Dissertationen zum Thema „Flippases“
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McDowell, Stephen C. „Lipid Flippases and Elemental Homeostasis Systems in Arabidopsis thaliana“. Thesis, University of Nevada, Reno, 2013. http://pqdtopen.proquest.com/#viewpdf?dispub=3566276.
Der volle Inhalt der QuelleMany molecules in living systems are present in charged forms, and these molecules are often highly regulated. The work presented in the following chapters addresses two main topics involving charged molecules using the model plant Arabidopsis thaliana: elemental homeostasis and lipid flippases. The study of elemental homeostasis is referred to as ionomics and is the topic of Chapter II. P4-ATPases are thought to be the principle class of proteins with lipid flippase activity and are the topics of Chapter III and Chapter IV.
Plants, especially seed crops, are an important source of mineral nutrition in the human diet and are thus important targets for biofortification and toxic element exclusion. Here, we report the results of a pilot ionomic screen in which we quantified the concentrations of 14 elements in Arabidopsis seeds. To identify conditional ionomic phenotypes, plants were grown under four different soil conditions: standard, or modified with NaCl, heavy metals, or alkali. To help identify the genetic networks regulating the seed ionome, elemental concentrations were evaluated in mutants corresponding to 760 genes as well as 10 naturally occurring accessions. The frequency of ionomic phenotypes observed in the mutant screen supports an estimate that up to 11% of the Arabidopsis genome encodes proteins of functional relevance to the seed ionome. A subset of mutants were analyzed with two independent alleles, providing five examples of genes important for regulation of the seed ionome: SOS2, ABH1, CCC, At3g14280, and CNGC2. Reproducible ionomic differences were also observed between the Col-0 reference accession and eight of the other nine accessions screened. Significantly, all 15 mutants with reproducible ionomic phenotypes showed at least one change under standard soil conditions. This suggests that the sole use of a standard growth environment might be the most effective strategy for continued reverse-genetic efforts to identify genes that impact the Arabidopsis seed ionome. Nonetheless, each soil modification had a unique impact on the Col-0 seed ionome and elicited several conditional phenotypes in both the mutant and accession screens, indicating that seed elemental homeostasis is sensitive to soil conditions. Together, the results of this study establish that elemental analysis is a sensitive approach to identify genes and environmental conditions that impact elemental accumulation in Arabidopsis seed.
By flipping lipids between membrane leaflets, P4-ATPases are thought to help create and maintain asymmetry in biological membranes. Lipid asymmetry between membrane leaflets has been implicated in a wide range of biological processes including: vesicular trafficking, cell signaling, modulation of membrane permeability, protein recruitment, and regulation of protein activity. Additionally, one P4-ATPase, Neo1p, is essential in yeast. In Arabidopsis thaliana, 12 P4-ATPases have been identified: Aminophospholipid ATPase 1 (ALA1) to ALA12. However, very little is known about P4-ATPases in the context of plant systems.
Of the 12 ALA isoforms, only ALA3 has been extensively studied. Previous studies have shown that loss of ALA3 results in pleiotropic phenotypes affecting root, shoot, and reproductive development. Here, we expand on the previous studies by showing that multiple phenotypes for ala3 mutants are strongly sensitive to growth conditions. We also expand on the ala3 pollen phenotype by identifying three points of defect in ala3 pollen tubes: delayed germination, slow growth, and reduced overall length. Furthermore, we show that ala3 pistils have reduced ovule production, thus providing the first evidence of a female reproductive defect in ala3 mutants. Together, these results support a model in which ALA3 functions in multiple cell types and is critical to plants for development and adaptation to varied growth conditions.
Two other ALA isoforms, ALA6 and ALA7, were also examined in this study. We provide in-vitro and in-vivo evidence that ALA6 and ALA7 are important for rapid, sustained pollen tube growth. Expression of fluorescently labeled ALA6 fusion proteins indicates that the subcellular localization of ALA6 includes the plasma membrane and highly mobile endomembrane structures. We also show that staining by lipophilic FM dyes is reduced by ∼10-fold in ala6-1/7-2 pollen tubes relative to wild-type, suggesting differences in plasma membrane composition. Furthermore, tandem mass spectroscopy analysis revealed significant differences between the lipid compositions of ala6-1/7-2 and wild-type pollen grains, both in the concentrations of different headgroups and in the average number of double bonds present within acyl side chains. Together, these results support a model in which ALA6 and ALA7 function to directly or indirectly regulate the distribution and concentration of lipids in pollen and are thus critical for pollen fitness.
Lamy, Anaïs. „Lipid Flippases from Plasmodium Parasites : from Heterologous Production towards Functional Characterization“. Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS447/document.
Der volle Inhalt der QuelleMalaria is a devastating disease caused by a parasite of the genus Plasmodium. Due to the spread of strains resistant to current antimalarial drugs, it is necessary to understand essential physiological functions of the parasite in order to find new drug targets. Membrane transport proteins are an important class of drug targets in humans, as they perform essential physiological roles of the cell. However, for Plasmodium parasites, just a few membrane transporters have been biochemically described. Recent gene-deletion studies in malaria mouse models have shown that the Plasmodium P4-ATPase, or lipid flippase, ATP2 is essential for the parasite. In eukaryotes, the phospholipid translocation activity of P4-ATPases is needed to maintain the asymmetric distribution of membranes, a key element in many essential processes like vesicle budding or apoptosis. Lipid flippases form heteromeric complexes with members of the Cdc50 protein family, also found in the genomes of Plasmodium parasites. To understand the functional role of these still putative transporters during malaria infection we need to study their transport mechanism and identify their substrate(s). We have conducted the heterologous expression in Saccharomyces cerevisiae of ATP2 in complex with the Cdc50 subunits from three different Plasmodium species. We succeeded to co-express the ATP2 ortholog of P. chabaudi (PcATP2) and the related putative PcCdc50 proteins. By co-immunoprecipitation and Fluorescence-detection Size Exclusion Chromatography, we have managed to identify the Cdc50 β-subunit that associates to PcATP2: PcCdc50.1. We then purified the complex PcATP2/PcCdc50.1 using immobilized nanobodies that recognize the GFP fused at the C-terminal end of PcATP2 and we initiated the functional characterization using ATPase and phosphorylation activity assays
Basante-Bedoya, Miguel Angel. „Transporteurs lipidiques dans la morphogenèse du champignon pathogène opportuniste de l’Homme Candida albicans“. Electronic Thesis or Diss., Université Côte d'Azur, 2021. http://theses.univ-cotedazur.fr/2021COAZ6030.
Der volle Inhalt der QuelleCandida albicans is a human opportunistic fungal pathogen that can cause superficial or systemic infections; its ability to change from an ovoid to a filamentous form is associated with its virulence. During this highly polarized filamentous growth, an accumulation of vesicles (Spitzenkörper), characteristic of filamentous fungi, as well as a polarized distribution of lipids, such as ergosterol, phosphorylated derivatives of phosphatidylinositol (PI(4)P, PI(4,5)P2) and phosphatidylserine (PS) is observed at the apex of filaments. However, the importance of the asymmetry of these lipids in the membrane bilayer is not completely understood. Flippases (P4-ATPases) transport lipids across the membrane bilayer to generate and maintain its asymmetry. C. albicans has 5 flippases, including Drs2 which is critical for filamentous growth and phosphatidylserine (PS) distribution. Furthermore, a drs2 deletion mutant is hypersensitive to fluconazole and copper. We show here that such a mutant is also critical to virulence in a mouse model of systemic infection. To clarify the role of Drs2 during C. albicans filamentous growth, we studied the distribution of this ATPase, as well as that of key lipids and regulators, during the initiation and maintenance of this growth process. We also characterized point mutants of Drs2, analogous to those altered for PS transport in S. cerevisiae. In addition, we examined the importance of other flippases, such as Dnf1-3, in invasive growth and the role of lipid transporters belonging to the oxysterol binding protein (Osh) family. Our results indicate in particular that Drs2 plays a unique role in the maintenance of invasive filamentous growth of C. albicans, which appears to be more critical after the first septum formation, and that an interaction between Drs2 and Osh4, via PI(4)P, plays an essential role during invasive filamentous growth
Ezanno, Pierre. „Flippase, tension mécanique et mécanosensibilité“. Paris 6, 2009. http://www.theses.fr/2009PA066167.
Der volle Inhalt der QuelleMembrane proteins and soluble proteins are sensitive to their environment. The lateral mechanical tension in membrane via lipids is a physico-chemical parameter of membrane protein environment. The flippase (a membrane protein) can modulate this tension creating an asymmetry of lipids populations between both of membrane leaflets. Flippase from erythrocytes (a Mg ATP dependent ATPase) is, in this thesis, used unpurified: from its native membrane. A mechanosensitive membrane protein changes conformation according to the mechanical tension in the membrane; for example, the MscL (Mechano sensitive channel Large conductance). The flippase is still active in giant membrane systems used, despite a partial dehydration step of membrane (required step in making giant liposomes). An addition of MG ATP triggers the flippase activity which is detected by liposome shape changes. Then, the lateral mechanical tension is triggered in a membrane containing the flippase and the MscL the opening of which is monitored by electrophysiology. In the presence of flippase activity, the MscL’s behaviour is modified: the required tension to open the channel seems to be lowered
Dieudonne, Thibaud. „Functional and Structural Characterization of Lipid Flippases : The Yeast Drs2p/Cdc50p and the Disease-Related Human Atp8b1/Cdc50a Complexes Structure and Autoregulation of a P4-ATPase Lipid Flippase Screening of Detergents for Stabilization of Functional Membrane Proteins High phosphatidylinositol 4-phosphate (PI4P)-dependent ATPase activity for the Drs2p-Cdc50p flippase after removal of its N- and C-terminal extensions Slow Phospholipid Exchange between a Detergent-Solubilized Membrane Protein and Lipid-Detergent Mixed Micelles: Brominated Phospholipids as Tools to Follow Its Kinetics“. Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASS023.
Der volle Inhalt der QuelleLiving cells are surrounded by membranes organized in bilayers, separating the intracellular medium from the extracellular environment. A hallmark of eukaryotic membranes from the late secretory/endocytic pathways is the asymmetric distribution of phospholipids between the two leaflets. Indeed, phosphatidylcholine (PC) and sphingolipids (SL) are mainly found in the outer leaflet whereas phosphatidylserine (PS) and phosphatidylethanolamine (PE) are sequestered in the inner leaflet. This asymmetry is maintained thanks to different membrane lipid transporters. Among them, flippases, which are transporters fueled by ATP hydrolysis, translocate lipids from the outer to the inner leaflet. Flippases belong to the P4-ATPase family and have been linked to several diseases. For instance, mutated forms of a human P4-ATPase, ATP8B1, are responsible for intrahepatic cholestasis, a severe liver disease. In this thesis, we investigated the regulatory mechanism of two flippases, the yeast PS-specific flippase complex Drs2p/Cdc50p, and the human disease-related flippase complex ATP8B1/CDC50A. Both proteins were expressed in S. cerevisiae and purified for downstream functional characterization. Our results demonstrate that both flippases are tightly regulated by phosphoinositides and autoinhibited by their N- and C-terminal extensions
Johansson, Martin. „Instruktionsfilmer och det flippade klassrummet - Det flippade klassrummet, varför inte?“ Thesis, Malmö universitet, Fakulteten för lärande och samhälle (LS), 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:mau:diva-34640.
Der volle Inhalt der QuelleAndersson, Hanna. „Den flippade läxan : En systematisk litteraturstudie av läxor i det flippade matematikklassrummet“. Thesis, Linköpings universitet, Matematik och tillämpad matematik, 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-153116.
Der volle Inhalt der QuelleThe purpose of this study is to investigate homework given in the flipped mathematics classroom. One of the characteristics of the “flipped classroom” is that traditional lectures are not placed in class time. Direct instruction is instead given as homework, “flipped homework”, often in the form of video lectures. The literature review is based on nine articles and focuses on the design of flipped mathematics homework, pupil’s views of the method, and the possible advantages and disadvantages of flipped homework in relation to traditional homework. There is still a lack of research done on “flipped homework”, which makes it difficult to draw any general conclusions. However, the results indicate that the teaching method may have some advantages, including that video lectures gives the students a greater responsibility for their own learning, and that the fixed time of the video have the potential to reduce the difference in time spent by different students on the same homework.
Villazana-Kretzer, Diana L. „Giardia lamblia genomic and molecular analyses of flippase /“. To access this resource online via ProQuest Dissertations and Theses @ UTEP, 2008. http://0-proquest.umi.com.lib.utep.edu/login?COPT=REJTPTU0YmImSU5UPTAmVkVSPTI=&clientId=2515.
Der volle Inhalt der QuellePeters, Ida. „Det flippade klassrummet : ur ett elevperspektiv“. Thesis, Högskolan Kristianstad, Sektionen för lärande och miljö, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:hkr:diva-13239.
Der volle Inhalt der QuelleNaito, Tomoki. „Phospholipid Flippase Activity and Cellular Function of Class 5 P4-ATPases“. 京都大学 (Kyoto University), 2017. http://hdl.handle.net/2433/225530.
Der volle Inhalt der QuelleTsuchiya, Masaki. „Studies on the functional role of phospholipid flippase in myotube formation“. Kyoto University, 2018. http://hdl.handle.net/2433/235092.
Der volle Inhalt der QuelleMARCOVICH, IRENE. „Structural studies of enzymes: a bacterial flippase and a human kinase“. Doctoral thesis, Università degli Studi di Trieste, 2020. http://hdl.handle.net/11368/2961530.
Der volle Inhalt der QuelleQuesta tesi di dottorato include il lavoro di ricerca su due progetti diversi riguardanti l’espressione, la purificazione e la caratterizzazione di due enzimi. Il primo progetto è stato svolto all’Università di Trieste sotto la supervisione della professoressa Rita De Zorzi ed era volto alla caratterizzazione strutturale della O antigen flippasi Wzx da Pseudomonas aeruginosa. Wzx è una proteina integrale di membrana che si trova nella membrana interna dei batteri gram-negativi ed è coinvolta nella biosintesi del lipopolisaccaride (LPS). P. aeruginosa è un patogeno opportunista e uno delle principali cause delle infezioni nosocomiali, è inoltre particolarmente difficile da eradicare a causa della resistenza del batterio agli antibiotici. Essendo Wzx un enzima fondamentale per la formazione della parete cellulare, questa proteina potrebbe essere un promettente bersaglio per una nuova classe di antibiotici e di adiuvanti degli antibiotici. Le procedure di espressione e purificazione di Wzx sono state ottimizzate portando a una considerevole resa della proteina di membrana utilizzabile per gli studi cristallografici. Sono stati ottenuti dei piccoli cristalli in condizioni differenti che, considerando la differenza dei parametri di cella unitaria, sono stati ottimizzati. Finora la qualità dei cristalli non ha permesso la determinazione della struttura. Parallelamente, il folding e la stabilità della proteina sono stati investigati con il dicroismo circolare e la spettroscopia a fluorescenza. Inoltre, le spettroscopie IR e Raman hanno fornito informazioni sul processo di degradazione di Wzx. Lo stato oligomerico della proteina è stato investigato con il microscopio elettronico utilizzando la tecnica del negative staining. Il secondo progetto è stato svolto nel Laboratorio di Biologia Strutturale di Elettra Sincrotrone (Trieste) sotto la supervisione della dottoressa Paola Storici. Questo progetto era volto alla determinazione strutturale del complesso tra la Glicogeno Sintasi Chinasi 3 (GSK3-) e il suo inibitore SR90, sintetizzato nel laboratorio della dottoressa Stephanie Federico all’Università di Trieste. GSK3- catalizza l’addizione di un gruppo fosfato a specifiche proteine bersaglio e fa parte dei meccanismi di regolazione di molteplici vie metaboliche tra cui le cascate di segnale associate ai disordini neurodegenerativi. Visto che un’inibizione di questa chinasi può essere benefica per il trattamento di queste patologie, l’inibitore SR90 è stato progettato per legarsi covalentemente con GSK3-. Inoltre, SR90 è in grado di inibire un’altra chinasi, la casein chinasi1 (CK1-), competendo con il legame dell’ATP. Anche CK1- è coinvolta nelle patologie neurodegenerative e fosforila I substrati di GSK3- agendo come chinasi iniziatrice. Tre costrutti sono stati clonati ed espressi in cellule d’insetto: la proteina intera e due costrutti troncati, corrispondenti ai residui 25-393 e 35-386 rispettivamente in cui sono stati rimossi i terminali a causa della loro flessibilità che può pregiudicare la cristallizzazione. L’attività dell’inibitore sulla chinasi è stata investigata con il Thermal Shift Assay per determinare l’effetto del legame sulla stabilità della proteina. Visto che il composto si è dimostrato in grado di stabilizzare la conformazione proteica, sono state effettuate delle prove di cristallizzazione su GSK3- 35-386 complessato con SR90. I dati di diffrazione dei raggi X sui cristalli proteici sono stati raccolti al Sincrotrone ottenendo dati a una risoluzione 2.3 Å. L’analisi della mappa di densità elettronica ha dimostrato la formazione di un legame covalente tra la proteina e l’inibitore.
Ekelund, Gustaf. „Stöd och stimulans i det flippade klassrummet“. Thesis, Malmö universitet, Fakulteten för lärande och samhälle (LS), 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:mau:diva-30561.
Der volle Inhalt der QuelleBotella, César. „Rôle d'une ATPase de type P4 dans l'homéostasie des glycérolipides membranaires chez Arabidopsis thaliana“. Thesis, Université Grenoble Alpes (ComUE), 2016. http://www.theses.fr/2016GREAV019/document.
Der volle Inhalt der QuelleIn a eukaryotic cell, each membrane compartment has a specific lipid composition, regulated according to physiological and environmental conditions. This lipid homeostasis results from coordination of lipid synthesis, conversion, degradation and trafficking. Whereas most enzymes involved in lipid metabolism are now identified, most steps of lipid transport remain to be characterized. We focus on the chloroplast lipid homeostasis, particularly on galactolipid homeostasis, essential lipids of photosynthetic membranes. This lipids are synthetized within the chloroplast's envelope. However, a majority of galactolipids derived from phosphatidylcholine which is synthetized in the endoplasmic reticulum. The delocalization of this synthesis pathway underline the importance of the associated lipid trafficking.Transcriptomic studies have highlighted that ALA10 is overexpressed in condition modifying the galactolipids synthesis such as the chemical inhibition of MGDG synthesis by the galvestine-1, or during phosphate starvation.The aim of this thesis is to characterize ALA10, analyzing its role concerning this lipid trafficking and the chloroplastic galactolipid homeostasis.To understand ALA10's role, we firstly have used GFP fusion to determine its subcellular localization and analyzed lipid composition of different plant lines expressing ALA10 at different levels. Lipid analysis show that ALA10 boosts galactolipid synthesis and limits endoplasmic reticulum located phosphatidylcholine desaturation. We searched ALA10's potential partners in order to explain this effects and studied their interaction using a bimolecular fluorescence complementation approach. We determined that ALA10 interacts with ALIS1 and ALIS5, two beta subunit potentially necessary for ALA10's localization and function, and confirmed the colocalization of these proteins with ALA10 using GFP/CFP fusions. ALA10 with ALIS5 can localize within the endoplasmic reticulum in close proximity to chloroplast, or near the plasma membrane with ALIS1. We have also determined that ALA10 interacts with a fatty acid desaturase, FAD2 and with an E3-ubiquitine ligase PUB11. FAD2 interaction confirms the link between ALA10 and phosphatidylcholine desaturation.Then we have studied the ALIS1 and ALIS5 effect on ALA10 function using KO lines for these proteins or overexpressor lines in conjunction with ALA10 overexpression. Electron microscopy observation revealed that the chloroplast morphology and their relations with endomembrane system are modified depending of the ALIS expressed with ALA10. Lipid analysis on KO lines confirms an impact of ALA10 on galactolipids homeostasis as well as in phosphatidylcholine desaturation. This effect appears to be variable depending of the photoperiod or the circadian rhythm indicating a post traductional regulation of ALA10. The role of PUB11 in this regulation have been studied.Finally this study reveal that, in chlorophyll cells, the endoplasmic reticulum phospholipid flippase ALA10 is involved in the desaturation process of phosphatidylcholine. Its activity stimulates galactolipid synthesis and activates biogenesis of photosynthetic membranes, probably by promoting lipids exchange between chloroplasts and endoplasmic reticulum
Yilmaz, Burcin, und Isabelle Österberg. „Det flippade klassrummet : En fenomenografisk studie av tolv elevers uppfattningar“. Thesis, Örebro universitet, Institutionen för humaniora, utbildnings- och samhällsvetenskap, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:oru:diva-45281.
Der volle Inhalt der QuelleHäggkvist, Häggkvist. „Varför läxor? : En studie om några mellanstadielärares tankar om och syfte med läxor“. Thesis, Umeå universitet, Institutionen för språkstudier, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-103329.
Der volle Inhalt der QuelleKubelt, Janek. „Investigations on the rapid transbilayer movement of phospholipids in biogenic membranes“. Doctoral thesis, [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=972641653.
Der volle Inhalt der QuelleAndersson, Hanna. „Att flippa läxan – En kvalitativ intervjustudie om lärares erfarenheter av flippade läxor i matematikundervisningen“. Thesis, Linköpings universitet, Matematik och tillämpad matematik, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-167930.
Der volle Inhalt der QuelleThis interview study addresses the use of homework in the flipped mathematics classroom, so-called flipped homework. Flipped classroom is an educational method that is characterized by the removal of some traditional teaching from the classroom. Instead, students prepare for lessons by, for example, watching a video lecture online. Then the in-class time is spent processing the lectures contents. The purpose of this study is to investigate one of the more central components of this method, the homework, in a Swedish context. In the study four mathematics teachers from different parts of Sweden were interviewed about their experiences working with the method. The results show that the Swedish teachers experiences of the method seem to correspond relatively well with international research. Among other things they talked about the possibility for the flipped homework to increase the availability of the material, and the difficulties emerging from the fact that some students don’t complete their homework.
Marainen, Helen. „Formativ bedömning i matematikundervisning : fallstudie i en studieförberedande gymnasieklass“. Thesis, Umeå universitet, Institutionen för naturvetenskapernas och matematikens didaktik, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-126079.
Der volle Inhalt der QuelleAzouaoui, Hassina. „Étude structurale et fonctionnelle d’un transporteur de lipides « une flippase » de la levure S. cerevisiae : l’ATPase P4 Drs2p et sa sous unité-associée Cdc50p“. Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLS224/document.
Der volle Inhalt der QuelleMaintenance of phospholipid asymmetry in eukaryotic cell membranes is essential for cellular integrity and function. P4-ATPases, from the P-type ATPases family, are energy-dependent transporters, together with their CDC50 accessory subunits couple ATP hydrolysis to lipid transport from the exoplasmic to cytoplasmic leaflet to maintain membrane asymmetry.Drs2p is one of these P4-ATPases in the yeast Saccharomyces cerevisiae. Drs2p is localised in trans-Golgi (TGN) membranes in association with its binding partner Cdc50p, which contributes to the correct addressing of Drs2p and probably in the catalyzed transport by Drs2p. Drs2p transport principally phosphatidylserine (PS) in TGN membranes. The PS is important for a several signalling pathways, for example, in apoptosis and recruitment of the proteins implied in various essential cellular process, so, it's very important to understand the mechanism that establishes this asymmetry.To gain in comprehension of molecular mechanism of lipid transport, robust protocols for expression and purification are required. In this work, we present a procedure for high-yield co-expression of Drs2p and Cdc50p. The purification of Drs2p and Cdc50p is achieved in a single step by affinity chromatography on streptavidin beads, yielding, 1-2 mg purified Drs2p/Cdc50p per liter of culture. This procedure allows purification of the complex Drs2p/Cdc50p with stoichiometry to 1:1. Our complex is functional, overal ATP hydrolysis by the complex is dependent of PS, favourite substrate of Drs2p. This hydrolyze is critically dependent on the presence of PI4P, a phosphoinositide involved in regulation of membrane trafficking.Like many P-type ATPases auto-regulate their activity, we examined the possibility that P4-ATPases are auto-regulated. In this work, we use directed mutagenesis and limited proteolysis associated with mass spectrometry for identify peptides. We show that limited proteolysis of a purified complex Drs2p/Cdc50p resulted in up to a 30-50 fold increase of it ATPase activity, which however remained dependent on PI4P. Using thrombin as the protease, Cdc50p remained intact and in complex with Drs2p, which was cleaved at two positions, namely after R104 and after R1290. Our results therefore reveal that trimming off appropriate regions of the terminal extensions of Drs2p can increase its ATPase activity in the presence of PI4P by an enormous factor, thereby identifying a role of N and/or C-terminal extensions in auto-inhibition of Drs2p.Our results open perspectives on the structural and the functional characterization of the lipid transport mechanism by the complex Drs2p/Cdc50p. Furthermore, our procedures open up the possibility of studying the molecular bases of the pathologies associated with the mutations of human P4-ATPases
Pohl, Antje Heide. „Lipid transport by ABC proteins“. Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2002. http://dx.doi.org/10.18452/14784.
Der volle Inhalt der QuelleIn eukaryotic cells, the lipid species are frequently distributed asymmetrically between the plasma membrane leaflets. Phosphatidylserine (PS), in particular, often exhibits a distinct transverse asymmetry, being restricted almost exclusively to the inner leaflet. In the past years, several proteins were suggested to transport lipids between the leaflets of a membrane, and to potentially influence transverse lipid asymmetry and related cell properties. This thesis focuses on outward transport of fluorescent (C6-NBD-) lipid analogs and endogenous lipids by the Multidrug Resistance 1 P-Glycoprotein (MDR1 Pgp), a member of the ATP binding cassette (ABC) transporter superfamily. Interestingly, MDR1 Pgp has been suggested to exhibit an unusually broad substrate specificity. Here, the anionic PS was of particular concern, although previously reported not to be an MDR1 Pgp substrate. In a human gastric carcinoma cell line (EPG85-257) overexpressing MDR1, outward transport of phosphatidylcholine, phosphatidylethanolamine, glucosylceramide and sphingomyelin analogs via MDR1 Pgp was confirmed using fluorescence spectroscopy. In addition, decreased accumulation of analogs of diacylglycerol and ceramide suggest MDR1 Pgp mediated transport of these lipid species. Upon intracellular labelling with C6-NBD-PS using a novel approach, significantly increased outward transport of this analog in MDR1 overexpressing cells could be attributed to MDR1 Pgp by employing specific inhibitors. In a flow cytometry setup, the exposure of endogenous PS on the outer plasma membrane leaflet was significantly elevated in MDR1 overexpressing cells compared to controls. Reduction of PS exposure by an MDR1 Pgp inhibitor suggests transport of endogenous PS by MDR1 Pgp. Transport of C6-NBD-PS was furthermore characterized here in four additional cell lines of different species and tissue origin with varying synthesis levels of MDR1 Pgp. Besides MDR1 Pgp, the ABC half-size transporter Breast Cancer Resistance Protein (BCRP) is possibly also involved in transport of C6-NBD-PS and in increased exposure of endogenous PS, as found in a BCRP overexpressing EPG85-257 subline.
Schreiber, Gabriele. „Aufreinigung und funktionelle Charakterisierung der peroxisomalen ABC-Transporter Pxa1p-Pxa2p aus Saccharomyces cerevisiae“. Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2007. http://dx.doi.org/10.18452/15711.
Der volle Inhalt der QuelleThe 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.
Jacquot, Aurore. „Co-expression et caractérisation fonctionnelle d'un transporteur de lipides (une " flippase ") de la levure S. cerevisiae : l'ATPase P4 Drs2p, en complexe avec sa sous-unité associée Cdc50p“. Phd thesis, Université Paris Sud - Paris XI, 2012. http://tel.archives-ouvertes.fr/tel-00938337.
Der volle Inhalt der QuelleJacquot, Aurore. „Co-expression et caractérisation fonctionnelle d’un transporteur de lipides (une « flippase ») de la levure S. cerevisiae : l’ATPase P4 Drs2p, en complexe avec sa sous-unité associée Cdc50p“. Thesis, Paris 11, 2012. http://www.theses.fr/2012PA11T081/document.
Der volle Inhalt der QuelleTrans-Golgi membranes and plasma membranes of eukaryotic cells are asymmetric, with their cytosolic leaflet enriched in aminophospholipids (APLs: phosphatidylserine and phosphatidylethanolamine). Dissipation of this asymmetry is involved in many (patho)physiological processes. P4 ATPases are prime candidates for APL transport and for maintaining asymmetry across membranes. In addition, yeast deleted for P4 ATPases display membrane trafficking defects. Besides, CDC50 proteins have been shown to interact physically with P4 type ATPases, and this interaction is important for addressing the complex to the right destination, and possibly also for its function. To gain insight into the molecular mechanism of lipid transport by P4 ATPases, the goal of my thesis was to develop the co-expression, in yeast, of a functional P4 ATPase, Drs2p, together with its partner, Cdc50p. The strategy we developed allowed us to obtain a membrane fraction enriched in Drs2p (~3%), mainly in complex with Cdc50p. Functional characterization of the complex identified phosphatidylinositol-4-phosphate (PI4P), a major regulator of membrane trafficking, as a crucial component for rapid completion of the Drs2p/Cdc50p catalytic cycle. We also purified the complex in one step on streptavidin beads. Finally, we started investigating the potential auto-inhibitory roles of the C-terminus (as the C-terminus of Drs2p contains a PI4P binding site) and the N-terminus of Drs2p, by expressing various truncated versions of Drs2p. Our work sets the stage for detailed functional and structural characterization of the Drs2p/Cdc50p complex and its role in membrane traffic
Carlsson, Samuel. „Ett flippat klassrum : från ett yrkeslärarperspektiv“. Thesis, Högskolan i Skövde, Institutionen för hälsa och lärande, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-15153.
Der volle Inhalt der QuelleThe purpose of the study is to investigate how vocational teachers who teach industrial engineering education reflect around, flipped classroom as a teaching method. The method used in the study has been qualitative and three vocational teachers have been interviewed. Previous research on flipped classrooms and different learning styles forms the basis for the analysis.The result showed that flipped classrooms have many positive effects for both teachers and students. For many students, learning time is many times less compared with traditional teaching. Many teachers mix traditional classroom with flipped classroom and the variation allows students to learn new things more easily.My conclusion was that flipped classrooms are a popular teaching method of both teachers and students, but it is time-consuming for the teachers at the beginning and that there will be a change for the students who are expected to be more prepared before a lesson starts. The teacher and the students working with flipped classrooms must more or less be technically motivated for the model to work.
Galeano, Victor. „Flipped Classroom och Digitala Materiel : En Karaktärisering av Tre Naturvetenskapliga Ämneslärares Användning av Digitala Hjälpmedel“. Thesis, Linnéuniversitetet, Institutionen för fysik och elektroteknik (IFE), 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:lnu:diva-71504.
Der volle Inhalt der QuelleJing, Weidong. „Phospholipid Flippases in B cells and Platelets“. Phd thesis, 2019. http://hdl.handle.net/1885/165928.
Der volle Inhalt der QuelleKamani, Mustafa. „Novel Intrinsic and Extrinsic Approaches to Selectively Regulate Glycosphingolipid Metabolism“. Thesis, 2013. http://hdl.handle.net/1807/35860.
Der volle Inhalt der QuelleIslam, Salim Timo. „Structural and Functional Characterization of O-Antigen Translocation and Polymerization in Pseudomonas aeruginosa PAO1“. Thesis, 2013. http://hdl.handle.net/10214/7239.
Der volle Inhalt der QuelleBookmarks within the document have been provided for ease of access to a particular section in the body of the thesis. Each entry in the Table of Contents, List of Tables, and List of Figures has been "linked" to its respective position and as such can be clicked for direct access to the entry. Similarly, each in-text Figure or Table reference has been "linked" to its respective figure/table for direct access to the entry.
1.) Canadian Institutes of Health Research (CIHR) Frederick Banting and Charles Best Canada Graduate Scholarship doctoral award, 2.) CIHR Michael Smith Foreign Study Award, 3.) Cystic Fibrosis Canada (CFC) doctoral studentship, 4.) University of Guelph Dean's Tri-Council Scholarship, 5.) Ontario Graduate Scholarship in Science and Technology, 6.) Operating grants to Dr. Joseph S. Lam from CIHR (MOP-14687) and CFC