vill öka utnyttjandegraden maskinerna och minska ledtiderna. Av den anledningen var studiens syfte att utreda hur den interna transportens utnyttjandegrad kan ökas och ledtiden minskas med hjälp av en förbättrad truckorganisation. Studien avgränsades till att enbart studera den nuvarande truckorganisationen vid Gestamp Hardtech. Studien begränsas även till nuvarande lager- och maskinplaceringar i produktionen. Till en början studerades och illustrerades hela truckorganisationen, det vill säga fem olika truckområden i fabriken. Under studien fastställdes utnyttjandegraden på de maskiner som används frekvent. Utnyttjandegraden på de huvudsakliga truckarna i organisationen var ungefär 50 procent medan mindre belagda truckar hade en utnyttjandegrad på mindre än 10 procent. Vidare kontrollerades även den totala tiden maskiner står i väntan på truck samt arbetsuppgifternas frekvens tillsammans med tidsåtgången. Utöver detta utfördes även benchmarking vid två olika tillverkande företag för att fastställa hur de arbetar med truckorganisationer och truckpositioner. Analys av datainsamlingen visade att de truckpositioner fallföretaget använder baseras på ett begränsat geografisk område. Dock hanterar även samtliga positioner arbetsuppgifter som tvingar föraren att avvika från det huvudsakliga området.  Maskiner står i väntan på att bli betjänade av truck trots att utnyttjandegraden på resurserna är låg. Att truckförarna avviker från sitt huvudsakliga geografiska område överensstämmer inte med hur konkurrenterna organiserar sina interna transporter. Dessa avvikande rutter skapar även problem ifall en maskin skulle stanna i väntan på betjäning när föraren är utanför det geografiska området. Av den anledningen slås två närliggande truckområden samman för att sedan bryta ut de körningar som avviker från området. En truck hanterar allt material direkt mot maskiner på området och en annan truck hanterar materialhanteringen som avviker från området. I Gestamp Hardtechs fall handlar det om lager- och emballagehantering samt tömning av skrotbyttor. I teorin skulle trucken som hanterar maskinområdet maximalt beläggas till 90 procent medan trucken som hanterar avvikande körningar skulle beläggas till mindre än 50 procent. I verkligheten skulle båda truckarnas utnyttjandegrad bli lägre vilket gör det fullt möjligt att i framtiden addera arbetsuppgifter till avvikande truckområdet. Förslagsvis addera avvikande transporter från övriga truckområden eller annan form av skrottömning.

Die opportunistische Hefe Candida albicans ist in der Lage durch ein koordiniertes Zusammenspiel bestimmter zellulärer Eigenschaften sich unterschiedlichen Umweltbedingungen anzupassen und unterschiedliche Nischen innerhalb des menschlichen Wirts zu kolonisieren. Die Sekretion hydrolytischer Enzyme, wie Proteinasen und Phospholipasen, stellt eine wichtige Eigenschaft des Pilzes dar, die als wesentlicher Faktor für die Aufrechterhaltung der Pathogenität von C. albicans angesehen wird. Ein Schwerpunkt der hier vorliegenden Studie ist die funktionale Charakterisierung des caPLB5-Gens, eines neuen Mitglieds der insgesamt 5 Mitglieder umfassenden Phospholipase-B-Genfamilie. Im Gegensatz zu den gut untersuchten sekretorischen PLBs caPlb1p and caPlb2p scheint das caPlb5-Protein GPI-verankert und letztlich zellwandgebunden zu sein. Mittels Northernexpressions-Studien ließen sich in verschiedenen C.-albicans-Stämmen und unterschiedlichen Wachstumsbedingungen caPLB5-spezifische Transkripte nachweisen. Während des Hefe-Hyphe-Wechsels in Lee’s Medium zeigte sich interessanterweise eine differentielle Regulation der Gene caPLB5, caPLB1 and caPLB2. Durch Sequenzanalyse einzelner caPLB5-Allele konnte die Anwesenheit zweier unterschiedlicher Allele in C. albicans bei verschiedenen Stämmen nachgewiesen werden. Die gezielte Geninaktivierung beider Allele in zwei verschiedenen Stämmen resultierte in einer attenuierten Virulenz, was sich im Mausmodell für systemische Infektion anhand des Kolonisationsgrads des Wirtsgewebes messen ließ. Die Phänotypen sowohl der Nullmutanten als auch der caPLB5-Revertanten belegen, dass die Phospholipase B caPlb5p für die vollständige Virulenz des Pathogens benötigt wird und dabei eine Rolle bei der in vivo Organbesiedlung spielt. Diese Arbeit präsentiert zudem die Isolierung und Charakterisierung des ATP-Binding-Cassette-(ABC)-Transporter-Gens caMLT1 aus C. albicans. CaMlt1p zählt zur MRP/CFTR-Unterfamilie ATP-bindender Transportproteine, eine Proteinkategorie, die in diesem Pilz bislang noch nicht beschrieben wurde. Energiebetriebene Transportproteine der ABC-Superfamilie schleusen eine Vielzahl unterschiedlicher Substrate aktiv durch biologische Membranen und erfüllen dabei wichtige Funktionen im zellulären Metabolismus und in der Entgiftung. Das caMlt1-Protein zeigt hohe sequenzielle und strukturelle Ähnlichkeiten zu den vakuolaren Efflux-Pumpen Ycf1p und Bpt1p von S. cerevisiae. Durch genomische Markierung mit dem grün fluoreszierenden GFP-Protein konnte caMlt1p in der vakuolaren Membran lokalisiert werden. Northernblothybridisierungen belegten die Induzierbarkeit der Gentranskripte durch die metabolischen Gifte Cadmium und CDNB, beides Substrate der scYcf1-Pumpe. Obwohl diese Untersuchungen darauf hindeuten, dass caMlt1p ein Ortholog von scYcf1p sein könnte, zeigte sich bei dem Komplementationsversuch einer scycf1-negativen S.-cerevisiae-Mutante mit einer caMLT1-Genkopie keine Reversion des sensitiven Phänotyps gegenüber Cadmium oder CDNB. Auch wiesen die in dieser Arbeit konstruierten, camlt1-negativen Mutanten in C. albicans, die zur Identifizierung potentieller caMlt1p-Substrate eingesetzt wurden, keinen hypersensitiven Phänotyp gegenüber CdCl2, CDNB oder irgendeiner anderen getesteten inhibitorischen Substanz auf. CaMlt1p ist demzufolge kein funktionales Homolog von scYcf1p. Als vakuolar lokalisiertes Protein weist caMLT1 ein für diese Proteingruppe typisches Transkriptionsprofil auf. Die mRNA-Expression erfolgt dabei wachstumsphasenabhängig mit der höchsten Geninduktion während des Diauxic-Shifts, wenn ein Mangel an Glucose (und anderen Nährstoffen) im Medium entsteht. Eine generierte camlt1-Nullmutante war interessanterweise in einem murinen Peritonitismodell in ihrer Fähigkeit die Leber zu invadieren drastisch reduziert. Durch Reintegration einer funktionalen caMLT1-Genkopie konnte der Virulenzdefekt aufgehoben werden. CaMlt1p scheint in die Fähigkeit von C. albicans involviert zu sein an intestinale Organe adhärieren und Gewebebarrieren penetrieren zu können, möglicherweise durch Einbindung des Transporters in Stressantwort- und Detoxifikationsmechanismen. Beide Gene, caMLT1 und caPLB5, wurden auf zweierlei Weise inaktiviert: mittels einer klassischen Mutagenesemethode für C. albicans (dem URA3-Blaster-System im Ura--auxotrophen Stamm CAI4) und durch Entwicklung eines neuen dominanten Selektionssysstems. Die dominante Selektion basiert dabei auf der genomischen Insertion einer Einzelkopie eines mutierten caIMH3-Allels (MPAR), das Transformanten Resistenz gegenüber Mycophenolsäure (MPA) verleiht. Dieses System ermöglicht die genetische Manipulation von C. albicans Wildtypstämmen, wodurch die mühselige Konstruktion auxotropher und oft avirulenter Stämme nicht mehr nötig ist. / A coordinated interplay of certain traits enables the opportunistic yeast Candida albicans to adapt to different environmental conditions and to colonize different niches of the human host. Secretion of hydrolyzing enzymes, like proteinases and phospholipases, is an important characteristic of C. albicans which is considered to be integral to pathogenesis. This study focuses on the functional characterisation of the caPLB5 gene, a new member of the phospholipase B multigene family with five putative members. In contrast to the well characterized secretory PLBs caPlb1p and caPlb2p, the putative caPlb5-protein is likely to be GPI-anchored and ultimately bound to the cell wall. Northern expression studies showed caPLB5-specific transcripts in several strains of C. albicans under each growth condition tested. Interestingly, differential regulation of caPLB5, caPLB1 and caPLB2 could be detected during the yeast to hyphae transition in Lee’s medium. Sequence analysis of single caPLB5 allels resulted in the identification of two different alleles in several strains of C. albicans. The targeted gene disruption of both alleles in two different strains resulted in attenuated virulence as measured by host tissue colonization in a mouse model of systemic infection. The phenotypes expressed by null mutants and revertant strains of caPLB5 indicate that the phospholipase is required for complete virulence of this pathogen by playing a role for in vivo organ colonization. This study further presents the isolation and characterisation of the ABC-transporter gene caMLT1 in C. albicans belonging to the MRP/CFTR-subfamily of ATP-binding casette (ABC) transporters, a class of proteins so far not described in this fungus. Energy-driven transport proteins within the ABC-superfamily actively transport a wide variety of substances across biological membranes and fulfill important functions in cellular metabolism and detoxification. The protein encoded by the caMLT1 gene shows high similarities to the vacuolar efflux-pumps Ycf1p and Bpt1p of S. cerevisiae. Genomic tagging with the green fluorescent protein (GFP) revealed vacuolar membrane localization of caMlt1p. Northern hybridisation experiments documented the inducibility of gene transcripts by the metabolic poisons cadmium and CDNB, which are also substrates of the scYcf1-pump. While caMlt1p could be an orthologue of scYcf1p, complementation of a scycf1-negative S. cerevisiae mutant with a caMLT1-gene copy did not reverse the sensitive phenotype to these toxins. Moreover, the construction of camlt1-negative mutants in C. albicans allowed for screening of substrates putatively transported by caMlt1p. These null mutants showed no hypersensitive phenotype to neither CdCl2 nor CDNB or any other tested inhibitory substances, hence caMlt1p is not a functional homologue of scYcf1p. The caMLT1 mRNA expression pattern is typical for a vacuolar gene, showing an extensively growth phase dependent regulation with the highest gene induction during the diauxic transition when glucose (and other nutrients) becomes limited. Most interestingly, a generated mlt1 null mutant showed a dramatic reduction in liver invasion in a mouse peritonitis model. Reintegration of a functional caMLT1 gene copy reverted the virulence defect. CaMlt1p seems to be involved in the capability of C. albicans to adhere to the intestinal organs and penetrate tissue barriers putatively because of its involvement in mechanisms of stress response and detoxification. Both genes, caMLT1 and caPLB5, were inactivated by using a classical disruption method for C. albicans (the URA3-Blaster-system in Ura- auxotrophic strain CAI4) and by developing a new dominant selection system. Dominant selection is based on genomic insertion of a single copy of a mutated caIMH3 allel (MPAR) that renders transformants resistant to mycophenolic acid (MPA). Using this system, the cumbersome generation of auxotrophic strains, which are often avirulent, is obsolete, while C. albicans wild-type strains become amenable to genetic manipulation.

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.

Programmed cell death (PCD) is an essential biological process in animal development and tissue homeostasis that is necessary to ensure the physiological well-being of the organism. During PCD, phagocytes facilitate the selective removal of excess, damaged, and potentially deleterious cells, in a multi-step engulfment process. Genetic studies in Drosophila melanogaster, Caenorhabditis elegans, and mammals have identified two evolutionarily conserved signal transduction pathways that act redundantly to regulate engulfment: the CED-1/-6/-7 and CED-2/-5/-12 pathways. Of these cell death (CED) proteins, the ABC transporter CED-7 is the only protein reported to be required in both the engulfing cell and the dying cell. However, its function in the cell death process remains the most enigmatic and the ced-7 ortholog previously has not been identified in Drosophila. Homology searches revealed a family of putative ced-7 orthologs that encode transporters of the ABCA family in Drosophila. To determine which of these genes functions similarly to ced-7/ABCA1 in PCD, we analyzed their engulfment function in oogenesis, during which 15 germ cells in each egg chamber undergo programmed cell death and are removed by neighboring phagocytic follicle cells. It has been shown that genetically knocking down individual engulfment genes results in inefficient clearance of the germ cells, which then persist in late-stage egg chambers. Only two of the putative ced-7/ABCA1 genes are expressed significantly in the ovary, CG31731 and CG1718, and we have characterized these genes using transposon insertions, deficiencies, and RNAi knockdowns. Our genetic analysis reveals that CG31731 is necessary for germ cell clearance in the Drosophila ovary. Immunostaining shows that genetically knocking down CG31731 results in uncleared germ cells which persist in late-stage egg chambers. Altogether, our findings suggest that CED-7/ABCA1/CG31731 play evolutionarily conserved roles during engulfment.

P-glycoprotein (P-gp or ABCB1) is a membrane-bound active transporter belonging to the ABC protein superfamily. It is responsible for xenobioIc efflux and also contributes to multidrug resistance in diverse diseases including cancer and epilepsy. P-gp has been increasingly recognised as a potential target for future therapeutics. Although the protein has been studied for decades, understanding of the P-gp transport mechanism is still incomplete. Two P-gp orthologues, mouse (m) and human (h), were therefore expressed in yeasts and purified in the presence of the detergent, n-Dodecyl-β-D- Maltoside (DDM). Purified proteins were examined for aggregation and monodispersity via dynamic light scattering (DLS) and their thermal stability was determined by an assay using a thiol-specific dye (CPM). ATPase activity, measured in a detergent environment, showed that the proteins were active with a basal activity of 60 ± 4 and 35 ± 3 nmol/min/mg for mP-gp and hP-gp, respectively. Crystallisation trials were conducted in the presence of nucleotide. In meso crystallisation using commercial monoolein pre- dispensed plates yielded hexagonal crystal-like objects however they failed to diffract X- rays. P-gp samples were also subjected to cryo-EM where mP-gp in the post-hydrolytic (ADP-bound, vanadate-trapped) state provided the highest resolution dataset that led to a reconstruction of 3D density map at the resolution of 7.9 Å which showed an inward- facing conformation. Rigid-body model fitting unveiled densities that were not accounted for by the fitted model illustrating new features such as bound ADP, extended NBD1- TMD2 linker and alternative allocrite-binding sites. Ultimately, the knowledge of P-gp conformation alteration was enhanced and a refined alternating access mechanism of P- gp was proposed based upon information derived from this study.

In this dissertation, I employed a combination of in vitro and in silico techniques to characterize two bacterial membrane proteins: the MtrE efflux conduit from Neisseria gonorrhoeae and the AmtB ammonia transporter from Escherichia coli. MtrE is an efflux conduit located in the outer membrane of N. gonorrhoeae. It can form a protein complex with MtrC (a periplasmic adapter) and MtrD (an inner membrane active transporter). This tripartite machinery actively extrudes a diverse set of compounds from the periplasm to the exterior of the bacteria. The MtrCDE complex and homologous tripartite efflux pumps are amongst the major contributors to the emergence of super-resistant Gram-negative bacteria. The efforts to abolish the activity of these protein complexes have so far concentrated on the inhibition of the active pump, but had little clinical success to date. I carried out a combination of Planar Lipid Bilayer electrophysiology experiments, Molecular Dynamics simulations, and protein homology modeling on the MtrE and the MtrC proteins. I characterized the MtrE conduit as being slightly cation selective, as opposed to other homologous proteins which exhibit a much stronger selectivity. Additionally, I demonstrated that the opening of MtrE is modulated by the binding of the adapter protein MtrC. These results have a critical importance because they imply that the tripartite pump activity can be diminished not only by the MtrD inhibition, but by targeting the interface between MtrC and MtrE. The AmtB ammonia transporter is a protein embedded in the inner membrane of E. coli in situations of growth-limiting low levels of ammonia. The transport has been shown to be electrogenic in similar orthologous proteins, but the exact mechanism has so far remained elusive. The only inner pathway that has been identified in AmtB is lined by hydrophobic amino acids and for this reason it has been proposed to carry neutral ammonia. My Molecular Dynamics simulations reveal the opening of a water wire separated from the well known hydrophobic pore. This finding suggests a transport mechanism in which the neutral ammonia and the proton travel along different conduits. My simulations on several AmtB mutants gave additional important insights into the protein function. Notably my results explain the structural determinants of the switching of substrate from NH4+ to K+ observed for the H168D/H318E double mutant. Lastly, I identified several 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) binding sites on AmtB, which could possibly contribute to modulating the transport. Importantly, these in silico experiments were guided and confirmed by Solid Supported Membrane-electrophysiology measurements carried out in collaboration with the Javelle group. The data presented in this Thesis highlights the strength of a coordinated approach, in which experimental and computational findings direct, and integrate with each other.

I sin nuvarande form är det allmänt känt att bilismen är en bidragande orsak till den rådande klimatproblematiken. Något som sällan uppmärksammas i lika stor utsträckning är de negativa sidorna som bilismen har i stadskärnor. En av de städer i Sverige som har störst problem med dålig luftkvalitet orsakat av biltrafik är Uppsala. Här har många olika åtgärder vidtagits för att få bukt med problemen, utan önskad framgång. Med anledning av detta syftar föreliggande uppsats till att undersöka ett bilfritt centrum i Uppsala via tvingande styrmedel i form av förbud mot bilism. Uppsatsen syftar till att undersöka en sådan process kronologiskt med fokus på förutsättningar, genomförande och möjligheter. I uppsatsen presenteras en forskningsöversikt där även tre europeiska städer som har infört förbud mot bilism studeras. Vidare genomförs en kvalitativ fallstudie om situationen i Uppsala, vilken analyseras utifrån den tidigare forskningen och de tre studerade städerna. Resultaten tyder på att ett förbud mot bilism är intressant utifrån flera perspektiv. Till exempel uppfyller staden flera av de grundläggande förutsättningar som uppsatsen identifierar som väsentliga för att ett förbud ska kunna genomföras, och flera av de troliga positiva konsekvenserna av ett förbud är intressanta mot bakgrund av dagens problematik och kommunens hållbarhetsarbete.

Syftet med uppsatsen är att undersöka spårvägsprojektet i Uppsala. Studien ska identifiera de huvudsakliga faktorerna som har varit avgörande för valet av spårväg. Det studeras även hur projektets beslutsfattare resonerar kring en spårväg utifrån de tre hållbarhetsaspekterna och hur privatbilismen förväntas påverkas. Genom intervjuer med tjänstemän och politiker ska studiens frågeställningar besvaras. Intervjusvaren kompletteras med tidigare forskning inom ämnesområdet och styrdokument från Uppsala kommun. Resultatet visar att kapaciteten är en av de viktigaste faktorerna bakom valet av spårväg. I takt med den befolkningsökning som förväntas i Uppsala behöver staden en kapacitetsstark kollektivtrafik som klarar av att hantera dessa nya stora flöden. En annan viktig faktor bakom valet av spårväg är de miljömässiga fördelarna, till exempel som att spårvägen ska drivas på förnyelsebar el. Minskad privatbilism är inget uttalat mål med spårvägen, men biltrafiken förväntas ändå minska som en följd av att en spårväg anses vara attraktiv och får en hög medelhastighet i samband med den höga prioritering som spårvägen ska erhålla. Finansieringen anses vara en av de största utmaningar med att få spårvägen realiserad.

No description available.