In the broiler industry, chicks are often deprived of feed and water up to 48 h posthatch. This delayed access to feed (DAF) has been found to inhibit small intestinal development, compromising growth of the chick. To further understand the impact of DAF on small intestines at the molecular level, many developmental genes that regulate intestinal development were investigated. The objective of this study was to determine the effect of DAF on early posthatch broiler small intestinal morphology, which includes villus height (VH) and crypt depth (CD), and to quantify changes in regulatory genes, such as Olfactomedin 4 (Olfm4), Marker of Ki-67 (Ki-67), Peptide Transporter 1 (PepT1), and Mucin 2 (Muc2), in response to DAF. The Olfm4 mRNA can clearly identify stem cells in the intestinal crypt, which allows VH and CD to be measured, while Ki-67 marks the proliferating cells. The peptide transporter PepT1 is located on intestinal epithelial cells and plays a critical role in transporting di- and tripeptides. Muc2, which is secreted from goblet cells, forms mucus that lines the intestinal epithelial cells acting as a layer of protective coating. Cobb 500 chicks, hatching within a 12 h window, were randomly allocated into three experimental groups: control with no feed delay (ND), 24 h feed delay (D24), and 36 h feed delay (D36). Quantification of Olfm4, Ki-67, PepT1, and Muc2 mRNA abundance were investigated by quantitative PCR, in duodenum, jejunum, and ileum at 0 h, 24 h, 36 h, 72 h, 120 h, and 168 h posthatch. Additionally, localization of cells expressing each gene was visualized using in-situ hybridization at all listed times except 168 h posthatch. Statistical analysis was performed using JMP Pro 14, and significant differences between treatments within a collection day were determined by t-test and one-way ANOVA (P < 0.05). In the ND group, duodenal CD at 0 h was greatest compared to all other time points. With DAF, the duodenal VH of D36 chicks was lower at 36 h (P < 0.001) and 72 h (P = 0.002) compared to ND chicks. In the jejunum and ileum, the VH of D36 chicks was lower at 120 h (P = 0.005) and 72 h (P = 0.03), respectively, compared to ND chicks. In contrast, the VH of D24 chicks at 24 h was greater than ND (P = 0.004) in the jejunum. There was no difference between treatments by 168 h in all intestinal segments. The CD was also lower in DAF groups compared to ND but only in the jejunum and ileum. In contrast, duodenal CD was greater in D24 chicks at 24 h (P = 0.039) and in D36 chicks at 36 h (P < 0.0001) compared to ND chicks, but the difference was no longer significant by 72 h. The VH/CD ratio was lower in all three segments, except the ileum displayed a greater VH/CD ratio in D24 and D36 chicks at 24 h and 36 h, respectively, compared to ND chicks. The mRNA abundance of Olfm4 and Ki-67 was greater in DAF groups upon refeeding, but not until 120 h. The PepT1 mRNA abundance was greater in DAF groups while the abundance of Muc2 mRNA was lower. This difference in mRNA abundance level was more prominent in the duodenum and jejunum. From the analysis of number and distribution of goblet cells found in the upper half and lower half of the villi, expressed as a ratio (VU/VL), a greater ratio was observed in delayed groups compared to ND. In summary, while DAF resulted in altered small intestinal morphology with an effect more pronounced in D36 than D24 chicks, upon refeeding, some genes important to intestinal development were upregulated as a response to the treatment. / Master of Science / In the broiler industry, chicks are often deprived of feed and water up to 48 h posthatch. This delayed access to feed (DAF) was found to negatively impact small intestinal development, compromising their growth. The objective of this study was to determine the effect of DAF on early posthatch broiler small intestinal morphology and to observe the changes in regulatory genes, such as the stem cell marker, proliferating cell marker, absorptive cell marker, and mucus producing cell marker, in response to the DAF. To simulate the DAF condition in the broiler industry, chicks with no feed delay (ND) were tested against chicks with DAF for 24 h (D24) and 36 h (D36). Quantification and cell localization of these cell markers were investigated in the small intestines at early posthatch. In general, DAF chicks had lowered intestinal villus height and crypt depth compared to ND chicks. The mRNA abundance of markers for stem cells and proliferating cells were greater in DAF groups upon refeeding. The mRNA abundance of markers for absorptive cells was greater in DAF groups while the mRNA abundance of markers for mucus producing cells was lowered as a result of DAF. In summary, DAF negatively impacted small intestinal morphology and altered the regulation of some developmental genes important to early posthatch chick performance.

Black bears are omnivorous, and tend to be opportunistic feeders, in that they will eat what is readily abundant or available. The end-products of intestinal digestion are absorbed by the body through the action of transporter proteins expressed on the brushborder membrane of small intestinal epithelial cells. The goal of this study was to increase the understanding of the physiological processes associated with nutrient assimilation by black bears. Distribution and relative abundance of mRNA of a peptide transporter (PepT1), a glucose transporter (SGLT1), two AA transporters (NBAT, bo,+AT), and a digestive enzyme, aminopeptidase N (APN), in the intestinal tract of black bears were investigated. Ten bears were used for this study. For tissue collection, the intestine was removed from the animal and divided into five sections. Each collected section was opened longitudinally, rinsed in ice-cold PBS, and the mucosal scrapings were stored at -80&#61616;C. Total RNA was extracted and quantified by spectrophotometry. Abundance of PepT1, SGLT1, NBAT, bo,+AT, and APN mRNA was determined by performing Northern blots, using bear cDNA probes. Northern blot data were quantified by densitometric analysis, with the abundance of each gene expressed relative to GAPDH. Abundance of PepT1 (P < 0.05), APN (P < 0.05), and SGLT1 (P < 0.0001) changed quadratically from the proximal to the distal intestine with abundance being greatest in the midregion. Abundance of bo,+AT mRNA increased linearly (P < 0.05) from the proximal to distal intestine. Abundance of NBAT mRNA did not change among intestinal segments.The absolute number of molecules of mRNA/ng of total RNA for each gene was determined using Real-Time PCR. Similar to the Northern results, abundance of PepT1 (P < 0.0003), SGLT1 (P < 0.0003), and APN (P < 0.02) changed quadratically from the proximal to distal intestine with abundance being greatest in the mid-region, and bo,+AT mRNA increased linearly (P < 0.0001) from the proximal to distal intestine. NBAT mRNA abundance also increased linearly (P < 0.0001) from proximal to distal intestine. PepT1 mRNA was present at tenfold or greater levels than AA transporter mRNA in all segments of the intestine, suggesting that di- and tripeptides constitute the major form in which AAs are absorbed. NBAT and bo,+AT mRNA abundance was greater towards the distal portion of the intestine, suggesting their importance in salvaging remaining unabsorbed AAs.These results indicate that the mRNA of nutrient transporters examined and APN are differentially expressed throughout the gastrointestinal tract of black bears, suggesting their involvement in nutrient assimilation. / Master of Science

Francisella tularensis is a Gram-negative, facultative intracellular coccobacillus and the etiologic agent of tularemia for a wide variety of vertebrate and invertebrate animal species. Several species and subspecies of Francisella are currently recognized. However, the majority of infections are caused by F. tularensis subspecies tularensis (type A) and subspecies holarctica (type B). Given the low infectious dose, multiple transmission routes, severity of illness, and lack of licensed vaccines, F. tularensis has long been considered a potential biological weapon and is now classified as a category A select agent by the National Institutes of Health and the Centers for Disease Control and Prevention. The investigation of the mechanisms of pathogenesis by F. tularensis type A and B strains is hindered by the difficulty and lack of methods to mutate the putative genes that encode for virulence factors. New genetic tools have been developed that have enabled mutagenesis of F. tularensis type A and type B stains. However, site-specific mutations remain difficult to execute or these methods generate random mutations. In this study a novel method was developed to create site-directed mutations in a putative capsule biosynthesis locus to knock out encapsulation of the attenuated F. tularensis live vaccine strain. Two suicide vectors for mutagenesis of F. tularensis were constructed based on the commercial PCR cloning vector pSC-A. These vectors were created by inserting into the cloning site a kanamycin resistance gene boarded upstream by 1.3 kb of N-terminal DNA and downstream by 1.3 kb of C-terminal DNA that flanks the target gene. Cryotransformation was used to introduce the vectors into F. tularensis. Open reading frame (ORF) FTT0793, which may encode for an ABC transporter involved in capsule export, was initially selected for mutagenesis in order to generate a mutant that was nonencapsulated, but could still synthesize capsule and induce a host immune response. Mutagenesis of this gene was successful. However, phenotypic assays could not confirm that the mutant was nonencapsulated compared to the parent. Therefore, adjacent ORFs FTT0798 and FTT0799, which may encode for a galactosyl transferase and mannosyl transferase, respectively, were also deleted to completely knock out capsule synthesis. The resulting mutant appeared to be nonencapsulated as determined by negative staining transmission electron microscopy. In this study, a plasmid and method for generating allelic exchange mutants is reported, which should be useful for generating additional mutants of F. tularensis for use in clarifing the roles of specific genes. This vector is currently being used to make a nonencapsulated mutant of a virulent type A strain to determine the role of capsule in virulence. / Master of Science

The Swedish railway industry faces a predicted increase in demand, a demand shift in each railway product, new noise regulation, and the problem with attaining the right personnel. This creates new operating conditions that railway operators must adapt to. The planning process for allocating empty-wagons from one geographical area to another to fulfill customer demand is manually performed by three experienced Wagon Managers after the discontinuation of a fully automatic system called New VADIS. Green Cargo needs to streamline operations through a standardized empty-wagon allocation process to increase delivery precision, the utilization of the wagon fleet, and to promote cost-efficiency. The thesis will evaluate Green Cargo’s usage of wagons, provide a framework for how to standardize the allocation process, discuss benefits of wagon fleet rationalization, and expand upon the current method to eliminate flaws and internal waste. Literature reviews relating to the New VADIS project, intermodal load unit assignment, and transport fleet standardization were reviewed to provide the theoretical basis of the study. An evaluation of current KPIs resulted in the introduction of the Wagon Fleet Scorecard. Interviews with representatives from the Customer Service and Operations Center departments at Green Cargo formed the basis for necessary operative conditions needed to ensure the success of a future automatic allocation system. An attempt at solving the intermodal demand translation flaw of New VADIS was done through a new mixed-integer-linear programming optimization model for optimal loading of load units on ten intermodal wagon types. The model was evaluated on real-life production data from three intermodal terminals resulting in a significantly better assignment compared to the manual process, with an 11.26% shorter cumulative train length and 10.44% less loaded wagons. An indirect effect was the reduction of the cumulative tare weight of the departing loaded wagons with 11.49%. A new standardized process is presented that includes an operative implementation of the mathematical model using a common Visual Loading Plan. Furthermore, new allocation principles were formulated to maximize wagon capacity and fleet utilization as well as minimize allocation costs. The results indicate benefits towards Green Cargo’s continuous work with sustainable business practices. The proposed process results in a 1.5% reduction of Green Cargo’s total annual Scope 2 CO2 emissions through reduced energy consumption. It gives potential for extra revenue and reduces lean wastes from overproduction, unnecessary transports, extra processing, and waiting by providing better utilization of train capacities. It also results in enhanced social sustainability during loading operations at terminals by decreasing risk of human error, decreasing risks of wagon derailment, and by providing better conditions for terminal personnel to ensure proper load safety. / Den svenska järnvägsindustrin står inför en förutspådd efterfrågansökning, en efterfrågeförskjutning av varje järnvägsprodukt, ny bullerreglering och problem med att anställa personal med rätt kompetens. Detta skapar nya operativa förutsättningar som svenska järnvägsoperatörer måste anpassa sig till. Planeringsprocessen för att allokera tomma vagnar från ett överskottsområde till ett underskottsområde för att realisera kunders transportbehov utförs manuellt av tre erfarna Vagnledare, efter att det automatiserade systemet Nya VADIS avvecklades. Green Cargo behöver effektivisera verksamheten genom en standardiserad vagnförsörjningsprocess för att öka leveransprecisionen, nyttjandet av vagnsflottan och för att främja kostnadseffektivitet. Examensarbetet utvärderar Green Cargos användning av vagnar, tillhandahåller ett ramverk för hur vagnförsörjningsprocessen kan standardiseras, diskuterar fördelarna med en rationalisering av vagnflottan samt utökar den nuvarande metoden för att eliminera brister och internt slöseri. Litteraturgenomgångar relaterade till Nya VADIS-projektet, optimal lastning av intermodala lastbärare och standardisering av vagnflottan utfördes för att ge den teoretiska grunden för examensarbetet. En utvärdering av aktuella nyckeltal resulterade i skapandet av ett Wagon Fleet Scorecard. Intervjuer med representanter från avdelningarna Kundservice och Driftcenter på Green Cargo utgjorde grunden för nödvändiga operativa förutsättningar för att säkerställa framgången för ett framtida automatiskt vagnförsörjningssystem. Ett försök att lösa bristerna i den intermodal efterfrågeöversättningen i Nya VADIS genomfördes genom en ny linjär heltalsoptimeringsmodell för optimal lastning av lastbärare på tio intermodala vagntyper. Modellen utvärderades på verkliga produktionsdata från tre intermodala terminaler vilket resulterade i en betydligt mer optimal lastning jämfört med den manuella processen, med 11,26 % kortare ackumulerad tåglängd och 10,44 % färre lastade vagnar. En indirekt effekt var minskningen av den ackumulerade egenvikten av de avgående lastade vagnarna med 11,49 %. En ny standardiserad process presenteras som inkluderar en operativ implementering av den matematiska modellen med hjälp av en gemensam Visual Loading Plan. Vidare formulerades nya vagnstyrningsprinciper för att maximera nyttjandet av vagnars kapacitet, hela vagnflottans nyttjande samt minimera allokeringskostnaderna. Resultaten indikerar fördelar för Green Cargos kontinuerliga arbete med hållbara affärsmetoder. Den föreslagna processen innebär en 1,5 % minskning av Green Cargos totala årliga Scope 2 CO2 utsläpp genom minskad energiförbrukning. Det ger potential för extra intäkter och minskar lean slöserierna överproduktion, onödiga transporter, överarbete och väntan genom bättre nyttjande av tågkapaciteten. Det resulterar även i förbättrad social hållbarhet under lastning vid terminaler genom att minska risken för mänskliga fel, minska riskerna för urspårning av vagnar samt skapa bättre förutsättningar för terminalpersonal att säkerställa god lastsäkring.

No description available.

No description available.

Efficient control of sea lice is a major challenge for the sustainable production of farmed Atlantic salmon (Salmo salar (Linnaeus, 1758)). These marine ectoparasites feed on mucus, skin and blood of their hosts, thereby reducing the salmon’s growth rate and overall health. In the northern hemisphere, the most prevalent species is Lepeophtheirus salmonis (Krøyer, 1837). In 2006, global costs of sea lice infections are estimated to have exceeded €300 million, with the majority spent on a limited number of chemical delousing agents. Emamectin benzoate (EMB; SLICE®), an avermectin, has been widely used since its introduction in 2000, due to its convenient administration as an in-feed medication and its high efficacy against all parasitic stages of L. salmonis. However, over-reliance on a single or limited range of medicines favours the emergence of drug resistance and, as a result, the efficacy of this compound in treating L. salmonis has decreased in recent years, as reported from e.g. Chile, Norway, Scotland and Canada. Declining efficacy underlines the need for an improved understanding of the molecular mechanisms underlying EMB drug resistance in L. salmonis. Elucidation of these mechanisms would allow for improved monitoring tools, earlier detection of developing resistance, extended usability of current delousing agents and development of new parasiticides. The work described in this thesis sets out to examine the molecular mechanisms underlying EMB resistance in L. salmonis. In earlier studies, research in nematodes and arthropods has linked drug efflux transporters belonging to the family of ATP-binding cassette (ABC) transporters to ivermectin (IVM) resistance, a parasiticide with high chemical similarity to EMB. ABC transporters such as permeability glycoprotein (P-gp), transport a wide range of substrates, including drugs, and have been suggested to provide a potential molecular mechanism through which EMB resistance might be mediated in sea lice. As an example of such mechanisms, increased expression of P-gp is one of the causative factors for drug resistance in human cancer cells and avermectin resistance in nematode parasites such as Caenorhabditis elegans or Haemonchus contortus. Initial research involved screening for novel salmon lice P-gps that might contribute to EMB resistance. A novel P-gp, SL-PGY1, was discovered using a combined bioinformatic and molecular biological approach. The expression was compared in two well-characterised L. salmonis strains differing in their susceptibility to EMB (S = susceptible, R = resistant). Prior to EMB exposure, mRNA levels did not differ from each other, while, after 24 h exposure, a 2.9-fold increase in SL-PGY1 mRNA expression was observed in the R strain. SL-PGY1 appears not to be a major factor contributing to reduced EMB susceptibility, although it could play a role, as expression levels increased upon exposure to EMB. A further four additional drug transporters (ABC C subfamily) were also discovered showing high homology to multidrug-resistance proteins (MRP). The relative expression levels of each MRP was compared in the strains S and R, before and after exposure to EMB. No significant changes were found in their expression patterns. If ABC drug transporters mediate the efflux of EMB and thereby reduce the intracellular concentrations of the drug in exposed animals, the inhibition of those ABC drug transporters was expected to lead to higher intracellular levels of EMB. This could result in an enhanced toxic effect when EMB is co-administered with an inhibitor. Two known inhibitors of human P-gps and MRPs, cyclosporin A (CSA) and verapamil (VER), were co-administered with EMB. CSA increased the toxic effect of EMB in both tested strains, implying that the targets of CSA are expressed at comparable levels and that they may be part of the mechanism conferring EMB resistance. VER increased the toxic effect of EMB in the R strain, but had no significant effects on the S strain. This implies that the expression of factors inhibited by VER differs between the two L. salmonis strains. It is hypothesised that a number of ABC transporters with distinct, yet overlapping patterns of inhibitor specificity are affected by those inhibitors. The search for drug-resistance conferring genes was complemented with a systematic, genome-wide survey of ABC transporters in L. salmonis to find additional members of this important gene family. Next-generation high-throughput RNA sequencing (RNA-seq) was employed to assemble a reference transcriptome from pooled total RNA of salmon lice at different development stages. The transcriptome was assembled against the L. salmonis genome and annotated. Thirty-nine putative ABC transporters were found. Of further interest were transcripts of the subfamily B, C and G, as they contain drug-transporting ABC proteins. For the ABC B subfamily, one full (SL-PGY1) and three half transporter transcripts were found. Only full transporters are known to transport drugs and SL-PGY1 is apparently not a major factor contributing to EMB resistance. Fourteen ABCC sequences were found – 11 MRPs and 3 homologues to sulfonylurea receptors. Of interest are MRPs, as they contribute to drug detoxification in humans and invertebrates. Four MRPs had been identified previously and their expression ratios did not differ between S and R strain parasites. Seven sequences belonging to ABCG subfamily were found. However, none of the L. salmonis ABCG transcripts identified showed sufficient homology to known drug transporters in other species. With the currently limited understanding of the mechanisms conferring EMB resistance, monitoring the susceptibility of L. salmonis subpopulations is essential. Dose-response bioassays are currently widely used. Tests with pre-adult II or adult parasites requires relatively large numbers of parasites (~150) to conduct this type of bioassay, which may not always be available. Addressing this issue, we tested the feasibility of a single-dose bioassay (requiring fewer test animals than dose-response bioassays) to discriminate between L. salmonis strains with differing EMB susceptibility. This alternative approach uses time-course toxicity analysis, where the toxic effect of EMB is monitored over time. After clearly defining the effect criteria, we found that it is possible to discriminate between those L. salmonis strains. However, while requiring fewer test animals, time course toxicity analysis is more labour-intensive, but the alternative design can be suitable under certain circumstances. The work reported here has provided new knowledge concerning the mechanisms of EMB resistance in sea lice. Several novel putative drug transporters have been identified, an important first step toward unravelling the complex interactions of genes involved in EMB resistance in this commercially important parasite.

<p>Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer in part due to inherent resistance to chemotherapy, including the first-line drug gemcitabine. Gemcitabine is a nucleoside pyrimidine analog that has long been the backbone of chemotherapy for PDAC, both as a single agent, and more recently, in combination with nab-paclitaxel. Since gemcitabine is hydrophilic, it must be transported through the hydrophobic cell membrane by transmembrane nucleoside transporters. Human equilibrative nucleoside transporter-1 (hENT1) and human concentrative nucleoside transporter-3 (hCNT3) both have important roles in the cellular uptake of the nucleoside analog gemcitabine. While low expression of hENT1 and hCNT3 has been linked to gemcitabine resistance clinically, mechanisms regulating their expression in the PDAC tumor microenvironment are largely unknown. We identified that the matricellular protein Cysteine-Rich Angiogenic Inducer 61 (CYR61) negatively regulates expression of hENT1 and hCNT3. CRISPR/Cas9-mediated knockout of CYR61 significantly increased expression of hENT1 and hCNT3 and cellular uptake of gemcitabine. CRSIPR-mediated knockout of CYR61 sensitized PDAC cells to gemcitabine-induced apoptosis. Conversely, adenovirus-mediated overexpression of CYR61 decreased hENT1 expression and reduced gemcitabine-induced apoptosis. We demonstrate that CYR61 is expressed primarily by stromal pancreatic stellate cells (PSCs) within the PDAC tumor microenvironment, with Transforming Growth Factor- β (TGF-β) inducing the expression of CYR61 in PSCs through canonical TGF-β-ALK5-Smad signaling. Activation of TGF-β signaling or expression of CYR61 in PSCs promotes resistance to gemcitabine in an in vitro co-culture assay with PDAC cells. Our results identify CYR61 as a TGF-β induced stromal-derived factor that regulates gemcitabine sensitivity in PDAC and suggest that targeting CYR61 may improve chemotherapy response in PDAC patients.</p> / Dissertation

In den Firmicutes wird der Induktorausschluss (Katabolitrepression) durch das am Serin46 phosphorylierte HPr (PTS) vermittelt. Der genaue Mechanismus war jedoch unklar. Um diese Frage auf der Grundlage von isolierten Proteinen zu klären, wurde ein zum Escherichia coli Maltose-/Maltodextrin-ABC-Transporter homologes System aus Lactobacillus casei BL23 (MalE1-MalF1G1K12) als Modellsystem genutzt. Im Rahmen der Promotion wurde über isothermale Titrationskalorimetrie und Fluoreszenzspektroskopie gezeigt, dass das Bindeprotein MalE1 lineare und zyklische Maltodextrine, aber keine Maltose bindet. Experimentell ermittelte dreidimensionale Strukturen von MalE1 im Komplex mit diesen Zuckern belegten eine vergleichbar geschlossene Konformation und dienten zusätzlich als Grundlage, um die fehlende Maltosebindung zu erklären. Die Stimulierung der ATPaseaktivität des in Liposomen und Nanodiscs eingebauten Komplexes wurde jedoch hauptsächlich durch eine MalE1-Beladung mit linearen Maltodextrinen bewirkt. Eine bis zu 85 %ige Inhibierung der ATPaseaktivität durch P-Ser46-HPr belegte erstmals in vitro eine Interaktion von mehr als einem phosphorylierten Protein mit dem Transporter. Analog zum EIIAGlc-Inhibitor des homologen Systems aus E. coli wurden über Quervernetzungsexperimente und massenspektrometrische Analysen Interaktionen mit dem MalK1-Dimer als interagierende Komplexeinheit in der Nähe des Walker A-Motivs nachgewiesen. Über Fluoreszenzmessungen in Anwesenheit des ATP-Analogons TNP-ATP wurde eine unbeeinflusste ATP-Bindung und damit eine fehlende Blockade der γ-Phosphatbindestelle des Walker-A Motivs durch die Phosphorylgruppe von P-Ser46-HPr bestimmt. Die folgende Substitution verschiedener positiv geladener MalK1-Reste, die als potenzielle Interaktionsstellen für die Phosphorylgruppe fungieren könnten, identifizierte K63 in der Nähe des Walker A-Motivs als ersten möglichen Partner. Der genaue Mechanismus der Inhibierung bleibt jedoch unklar. / Catabolite repression is a global mechanism which controls the utilization of carbohydrates in bacteria. In Firmicutes HPr, a component of the phosphoenolpyruvate carbohydrate phosphotransferase system, prevents the uptake of less preferred sugars but only when it is phosphorylated at serine46. However the exact mechanism was unclear. To address this question the purified ATP-binding cassette transporter from Lactobacillus casei BL23 (MalE1-MalF1G1K12) was used as a model system, which is homologous to the Escherichia coli maltose/maltodextrin ABC importer. Isothermal titration calorimetry and fluorescence spectroscopy revealed that the binding protein MalE1 binds linear and cyclic maltodextrins but not maltose. Experimentally determined three-dimensional structures from MalE1 in complex with these sugars show a comparably closed conformation and served as a basis to explain the lack of maltose binding. The stimulation of the ATPase activity of the transporter incorporated in liposomes and nanodiscs however, was mainly caused by MalE1 loaded with linear maltodextrins. For the first time an inhibition of ATPase activity by P-Ser46-HPr up to 85 % and an interaction of more than one phosphorylated protein with the transporter was demonstrated. Analogous to the EIIAGlc inhibitor of the homologous system from E. coli, cross-linking experiments and mass spectrometric analyzes revealed interactions with the MalK1 dimer near the Walker A motif. Fluorescence measurements in the presence of the ATP analogue TNP-ATP, however, revealed an unaffected ATP binding and thus a lack of blockade of the γ-phosphate binding site (Walker A motif) by the phosphoryl group from P-Ser46-HPr. The following substitution of several positively charged MalK1 residues that could act as potential sites of interaction for the phosphoryl group, identified K63 near the Walker A motif as the first potential partner. The exact mechanism of inhibition, however, remains unclear.

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.