Une distribution lipidique hétérogène est essentielle à l’identité et fonction des organelles, mais l’échange par trafic vésiculaire tend à annuler cette distribution. Il existe donc des mécanismes qui assurent l’homéostasie des lipides. Les protéines Osh (S. cerevisiae) et les OSBP-Related Proteins (ORP, H. sapiens), sont des transporteurs de lipides. Osh4 est capable d’échanger de l’ergostérol contre le phosphatidylinositol-4-phosphate (PI4P), présent sur l’appareil de Golgi. Utilisant des outils fluorescents mesurant avec une précision inégalée le transport de stérol et de PI4P, nous démontrons qu’Osh4 transporte du stérol contre son gradient de concentration en utilisant l’énergie d’un gradient de PI4P. Un couplage au métabolisme du PI4P permettrait à Osh4 d’alimenter le Golgi avec du stérol, ainsi créant le gradient de stérol entre ces organelles. La protéine OSBP participe, via sa capacité à connecter la membrane du RE à celle du trans-Golgi, à la création de jonctions entre ces organelles. Nous avons montré qu’OSBP, par échange stérol/PI4P, utilise le PI4P pour transférer du cholestérol au Golgi, mais également pour autoréguler sa capacité à former les jonctions. Osh6 lie la phosphatidylsérine, nous permettant d’étudier un nouveau mécanisme d’échange. Nous avons résolu la structure cristallographique d’un complexe Osh6/PI4P et avons pu observer l’échange de ces deux ligands par Osh6 entre deux membranes. Cette étude nous permet de suggérer que l’échange de PI4P avec divers lipides, via les protéines Osh/ORP, serait un mécanisme général permettant aux cellules de maintenir le gradient lipidique entre le RE et les membranes tardives de la voie sécrétoire. / An uneven lipid distribution is essential for the function of eukaryotic organelles. However, exchange of material by vesicular trafficking has a tendency to perturb this distribution; mechanisms must though exist to ensure lipid homeostasis. Osh proteins (S. cerevisiae) and OSBP-Related Proteins (ORPs, H. sapiens), are lipid transfer proteins (LTPs). Osh4 is capable of exchanging ergosterol for phosphatidylinositol 4-phosphate (PI4P), found on the Golgi. Using novel fluorescent tools to measure with unprecedented precision the transport of sterol and PI4P, we find that Osh4 can transport sterol against its concentration gradient using the energy of a PI4P gradient. Coupled to phosphoinositide metabolism, this allows Osh4 to transport sterol to the trans-Golgi and create the sterol gradients observed between these organelles. OSBP participates in the creation of membrane contact sites (MCSs) via its capacity to connect ER membranes to those of the trans-Golgi. We have shown that it uses PI4P for transporting cholesterol from the ER to the trans-Golgi by sterol/PI4P counterexchange, hence also autoregulating its tethering activity. Finally, the identification of phosphatidylserine as a ligand for Osh6 allowed us to analyze the possible extrapolation of the PI4P counterexchange mechanism. We have solved the crystal structure of Osh6 in complex with PI4P and have been able to follow counterexchange of PI(4)P and PS in vitro. Concluding, our studies allow us to suggest a general mechanism for ORP/Osh-mediated counterexchange of PI4P for other lipids to maintain lipid gradients between the ER and late membranes of the secretory pathway.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A incidência de infecções fúngicas invasivas vem aumentando nos últimos anos. Estas infecções, em geral, apresentam altas taxas de mortalidade. A profilaxia com antifúngicos ainda é a estratégia mais comum na contenção da mortalidade e prevenção contra infecções fúngicas invasivas, porém, apresenta baixa eficiência, e relatos de resistência às drogas. Além disso, a terapia antifúngica é limitada a um pequeno grupo de drogas, como os polienos, azóis e equinocandinas. Desta forma, a busca de novos alvos de drogas é fundamental para o desenvolvimento de novos antifúngicos. Estudos in silico indicaram quatro genes como potenciais alvo de drogas em fungos patogênicos. Neste contexto, o objetivo deste trabalho foi verificar a expressão das proteínas codificadas por dois destes possíveis genes alvo, a proteína erg6, na fração microssomal, e trr1, na fração citosólica, em hifas de A. fumigatus. Visando alcançar este objetivo, foram primeiramente padronizadas todas as etapas de fracionamento celular visando isolar estas duas subfrações celulares de A. fumigatus. Posteriormente, foi otimizado o protocolo de extração e reidratação de proteínas microssomais bem como reidratação de proteínas citosólicas. Estes extratos foram submetidos a diferentes protocolos de fracionamento proteico em um sistema de eletroforese OFFGEL (OGE). Os resultados de Western immunoblot mostraram que estas duas proteínas, erg6 e trr1, são de fato expressas na fase filamentosa de A. fumigatus. O extrato proteico da fração microssomal submetido ao OGE em doze subfrações apresentou três subunidades da proteína erg6, reconhecidas pelo anticorpo monoclonal, com massas moleculares e pI distintos: uma subunidade de aproximadamente 79 kDa com pI entre 5,91 e 6,49, e outras duas subunidades de aproximadamente 35 kDa e 32 kDa, ambas com pI entre 6,49 e 7,08. A enzima erg6 foi descrita como um homotetrâmero em outros fungos. Porém, nossos resultados sugerem que, em A. fumigatus, a erg6 possui uma estrutura heterotetramérica. Quanto à proteína trr1, tanto no extrato total quanto nas frações resultantes do fracionamento em OGE, uma banda única de aproximadamente 40 kDa, com pI na faixa de 4,79 e 5,33, foi reconhecida pelo anticorpo policlonal. Desta forma, esta proteína parece ter uma estrutura homodimérica, assim como descrito em outros micro-organismos. / The invasive fungal infections incidence has increased in recent years. These infections usually presents high mortality rates. Antifungal prophylaxis remains the most common clinical strategy to decrease mortality and prevent invasive fungal infections, however, it has low efficiency and drug resistance reports. Furthermore, antifungal therapy is limited to a small group of drugs such as polyenes, azoles, and echinocandins. Thus, the search for new drug targets is imperative for the new antifungal agents development. In silico studies have indicated four genes as potential drug target in pathogenic fungi. In this context, our aim was to investigate the expression of two proteins encoded by two putative target genes, erg6 in the microsomal fraction, and trr1 in the cytosolic fraction of A. fumigatus hyphae. To achieve this goal, we first standardized all steps of cell fractionation to isolate these two fractions of A. fumigatus hyphae. Subsequently, was optimized the protein extraction and rehidratation protocols of these two subfractions, such as cytosolic proteins rehidratation. These extracts were submitted to different protocols for protein fractionation in an OFFGEL electrophoresis system (OGE). The Western immunoblot results showed that these two proteins, erg6 and trr1, are expressed in filamentous phase of A. fumigatus. The microsomal protein extract submitted to the OGE in twelve fractions, showed three erg6 protein subunits recognized by monoclonal antibody, with distincts molecular weight and pI: a subunit with approximately 79 kDa, with pI in the range of 5,91 and 6,49, and others two subunits with 35 kDa and 32 kDa, both with pI between 6,49 and 7,08. The enzyme erg6 was described as a homotetramer in other fungi, however, our results suggest that in A. fumigatus the erg6 has a heterotetrameric structure. Regarding trr1 protein, in both, total and fractionated (OGE) extracts, a single band of approximately 40 kDa, with pI in the range of 4.79 and 5.33, was recognized by the polyclonal antibody, suggesting that this protein appears to have a homodimeric structure, as described in other microorganisms.

The sterol biosynthetic pathway is essential for growth of all eukaryotic cells and the main target of antifungal agents. The emergence of resistance to these antifungals in an already ill patient population indicates a need to develop drugs that have a broad spectrum of activity among pathogenic fungi and have minimal patient toxicity. Squalene synthase is the first committed step in the sterol pathway and has been studied intensively for development of antifungal agents. While the overall architecture of this enzyme is identical throughout eukaryotes, it was shown that plant and animal genes cannot complement a squalene synthase knockout mutation in yeast unless the carboxy-terminal domain is swapped for one of fungal origin. This implies that there is a component of the fungal carboxy-terminal domain that is responsible for the complementation phenotype and that is unique to the fungal kingdom of life. To determine the role of the carboxy-terminal domain of squalene synthase in the sterol pathway, we used the yeast Saccharomyces cerevisiae with a squalene synthase knockout mutation and expressed squalene synthases originating from fungi, plants, and animals. In contrast to previous observations, all enzymes tested could partially complement the knockout mutation when the genes were weakly expressed. When induced, non-fungal squalene synthases could not complement the knockout mutation and instead led to the accumulation of carboxysterol intermediates, suggesting an interaction between squalene synthase and the downstream sterol C4-decarboxylase. Overexpression of a sterol C4-decarboxylase from any kingdom of life both decreased the accumulation of carboxysterol intermediates and allowed non-fungal squalene synthases to complement the squalene synthase knockout mutation. Using chimeric squalene synthases from each kingdom of life, the motif in the C-terminal domain responsible for preventing this toxicity was mapped to a kingdom-specific 26-amino acid hinge motif adjacent to the catalytic domain. Furthermore, over-expression of the carboxy-terminal domain alone containing a hinge motif from fungi, not from animals or plants, led to growth inhibition of wild-type yeast. Since this hinge region is unique to and highly conserved within each kingdom of life, this data provides evidence for the development of an antifungal therapeutic as well as for tools to develop an understanding of triterpene catalytic activity and identify similar motifs in other biosynthetic pathways.

No description available.

No description available.

This study was undertaken to determine the sensitivity of grape downy mildew (DM, Plasmopara viticola) and powdery mildew (PM, Erysiphe necator) to commonly used single-site fungicides in Virginia and nearby states. DM and PM isolates were collected from 2005 to 2007. In grape leaf disc bioassays, 92% of the DM isolates were QoI (azoxystrobin)-resistant, but none were resistant to mefenoxam. Eighty-two percent of the PM isolates were QoI-resistant, but none were resistant to boscalid and quinoxyfen. The frequency of the G143A point mutation, which confers high levels of QoI resistance, was quantified in DM and PM isolates by real-time PCR. Most of the QoI-resistant DM and PM isolates contained >95% of the 143A allele. QoI-sensitive DM isolates contained less than 1% of 143A. One out of 145 and 14 out of 154 QoI-resistant DM and PM isolates (able to grow on azoxystrobin concentration ï ³ 1 µg/ml), respectively, contained less than 1% 143A. Most PM isolates exhibited reduced sensitivity to five DMI fungicides when compared to a sensitive subgroup (n=9) and compared to published reports for unexposed populations; the resistance factor (median EC50 of the entire isolate collection divided by median EC50 of sensitive subgroup) was highest for tebuconazole (360) and myclobutanil (350), followed by triflumizole (79), triadimefon (61), and fenarimol (53). Sensitivities to all five DMI fungicides, but also azoxystrobin, were moderately to strongly correlated (pairwise r-values ranging from 0.60 to 0.88). / Master of Science in Life Sciences

Le complexe NADPH oxydase est un élément essentiel de l’immunité inné. Présent dans les cellules phagocytaires (neutrophile), sa fonction est de produire massivement, dans le phagosome, des anions superoxyde et générer ainsi des espèces encore plus réactives de l’oxygène qui vont détruire acides nucléiques, lipides et protéines des bactéries phagocytées. Le cœur membranaire catalytique du complexe NADPH oxydase est constitué d’un hétérodimère membranaire, le cytochrome b₅₅₈ (Cyt b₅₅₈). Après activation de celui-ci par les partenaires protéiques cytosoliques p47phox, p67phox, p40phox et Rac, une succession de réactions de transferts d’électron de part et d’autre de la membrane a lieu au sein du Cyt b₅₅₈ pour aboutir à la réduction du dioxygène de manière très contrôlée. Afin de mieux comprendre cette régulation, nous nous sommes d’abord intéressés aux stéreoisomères trans de l’acide arachidonique, activateur naturel de cet enzyme (cis), sur le fonctionnement de la NADPH oxydase et avons abordé cette étude parallèlement sur du Cytb₅₅₈ d’origine bovine présent dans des membranes de neutrophiles et dans des membranes de levures exprimant le Cytb₅₅₈ de manière hétérologue. Nous avons montré que la géométrie joue un rôle important sur l’activation du complexe enzymatique. Dans un deuxième temps, afin d’étudier le rôle de l’environnement membranaire sur le fonctionnement de la NADPH oxydase, nous avons déterminé les propriétés cinétiques et thermodynamiques de l’activité NADPH oxydase du Cytb₅₅₈ recombinant exprimé en levures, purifié, puis reconstitué en liposomes de composition lipidique variée. Après comparaison avec ces mêmes propriétés obtenues pour le Cytb₅₅₈ dans les membranes plasmiques et du réticulum endoplasmique de levures, nous avons montré que l’activité NADPH oxydase très sensible à la température peut être modulée par la composition et l’état physique de la membrane. / NADPH oxidase complex is a major actor of both antimicrobial host defense and inflammation by generating highly regulated superoxide anion, rapidly converted into reactive oxygen species (ROS). The NADPH oxidase complex consists of a heterodimeric integral membrane flavocytochrome b₅₅₈ and three cytosolic components p67phox, p47phox and p40phox, and the small GTP binding protein Rac. In response to a cellular stimulus, cytosolic proteins are recruited to the phagosomal membrane where they are assembled with the Cytb₅₅₈ to form the active NADPH oxidase. The aim of the work was to better understand the modulation of superoxide anion production by this enzyme. For this purpose, we performed experiments with both bovine neutrophil membranes and yeast membranes expressing the bovine recombinant Cytb₅₅₈. We first investigated the effect of the trans-isomerization of the cis-arachidonic acid, the activator of NADPH oxidase in vitro and showed that specific geometry of the activator plays an important role in the activation of the complex. We also studied the role of the membrane environment on the functioning of NADPH oxidase and determined the kinetics and thermodynamics of NADPH oxidase activity depending on the lipid composition of Cytb₅₅₈ proteoliposomes. Comparison with these properties obtained with recombinant Cytb₅₅₈ embedded into endoplasmic reticulum and plasma membranes, we showed that the NADPH oxidase activity is highly temperature dependent and can be modulated by the lipid environment and the physic state of the membrane.

<p>The main aim of this thesis was to characterize membrane-solute interactions using artificial membranes in immobilized liposome chromatography or capillary electrophoresis. The partitioning of a solute into a cell membrane is an essential step in diffusion across the membrane. It is a valid parameter in drug research and can be linked to the permeability as well as the absorption of drugs. Immobilized liposome chromatography was also used to study partitioning of lignin precursor models. Lignin precursors are synthesized within plant cells and need to pass the membrane to be incorporated into lignin in the cell wall.</p><p>In immobilized liposome chromatography, liposomes or lipid bilayer disks were immobilized in gel beads and the partitioning of solutes was determined. Capillary electrophoresis using disks as a pseudostationary phase was introduced as a new approach in drug partitioning studies. In addition, octanol/water partitioning was used to determine the hydrophobicity of the lignin precursor models.</p><p>Electrostatic interactions occurred between bilayers and charged drugs, whereas neutral drugs were less affected. However, neutral lignin precursor models exhibited polar interactions. Moreover, upon changing the buffer ionic strength or the buffer ions, the interactions between charged drugs and neutral liposomes were affected. Hydrophobic interactions were also revealed by including a fatty acid or a neutral detergent into the bilayer or by using a buffer with a high salt concentration. The bilayer manipulation had only a moderate effect on drug partitioning, but the high salt concentration had a large impact on partitioning of lignin precursor models.</p><p>Upon comparing the partitioning into liposomes and disks, the latter showed a more pronounced partitioning due to the larger fraction of lipids readily available for interaction. Finally, bilayer disk capillary electrophoresis was successfully introduced for partitioning studies of charged drugs. This application will be evaluated further as an analytical partitioning method and separation technique.</p>

The main aim of this thesis was to characterize membrane-solute interactions using artificial membranes in immobilized liposome chromatography or capillary electrophoresis. The partitioning of a solute into a cell membrane is an essential step in diffusion across the membrane. It is a valid parameter in drug research and can be linked to the permeability as well as the absorption of drugs. Immobilized liposome chromatography was also used to study partitioning of lignin precursor models. Lignin precursors are synthesized within plant cells and need to pass the membrane to be incorporated into lignin in the cell wall. In immobilized liposome chromatography, liposomes or lipid bilayer disks were immobilized in gel beads and the partitioning of solutes was determined. Capillary electrophoresis using disks as a pseudostationary phase was introduced as a new approach in drug partitioning studies. In addition, octanol/water partitioning was used to determine the hydrophobicity of the lignin precursor models. Electrostatic interactions occurred between bilayers and charged drugs, whereas neutral drugs were less affected. However, neutral lignin precursor models exhibited polar interactions. Moreover, upon changing the buffer ionic strength or the buffer ions, the interactions between charged drugs and neutral liposomes were affected. Hydrophobic interactions were also revealed by including a fatty acid or a neutral detergent into the bilayer or by using a buffer with a high salt concentration. The bilayer manipulation had only a moderate effect on drug partitioning, but the high salt concentration had a large impact on partitioning of lignin precursor models. Upon comparing the partitioning into liposomes and disks, the latter showed a more pronounced partitioning due to the larger fraction of lipids readily available for interaction. Finally, bilayer disk capillary electrophoresis was successfully introduced for partitioning studies of charged drugs. This application will be evaluated further as an analytical partitioning method and separation technique.

Nous démontrons qu'il est possible de former des bicouches fluides non phospholipides en milieu aqueux avec un mélange d'acide palmitique (PA), cholestérol (Chol) et sulfate de cholestérol (Schol) avec une proportion molaire de 30/28/42. Ces liposomes non phospholipidiques peuvent maintenir un gradient de pH (pHinterne 8 / pHexterne 6) sur une période 100 fois plus longue que les liposomes faits de 1-palmitoyl-2-oléoyl-sn-glycéro-3-phosphocholine (POPC) et de cholestérol (60/40 mol/mol). De plus, ces LUV non phospholipidiques protègent l'acide ascorbique d'un milieu oxydant (1 mM de fer (III)). Une fois piégé dans les liposomes, l'acide ascorbique présente une vitesse de dégradation similaire à celle obtenue en l'absence de fer(III). Ces performances illustrent la perméabilité exceptionnellement limitée de ces liposomes, ce qui implique qu'ils peuvent présenter des avantages comme nanocontenants pour certaines applications. D'autre part, des vésicules unilamellaires géantes (GUV pour Giant Unilamellar Vesicles) ont été formées à partir d'un mélange d'acide palmitique et de cholestérol (30/70 mol/mol). Ces GUV sont stables sur l'échelle de temps de semaines, elles ne s'agrègent pas et elles sont sensibles au pH. Afin d'établir la formation des GUV, l'imagerie par microscopie confocale à balayage laser a été utilisée. Deux sondes fluorescentes ont été utilisées: le rouge du Nile, une sonde hydrophobe qui s'insère dans le cœur hydrophobe des bicouches lipidiques, et la calcéine, une sonde hydrophile qui a été emprisonné dans le réservoir interne des GUV. Cette approche a permis l'observation des parois des GUV ainsi que de leur contenu. Ces résultats montrent la possibilité de former de nouveaux microcontenants à partir d'un mélange d'un amphiphile monoalkylé et de stérol. / First, we demonstrate that it is possible to form non-phospholipid fluid bilayers in aqueous milieu with a mixture of palmitic acid (PA), cholesterol (Chol), and cholesterol sulfate (Schol) in a molar proportion of 30/28/42. These non-phospholipid liposomes can sustain a pH gradient (pHinternal 8 / pHexternal 6) 100 times longer than LUVs made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and cholesterol (60/40 mol/mol). These non-phospholipid LUVs are shown to protect ascorbic acid from an oxidizing environment (1 mM Iron (III)). Once entrapped in these liposomes, ascorbic acid displays a degradation rate similar to that obtained in the absence of Iron (III). This ability illustrates the exceptionally limited permeability of these liposomes, indicating that they can present advantages as nanocontainers for some applications. Second, Giant Unilamellar Vesicles (GUVs) were formed from a mixture of palmitic acid and cholesterol (30/70 mol/mol). These GUVs were stable over weeks, did not aggregate, and were pH-sensitive. In order to establish their formation, confocal laser scanning microscopy imaging was carried out. Two fluorescent probes were used: Nile Red, a hydrophobic probe that inserted in the hydrophobic core of lipid bilayers, and calcein, a hydrophilic probe that was trapped in the GUV internal pool. This approach allowed observation of both the walls of the GUVs as well as their entrapped content. These results show the possibility to form novel microcontainers from a mixture of a monoalkylated amphiphile and sterols.