Tesi sul tema "Flippases"

Many 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.

Le paludisme est une maladie dévastatrice causée par un parasite du genre Plasmodium. Du fait de la propagation de souches résistantes aux actuels antipaludéens, il est nécessaire de comprendre les fonctions physiologiques essentielles du parasite afin de trouver de nouvelles cibles thérapeutiques. Les transporteurs membranaires sont une classe importante de cibles chez l'homme du fait de leur rôle physiologique essentiel pour la cellule. Cependant, chez les parasite du genre Plasmodium, seulement quelques transporteurs ont été biochimiquement caractérisés. Des études récentes de délétion de gènes dans un model murin ont montrées que l’ATPase de type P4, ou flippase, ATP2 de Plasmodium est essentielle pour le parasite. Chez les Eucaryotes, l’activité de translocation des lipides des ATPases de type P4 est nécessaire pour maintenir l’asymétrie des membranes, un élément clé dans de nombreux processus essentiels comme la formation de vésicules ou l’apoptose. Les flippases forment des complexes hétéromériques avec les protéines de la famille Cdc50 qui sont également trouvées dans le génome de Plasmodium. Pour comprendre le rôle fonctionnel de ces transporteurs putatifs durant l’infection par le parasite, nous avons besoin d’étudier leur mécanisme de transport et d’identifier leur (s) substrat (s). Nous avons entrepris l’expression hétérologue chez Saccharomyces cerevisiae d’ATP2, en complexe avec les sous unités Cdc50, de trois espèces différentes de Plasmodium. Nous avons réussi à co-exprimer l’orthologue ATP2 de P. chabaudi (PcATP2) et les sous unités PcCdc50 correspondantes. Par co-immunoprécipitation et une chromatographie d’exclusion stérique détectée par fluorescence, nous sommes parvenus à identifier la sous unité s’associant à PcATP2 : PcCdc50.1. Nous avons ensuite purifié le complexe PcATP2/PcCdc50.1 en utilisant des nanobodies reconnaissant la GFP fusionnée à l’extrémité C-terminale de PcATP2 et nous avons initié la caractérisation fonctionnelle avec des tests de phosphorylation et d’activité ATPasique
Malaria 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

Candida albicans est un champignon pathogène opportuniste de l'homme qui peut causer des infections superficielles ou systémiques; sa capacité à passer d'une forme ovoïde à une forme filamenteuse est associée à sa virulence. Pendant cette croissance filamenteuse hautement polarisée, une accumulation de vésicules (Spitzenkörper), caractéristique des champignons filamenteux, ainsi qu'une distribution enrichie de lipides, tels que l'ergostérol, les dérivés phosphorylés du phosphatidylinositol (PI(4)P, PI(4,5)P2) et la phosphatidylsérine (PS) est observée à l'apex des filaments. Cependant, l'importance de l'asymétrie de ces lipides dans la bicouche membranaire est méconnue. Les flippases (P4-ATPases) transportent les lipides à travers la bicouche membranaire pour générer et maintenir son asymétrie. C. albicans a 5 flippases, incluant Drs2 qui est critique pour la croissance filamenteuse et la distribution de phosphatidylsérine (PS). De plus, un mutant de délétion drs2 est hypersensible au fluconazole et au cuivre et nous montrons ici qu’un tel mutant est aussi critique à la virulence dans un modèle murin d'infection systémique. Pour préciser le rôle de Drs2 pendant la croissance filamenteuse de C. albicans, nous avons étudié la distribution de cette ATPase, ainsi que celle de lipides et régulateurs clés, pendant l'initiation et le maintien de ce processus de croissance. Nous avons également caractérisé des mutants ponctuels de Drs2, analogues à ceux altérés pour le transport de PS chez S. cerevisiae. De plus, nous avons examiné l’importance d'autres flippases, telles que Dnf1-3, dans la croissance filamenteuse invasive ainsi que le rôle de transporteurs de lipides appartenant à la famille des « oxysterol binding protein » (Osh). Nos résultats indiquent que Drs2 joue un rôle unique dans le maintien de la croissance filamenteuse de C. albicans, qui paraît particulièrement critique après la formation du premier septum, et indiquent qu’une interaction entre Drs2 et Osh4, via PI(4)P, joue un rôle essentiel pour la croissance filamenteuse
Candida 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

Membranaires ou solubles, les protéines sont sensibles à leur environnement. La tension mécanique latérale de membrane via les lipides est un paramètre physico-chimique de l’environnement des protéines membranaires. La flippase (une protéine membranaire) peut moduler cette tension en créant une asymétrie de population des lipides entre les deux feuillets d’une membrane. La flippase des érythrocytes (ATPase dépendante du Mg ATP) est, dans cette thèse, utilisée non purifiée : à partir de sa membrane native. Une protéine membranaire mécanosensible change de conformation en fonction de la tension mécanique dans la membrane. Le MscL (Mechano sensitive channel Large conductance) en est un exemple. La flippase reste active dans les systèmes membranaires géants utilisés, malgré une étape de déshydratation partielle de la membrane (étape nécessaire à la formation de liposomes géants). Un ajout de Mg ATP déclenche l’activité flippase qui est détectée par un changement de forme de liposomes. Ensuite, la tension mécanique latérale a été déclenchée dans une membrane qui contient la flippase et aussi le MscL dont le changement de conformation est observé en électrophysiologie. En présence d’activité flippase, le comportement du MscL est modifié : la tension requise d’ouverture semble plus basse
Membrane 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

Les cellules sont entourées de membranes lipidiques organisées en bicouche séparant ainsi le milieu intracellulaire du milieu extérieur. L’une des caractéristiques des cellules eucaryotes est de posséder une distribution asymétrique des lipides constituants les membranes de la voie sécrétoire. En effet, dans ces membranes, la phosphatidylcholine (PC) et les sphingolipides (SL) sont majoritairement retrouvés sur le feuillet externe alors que la phosphatidylsérine (PS) et la phosphatidyléthanolamine (PE) sont séquestrées sur le feuillet interne. Cette asymétrie est maintenue grâce à la présence de transporteurs de lipides. Parmi ces transporteurs, on retrouve les flippases, qui grâce à l’énergie apportée par la consommation d’ATP, transportent les lipides du feuillet interne vers le feuillet externe. Les flippases appartiennent à la superfamille des ATPases de type P et ont été reliées à différentes pathologies humaines lorsqu’elles sont mutées. Par exemple, des mutations du gène ATP8B1 sont responsables d’une forme de cholestase intrahépatique, une maladie hépatique sévère. Dans cette thèse, nous avons étudier le mécanisme de régulation de deux flippases : la flippase de levure PS spécifique Drs2p/Cdc50p ainsi que la flippase humaine ATP8B1/CDC50A. Les deux flippases ont été exprimées dans la levure de bière S. cerevisiae et purifiées afin de réaliser leur caractérisation fonctionnelle. Nos résultats montrent que les deux flippases sont régulées par des phosphoinositides et auto-inhibées par leurs extrémités N- et C-terminales
Living 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

Det flippade klassrummet är en relativt ny undervisningsmetod som grundar sig på att eleverna förbereder sig inför lektionen genom att ta del av det kursmoment som läraren i vanliga fall skulle gå igenom på lektionen. Vanligtvis sker detta genom att de får se en instruktionsfilm som läraren gjort. På så sätt frigörs tid till annan aktivitet på lektionen, såsom hjälp med läxor eller grupparbeten. Det flippade klassrummet har fått en hel del publicitet och instruktionsfilmerna som är en produkt av metoden finns det många av t.ex. Youtube.Syftet med detta examensarbete är att undersöka matematiklärares förhållningssätt och inställning till det flippade klassrummet och instruktionsfilmer, och om vilka faktorer som de anser vara de viktigaste begränsningarna för det flippade klassrummet. För detta användes en kvalitativ metod med semistrukturerade intervjuer som sedan transkriberades. Intervjuerna analyserades sedan och tolkades med hjälp av ramfaktorteorin och en teori om teknikintegrering i undervisningen. Det största hindret som lärarna såg med det flippade klassrummet var att metoden ställde stora krav på att eleverna förberedde sig inför lektionen, vilket fyra av fem lärare inte trodde att deras elever skulle kunna hantera. En av lärarna ansåg istället att det största hindret av att det var ett stort tidskrävande projekt att börja producera instruktionsfilmer.

Syftet med denna litteraturstudie är att redogöra för läxans roll i det flippade matematiklassrummet. Arbetsmetoden ”Flippat klassrum” karaktäriseras av en förflyttning av traditionella föreläsningar ut ur klassrummet. Direkta instruktioner ges istället som läxa, ”flippad läxa”, ofta i form av videoföreläsningar. Litteraturstudien baserar sig på nio artiklar och behandlar den flippade matematikläxans utformning, elevers åsikter om arbetsmetoden, och vilka fördelar respektive nackdelar flippad läxa har i förhållande till traditionell läxa. ”Flippad läxa” är fortfarande är ett relativt outforskat begrepp, vilket gör det svårt att dra generella slutsatser. Studiens resultat tyder dock på att metoden kan ha flera fördelar, bland annat i att videoföreläsningar som läxa ger eleverna ett större ansvar för sitt eget lärande, och att videons bestämda speltid har potential att minska skillnaden i den tid, som olika elever använder för att göra samma läxa.
The 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.

Studien, som utfördes på en kommunal gymnasial och vuxenutbildning, syftar till att undersöka elevers uttryckta uppfattningar av fenomenet "det flippade klassrummet". Det flippade klassrummet är ett arbetssätt som innebär att det som traditionellt sker i klassrummet nu äger rum i hemarbetet och tvärtom. Informanterna, eleverna, fick först uppleva det flippade klassrummet genom att deltaga i lektioner planerade utifrån arbetssättet. Därefter intervjuades de i olika former. Intervjuerna transkriberades och analyserades vilket genererade fem olika kategorier där eleverna på skilda sätt uttrycker sig om det flippade klassrummet. Den första kategorin fick benämningen "flippa klassrummet – teknik och lärande" där uttryckta uppfattningar som berör användningen av den digitala tekniken sorterats in. Den andra kategorin som uppkom vid analysen behandlar uttryckta uppfattningar där eleverna diskuterar hur det flippade klassrummet leder till en anpassning till samhällsförändring. Den tredje kategorin benämns "flippa klassrummet-leder till förändrat klassrumsklimat" där elever uttrycker att det flippade klassrummet erbjuder ett annat klassrumsklimat, i detta fall exempelvis gällande struktur och diskussioner. Den fjärde kategorin berör elevsvar där eleverna uttrycker att det flippade klassrummet leder till effektivt lärande, både i form av direkta effekter men även indirekta effekter. I den femte och sista kategorin behandlar elevsvar där eleverna uttrycker sig om hur det flippade klassrummet inverkar på dem, i både positiv och negativ mening. Resultatet visar att eleverna överlag uttrycker sig positivt om arbetssättet och påpekar både möjligheter och svårigheter. Dessa uttryckta uppfattningar kan och bör tas i beaktning av undervisande lärare.

Questa 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.
Questa 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.

Syftet med denna studie är att undersöka elever och lärares syn på hur arbetsmetoden Flippat klassrum påverkar elevernas möjlighet till stöd och stimulans i matematikämnet. Skolverket (2011:10) har satt upp ambitiösa mål och skriver i Lgy11 att alla de anställda i skolan ska ”ge stöd och stimulans till alla elever så att de utvecklas så långt som möjligt”. Studien bygger på intervjuer med tre lärare och fyra elever och resultatet visar på både stor potential hos arbetsmetoden, men också på en tungrodd verklighet i behov av tydlig struktur. I studien deltar både lärare som aktivt flippar sitt klassrum men också lärare som valt att lämna arbetsmetoden vilket innebär att studien får en intressant dynamik.

Dans les cellules eucaryotes, chaque membrane a une composition lipidique qui lui est propre. La composition des membranes est finement régulée en fonction des conditions environnementales et physiologiques de la plante. Cette homéostasie lipidique est le résultat des processus de synthèse, conversion, dégradation et de trafic des lipides. Si la majorité des enzymes impliqués dans le métabolisme des lipides est identifiée, la majorité des mécanismes de transferts intermembranaires des lipides reste à caractériser. Nous nous concentrons sur l'homéostasie lipidique des chloroplastes et plus particulièrement celle des galactolipides, lipides essentiels des membranes photosynthétiques. Les galactolipides sont synthétisés au niveau de l'enveloppe des chloroplastes. Cependant, une grande partie des galactolipides proviennent de la phosphatidylcholine, elle-même synthétisée dans le réticulum endoplasmique. Cette délocalisation de la voie de synthèse sur deux compartiments souligne l'importance de l’étape de transfert lipidique associé.Des études transcriptomiques ont montré qu'ALA10, une ATPase de type P4, flippase de phospholipides, est surexprimée dans des conditions faisant varier la synthèse des galactolipides, telles que l'inhibition chimique des MGDG synthases par la galvestine-1 ou la carence de phosphate.Le but de cette thèse est de caractériser ALA10 et d'analyser son rôle dans ce trafic lipidique et dans l’homéostasie des galactolipides chloroplastiques.Pour comprendre le rôle d'ALA10, nous avons d'abord étudié sa localisation subcellulaire à l'aide d'une fusion traductionnelle avec la GFP et effectué des analyses lipidiques de différentes lignées exprimant ALA10 à différents niveaux. L’analyse de la composition lipidique indique qu'ALA10 stimule la synthèse des galactolipides et limite la désaturation de la phosphatidylcholine dans le réticulum endoplasmique. Nous avons recherché les partenaires protéiques potentiels d'ALA10 permettant d'expliquer ces effets et utilisé une approche de complémentation de fluorescence bimoléculaire afin d'étudier ces interactions. Nous avons pu déterminer qu'ALA10 interagit avec ALIS1 et ALIS5, deux sous-unités beta potentiellement nécessaires à la localisation et à la fonction d'ALA10, et confirmer leurs colocalisation avec ALA10 à l'aide de fusions GFP/CFP. ALA10 peut être localisée dans le réticulum endoplasmique à proximité des chloroplastes avec ALIS5 ou au niveau de la membrane plasmique avec ALIS1. Nous avons aussi pu déterminer qu'ALA10 interagit avec l’acide gras désaturase, FAD2, et une E3-ubiquitine ligase, PUB11. L''interaction avec FAD2 confirme un lien entre ALA10 et la désaturation de la phosphatidylcholine.Nous avons ensuite étudié l'effet d'ALIS1 et d'ALIS5 sur la fonction d'ALA10 en utilisant des lignées n’exprimant pas ces protéines ou les surexprimant avec ALA10. L'observation en microscopie électronique a révélé que la forme des chloroplastes et leurs relations avec le système endomembranaire sont modifiées en fonction de l'ALIS coexprimée avec ALA10. Les analyses lipidiques effectuées sur les plantes mutantes confirment un effet d’ALA10 sur l’homéostasie des galactolipides et la désaturation de la phosphatidylcholine. Les résultats suggèrent plusieurs fonctions d'ALA10, dépendantes de l’ALIS. Cet effet apparait variable en fonction de la photopériode ou du rythme circadien et indiquent une régulation post traductionnelle d'ALA10. Le rôle de PUB11 dans cette régulation a été exploré.Au final, cette étude révèle que, dans les cellules chlorophylliennes, ALA10, une flippase de phospholipides du réticulum endoplasmique, est impliquée dans la dynamique de désaturation de la phosphatidylcholine. Son activité stimule la synthèse des galactolipides et active la biogénèse des membranes photosynthétiques, probablement, en favorisant les échanges de lipides entre le chloroplaste et le réticulum endoplasmique
In 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

The aim of my study is to get an idea of how some teachers use homework, what they consider a good homework is, and what considerations they take to the students' home circumstances. I have made a qualitative interview study with four middle school teachers. The results of my study show that teachers can flip the classroom and use so-called preparatory tasks. The time saving aspect is a strong reason they choose that model. That students are able to practice their own responsibility and perform meaningful homework seems to also be in focus, but the biggest reason they use flipped classrooms seems to be because it is a more equivalent homework then traditional homework, and that students and their guardians also appreciate the flipped classroom model as they become more involved in their children's schoolwork.

Denna intervjustudie behandlar användningen av läxor i det flippade matematikklassrummet, så kallade flippade läxor. Flippat klassrum är en pedagogisk arbetsmetod som karaktäriseras av att traditionella föreläsningar flyttas ut ur klassrummet. Istället förbereder sig eleverna inför lektionen genom att till exempel se en videogenomgång online, för att sedan bearbeta genomgångens innehåll under lektionstid. Syftet med denna studie är att undersöka en av metodens mer centrala komponenter, läxan, i en svensk kontext.  Studien innefattar intervjuer med fyra matematiklärare från olika delar av Sverige, tre av dessa arbetar på  gymnasieskolor och en arbetar på högstadieskola, Intervjuare behandlar hur de arbetar med läxor i det flippade matematikklassrummet. Resultatet visar att svenska lärares erfarenheter tycks stämma relativt väl överens med internationell forskning. Bland annat i den flippade läxans möjligheter att öka materialets tillgänglighet, men även i att det finns svårigheter i att vissa elever inte gör läxan.
This 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.

I föreliggande arbete studeras hur formativ bedömning har införts i matematik-undervisningen i en studieförberedande gymnasieklass. Vid formativ bedömning kartlägger man vad eleverna redan kan för att sedan använda denna information för att förbättra undervisningen och lärandet. Frågeställningarna har varit hur läraren anpassar undervisningen för elevers olika behov, hur matematikundervisningen upplevs ur ett elevperspektiv samt vilken uppfattning läraren har om elevernas måluppfyllelse. Webbaserade genomgångar som hemläxa och webbverktyg används vid undervisningen. Studien är en fallstudie som består av deltagande observationer, intervjuer och en enkät. Resultatet visar att eleverna vid lärande använder metakognitiva strategier samt är väl medvetna om mål och kriterier. Eleverna är i stort sett nöjda med återkopplingen. När det gäller elevernas måluppfyllelse går det inte att dra generella slutsatser, men undervisningen blir enligt läraren mer likvärdig då alla elever kan se de webbaserade genomgångarna och inte är beroende av föräldrarnas stöd.

Les ATPases-P4 sont des transporteurs membranaires couplant l'hydrolyse de l'ATP au transport de lipides dans les membranes cellulaires eucaryotes. Avec leurs partenaires, les protéines CDC50, les ATPases-P4 transportent les phospholipides, en particulier la phosphatidylsérine (PS) et la phosphatidyléthanolamine (PE), du feuillet exoplasmique au feuillet cytosolique des membranes, assurant ainsi le maintien de l'asymétrie membranaire.Drs2p est l'une des cinq ATPases-P4 de la levure Saccharomyces cerevisiae. Elle est localisée dans les membranes du trans-Golgi (TGN), et elle a comme partenaire la protéine Cdc50p, qui est nécessaire à l'adressage correct et probablement au transport catalysé par Drs2p. Drs2p est principalement responsable du transport de la phosphatidylsérine (PS) dans les membranes du TGN et son activité est essentielle pour le maintien de la PS dans le feuillet cytosolique de ces membranes. En raison du rôle crucial de la PS dans de nombreuses voies de signalisation, aussi bien à l’extérieur (au cours de l’apoptose par exemple) qu’à l’intérieur de la cellule (par le recrutement de protéines impliquées dans des processus cellulaires essentiels), il est important de comprendre le mécanisme par lequel l’asymétrie de la PS est établie.Afin de progresser dans la compréhension du mécanisme moléculaire du transport de lipides, nous avons mis au point une procédure qui nous a permis de co-exprimer Drs2p et Cdc50p dans Saccharomyces cerevisiae. La purification de Drs2p par chromatographie d'affinité sur résine streptavidine a permis d'obtenir une fraction purifiée contenant très majoritairement Drs2p et Cdc50p, à raison de 1-2 mg/L de culture. Les deux protéines sont sous forme de complexe avec une stœchiométrie d'association de 1:1. Le complexe purifié est fonctionnel, et présente une activité d’hydrolyse de l’ATP stimulée par son substrat, la PS. Cette stimulation n’est cependant possible qu’en présence de PI4P, un phosphoinositide impliqué dans la régulation du trafic membranaire.De par leur rôle crucial dans le maintien de l'asymétrie membranaire, les ATPases-P4 ne peuvent qu'être régulées. Comme de nombreuses ATPases de type P sont soumises à une auto-régulation de leur activité, nous avons examiné la possibilité d’une telle auto-régulation dans le cas des ATPases P4. Pour ce faire, une approche par mutagenèse dirigée et protéolyse ménagée associée à l’identification par spectrométrie de masse des peptides ont été effectuées. La protéolyse ménagée du complexe purifié Drs2p/Cdc50p montre une activité ATPasique dépendante au PI4P de 30-50 fois plus importante. La protéolyse par la thrombine engendre un Drs2p dépourvu d'une partie N-terminale (R104) et d'une partie C-terminale (R1290) qui reste toujours associé à Cdc50p. Ce résultat montre qu'une coupure appropriée au niveau des extrémités terminales de Drs2p peut augmenter de façon significative, en présence du PI4P, l'activité ATPasique du complexe, nous amenant ainsi à identifier un rôle auto-inhibiteur des extrémités N- et/ou C-terminales de Drs2p.Ce travail ouvre des perspectives quant à la caractérisation structurale et fonctionnelle du mécanisme de transport de lipides par le complexe. Par ailleurs, il laisse entrevoir la possibilité d’étudier les bases moléculaires des pathologies associées aux mutations de certaines ATPases P4 humaines
Maintenance 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

In eukaryotischen Zellen sind die Lipidspezies häufig asymmetrisch zwischen den Hälften der Plasmamembran verteilt. Insbesondere Phosphatidylserin (PS) weist oft eine ausgeprägte transversale Asymmetrie auf, da es fast ausschliesslich auf die innere Hälfte der Plasmamembran beschränkt ist. In den letzten Jahren wurden mehrere Proteine diskutiert, die Lipide zwischen den Membranhälften transportieren und möglicherweise die transversale Lipidasymmetrie sowie damit verbundene Zelleigenschaften beeinflussen. Im Mittelpunkt der vorliegenden Promotion steht der Auswärtstransport fluoreszierender (C6-NBD-) Lipid-Analoga und endogener Lipide durch das Multidrug Resistance 1 P-Glycoprotein (MDR1 Pgp), das der ATP Binding Cassette (ABC) Transporter Superfamilie angehört. Interessanter Weise wird für MDR1 Pgp eine ungewöhnlich breite Substratspezifität angenommen. Das anionische Lipid PS war hier von besonderem Interesse, obgleich es in vorhergehenden Arbeiten nicht als MDR1 Pgp Substrat betrachtet wurde. Der Auswärtstransport von Phosphatidylcholin-, Phosphatidylethanolamin-, Glucosylceramid- und Sphingomyelin-Analoga durch MDR1 Pgp konnte in einer humanen Magenkarzinomlinie (EPG85-257), die MDR1 überexprimiert, mittels Fluoreszenzspektroskopie bestätigt werden. Zudem legt die verringerte Akkumulation von Diacylglycerol- und Ceramid-Analoga den Transport dieser Lipidspezies durch MDR1 Pgp nahe. Im Anschluß an die intrazelluläre Markierung mit C6-NBD-PS mittels eines neuen Verfahrens konnte der signifikant erhöhte Auswärtstransport dieses Analogons in MDR1 überexprimierenden Zellen durch Verwendung spezifischer Inhibitoren MDR1 Pgp zugeschrieben werden. In flusscytometrischen Versuchen war die Exponierung von endogenem PS auf der äusseren Membranhälfte von MDR1 überexprimierenden Zellen signifikant höher als in Kontrollzellen. Verringerung der PS-Exponierung durch einen Inhibitor von MDR1 Pgp deutet auf den Transport von endogenem PS durch MDR1 Pgp hin. Zusätzlich wurde hier der Transport von C6-NBD-PS in vier weiteren Zellinien mit verschiedener Spezies- und Gewebezugehörigkeit charakterisiert, die unterschiedliche Mengen an MDR1 Pgp synthetisieren. Wie Experimente in einer BCRP überexprimierenden EPG85-257-Sublinie nahelegen, ist ausser MDR1 Pgp möglicherweise ebenfalls der ABC Halb-Transporter Breast Cancer Resistance Protein (BCRP) am Transport von C6-NBD-PS und an der verstärkten Exponierung von endogenem PS beteiligt.
In 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.

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.

Les membranes plasmiques et les membranes du trans-Golgi des cellules eucaryotes présentent une asymétrie des lipides qui les composent, avec les aminophospholipides (APLs : phosphatidylsérine et phosphatidyléthanolamine) enrichis dans le feuillet cytosolique. La dissipation de cette asymétrie est impliquée dans de nombreux processus (patho)physiologiques. Plusieurs études suggèrent que les ATPases P4 sont les candidats les plus probables pour le transport des APLs et le maintien de leur distribution asymétrique ; leur délétion dans la levure inhibe le trafic membranaire. En outre, des études ont montré que les ATPases P4 interagissaient avec les protéines de la famille CDC50 ; cette interaction est essentielle pour l'adressage et peut-être aussi la fonction des ATPases P4. Afin de contribuer à la compréhension du mécanisme de transport des lipides par les ATPases P4, l'objectif de ce travail a été de mettre au point la co-expression fonctionnelle, dans la levure, de l'ATPase P4 Drs2p et de sa protéine partenaire Cdc50p. Nous avons obtenu une fraction membranaire enrichie à 3% avec la protéine Drs2p, majoritairement en complexe avec Cdc50p. L'étude fonctionnelle du complexe nous a permis de mettre en évidence un rôle crucial du phosphatidylinositol-4-phosphate (PI(4)P), un important régulateur du trafic membranaire, au cours d'une étape particulière du cycle catalytique. Nous avons également développé un protocole de purification sur résine streptavidine du complexe Drs2p/Cdc50p. Enfin, comme un site potentiel d'interaction avec le PI(4)P est présent sur l'extrémité C-terminale de Drs2p, nous avons engendré différentes constructions de Drs2p, dans lesquelles une partie de l'extrémité C-terminale a été délétée ; dans une autre construction, l'extrémité N-terminale a également été délétée. Notre travail ouvre la voie à la caractérisation fonctionnelle et structurale détaillée du complexe Drs2p/Cdc50p, et à l'étude du rôle du transport de lipides dans le trafic membranaire.

Les membranes plasmiques et les membranes du trans-Golgi des cellules eucaryotes présentent une asymétrie des lipides qui les composent, avec les aminophospholipides (APLs : phosphatidylsérine et phosphatidyléthanolamine) enrichis dans le feuillet cytosolique. La dissipation de cette asymétrie est impliquée dans de nombreux processus (patho)physiologiques. Plusieurs études suggèrent que les ATPases P4 sont les candidats les plus probables pour le transport des APLs et le maintien de leur distribution asymétrique ; leur délétion dans la levure inhibe le trafic membranaire. En outre, des études ont montré que les ATPases P4 interagissaient avec les protéines de la famille CDC50 ; cette interaction est essentielle pour l’adressage et peut-être aussi la fonction des ATPases P4. Afin de contribuer à la compréhension du mécanisme de transport des lipides par les ATPases P4, l’objectif de ce travail a été de mettre au point la co-expression fonctionnelle, dans la levure, de l’ATPase P4 Drs2p et de sa protéine partenaire Cdc50p. Nous avons obtenu une fraction membranaire enrichie à 3% avec la protéine Drs2p, majoritairement en complexe avec Cdc50p. L’étude fonctionnelle du complexe nous a permis de mettre en évidence un rôle crucial du phosphatidylinositol-4-phosphate (PI(4)P), un important régulateur du trafic membranaire, au cours d’une étape particulière du cycle catalytique. Nous avons également développé un protocole de purification sur résine streptavidine du complexe Drs2p/Cdc50p. Enfin, comme un site potentiel d’interaction avec le PI(4)P est présent sur l’extrémité C-terminale de Drs2p, nous avons engendré différentes constructions de Drs2p, dans lesquelles une partie de l’extrémité C-terminale a été délétée ; dans une autre construction, l’extrémité N-terminale a également été délétée. Notre travail ouvre la voie à la caractérisation fonctionnelle et structurale détaillée du complexe Drs2p/Cdc50p, et à l’étude du rôle du transport de lipides dans le trafic membranaire
Trans-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

Syftet med studien är att undersöka hur yrkeslärare som undervisar på industritekniska utbildningar reflekterar kring, flippade klassrum som undervisningsmetod. Metoden som använts i studien har varit kvalitativ och tre stycken yrkeslärare har intervjuats. Tidigare forskning om flippade klassrum och olika lärstilar ligger till grund för analysen.Resultatet visade att flippade klassrum har många positiva effekter för både lärare och elever. För många elever blir inlärningstiden många gånger mindre jämfört med traditionell undervisning. Många lärare blandar traditionell undervisning med flippade klassrum och variationen gör att eleverna lättare kan lära sig nya saker.Min slutsats blev att flippade klassrum är en omtyckt undervisningsmetod av både lärare och elever, men att det är tidkrävande för lärarna i början och att det blir en omställning för eleverna som förväntas vara mer förberedda innan en lektion startar. Läraren och eleverna som arbetar med flippade klassrum måste mer eller mindre vara teknikintresserade för att modellen ska fungera.
The 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.

IT- och IKT-kompetens utgör en stor del av såväl internationell som svensk utbildningsforskning och enda sedan 60-talet har regeringen gjort satsningar med ändamål att förbättra lärares och elevers kompetens inom dessa områden. Den internationella strävan är att läroplaner införlivar mål för förbättrad IT- och IKT-kompetens, för att förbereda elever inför en arbetsmarknad som alltmer kräver teknologiska kompetenser. I den svenska forskningen har det visat sig att användningen av IT i den svenska skolan har ökat märkbart de senaste åren. PISA-undersökningen har visat att Sverige har låga resultat i problemlösning och att dessa resultat samvarierar med hög användning av IT. Vidare kompetensutveckling behövs och lärare bör anpassa sin undervisning utefter de teknologiska verktygens närvaro i den svenska skolan. Flipped Classroom är en undervisningsmetodik som innebär en betydande omordning av aktiviteter i den klassiska undervisningen, med avseende på vad som sker i klassrummet och vad som sker utanför klassrummet. Vidare ämnar metodiken att tydligt integrera digitala materiel i undervisningen. Framtida forskning bör undersöka hur Flipped Classroom-lärare väljer att utforma deras videoföreläsningar och övriga datorbaserade instruktioner. Detta examensarbete söker finna svar på hur och i vilket syfte Flipped Clasroom-lärare i de naturvetenskapliga ämnena använder digitala materiel i sin flippade undervisning samt ge en överblick av dessa. Tre naturvetenskapliga ämneslärare som använder Flipped Classroom har intervjuats. Det är alltså intervjuerna som utgör den empiriska grunden för detta arbete. Vidare innefattar detta arbete en konstruktion av en lämplig modell för att karaktärisera Flipped Classroom-lärares användning av digitala materiel. Karaktäriseringen indikerar att övervägande del av de digitala materielen används för datorbaserad instruktion utanför klassrummet. Totalt sett utgör 57 % datorbaserad instruktion och 30 % elev-centrerade aktiviteter i klassrummet. Resterande 13 % består av digitala materiel som till övervägande del består i mjukvaror för automatisk rättning av prov. För de intervjuade lärarna gäller att övervägande del av samtliga materiel används för att förmedla det centrala innehållet i respektive kurser. Men individuella skillnader finns. Totalt sett så utgör ungefär 52 % av samtliga materiel, användingsområden som ämnar att elever antingen ska återge kunskap eller lagra den i långtidsminnet. Det visar sig också att större delen av dessa användningsområden samtidigt innebär omfattande förändringar av undervisningsmoment.

The asymmetric distribution of phospholipids in the plasma membrane is generated and maintained through the action of phospholipid flippases in resting cells, but becomes disrupted in apoptotic cells and activated platelets, resulting in phosphatidylserine (PS) exposure on the cell surface. Exposure of PS is indispensable for the clearance of apoptotic cells and promotion of blood coagulation. Stable PS exposure during apoptosis requires inactivation of flippases to prevent PS from being re-internalised. In addition to the plasma membrane, flippases also function in generating phospholipid asymmetry in intracellular membranes and play critical roles in vesicle-mediated protein trafficking. In my PhD I investigated the biochemical properties and physiological roles of the flippases ATP11C and ATP8A1. Using ATP11C-deficient mice, I demonstrated that: 1) ATP11C mediated significant flippase activity in murine B cell subsets. Loss of ATP11C resulted in a defective internalization of PS and phosphatidylethanolamine (PE) in comparison to control cells. The diminished flippase activity caused increased PS exposure on viable pro-B cells freshly isolated from the bone marrow of ATP11C-deficient mice, which was corrected upon a 2-hour resting period in vitro. These findings identified ATP11C as an aminophospholipid translocase in B cell lineages and suggested that the temporary increased PS accumulation on the surface of pro-B cells caused by impaired flippase activity contributed to the B cell lymphopenia observed in ATP11C-deficient mice. 2) Loss of ATP11C resulted in reduced life span and increased size of platelets but did not induce thrombocytopenia or affect platelet function. Interestingly, I found that ATP11C did not mediate the flippase activity in platelets as evident by the normal flippase activity in mutant platelets and absence of protein expression of ATP11C in wild type platelets. These results suggested that ATP11C was not a contributing flippase in murine platelets and ATP11C likely regulated cell extrinsic factors that were required for the survival of platelets in the peripheral blood. Together, these findings highlighted a fundamental difference between the action of ATP11C in leukocytes and platelets. Moreover, I identified that flippase ATP8A1was highly expressed in both murine and human platelets but was not present in the plasma membrane. ATP8A1 was cleaved by the cysteine protease calpain during apoptosis, and the cleavage was prevented indirectly by caspase inhibition, involving blockage of calcium influx into platelets and subsequent calpain activation. In contrast, in platelets activated with thrombin and collagen and exposing PS, ATP8A1 remained intact. These data revealed a novel mechanism of flippase cleavage and suggested that flippase activity in intracellular membranes differed between platelets undergoing apoptosis and activation. Collectively, these findings extended our understanding on the role of flippases in B cell development and important mechanisms of platelet survival and function.

Glycosphingolipid (GSL) metabolism is a complex process involving proteins and enzymes at distinct locations within the cell. Mammalian GSLs are typically based on glucose or galactose, forming glucosylceramide (GlcCer) and galactosylceramide (GalCer). Most GSLs are derived from GlcCer, which is synthesized on the cytosolic leaflet of the Golgi, while all subsequent GSLs are synthesized on the lumenal side. We have utilized both pharamacological and genetic manipulation approaches to selectively regulate GSL metabolism and better understand its mechanistic details. We have developed analogues of GlcCer and GalCer by substituting the fatty acid moiety with an adamanatane frame. The resulting adamantylGSLs are more water-soluble than their natural counterparts. These analogues selectively interfere with GSL metabolism at particular points within the metabolic pathway. At 40 µM, adaGlcCer prevents synthesis of all GSLs downstream of GlcCer, while also elevating GlcCer levels, by inhibiting lactosylceramide (LacCer) synthase and glucocerebrosidase, respectively. AdaGalCer specifically reduces synthesis of globotriaosylceramide (Gb3) and downstream globo-series GSLs. AdaGalCer also increases Gaucher disease N370S glucocerebrosidase expression, lysosomal localization and activity. AdaGSLs, therefore, have potential as novel therapeutic agents in diseases characterized by GSL anomalies and as tools to study the effects of GSL modulation. Two predominant theories have been developed to explain how GlcCer accesses the Golgi lumen: one involving direct translocation from the cytosolic-to-lumenal leaflet of the Golgi by the ABC transporter P-glycoprotein (P-gp, ABCB1, MDR1), and the other involving retrograde transport of GlcCer by FAPP2 to the ER, followed by entry into the vesicular transport system for Golgi lumenal access. To examine the in vivo involvement of P-gp in GSL metabolism, we generated a knockout model by crossbreeding the Fabry disease mouse with the P-gp knockout mouse. HPLC analyses of tissue Gb3 levels revealed a tissue-specific reduction in MDR1/Fabry mice. TLC analyses, however, did not show such reduction. In addition, we performed a gene knockdown study using siRNA against P-gp and FAPP2. Results show these siRNA to have distinct effects on GSL levels that are cell-type specific. These results give rise to the prospect of unique therapeutic approaches by targeting P-gp or FAPP2 for synthesis inhibition of particular GSL pathways.

Heteropolymeric O antigen (O-Ag)-capped lipopolysaccharide is the principal constituent of the Gram-negative bacterial cell surface. It is assembled via the integral inner membrane (IM) Wzx/Wzy-dependent pathway. In Pseudomonas aeruginosa, Wzx translocates lipid-linked anionic O-Ag subunits from the cytoplasmic to the periplasmic leaflets of the IM, where Wzy polymerizes the subunits to lengths regulated by Wzz1/2. The Wzx and Wzy IM topologies were mapped using random C-terminal-truncation fusions to PhoALacZα, which displays PhoA/LacZ activity dependent upon its subcellular localization. Twelve transmembrane segments (TMS) containing charged residues were identified for Wzx. Fourteen TMS, two sizeable cytoplasmic loops (CL), and two large periplasmic loops (PL3 and PL5 of comparable size) were characterized for Wzy. Despite Wzy PL3–PL5 sequence homology, these loops were distinguished by respective cationic and anionic charge properties. Site-directed mutagenesis identified functionally-essential Arg residues in both loops. These results led to the proposition of a “catch-and-release” mechanism for Wzy function. The abovementioned Arg residues and intra-Wzy PL3–PL5 sequence homology were conserved among phylogenetically diverse Wzy homologues, indicating widespread potential for the proposed mechanism. Unexpectedly, Wzy CL6 mutations disrupted Wzz1-mediated regulation of shorter O-Ag chains, providing the first evidence for direct Wzy–Wzz interaction. Mutagenesis studies identified functionally-important charged and aromatic TMS residues localized to either the interior vestibule or TMS bundles in a 3D homology model constructed for Wzx. Substrate-binding or energy-coupling roles were proposed for these residues, respectively. The Wzx interior was found to be cationic, consistent with translocation of anionic O-Ag subunits. To test these hypotheses, Wzx was overexpressed, purified, and reconstituted in proteoliposomes loaded with I−. Common transport coupling ions were introduced to “open” the protein and allow detection of I− flux via reconstituted Wzx. Extraliposomal changes in H+ induced I− flux, while Na+ addition had no effect, suggesting H+-dependent Wzx gating. Putative energy-coupling residue mutants demonstrated defective H+-dependent halide flux. Wzx also mediated H+ uptake as detected through fluorescence shifts from proteoliposomes loaded with pH-sensitive dye. Consequently, Wzx was proposed to function via H+-coupled antiport. In summary, this research has contributed structural and functional knowledge leading to novel mechanistic understandings for O-Ag biosynthesis in bacteria.
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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