Tesis sobre el tema "Lipid transporters"
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1
Raggers, René John. "Lipid translocation by multidrug transporters". [S.l. : Amsterdam : s.n.] ; Universiteit van Amsterdam [Host], 2001. http://dare.uva.nl/document/60218.
2
Alrosan, Amjad. "Characterization of Interacting Partners of ABC Lipid Transporters". Thesis, University of Sydney, 2020. https://hdl.handle.net/2123/22999.
Resumen
This thesis outlines a series of experimental approaches that aim to investigate the roles of the co-factor Alix (apoptosis-linked gene 2-interacting protein X), the deubiquitinase USP9X (Ubiquitin-specific protease 9, X-linked), and sterols, including cholesterol synthesis intermediates and oxysterols, in the post-translational regulation of ATP-binding cassette (ABC) lipid transporters, ABCG1 and ABCG4. Alix and USP9X were identified in our laboratory using peptide mass spectrometry analysis as binding partners of ABCG1. The experiments presented in the following chapters characterize the effects of Alix, USP9X as well as sterols on the ABC transporters’ protein expression. Chapter 1 provides a general introduction about atherosclerosis and the important roles of ABC lipid transporters, including ABCA1 and ABCG1, in mediating cellular cholesterol balance through the reverse cholesterol transport (RCT) pathway. It presents also a general overview of Alzheimer’s disease (AD), and the suggested roles of the ABCA1, ABCG1 and ABCG4 transporters on AD progression. In addition, it describes the transcriptional and post-translational regulatory pathways of ABCA1, ABCG1 and ABCG4. This Chapter also outlines the roles of oxidized metabolites of cholesterol, i.e.‘oxysterols’, in atherosclerosis and AD. As an additional topic, it describes the roles of E3-ubiquitin ligases as well as deubiquitinases on the stability of proteins, such as the ABC lipid transporters, through the ubiquitin proteasomal pathway (UPS). Finally, this Chapter highlights the potential for therapeutic targeting of the UPS, and describes the hypotheses and aims of this thesis. In Chapter 2, materials and methods that are common to all the data chapters as described, including cell culture, optimization of siRNA transfection, preparation of cell lysates, SDS-PAGE and Western blotting, immunoprecipitations and cholesterol efflux experiments. Chapter 3 describes experiments investigating the role of the adaptor protein, Alix, as a potential cofactor between the E3-ubiquitin ligase NEDD4-1 (Neural precursor cell-expressed developmentally down regulated gene 4) and its target, ABCG1. NEDD4-1 is a HECT-domain E3-ubiquitin ligase that was previously identified in our laboratory as a regulator of ABCG1/ABCG4 protein expression (Aleidi, Howe et al. 2015). The experiments presented in this Chapter demonstrate that Alix and NEDD4-1 are co-expressed in a number of brain cell lines including astrocytes and neurons where ABCG1 is expressed. ABCG1 protein levels were significantly increased after knockdown of Alix when ABCG1 overexpressing CHOK1 cells were incubated only with full serum media (i.e. high cholesterol content). Interaction of Alix, NEDD4-1 and ABCG1 was established using a co-immunoprecipitation (co-IP) approach. These observations confirm that Alix is a novel player in regulating ABCG1 expression and facilitates the interaction between NEDD4-1 with ABCG1. Chapter 4 describes experiments investigating the role of Alix in the NEDD4-1 mediated regulation of ABCG4 protein, a close relative of ABCG1. Unlike ABCG1 that was increased after Alix knockdown only when the cells were maintained in full serum media (Chapter 3), Alix knockdown resulted in an upregulation of ABCG4 expression when ABCG4 overexpressing CHOK1 cells were incubated with full as well as low serum media (i.e. high and low cholesterol content). Like ABCG1, Alix and NEDD4-1 were co-immunoprecipitated with ABCG4 using a co-IP approach. These observations confirm that Alix regulates ABCG4 expression by being a potential co-factor between NEDD4-1 and ABCG4. Chapter 5 demonstrates experiments examining the effect of deubiquitinases (DUBs) and specifically USP9X, on ABCG1 and ABCG4 protein levels. Firstly, experiments presented indicated that the use of the general DUB inhibitor PR-619 produced toxic side effects on our cell models. However, treatment of these cells with WP1130, which is a more selective inhibitor for specific DUBs, including USP9X, significantly reduced ABCG1 protein levels in ABCG1 overexpressing CHOK1 cells. Further experiments described the effect of USP9X knockdown on ABCG1 protein levels, however these experiments were complicated by the fact that inconsistent results were found between three independent USP9X siRNA primers, each being effective in reducing USP9X protein expression. Overall, these experiments indicated a role for deubiquitinases in the regulation of ABCG1 protein expression, but were not able to confirm that this was solely due to USP9X. Chapter 6 outlines experiments investigating the role of sterols, including cholesterol, oxysterols and cholesterol synthesis intermediates in the regulation of ABCG1 and ABCG4. Incubating ABCG1 overexpressing CHOK1 cells with cholesterol, oxysterols and cholesterol synthesis intermediates enhanced ABCG1 protein levels, with the highest increase after the addition of Liver X Receptor (LXR) ligands, specifically 24S-OH. However, ABCG4 stabilization was enhanced when ABCG4 overexpressing CHOK1 cells were incubated with cholesterol as well as cholesterol synthesis intermediates, including desmosterol, lathosterol and lanosterol. Consequently, ABCG1 and ABCG4 are regulated differently at the post-translational levels by oxysterols and cholesterol synthesis intermediates. The toxicity of sterols on cells, assessed using a variety of methods, made overall conclusions complicated and indicated that further experiments are warranted. In conclusion, the work presented in this thesis shows promising regulators of cholesterol homeostasis in cells. It describes newly discovered roles for the cofactor Alix, inhibition of deubiquitinases, and sterols in the regulation of ABCG1 and ABCG4 protein expression. Unravelling these pathways by which cells can fine-tune their lipid transporter expression and hence cellular lipid levels provides avenues in future to find novel therapeutic targets for treatments in the setting of atherosclerosis and AD.
3
Charalambous, Kalypso Nicola. "The effect of lipid bilayer composition on small multidrug resistance transporters". Thesis, University of Bristol, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.432740.
4
Wei, Liang Shen. "Drug and lipid interactions on the ABC transporters ABCG1 and MsbA". Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609340.
5
Frangos, Zachary Joseph. "Investigating the molecular mechanism of bioactive lipid inhibitors of GlyT2". Thesis, The University of Sydney, 2022. https://hdl.handle.net/2123/28029.
Resumen
Lipid modulation of glycine transport has attracted significant attention as a potential analgesic pathway for neuropathic pain conditions. An array of bioactive lipids that potently inhibit GlyT2 have recently been developed with one compound, C18 ω9 D-lysine (IC50 = 48 nM), demonstrating metabolic stability and analgesic properties in a neuropathic pain model. Despite this, the clinical viability of these bioactive lipids is limited due to their significant lipophilicity preventing effective clearance from the CNS. The first aim of my study was to perform structure activity relationships on second-generation bioactive lipids to identify a potent structural framework from which to reduce the lipophilicity of these compounds. In addition, I also sought to deepen our understanding of the molecular mechanisms that underlie the inhibitory activity of these lipids. The reversibility of oleoyl-L-carnitine has been shown to be enhanced by co-application of the cholesterol sequestering agent methyl-β-cyclodextrin (MβCD), suggesting a modulatory role of cholesterol on the activity of these GlyT2 inhibitors. The second aim of my study was to examine the influence of membrane cholesterol on the activity of bioactive lipid inhibitors of GlyT2. To achieve my first two aims wild-type and mutant GlyT2 transporters were expressed in Xenopus laevis oocytes with glycine transport and bioactive lipid inhibition measured using two-electrode voltage clamp electrophysiology.
18 bioactive lipids were synthesised, and their inhibitory activity was examined at GlyT2 and the closely related GlyT1. The double bond of the acyl lipid tail was exchanged for a phenylene ring substituted in the ortho, meta or para position and conjugated to various amino acid headgroups and tail lengths. In terms of potency, I found a tail substitution preference of meta > ortho > para and a headgroup preference for positively charged amino acids. There was also an optimal tail length for conferring potency (C18 > C16 > C14 > C12) with the most potent compound identified in this series, 8-8 meta-phenylene L-lysine (IC50 = 32 nM), demonstrating similar potency to its acyl equivalent. Molecular modelling of these compounds predicts that potency is dependent on the ability of the tail group to insert deeply into its binding site, highlighting the importance of this region, and suggesting that further optimisation of the physicochemical properties should focus on the lipid headgroup.
Treatment with MβCD suggests membrane cholesterol plays an important role in modulating the activity of bioactive lipids as cholesterol depletion decreased the potency and enhanced the reversibility of inhibition. A combination of molecular dynamics and site-directed mutagenesis studies revealed that this interaction is mediated via association of cholesterol, following bioactive lipid binding, to a conserved cholesterol site on neurotransmitter transporters formed by TMs 1, 5 and 7. In this site, cholesterol adopts a unique, flipped orientation and closely associates with the terminal end of the lipid tail. I propose that this interaction produces a complementary mechanism of action that enhances potency and alters the dissociation pathway from the lipid binding site, increasing reversibility. Due to the conserved nature of this site, it is likely that this modulation could readily be applied to other family members.
Currently the library of bioactive lipid inhibitors of GlyT2 consists of approximately 80 compounds, with only one examined in vivo. As this library continues to expand, it becomes increasingly important to explore alternative in vivo models that can identify lead compounds to test in neuropathic pain models. The final aim of my study was to examine the viability of zebrafish larvae as an in vivo model to test the activity of these compounds. This was achieved by investigating the role of glycinergic neurotransmission in larval zebrafish escape responses. Knockdown of zGlyT2 induced an impaired escape response which could be rescued by expression of hGlyT2, indicating the importance of glycinergic signalling in this response. Application of the bioactive lipid C18 ω9 D-lysine was able to re-establish this phenotype in larvae ectopically expressing hGlyT2, suggesting that zebrafish larvae are a viable alternative to test the in vivo activity of bioactive lipid GlyT2 inhibitors and identify lead compounds for analgesia studies.
The structure activity relationships performed here, in combination with the molecular mechanisms uncovered, will help drive the rational design of subsequent GlyT2 inhibitors that can be readily assessed in vivo.
6
Aleidi, Shereen Mohammad Suleimann. "Characterization of the Post-Translational Regulation of the ABCA1 and ABCG1 Lipid Transporters by E3 Ligases". Thesis, The University of Sydney, 2016. http://hdl.handle.net/2123/15477.
Resumen
This thesis present a series of experimental approaches that aim to characterize the roles of three E3 ubiquitin ligase enzymes in the post-translational regulation of two ABC lipid transporters; ABCA1 and ABCG1. These E3 ligases, namely HECTD1 ( Homologous to E6Ap C-terminus Domain 1), HUWE1 (HECT, UBA, and WWE domain containing 1), and NEDD4-1 (Neural precursor cell-expressed developmentally down regulated gene 4), were identified during a previous work in our laboratory using peptide mass spectrometry analysis as associate proteins with ABCG1. The experiments presented in the following chapters investigate the effect of these ligases on the ABC transporters' protein expression and cholesterol activity. In addition, the possible mechanism underlying the mode of interaction between these ligases and the ABC transporters is also investigated in attempt to provide a more specific approach to future targeting strategies. Chapter1 provides an overview of atherosclerosis and the essential roles of ABCA1 and ABCG1 lipid transporters in maintaining cellular lipid homeostasis and mediating the macrophage reverse cholesterol transport (RCT) pathway. This chapter also presents a general overview regarding the developed anti-atherogenic therapeutic strategies, including those that attempted to upregulate these ABC transporters, although they were precluded due to drawbacks, indicating the need for a novel specific therapeutic strategy. In addition, it discusses the different regulatory pathways of ABCA1 and ABCG1, in particular the ubiquitin proteasomal pathway, and the role of E3 ligases that determine the specificity of the overall pathway. Chapter 2 describes the main experimental procedures that have been used throughout this thesis, including optimization of siRNA silencing as well as transient overexpression of the ligases in cultured cells, measurement of the ABC transporters' protein expression, immunoprecipitation approaches that detect the interaction between each ligase and ABC transporter, and cholesterol efflux assays that measure the activity of the transporter in exporting cholesterol from cultured cells to extracellular acceptors. Chapter 3 presents experiments investigating the role of the first E3 ligase, HECTD1, in the regulation of ABCA1 and ABCG1. These experiments propose that this newly discovered E3 ligase specifically regulates ABCA1 expression and activity, in a manner that is independent of any change in ABCG1. Silencing of HECTD1 substantially increased ABCA1 expression and cholesterol efflux from THP-1 macrophages to apoA-I, without affecting the ABCG1-mediated cholesterol export to HDL from these cells. The role of the other E3 ligases, HUWE1 and NEDD4-1, are addressed via experiments presented in chapter 4. These two E3 ligases seemed to have an additive effect on ABCG1 protein expression and activity, but not ABCA1. Simultaneous silencing of both ligases significantly increased ABCG1 protein levels and substantially more than the individual silencing. These effects on ABCG1 protein levels were translated to upregulation of the transporter activity. Silencing of both ligases significantly increased the ABCG1- mediated cholesterol export to HDL from cells overexpressing the transporter as well as from THP-1 macrophages. Reciprocally, overexpression of both ligases resulted in a significant decrease in ABCG1 protein levels, confirming their roles in the regulation of the transporter degradation. Chapter 5 illustrates a research strategy used to understand how NEDD4-1 can interact with ABCG1 and also to identify potential candidate adaptor proteins that may perhaps mediate this interaction. This strategy involved looking for specific proline-rich consensus sequences, which are essential for NEDD4-1 interaction with its substrate, in the original mass spectrometry peptide hits. Out of twenty three potential candidates that were investigated, four candidates fulfilled the requirements to act as an adaptor proteins. These findings highlight the probability that one of the identified candidate proteins might recruitNEDD4-1 to ABCG1 and thus facilitate ABCG1 ubiquitination. In conclusion, the work presented in this thesis explores the roles of three E3 ubiquitin ligases in the post-translational regulation of ABC lipid transporters. Identifying these ligases as specific molecular determinates for ABCA1 and ABCG1 ubiquitination and subsequent degradation would provide a new insight into the post-translational regulation of these lipid transporters, as well as into the discovery of a selective therapeutic approach to promote their upregulation.
7
Chantemargue, Benjamin. "In silico investigation of xenobiotic interactions with lipid bilayers and ABC membrane transporters, the case of ABCC4/MRP4". Thesis, Limoges, 2018. http://www.theses.fr/2018LIMO0077/document.
Resumen
L’appréhension des mécanismes d’action biologiques des protéines membranaires nécessite de comprendre les interactions des xénobiotiques avec ces protéines et avec les membranes lipidiques. Les méthodes expérimentales sont parfois coûteuses et ne permettent d’obtenir que des informations partielles sur les interactions xénobiotiques-membrane-protéine. La modélisation moléculaire est une sérieuse alternative. Les simulations de dynamique moléculaire et de dynamique biaisées ont ouvert de nombreuses perspectives en permettant de décrire ces interactions moléculaires à l’échelle atomique. Grâce à des simulations de dynamique moléculaire, nous avons été capables de construire un modèle de transporteur humain ABC : ABCC4/MRP4. Cette protéine a été choisie pour sa présence dans le rein, notamment, et son importance clinique. Nous avons évalué l’influence du cholestérol sur cette protéine. L’étude de domaines spécifiques et l’impact d’un polymorphisme a été reliée à l’activité de transport de cette protéine. Nous avons également étudié l’interaction de xénobiotiques avec ce transporteur humain. Le cycle de transport des transporteurs ABC a été examiné afin de comprendre leur fonctionnement. L’incorporation de cholestérol a montré un impact significatif sur la protéine humaine ABCC4/MRP4 et sur les xénobiotiques étudiés. L’importance de domaines constituant la protéine ABCC4/MRP4 ainsi que l’importance de résidus individuels a clairement été prouvée. Nous avons également pu observer des intermédiaires du cycle de transport d’un transporteur ABC conjointement avec des changements structurauxUnderstanding the biological mechanisms of action of membrane proteins requires the comprehension of the interactions of xenobiotics with these proteins and with lipid membranes. Experimental methods are often demanding and only partially respond to xenobiotic-membrane-protein interactions. In silico molecular modeling is a serious alternative to tackle these issues. Molecular dynamics (MD) and biased dynamics simulations have opened many perspectives by providing an atomistic description of these intermolecular interactions. Using MD simulations, we built a model of the human ABC ABCC4/MRP4 transporter. We explored the influence of cholesterol on this protein as well as the impact of a polymorphism known to shut down the transport activity of this protein. We also studied the interaction of xenobiotics with this human transporter. The transport cycle of the ABC transporters was investigated in an attempt to better understand how it works.Interactions between lipid membranes and xenobiotics were explored by examining their ability to incorporate lipid membranes. Lipid mixtures with cholesterol showed a significant impact on the human protein ABCC4/MRP4 and on the xenobiotics studied. The importance of regions, domains constituting the ABCC4/MRP4 protein as well as the importance of specific residues has been clearly demonstrated. We also observed intermediates in the transport cycle of an ABC transporter in conjunction with structural changes occurring during this cycle
8
Basante-Bedoya, Miguel Angel. "Transporteurs lipidiques dans la morphogenèse du champignon pathogène opportuniste de l’Homme Candida albicans". Electronic Thesis or Diss., Université Côte d'Azur, 2021. http://theses.univ-cotedazur.fr/2021COAZ6030.
Resumen
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 filamenteuseCandida 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
9
Lacabanne, Denis. "Solid-state NMR studies of the ABC transporter BmrA in its lipid environment". Thesis, Lyon, 2017. http://www.theses.fr/2017LYSE1243/document.
Resumen
Les transporteurs à ATP binding cassette (ABC) peuvent transporter une grande variété de substrats utilisant l'ATP-Mg2+ comme source d'énergie. Ces transporteurs sont présents dans toutes les formes de vie et sont impliqués dans la résistance aux médicaments, comprenant les anticancéreux et les antibiotiques. Mes travaux de thèse se concentrent sur le transporteur BmrA (130 kDa) de Bacillus subtilis utilisé en tant que système modèle et homologue de la P-glycoprotéine humaine impliquée dans la multirésistance aux anticancéreux. Dans ces travaux nous montrons que la reconstitution de cette protéine dans les lipides de Bacillus subtilis répond aux deux exigences centrales pour RMN: haut rapport signal sur bruit et la stabilité de l'échantillon sur une période de plusieurs années. Les spectres obtenus indiquent une protéine bien repliée et une préparation très homogène, comme en témoignent les lignes de résonance étroites et la dispersion du signal typique de la distribution de structure secondaire attendue de la protéine membranaire. Nous avons adapté la méthode GRecon utilisée dans les études de microscopie électronique pour la reconstitution des protéines membranaires pour la RMN à l'état solide. Nous avons suivi en détail la reconstitution du transporteur ABC BmrA par dialyse comme référence, et établi des conditions optimales de reconstitution en utilisant un gradient combiné de saccharose / cyclodextrine / lipide caractérisant GRecon. Les spectres RMN de l‘échantillon reconstitué par GRecon sont très similaire à ceux obtenus précédemment sur des échantillons reconstitués par dialyse. La préparation d'échantillons par GRecon présente un gain de temps de près d'un ordre de grandeur. Afin d'étudier les états ouvert vers l'intérieur (inward-facing IF) et ouvert vers l'extérieur (outward-facing OF) du transporteur, nous avons développé un protocole reproductible et quantitatif induisant l'état OF. Nous avons enregistré des spectres bidimensionnels RMN à l'état solide avec différents temps de mélange (20 et 200 ms) afin de suivre les changements des déplacements chimiques et d'identifier les résidus par des corrélations séquentielles. L'apparition très apparente de nouveaux signaux concomitants à la grande amplitude des perturbations de déplacement chimique (CSP) met en évidence l'importante flexibilité et les changements conformationnels de la protéine en présence d'ATP: Mg2+. Afin d'identifier les résidus apparaissant dans les spectres, nous avons utilisé le remplacement paramagnétique du co-facteur Mg2+ par du Mn2+. Cette méthode a révélé que les acides aminés apparaissant dans les spectres sont situés à proximité du site de liaison de l'ATP. En outre, les mesures EPR ont confirmé l'état fermé de la protéine en identifiant la distance correspondant à 1,8 nm entre deux atomes de Mn2+. Nous avons étudié les différences conformationnelles entre l'état IF et OF de BmrA. L'observation de nombreux CSP, ainsi que l'apparition de nouveaux signaux sont observés pour un mutant ne pouvant pas hydrolyser l'ATP, indiquant que l'hydrolyse n'est pas nécessaire pour la transition IF à OF dans BmrA. Nous avons également analysé le mécanisme lié au motif X-loop décrit comme étant impliqué dans la communication entre deux domaines de la protéine. Nous avons observé pour une protéine mutante dans laquelle le transport est aboli mais qui reste ATPase active, une transition incomplète puisque seul un sous-ensemble de CSPs est observé, ainsi qu'un manque de rigidification. Ces mesures suggèrent que la flexibilité semble être le point central dans la transmission des changements conformationnels nécessaires de la partie motrice à la partie d'exportation de molécules. Ces observations montrent que ce système serait semblable à un moteur tournant à plein régime qui ne serait pas connecté de manière rigide à un arbre de transmission le reliant au système de transportATP binding cassette (ABC) transporters can translocate a variety of molecules by coupling drug/lipid efflux with an ATP-Mg2+ fuelled engine. They are found in all forms of life and they are involved in a number of drug resistances including anti-cancer drugs and antibiotics. My studies focus on the drug exporter BmrA (130 kDa) from Bacillus subtilis as a model system and homologue of the human P-glycoprotein that is involved in multidrug resistance in cancer. We show that the reconstitution of this protein in lipids from Bacillus subtilis at a lipid-protein ratio of 0.5 m/m allows an optimal protein insertion into lipid bilayer as well as it complies with the two central NMR requirements: high signal-to-noise in the spectra and sample stability over a time period of years. The obtained spectra point to a well-folded protein and a highly homogenous preparation, as witnessed by the narrow resonance lines and the signal dispersion typical of the expected secondary structure distribution of the membrane protein. In the same time, we adapted the GRecon method used in electron microscopy studies for membrane protein reconstitution to the needs of solid-state NMR sample preparation. We followed in detail the reconstitution of the ABC transporter BmrA by dialysis as a reference, and established optimal reconstitution conditions using the combined sucrose/cyclodextrin/lipid gradient characterizing GRecon. NMR spectra recorded on a sample produced by GRecon showed a highly similar fingerprint as those recorded previously on samples reconstituted by dialysis. GRecon sample preparation presents a gain in time of nearly an order of magnitude for reconstitution. In order to study the inward-facing (IF) and the outward-facing (OF) state of the transporter, we developed a reproducible and quantitative protocol of ATP:Mg2+:VO43- addition inducing the OF state. We used selectively labelled samples obtained by the addition of natural abundance residues in the bacterial medium in order to reduce the number of signals in the spectra of this large protein. We recorded solid-state NMR two-dimensional spectra with different mixing times (20 and 200 ms) in order to follow chemical shift changes and identify residues by sequential correlations. The very noticeable apparition of new signals concomitant with the large amplitude of chemical shift perturbations (CSPs) highlight the important flexibility and conformational changes of the protein in presence of ATP:Mg2+:VO43- substrate. In order to identify the residues appearing in the spectra, we use paramagnetic replacement by Mn2+ of the Mg2+ acting as a co-factor in the active site. The paramagnetic relaxation enhancements caused the Mn2+ revealed that the amino acids appearing in the spectra are located in proximity to the ATP binding pocket. Besides, EPR measurements confirmed the closed state of the protein by identifying the corresponding 1.8 nm distances between two Mn2+. We investigate on the conformational differences identified between the IF and OF state in the ABC transporter BmrA reconstituted in its natural lipids. The observation of numerous CSPs, as well as the apparition new signals are observed for a hydrolysis-incompetent mutant on addition of ATP, indicating that hydrolysis is not required for the IF to OF transition in BmrA. We also analyze the mechanistic of the X-loop motif described to be involved in the communication between two domains of the protein. We observe for a mutant protein in which transport is abolished, but which remains ATPase active, an incomplete transition since only a subset of CSPs is observed, as well as lack of rigidification. This suggests that the change in dynamics might be central for transmitting the relevant conformational changes to the part of the protein driving transport, concomitant of an engine which is turning an input shaft, but which fails to connect in a rigid manner, trough adequate gears, with the output shaft driving the pump
10
Mathey, Aline. "Nouvelles stratégies thérapeutiques et chimiorésistance : molécules bioactives et métabolisme lipidique". Electronic Thesis or Diss., Bourgogne Franche-Comté, 2023. http://www.theses.fr/2023UBFCI014.
Resumen
De nombreux échecs thérapeutiques persistent à cause de mécanismes d’adaptation et de résistance tumorale, tels que l’insensibilité aux signaux de mort cellulaire, la surexpression de transporteurs d’efflux de drogues (i.e. les transporteurs ABC caractéristiques du phénotype de résistance multiple aux drogues ou MDR), des mutations dans les voies de détection et de réparation des dommages à l’ADN ou encore une reprogrammation du métabolisme énergétique. Plus particulièrement, de plus en plus d’études suggèrent que des dérégulations du métabolisme des lipides de la mitochondrie, les cardiolipines, jouent un rôle dans la progression et l’agressivité tumorale, représentant une cible thérapeutique attractive depuis ces dernières années. De ce fait, de nouveaux protocoles thérapeutiques innovants reposant sur l’utilisation de molécules bioactives de faible toxicité sont apparus afin de surmonter la chimiorésistance, réduire la toxicité, les effets secondaires et potentialiser l’efficacité de l’agent chimiothérapeutique afin de prolonger l’espérance de vie et améliorer la qualité de vie des patients. Concernant la première partie de ce projet, nous avons démontré pour la première fois la capacité du xanthohumol, un flavonoïde prénylé extrait du houblon, à restaurer l’induction de dommages à l’ADN dans des cellules de cancer colorectal et à sensibiliser ces dernières à un agent anticancéreux couramment utilisé en clinique, le SN38. Nous avons également montré la capacité de deux huiles essentielles extraites d’Apiacées de Tunisie, Pituranthos chloranthus et Teucrium ramosissimum, à restaurer la sensibilité de cellules de sarcome utérin présentant le phénotype MDR, notamment à la doxorubicine, médiée par l’induction de l’apoptose, la diminution de la surexpression et de l’activité du transporteur ABC P-gp. Les travaux issus de la seconde partie de ce projet ont permis d’apporter une meilleure compréhension des relations existantes entre le métabolisme des cardiolipines et la chimiorésistance grâce à l’identification d’altérations du contenu, de la composition en cardiolipines et de facteurs moléculaires clés qui représentent de nouvelles cibles thérapeutiques potentielles afin de restaurer la sensibilité des tumeurs aux chimiothérapiesMany therapeutic failures persist due to adaptation and tumor resistance mechanisms, such as insensitivity to cell death signals, the overexpression of drug efflux transporters (i.e. ABC transporters involved in the multidrug resistance phenotype or MDR), mutations in DNA damage detection and repair pathways or reprogramming of energy metabolism. More specifically, an increasing number of studies suggest that deregulations of mitochondrial lipid metabolism, cardiolipins, play a role in tumor progression and aggressiveness, thereby representing an attractive therapeutic target in recent years. As a result, new innovative therapeutic protocols based on the use of bioactive molecules of low toxicity have appeared in order to overcome chemoresistance, reduce toxicity, side effects and potentiate the effectiveness of the chemotherapeutic drug in order to extend the life expectancy and enhance the quality of life of patients. Concerning the first part of this project, we demonstrated for the first time the ability of xanthohumol, a prenylated flavonoid extracted from hops, to restore the induction of DNA damage in colorectal cancer cells and to sensitize the latter to a commonly used clinically anticancer agent, SN38. We have also shown the ability of two essential oils extracted from Apiaceae in Tunisia, Pituranthos chloranthus and Teucrium ramosissimum, to restore the sensitivity of uterine sarcoma cells presenting the MDR phenotype, in particular to doxorubicin, mediated by the induction of apoptosis, the decrease in the overexpression and activity of the ABC transporter P-gp. The work resulting from the second part of this project provided a deeper insight into the existing relationships between cardiolipin metabolism and chemoresistance thanks to the identification of alterations in content, composition of cardiolipins and key molecular factors which represent new potential therapeutic targets in order to restore the sensitivity of tumors to chemotherapies
11
Azouaoui, Hassina. "Étude structurale et fonctionnelle d’un transporteur de lipides « une flippase » de la levure S. cerevisiae : l’ATPase P4 Drs2p et sa sous unité-associée Cdc50p". Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLS224/document.
Resumen
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 humainesMaintenance 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
12
Wang, Yuhuan. "ROLES OF ABCG5 ABCG8 CHOLESTEROL TRANSPORTER IN LIPID HOMEOSTASIS". UKnowledge, 2015. http://uknowledge.uky.edu/pharmacy_etds/50.
Resumen
The ABCG5 ABCG8 (G5G8) sterol transporter promotes cholesterol secretion into bile and opposes dietary sterol absorption in the small intestine. An emerging body of literature suggests that G5G8 links sterol flux to various risk factors for metabolic syndrome (MetS) and nonalcoholic fatty liver disease (NAFLD). Therapeutic approaches that accelerate G5G8 activity may augment reverse cholesterol transport (RCT) and provide beneficial effects in the prevention and treatment of cardiovascular and liver disease.
Mice lacking leptin (ob/ob) or its receptor (db/db) are obese, insulin resistant in part due to the reduced levels of hepatic G5G8 and biliary cholesterol. The underlying mechanisms responsible for the reduced G5G8 protein expression in these mice may provide a clue to the drug development for this target. My studies show that neither acute leptin replacement nor liver-specific deletion of leptin receptor alters G5G8 abundance or biliary cholesterol. Similarly, hepatic vagotomy has no effect on G5G8 expression. Conversely, expression of the ER chaperone, GRP78, rescues G5G8 in db/db mice.
Previous studies suggest an interdependent relationship between liver and intestine for cholesterol elimination. A combination therapy that increases G5G8-mediated biliary cholesterol secretion and simultaneously reduces intestinal absorption is likely to act additively in cholesterol elimination. My studies show that treatment with ursodiol (Urso) increases hepatic G5G8 protein and both biliary and fecal sterols in a dose-dependent manner. Ezetimibe (EZ), a potent inhibitor of intestinal cholesterol absorption, produces an additive and dose-dependent increase in fecal sterol excretion in the presence of Urso. However, the stimulatory effects of both Urso and Urso-EZ are not G5G8-dependent.
Beyond increasing G5G8 protein expression and biliary cholesterol secretion, my studies also show that Urso stimulates ileal FGF15 expression in mice. Our data of the stimulated ileal FGF15 expression in LIRKO and reduced hepatic G5G8 protein levels in Atsb KO mice both indicate the previous unrecognized role of FGF15/19 in the regulation of G5G8 and its activity. Indeed, this is subsequently confirmed by our results from the direct test of recombinant human FGF19 on G5G8. Thus, FGF15/19 may provide an alternative strategy in drug development to target G5G8 activity and accelerate cholesterol elimination.
13
McIlwain, Benjamin Carl. "Pharmacology and Lipid Sensitivity of the Prokaryotic Aspartate Transporter, GltPh". Thesis, The University of Sydney, 2016. http://hdl.handle.net/2123/15002.
Resumen
The aspartate transporter from Pyroccocus horikoshii, GltPh, is a model for the structure of the SLC1 family of amino acid transporters. Crystal structures of GltPh provide insight into mechanisms of ion coupling and substrate transport; however, structures have been solved in the absence of a lipid bilayer so they provide limited information regarding interactions that occur between the protein and lipids of the membrane. In this thesis, I have investigated the effect of the lipid environment on aspartate transport by reconstituting GltPh into liposomes of defined lipid composition. Here I show that the rates of aspartate transport and the transmembrane orientation of GltPh are influenced by the primary lipid in the liposomes. In phosphatidylethanolamine (PE) liposomes, I observe the highest rates of transport and show that 85% of the transporters are oriented right-side out, whereas in trimethyl PE liposomes, 50% of transporters are right-side out, and I observe a 4-fold reduction in transport rate. Differences in orientation could only partly explain my observations. Crystal structures of GltPh revealed a tyrosine residue that I propose interacts with lipid headgroups during the transport cycle. Based on site-directed mutagenesis, I propose that a cation-π interaction between Y33 and the lipid headgroups can influence the conformational flexibility of the trimerisation domain, and thus the rate of transport. These results provide a specific example of how interactions between membrane lipids and membrane-bound proteins can influence function, and highlight the importance of the role of the membrane in transporter function. Pharmacological inhibitors of GltPh can also provide valuable tools to understand the molecular basis for substrate and inhibitor specificity and selectivity of SLC1A members, but at present few inhibitors of GltPh have been identified. In this thesis I have screened a library of commercially available aspartate analogues and identified new transportable and non- transportable GltPh inhibitors. I have explored the inhibition profile of GltPh by utilising a thiol-modification assay that isolates sided populations of the transporters reconstituted in liposomes, to determine whether any members of the library displayed preference for either the inwardly- or outwardly-directed binding sites. Also in this thesis I identify several β- carbon substituted molecules that display a strong preference for the outwardly-directed binding site of GltPh. In addition, I have identified a new inhibitor of the SLC1A family that has a preference for the inwardly-directed binding site.
14
Mostyn, Shannon. "HEADS OR TAILS: LIPID INHIBITORS OF THE GLYCINE TRANSPORTER, GLYT2". Thesis, The University of Sydney, 2018. http://hdl.handle.net/2123/18939.
Resumen
Membrane proteins are influenced by the dynamic lipid membrane environment, which can impart stability, mediate protein interactions, and provide highly selective contacts essential for function. Membrane proteins can also bind endogenous lipid ligands or are able to be allosterically modulated by lipids, many of which are thought to access their specific binding sites via the cell membrane. N-arachidonyl glycine (NAGly) is a bioactive lipid that is found in its highest concentrations within the spinal cord and may play an important role in endogenous regulation of glycinergic neurotransmission and pain perception through inhibition of the glycine transporter, GlyT2. In addition to NAGly, a number of lipid inhibitors of GlyT2 have been identified. These compounds are comprised of a long flexible unsaturated acyl tail conjugated to an amino acid or amino acid derivative head group. The aims of my study were two-fold; first to identify new, more potent, lipid inhibitors and develop a structure activity relationship for these compounds; and second, to elucidate the molecular mechanisms of inhibition. Wild type and mutant GlyT2 transporters were expressed in Xenopus laevis oocytes with glycine transport and the subsequent inhibition of transport measured using two-electrode voltage clamp electrophysiology, and radiolabelled uptake of glycine. A library of 55 N-acyl amino acids with varying head and tail groups were synthesised and tested at both GlyT2 and the closely related glycine transporter, GlyT1. Two distinct groups of compounds were tested: the first group maintaining a glycine head group and altering the lipid tail; and the second conjugating the [C18 ω9] oleoyl tail to amino acids with varying properties. I found the lipid constituent of the acyl-glycine analogues is essential for specific interactions and the mechanism of inhibition and is not merely a non-selective, sticky adjunct. There was an ideal chain length, with an order of potency C18 > C16 > C14, and stringently defined double bond conformation and position. Conservative differences between compounds are sufficient to impart or remove inhibitory activity which validates highly specific binding to a subtype specific, allosteric pocket. While changing the tail did not greatly alter potency, analogues where the head group was altered significantly influenced apparent affinity. Acyl amino acids containing an aromatic or positively charged side chain conferred the highest apparent affinity, with C16 ω3 L-Lys possessing the highest potency (10.7 nM). 12 compounds inhibited GlyT2 < 100nM, and one of these inhibitors, oleoyl D-Lys, is also metabolically stable and produces analgesia in a rat model of neuropathic pain. Mutagenesis of extracellular loop 4 (EL4), and transmembrane helices TM5 and TM8 suggest that the allosteric binding site is comprised of a cluster of aromatic residues which may strongly coordinate aromatic or positively charged head groups of the most potent analogues. Additionally, changing the properties of a membrane facing residue alters the otherwise slow washout of lipids. From these results, in addition to dynamic docking studies, it is proposed that acyl amino acids may first diffuse into the lipid bilayer and interact with regions of GlyT2 at the protein-membrane interface. Acyl amino acids then access their final binding site formed by aliphatic and aromatic residues from TM5, TM8, and EL4. It has previously been shown that EL4 undergoes important conformational changes in this family of transporters, where EL4 shifts into the outward facing vestibule to occlude the extracellular side and continue the transport cycle. Acyl amino acids may therefore inhibit GlyT2 by stabilising EL4 in a conformation that does not favour transport. The combination of structure-activity studies with molecular insights provides key information on the mechanism of inhibition which will drive further generation of GlyT2 inhibitors for the treatment of neuropathic pain.
15
Taylor, Andrew Mark. "Biophysical studies on the human erythrocyte anion transporter, band 3". Thesis, University of Oxford, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.360571.
16
Krupski-Romsicki, Yolanda. "Modulation of the function of the P-glycoprotein multidrug transporter by lipid environment". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0001/NQ43271.pdf.
17
Quazi, Faraz. "The role of photoreceptor ABC transporter ABCA4 in retinal and lipid transport and Stargardt macular degeneration". Thesis, University of British Columbia, 2013. http://hdl.handle.net/2429/45626.
Resumen
ABCA4 is a member of the superfamily of ATP-binding cassette (ABC) transporters implicated in the clearance of retinoids from photoreceptor outer segments and associated with Stargardt macular degeneration. Stargardt patients display lipofuscin deposits and photoreceptor degeneration that invariably lead to the loss of central vision. In biochemical and knockout mice studies, ABCA4 has been implicated in the transport of N-retinylidene-PE, the Schiff base conjugate formed from all-trans retinal and phosphatidylethanolamine (PE). The principal challenge in assessing direction and transport has been impeded by the unstable hydrophobic nature of the proposed substrate. As part of this study, a novel biochemical transport assay was developed and used to show that ABCA4 actively flips N-retinylidene-PE from the lumen to the cytosolic side of membranes. This is the first mammalian ABC transporter shown to function as an importer. 11-cis retinal delivered in excess to photoreceptors is a defining cause of lipofuscin/A2E formation. Purified ABCA4 transports 11-cis retinylidene-PE. By HPLC analysis, 11-cis retinal also showed isomerization to all-trans retinal with PE present in discs. Thus, ABCA4 insures the complete removal of N-retinylidene-PE conjugates from the disc-lumen, thereby preventing accumulation of lipofuscin precursors including A2PE. Stargardt mutants which showed reduced functionality were rescued by allosteric enhancement of ATPase with dronedarone, an anti-arrhythmic drug, acting in concert with an increase in N-retinylidene-PE transport. Additionally, protein misfolding was selectively restored by 4-phenylbutyrate, a chemical chaperone. Genetic mutations in several ABCA subfamily transporters cause severe-inherited lipid disorders. The structure-function relationships underlying substrate specificity remain unclear. ABCA1 linked to Tangier disease has been implicated in the export of cholesterol and phospholipids to the apolipoproteinA-I acceptor by cell-based assays. In this study, biochemical analysis indicated that purified and reconstituted ABCA1 actively flipped phosphatidylcholine, phosphatidylserine, and sphingomyelin from the cytoplasmic to the lumenal side of membranes, while ABCA7 preferred phosphatidylserine. In contrast, ABCA4 transported PE in the reverse direction. Tangier and Stargardt mutants showed reduced lipid transport activities. This thesis demonstrates the importance of ABCA4 as a unique ABC importer, reports the first reconstitution of phospholipid flippase activity for ABCA transporters, and identifies several compounds as potential therapeutic drugs for Stargardt disease.
18
Butchbach, Matthew E. R. "Regulation of glutamate transport by GTRAP3-18 and by lipid rafts". Connect to this title online, 2003. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1054650123.
Resumen
Thesis (Ph. D.)--Ohio State University, 2003.Title from first page of PDF file. Document formatted into pages; contains xviii, 160 p.; also includes graphics Includes bibliographical references (p. 132-160). Available online via OhioLINK's ETD Center
19
Sasaki, Mayumi. "Cloning of ABCA17, a novel rodent sperm-specific ABC (ATP-binding cassette) transporter that regulates intracellular lipid metabolism". Kyoto University, 2007. http://hdl.handle.net/2433/135646.
20
Sands, Eric R. "Cloning and Expression of Thermophilic, Mesophilic, and Psychrophilic Zn2+ Transporting ATPases". Digital WPI, 2006. https://digitalcommons.wpi.edu/etd-theses/699.
Resumen
Protein folding and stability are essential for protein function. Changes in these characteristics can lead to altered physiological states and to the development of certain pathologies. While extensive research has focused on the stability of soluble proteins, membrane protein stability has received much less attention. Understanding the stability of membrane proteins can provide insight into folding mechanisms and the etiology of various pathologies. The purpose of this project is to prepare molecular tools to perform comparative studies of homologous membrane proteins that are found in various environments. To this end, thermophilic (Pyrococcus abyssi), mesophilic (Escherichia coli), and psychrophilic (Exiguobacterium 255-15) transmembrane Zn2+ transporting ATPases were cloned, expressed, and functionally characterized to correlate thermostability with optimal functional temperatures. In addition, the lipid environments and composition (rigid or fluid lipids) may also be involved in determining the stability of membrane proteins. Toward exploring the role of extremophilic lipids, Archaeoglobus fulgidus and Thermotoga maritima were grown and lipids were extracted. Availability of these molecular tools will enable physical-chemical studies toward understanding the structural factors that determine functional stability.
21
Snider, Shayne. "Choline Transport Links Phospholipid Metabolism and Inflammation in Macrophages". Thesis, Université d'Ottawa / University of Ottawa, 2017. http://hdl.handle.net/10393/35715.
Resumen
Choline is necessary for the synthesis of phosphatidylcholine (PC), the predominant phospholipid species and an important lipid intermediate. Macrophages, critical mediators of innate immunity, have been implicated in lipid dysregulation associated with metabolic disease. Despite the importance of choline in lipid metabolism, few studies have investigated the relationship between choline metabolism and inflammation. My research revealed that macrophage polarization increased choline metabolism and the expression of the choline transporter CTL1. In addition, choline deficient macrophages showed altered cytokine secretion, suggesting choline metabolism may play an important role in regulating the immune response. This study also describes the generation of a novel CTL1-/- mouse, which showed decreased choline uptake and incorporation into lipids. As an in vivo model for choline deficiency, CTL1-/- mice represent an important model for the future study of choline metabolism. Altogether, these findings suggest an important relationship exists between choline metabolism and inflammation.
22
Johnson, Soraya Sarah. "Control of the protein and lipid content of the plasma membrane by ATP-binding cassette transporter proteins in S. Cerevisiae". Diss., University of Iowa, 2010. https://ir.uiowa.edu/etd/825.
Resumen
Pdr5 and Yor1 are two ATP-binding cassette transporters regulated by the pleiotropic drug resistance (PDR) network in the yeast Saccharomyces cerevisiae. Recent work from another group demonstrated that a pdr5Δ yor1 strain confers remarkable resistance to a sphingolipid intermediate, phytosphingosine (PHS), which was surprising as loss of these transporters normally leads to elevated drug sensitivity.
PHS is toxic to the cell at high levels due to mislocalization of nutrient permeases, such as the high affinity tryptophan transporter, Tat2. Although the above study suggested that this resistance was due to increased expression of Rsb1, a known mediator of PHS tolerance, this was not reproducible in our hands and we sought to identify other determinants for this phenotype. The work presented here demonstrates that the pdr5Δ yor1 strain exhibits delayed turnover of Tat2 and an increase in tryptophan uptake, which we postulate is due to changes membrane asymmetry resulting in decreased endocytosis. Conversely, cells lacking Rsb1 showed a decrease in tryptophan import and increased Tat2 turnover, independent of endogenous PHS levels. Rsb1 has a predicted 7 transmembrane (7TM) topology, which argues against the idea that Rsb1 functions directly in PHS transport, as there are currently no known transporters with this topology. These data suggest that Rsb1 and Pdr5/Yor1 function in regulation of endocytosis of Tat2, and possibly other membrane proteins.
Ethyl methanesulfonate mutagenesis of the pdr5Δ yor1 strain and a candidate gene approach were alternative methods used to identify mediators of PHS tolerance in this strain. Inconsistent results from PHS selection led to the discovery that the pdr5Δ yor1 strain was also robustly resistant to the sphingolipid biosynthesis inhibitor, Aureobasidin A (AbA), which was subsequently used for analysis. These approaches revealed several genes, including Gda1, Mss4, and Ypk1 that are important for AbA tolerance in the pdr5Δ yor1 strain. Many of these determinants play a role in cell wall integrity, suggesting that loss of Pdr5 and Yor1 may lead to activated cell wall integrity pathways resulting in altered cell wall structure.
23
Jain, Deepak M. "Effect of corn fibre oil and its constituents on cholesterol metabolism and intestinal sterol transporter gene expression in hamsters". Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=98732.
Resumen
The cholesterol-lowering effect of corn fiber oil, obtained from the seed coats of corn kernels, has been reported previously. Corn fiber oil contains phytosteryl fatty acyl esters, ferulate phytostanyl esters, and free phytosterols. To date, however, no studies have examined the cholesterol-lowering efficacy of ferulate phytostanyl esters. Moreover, although plant stanols and sterols have been established as cholesterol-lowering agents over the past five decades, their exact mechanisms of action are not clearly understood. One of the possible mechanism is that plant sterols/stanols disrupts the normal sub-cellular cholesterol absorption by down-regulation of the influx sterol transporters such as the Niemann pick C1 like 1(NPC1L1) protein and/or up-regulation of efflux sterol transporters such as the ATP binding cassette (ABC) G5 and ABCG8 protein. Hence, the objectives of this thesis were to assess the efficacy of corn fiber oil, ferulate phytostanyl esters and their parent compounds including sitostanol and ferulic acid, on plasma cholesterol levels. Further, objectives were to investigate their impact on parameters of cholesterol kinetics and gene expression of sterol transporters to obtain insight into their role in genetic control of regulation of cholesterol flux. Results of this experiment demonstrate that the hypocholesterolemic effect of corn fiber oil is mostly due to sitostanol, while esterification of ferulic acid and sitostanol yields no apparent synergistic cholesterol lowering effect. Present data exhibited a cholesterol absorption lowering effect of corn fiber oil and sitostanol and suggest that this effect may be due to up-regulation of intestinal enterocyte efflux sterol transporters such as ABCG5 and ABCG8.
24
Lima, Alex da Silva. "Uso da microscopia eletroquímica de varredura (SECM) no estudo de sistemas micelares e do transporte de espécies químicas através de membranas lipídicas". Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/46/46136/tde-01102015-084001/.
Resumen
A presente tese versa sobre resultados obtidos na aplicação da microscopia eletroquímica de varredura no estudo de sistemas micelares e no estudo de bicamadas lipídicas. Os estudos envolvendo sistemas micelares foram realizados utilizando a SECM no modo substrato-gerador/microeletrodo-coletor. Neste modo de operação, um microeletrodo de platina foi posicionado próximo a um substrato de platina e utilizado para monitorar espécies eletrogeradas nesse substrato. Conhecendo o tempo necessário para a espécie eletrogerada difundir do substrato até o microeletrodo, foi possível aplicar a equação de Einstein-Smoluchowski para determinar o coeficiente de difusão da espécie eletroativa e de micelas de surfactantes. Como as micelas não são eletroativas, o ferroceno eletrogerado no substrato e incorporado nas micelas foi utilizado como sonda para a estimativa do tempo de difusão. Os resultados obtidos para o surfactante brometo de tetradecil trimetil amônio (C14TABr) corroboram dados reportados na literatura, demonstrando a utilidade da metodologia proposta no estudo de sistema micelares. Também foram realizados experimentos envolvendo micelas do surfactante cloreto de 1-alquil-3-metilimidazólio, CxMelmCl (x = 10, 12, 14, 16) e com os resultados obtidos foi possível evidenciar o efeito da cadeia carbônica no coeficiente de difusão das espécies. Os experimentos envolvendo a permeação de substâncias através de bicamadas lipídicas foram realizados em duas etapas. Os primeiros ensaios foram realizados utilizando modelo de membrana semipermeável (papel celofane) com o intuito de verificar a aplicabilidade da SECM no monitoramento de espécies eletroativas que permeiam através da membrana. Na segunda etapa, apresentou-se metodologia para a obtenção de microfuros em folhas de poliestireno utilizados para a formação das bicamadas lipídicas, assim como detalhes sobre a construção da célula de medidas utilizadas nos experimentos de permeação. Foram realizados experimentos envolvendo o uso de bicamadas lipídicas planas obtidas pelo método de Miller preparadas com lecitina de soja. Esses experimentos foram realizados com o intuito de avaliar a estabilidade e para verificar a permeabilidade de algumas substâncias nas bicamadas formadas. Os experimentos de permeação foram realizados posicionando um microeletrodo próximo à membrana, com posterior detecção amperométrica da espécie eletroativa que atravessa a membrana.This thesis shows results on the use of scanning electrochemical microscopy in the study of micellar systems and lipid bilayers. Studies involving micellar systems were performed by using SECM in the substrate-generator/tip-collector mode. In this operation mode a platinum microelectrode was positioned close to a platinum substrate and used to monitor electrogenerated species on this surface. Taking into account the time for the electrogenerated species to diffuse from the substrate to the microelectrode, the diffusion coefficient of the electroactive species and of the micelles can be calculated by applying the Einstein-Smoluchowski equation. As micelles are not electroactive, ferrocene electrogenerated on the substrate and incorporated into the micelles was used as a probe to estimate the diffusion time. The results obtained for tetradecyl trimethyl ammonium bromide (C14TABr) corroborate those reported in the literature, demonstrating the applicability of the proposed methodology in the study of micellar systems. Experiments with micelles obtained from 1-alkyl-3-methylimidazolium, CxMelmCl (x = 10, 12, 14, 16) chloride surfactants were also performed and results showed the effect of the carbon chain in the diffusion coefficient. Experiments involving the permeation of chemical species through lipid bilayers were carried out in two steps. A membrane model (cellophane) was preliminary used in order to investigate the possibility of using SECM as a tool for monitoring the permeation of electroactive species through the membrane. Then, a methodology for obtaining microholes in polystyrene sheets used to form lipid bilayers was presented, as well as details about the design of an electrochemical cell used in the permeation experiments. Experiments involving the use of planar lipid bilayers obtained by the method of Miller prepared using soybean lecithin were performed. These experiments were carried out in order to evaluate the stability and to check the permeation of some substances through the prepared bilayers. Permeation experiments were performed by placing the microelectrode close to the membrane with subsequent amperometric detection of any electroactive species that cross the membrane
25
Kaniti, Archana. "Etudes du mode d'action antipaludique de nouveaux bis-cations". Thesis, Montpellier 2, 2010. http://www.theses.fr/2010MON20154.
Resumen
Dans cette thèse, j'ai essayé d'identifier le mode d'action de composés biscationiques récemment synthétisés et leurs interactions avec les parasites résistants à la chloroquine. Les activités de divers représentants des composés ammonium bisquaternaire, des amidines alkyl (provenant du groupe de professeur Vial, Montpellier, France), des amidines bisbenzyl (provenant du groupe Chimie de l'Université de Liverpool, Royaume-Uni) ont été comparés à la chloroquine et la pentamidine. Leurs potentiels de résistance croisée avec la chloroquine ont également été étudiés. Dans ce but, deux lignées cellulaires modifiées génétiquement par échange allélique, C3Dd2 et C2GCO3 furent utilisées.Parmi les amidines bisbenzyl, une série de composés appartenant aux guanidines, thiazoles et triazoles ont été criblés pour leur activité contre des souches résistantes et sensibles à la chloroquine chez Plasmodium falciparum. Une hypersensibilité significative est observée pour les amidines bisbenzyl parmi les isolats affichant un PfCRT mutant. Aucune différence n'est observée pour les composés provenant du groupe Vial. Pour comprendre le mode d'action et le rôle de PfCRT, j'ai réalisé des expériences de fixation compétitives (competitive binding') et de cristallisation d'hème. Tous les composés ont montré à différents degrés des interactions avec l'hème, cependant il fut observé que leur activité ne corrélait pas avec l'inhibition de la cristallisation d'hème. Une des raisons possibles à cela est que les différences structurales peuvent jouer un rôle important dans le transport du composé. De plus, j'ai étudié l'effet du pH sur l'activité des composés en utilisant les lignées cellulaires modifiées génétiquement par échange allélique afin d'observer l'effet du gradient de proton sur le transport de la chloroquine et de la pentamidine. Des différences significatives de l'activité de la chloroquine furent observées chez les deux souches. Malgré les valeurs de pKa élevées pour la pentamidine, il y avait une différence significative dans la sensibilité pour ce composé chez les souches quand le pH a changé.Car les diamidines requièrent des transporteurs pour traverser les barrières membranaires et qu'un possible transporteur de choline a été caractérisé chez Plasmodium falciparum, j'ai également réalisé des études initiales sur la caractérisation moléculaire de ce transporteur. Un gène qui encode une protéine chez P. falciparum avec une similarité significative aux eucaryotes supérieurs fut identifié en utilisant des analyses bioinformatiques et fut employé dans une transformation et des études analyses fonctionnelles.En conclusion, ce travail suggère qu'il est possible d'utiliser de nouveaux amidines bisbenzyl pour cibler spécifiquement les souches résistantes à la quinoléine chez Plasmodium falciparum, arborant des allèles de PfCRT mutantes. En adhérant à cette hypothèse et sachant que les deux classes de composés fixent la même cible non parasitaire (soit l'hème), il serait possible de créer rationnellement une combinaison de composés quinoléine / diamidine. Ainsi, les souches résistantes à un des deux composés seraient plus sensibles à l'autre partenaire, retardant ainsi l'apparition de résistanceIn this thesis I have attempted to subject the issues of mode of action of recently synthesized bis cationic compounds and their possible interactions with chloroquine resistance. Antimalarial activities of representatives of various bis quarternary ammonium compounds, alkyl amidines (received from Dr.Vial group, Montpellier) and of bisbenzyl amidines (received from Chemistry group, Liverpool) activity have been investigated with chloroquine and pentamidine and looked for cross resistance with chloroquine. For this purpose two genetically modified allelically exchanged cell lines C3Dd2 and C2GCO3 modified on the chloroquine resistance-related PfCRT (P.falciparum chloroquine Resistance Transporter) gene were used. Among the benzyl amidines, a significant hypersensitivity tobis benzyl amidines was observed among the isolates bearing the mutant PfCRT. No such difference is observed for the bisalkyl amidines. To understand the mode of action and role of PfCRT, competitive binding assay to heme (which may mediate the well-known cellular accumulation of the compounds) and effect on heme crystallization assays (which is involved in the toxic effect against the intracellular parasite) were performed. All these compounds were shown to interact with heme in various degrees. Their activity was observed not to be correlating with heme crystallization inhibition. This is likely due to the structural differences between the compound which discriminate the compounds in the transport of the compound to the parasite and their mechanism of antimalarial activities. In addition I have studied the effect of pH on the pharmacological activity of the drugs using allelically exchanged genetically modified cell lines (for PfCRT) to characterize the importance of proton gradient on the transport of chloroquine and pentamidine to the intracellular parasite. Significant difference (reduced antimalarial activity with increased pH) in the activity of chloroquine was observed for both the strains. Despite of the high pKa values for pentamidine, there was significant difference in the sensitivity of the strains to this compound, when the pH is changed. As both the diamidines and choline analogs require transporters to cross the membrane barriers and enter the parasite where they accumulate I have also performed initial studies on the molecular characterization of a potential carrier in P.falciparum. Using basic bioinformatic tools, a gene encoding a P.falciparum protein with significant similarity to higher eukaryotes choline transporter was identified and preliminary work for its functional analysis was performed. In conclusion, this work establishes substantial differences between the various classes of bis-cationic compounds essentially (based on benzamidine and choline-analogs alkylkamidine series) concerning their interaction with the infected erythrocyte and their antimalarial activity. The series are diffentallly affected by the PfCRT mutation and the chloroquine resistance. Results suggest that it may be possible to use novel bisbenzyl amidines to specifically target quinoline resistant Plasmodium falciparum malaria, harbouring mutant pfcrt alleles. Taking this idea further and since both classes of compound target the same non-parasite target (heme), it may even be possible to rationally design a quinoline / diamidine drug combination, in which isolates resistant to one partner drug become more sensitive to the other partner, thus delaying the onset of resistance
26
Li, Hao. "In vitro Studies of Genodermatoses Affecting Cytoskeletal Integrity and Lipid Processing in Human Epidermis : Pathogenic Mechanisms and Effects of Retinoid Therapy". Doctoral thesis, Uppsala universitet, Institutionen för medicinska vetenskaper, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-172863.
Resumen
Autosomal dominant epidermolytic ichthyosis (EI) is a rare disease characterized by intra-epidermal blistering due to mutations in either of two keratin genes, KRT1 and KRT10, expressed by suprabasal keratinocytes. Autosomal recessive congenital ichthyosis (ARCI) is a non-blistering, hyperkeratotic disease caused by mutations in one of the following genes: ABCA12, ALOX12B, ALOXE3, TGM1, CYP4F22, NIPAL4 and SLC27A4, which are all essential for skin barrier homeostasis. ARCI and EI often respond well to treatment with retinoids, but the mechanism of action is unclear. The aim of this thesis was to increase the knowledge of pathogenic pathways in ichthyosis and to find new explanations to the effect of retinoids. In vitro studies of immortalized keratinocytes from EI patients showed an abnormal keratin aggregation after heat stress, that could be partially inhibited by pre-treatment with all-trans retinoic acid (ATRA) or retinoic acid receptor α-agonists. ATRA treatment also reduced the relative expression of mutated vs wildtype KRT10. The clearance of ATRA in human keratinocytes was found to be mediated by CYP26B1. In skin biopsies from ARCI patients, immunofluorescence analysis of 12R-LOX, eLOX-3, TGM1, ichthyin and FATP4 showed altered expression, not only of the mutated protein, but also of the other proteins. These observations are consistent with a feedback regulatory mechanism by which the loss of one protein results in an up-regulation of other proteins. Furthermore, 12R-LOX, eLOX-3 and TGM1 were intimately co-localized in stratum corneum, as were ichthyin and FATP4, suggesting that the proteins are linked to the same metabolic pathway. When treated with a CYP26 inhibitor known to raise the endogenous ATRA level of the skin, two patients with NIPAL4 mutations, initially exhibiting increased co-localization signals for 12R-LOX and eLOX-3, displayed normalized lipoxygenase expressions and showed clinical improvement. In conclusion, mechanisms are proposed by which pathogenic keratin aggregations in EI and epidermal protein deficiencies in ARCI patients may be mitigated by retinoids. Furthermore, the vivid crosstalk between proteins incriminated in ARCI suggests that these enzymes operate along a common metabolic pathway essential for producing barrier lipids in stratum corneum. Any abrogation of this production may cause barrier failure, hence resulting in a compensatory hyperkeratosis characteristic of congenital ichthyosis.
27
Rodrigues, Alice Cristina. "Efeito da atorvastatina sobre a atividade funcional e expressão de transportadores de membrana do tipo ABC e SLC". Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/9/9136/tde-19052009-200620/.
Resumen
Os transportadores de membrana do tipo ATP Binding Cassette (ABC) e solute carriers (SLC) regulam a homeostase intracelular de fármacos, modificando a biodisponibilidade e possivelmente a eficácia terapêutica. A variabilidade na resposta a hipolipemiantes, como as vastatinas, tem sido associada a vários fatores genéticos e ambientais. Com a finalidade de avaliarmos os mecanismos de regulação da expressão dos transportadores pela atorvastatina, a expressão de RNAm de transportadores ABC (ABCB1, ABCG2 e ABCC2) e SLC (SLCO1B1, SLCO2B1 e SLC22A1) foi avaliada por RT-PCRq em células mononucleares do sangue periférico (CMSP) de 18 indivíduos normolipidêmicos (NL) e 22 pacientes hipercolesterolêmicos (HC) tratados com atorvastatina (10mg/dia/4 semanas). A possível associação entre o polimorfismo ABCB1 C3435T e a expressão de RNAm também foi avaliada. Os estudos in vitro foram realizados com as células das linhagens HepG2 e Caco-2. Foram avaliados os efeitos da atorvastatina na ativação de fatores de transcrição (NF-kappaB, NF-Y, c-jun, SP-1 e PXR) por ensaio de mobilidade eletroforética retardada em gel de poliacrilamida (EMSA) e na meia-vida do RNAm do gene ABCB1 por RT-PCRq, e a expressão e atividade funcional da proteína ABCB1 por Western blot, imunohistoquimica e citometria de fluxo. A proteina ABCB1 foi localizada por imunohistoquimica na membrana apical do canalículo biliar das celulas HepG2 e na membrana apical das Caco-2. O tratamento das células HepG2 com atorvastatina causou redução da expressão de RNAm do gene ABCB1 e aumento na expressão dos genes ABCG2 e ABCC2. Esses efeitos foram dose e tempo dependentes. O tratamento com atorvastatina das células Caco-2 não modificou a expressão dos transportadores de efluxo após 30 a 120 min. Nas células HepG2, as concentrações de 10 e 20 M de atorvastatina causaram diminuição da expressão de ABCB1 (0 µM: 1,00 ± 0,06; 10 µM: 0,69 ± 0,25, p< 0,05; 20 µM: 0,69 ± 0,06, p< 0,05). A atividade da ABCB1, avaliada pelo efluxo de Rh123, mostrou-se estar reduzida em 41% nas células HepG2, após tratamento com atorvastatina 20 µM. Embora a diminuição da expressão do ABCB1 não tenha sido decorrente de uma menor ativação transcricional, avaliada indiretamente por EMSA, estudos de mecanismos de regulação pós-transcricionais, revelaram que a atorvastatina diminui a estabilidade de RNAm do gene ABCB1. Esse resultado parece estar de acordo com o ocorrido nas CMSP, já que o tratamento com atorvastatina diminuiu a expressão de RNAm do gene ABCB1 nos indivíduos HC. Essa modulação, no entanto não está associada à presença do polimorfismo ABCB1 C3435T. Em relação aos transportadores de captação, a expressão do SLC22A1 nas células Caco-2 diminui após tratamento com atorvastatina por 30 min e não foi modificada nas células HepG2. Já o gene SLCO2B1 encontrou-se muito aumentado após 24 h de tratamento nas células HepG2. Estudos in vivo nas CMSP, mostrou que a expressão de mRNA basal dos transportadores nos HC foi 10 vezes maior que nos NL e diminuiu após tratamento com atorvastatina nos HC. Com os resultados obtidos podemos sugerir que diferenças no efeito da atorvastatina nos tipos celulares podem ser em decorrência da expressão tecido-específica de fatores de transcrição. No modelo de hepatócito, HepG2, a atorvastatina é um inibidor do transporte mediado pela ABCB1 e é capaz de diminuir a síntese e a função da ABCB1, via aumento da degradação de RNAm do gene ABCB1. Em conseqüência ocorre uma redução do efluxo pelo sistema biliar, causando aumento da concentração intracelular. Ainda, podemos concluir que em CMSP o colesterol pode ser o responsável pela modulação dos genes dos transportadores de membrana e que isso pode implicar em diferenças na eficácia da atorvastatina.Specific membrane transporters have a significant impact on drug absorption and disposition. Most of them belong to two super-families, ABC (ATP-binding cassette) and SLC (solute-linked carrier). Statins are important therapeutic agents in the management of hypercholesterolemia, and considerable inter-individual variation exists in response to its therapy. The effects of atorvastatin expression of efflux (ABCG2 and ABCC2) and uptake (SLCO1B1, SLCO2B1 and SLC22A1) drug transporters were investigated by qPCR in Caco-2 and HepG2 cell lines and in peripheral blood mononuclear cells (PBMCs) of eighteen normolipidemic (NL) and twenty two hypercholesterolemic (HC) individuals treated with atorvastatin (10mg/day/4 weeks). The possible involvement of ABCB1 C3435T polymorphism in ABCB1 mRNA expression was also evaluated. In vitro studies with the cell lines HepG2 and Caco-2 were also performed. The effect of atorvastatin on the activation of the promoter of ABCB1 by transcription factors (NF-kappaB, NF-Y, c-jun, SP-1, and PXR) was evaluated by electrophoretic mobility shift assay (EMSA), and ABCB1 mRNA half-life were measured by PCRq. The expression and functional activity of ABCB1 were investigated by Western blot, imunohistochemistry and flow cytometry. Immunohystochemical analysis revealed that ABCB1 is located at the apical membrane of the bile canaliculi in HepG2, and in apical membrane of Caco-2 cells. Atorvastatin treatment of HepG2 cells caused a decreased in ABCB1 and an increase in ABCC2 and ABCG2 transcript levels. These effects were time and dose-dependent. Treatment of Caco-2 cells did not present any differences in efflux transporters mRNA levels. Treatment of HepG2 cells with 10 and 20 M atorvastatin caused a reduction on ABCB1 expression (0 µM: 1,00 ± 0,06; 10 µM: 0,69 ± 0,25, p< 0,05; 20 µM: 0,69 ± 0,06, p< 0,05), and a 41% decrease in ABCB1-mediated efflux of Rhodamine123 (p < 0.01). Although reduced ABCB1 mRNA expression was not due to any repressor protein suppressing ABCB1 promoter activation, mRNA stability studies revealed that mRNA stability of ABCB1 was markedly decreased by atorvastatin treatment (2h versus 7h for control). In agrrement with these results, in PBMCs of HC individuals, atorvastatin treatment also reduced ABCB1 mRNA expression. However, the down-regulation was not associated with the presence of 3435T allele. For the uptake transporters, atorvastatin decreased SLC22A1 transcript levels after 30min-treatment and it was not regulated in HepG2. On the other hand, SLCO2B1 was up-regulated after 24h-treatment of HepG2 cells. In vivo studies with PBMCs revealed that during hypercholesterolemia all the drug transporters analyzed were increased almost 10-fold (p< 0.05), and after atorvastatin therapy the efflux and uptake transporters transcript levels were all down-regulated. These findings suggest that atorvastatin exhibits differential effects on mRNA expression of drug transporters depending on the cell type, which may be related to tissue-specific expression of transcription factors. Atorvastatin leads to decreased ABCB1 function and synthesis in HepG2 cells by increasing degradation of ABCB1 mRNA. Therefore, inhibition of ABCB1 may reduce atorvastatin elimination via bile, increasing its cellular concentrations. We also may suggest that in PBMCs cholesterol modulates mRNA expression of drug transporters, and this may contribute to the variability of response to atorvastatin.
28
Al-Khfajy, Wrood Salim Dawood. "Role Of Transmembrane 141 in Cholesterol Metabolism". Kent State University / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=kent1416142859.
29
Flinois, Thomas. "Modification d'électrodes par des films redox-actifs, des lipides et des transporteurs ioniques membranaires : vers l'élaboration d'une pile biomimétique". Thesis, Rennes 1, 2019. http://www.theses.fr/2019REN1S093.
Resumen
En s'inspirant des processus transmembranaires de transport d'ions à l'œuvre dans les cellules vivantes, la conception d'une pile biomimétique peut être envisagée. Le principe de cette pile est d'utiliser les gradients de concentration en ions ou en protons induits par la protéine NhaA, un antiport transmembranaire Na+/2H+, pour générer de l'énergie électrique. Cette thèse contribue à l'élaboration de cette pile à travers des modifications d'électrodes permettant de détecter l'activité de transporteurs ioniques à l'interface électrode / membrane biomimétique. Une revue bibliographique des aspects fondamentaux des membranes cellulaires et du principe de la pile est présentée. Des électrodes modifiées par électrodéposition de dérivés d'acides salicyliques ont permis l'obtention de films redox-actifs sensibles aux variations de pH ou à la concentration en ions monovalents et monoatomiques. La stabilité du dépôt lipidique sur électrode a été significativement augmentée par l'électro-greffage de la 4-decylaniline. Ce dépôt lipidique stable dans le temps permet d'obtenir des membranes biomimétiques et l'insertion de transporteurs ioniques. L'activité des transporteurs ioniques insérés dans les membranes biomimétiques est suivi à travers les variations de potentiel dépendant du pH ou de la concentration en ions sodium ou potassium à l'interface électrode/membraneBased on the transmembrane ion transport processes of living cells, the design of a biomimetic fuel cell can be considered. Its principle relies on ion or proton concentration gradients induced by the protein NhaA, a transmembrane Na+/2H+ antiport, to generate electrical energy. This thesis contributes to the development of this device through electrode modifications to detect the activity of ionic transporters at the electrode/biomimetic membrane interface.A bibliographic review of the fundamental aspects of cell membranes and of the biomimetic fuel cell principle is presented. Electrodes modified by electrodeposition of salicylic acid derivatives have yielded redox-active films sensitive to pH changes or monovalent and monoatomic ions concentration. The stability of the lipid deposit onto the electrodes was significantly increased by the electrografting of 4-decylaniline. This anchored and stable lipid deposit makes it possible to obtain biomimetic membranes allowing the insertion of ionic transporters. The activity of the ion carriers inserted into the biomimetic membranes has been monitored through the redox-active films' potential that is dependent on pH or on sodium or potassium ions concentrations at the electrode/membrane interface
30
Hakizimana, Pierre. "Phosphatidylethanolamine regulates the function and the structure of LmrP, a bacterial multidrug transporter protein associated to antibiotic resistance". Doctoral thesis, Universite Libre de Bruxelles, 2008. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210486.
Resumen
The multidrug transporter LmrP, member of the major facilitator superfamily (MFS), confers L. lactis and recombinant E. coli cells resistance to an array of cytotoxic compounds including antibiotics. LmrP mediates drug extrusion from the plasma membrane by an electrogenic proton/drug exchange reaction, whereby a positively charged substrate may move towards the external medium in exchange for two or more protons moving towards the cytoplasm. Recent studies have suggested that MFS transporters require phosphatidylethanolamine (PE) for function and proper topology. However, the specificity of the PE requirement, as well as the contribution of the electrochemical gradient (the driving force of the substrate transport) to this lipid requirement was not addressed. Here we report a new approach for addressing PE specific requirement for the function and the structure of membranes transporters. We used methyl-PE and dimethyl-PE analogs of PE to show that only replacement of the three hydrogens by methyl moieties leads to changes in the biochemical and biophysical properties of the reconstituted protein. This suggests that LmrP does not depend on the bulk properties of the phospholipids tested but solely on the hydrogen bonding ability of the headgroup. We then show that a single point mutation in LmrP, D68C, is sufficient to recapitulate precisely every biochemical and biophysical effect observed when PE is replaced by phosphatidylcholine (PC) ( including energy transfer between the protein tryptophan residues and the lipid headgroups). We conclude that the negatively charged Asp-68 is likely to participate in the interaction with PE and that such interaction is required for proton gradient sensing, substrate binding, and transport. Because Asp-68 belongs to a highly conserved motif in the Major Facilitator Superfamily (which includes LacY and EmrD), this interaction might be a general feature of these transporters that is involved in proton gradient sensing and lipid dependence.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished
31
Freire, Camilla Camerino Santana Davino. "Metabolismo de lipídeos em inseto coleóptero: digestão e transporte de ácidos graxos". Universidade Federal de Alagoas, 2018. http://www.repositorio.ufal.br/handle/riufal/3421.
Resumen
Coleoptera is an order of insects well known as beetles. Most coleopteran species are phytophagous insects and for this reason are essential to crop and storage pests such as the Tribolium castaneum. Lipid metabolism is vital for the biological functions of insects, playing a role in the generation of metabolic energy and other cellular processes. Fatty acid transport proteins (FATPs) play a crucial role in the transport of extracellular fatty acids to cells, have a conserved sequence between species and are involved in the synthesis of hormones and pheromones. Recent studies show that silencing the gene for FATPs through interfering RNAi techniques (RNAi) in insects affects fatty acid uptake and pheromone synthesis. This work aims to characterize proteins homologous to FATPs present in the genome of Tribolium castaneum, to evaluate the gene expression in tissues, developmental stages and insects treated with Orlistat and to evaluate the effect of FATP silencing on energy metabolism. Bioinformatics analyzes were performed with the amino acid sequences, and real-time PCR evaluated the gene expression. The effects of the drug Orlistat were evaluated through qPCR and analysis of nutritional index. The search for sequences in the T. castaneum genome revealed two sequences of proteins homologous to FATPs and bioinformatic analysis was performed. The study of the gene expression of FATPs by qPCR demonstrated more significant expression of the two genes in the fat body of larvae and many expressions in all stages of development of the insect, with higher expression in the pupa stage. The effects of Orlistat on the expression of FATPs evidenced the influence of diet composition on the regulation of the gene expression of these proteins. Gene silencing of TcasFATP was achieved, but no direct effects on the energetic dynamics of the larvae were observed since triacylglycerol levels, and β-oxidation rates were not affected. Thus, more detailed studies with the use of gene silencing will be necessary to characterize FATPs better and elucidate their role in insect energy metabolism.FAPEAL - Fundação de Amparo à Pesquisa do Estado de Alagoas
Os coleópteros constituem uma ordem de insetos bastante conhecidos como besouros. A maioria das espécies de coleóptero são insetos fitófagos e por esta razão constituem importantes pragas de culturas e de armazenamento, como o Tribolium castaneum. O metabolismo de lipídeos é importante para as funções biológicas de insetos, exercendo papel na geração de energia metabólica e em outros processos celulares. As proteínas transportadoras de ácidos graxos (FATPs) exercem papel crucial no transporte de ácidos graxos extracelulares para as células, possuem sequência conservada entre as espécies e estão envolvidas na síntese de hormônios e feromônios. Estudos recentes mostram que o silenciamento do gene para FATPs através de técnicas de RNA de interferência (RNAi) em insetos afetam a absorção de ácidos graxos e a síntese de feromônio. Este trabalho tem como objetivo caracterizar proteínas homólogas à FATPs presentes no genoma de Tribolium castaneum, avaliar a expressão gênica nos tecidos, fases de desenvolvimento e em insetos tratados com Orlistate e avaliar o efeito do silenciamento de FATPs no metabolismo energético. Foram realizadas análises bioinformáticas com as sequências de aminoácidos e a avaliação da expressão gênica foi realizada por PCR em tempo real. Os efeitos do fármaco Orlistate foram avaliados através de qPCR e análise dos índices nutricionais. A busca de sequências no genoma do T. castaneum revelou duas sequências de proteínas homólogas à FATPs e a análise bioinformática foi realizada. O estudo da expressão gênica de FATPs por qPCR demonstrou maior expressão dos dois genes no corpo gorduroso de larvas e expressão considerável em todos os estágios de desenvolvimento do inseto, com maior expressão no estágio de pupa. Os efeitos do Orlistate na expressão das FATPs evidenciaram a influência da composição da dieta na regulação da expressão gênica dessas proteínas. O silenciamento gênico de TcasFATP foi alcançado, mas não foram observados efeitos diretos na dinâmica energética das larvas, pois os níveis de triacilglicerol e as taxas de β-oxidação não foram afetadas. Dessa forma, estudos mais detalhados com uso do silenciamento gênico serão necessários para melhor caracterização funcional das FATPs e elucidação do seu papel no metabolismo energético do inseto.
32
Ohlwein, Nina. "Endocytosis controlled by monolayer area asymmetry". Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2011. http://dx.doi.org/10.18452/16403.
Resumen
Endozytose erfordert hohe Membrankrümmung und führt zu Flächenänderungen der Membranhälften. Dies kann durch eine Oberflächendifferenz zwischen den Schichten initiiert werden, die durch geänderte Lipidzusammensetzungen hervorgerufen werden kann. Daher wurde die Hypothese aufgestellt, dass Lipid-Transporter zu Beginn der Endozytose für veränderte Flächenverhältnisse verantwortlich sind. Um den Einfluss veränderter Flächen auf Endozytose zu untersuchen, wurden die Oberflächenverhältnisse der Membran durch Zugabe von Phospholipiden verändert und anschließend Endozytose gemessen. Abhängig von der Sorte wurden die Lipide nur in die äußere Schicht eingebaut oder auch auf die innere Seite transportiert, wodurch die entsprechende Seite vergrößert wurde. Die Zugabe verschiedener Aminophospholipide, die auf die innere Membranseite transportiert werden, führte zu gesteigerter „bulk flow“ Endocytose in K562-Zellen. Darüber hinaus deuten die Ergebnisse darauf hin, dass Clathrin-vermittelte Endozytose von Hep2-Zellen ebenfalls stimuliert wurde. Umgekehrt hatte die Zugabe von Lipiden, die auf der äußeren Hälfte bleiben, reduzierte „bulk flow“- oder Clathrin-vermittelte Endozytose in verschiedenen Zelllinien zur Folge. Bemerkenswert ist, dass auch Clathrin-vermittelte Endozytose durch die Lipidzugabe beeinflusst wurde, obwohl gerade in diesem Weg viele Proteine involviert sind, die Krümmung induzieren können. Dies passt zu einem neuen Modell wie Lipidtransporter in Endozytose involviert sind. Durch den Transport von Lipiden und die zusätzliche Interaktion mit Endozytoseproteinen, könnten diese Transporter zwei Mechanismen zur Erzeugung von Krümmung miteinander verbinden: Membrankrümmung induziert durch eine Flächenasymmetrie zwischen den Membranhälften und durch Wechselwirkung mit Proteinen. Die Ergebnisse dieser Arbeit deuten darauf hin, dass die für Endozytose notwendige Krümmung durch die durch Lipidtransport induzierte Flächenasymmetrie der Membranschichten unterstützt wird.Endocytic engulfment requires high local membrane curvature and causes significant area changes of the membrane leaflets. This can be initiated by differences between the surface areas of the two monolayers related to leaflet specific modulation of lipid composition. Thus, it was proposed that lipid translocators, pumping phospholipids from the outer to the inner leaflet, account for monolayer area asymmetry as an early step in endocytosis. To elucidate the influence of this asymmetry on endocytosis, surface area relation was altered by adding exogenous phospholipids to living cells and changes in endocytic activity were quantified. Depending on the lipid species, exogenous lipids were only incorporated into the outer layer or subsequently translocated across the plasma membrane thereby increasing either the outer or inner surface area. Addition of different analogues of aminophospholipids, which are translocated to the inner leaflet, led to an enhancement of bulk flow endocytosis in K562 cells. Moreover, our data indicate that clathrin-mediated endocytosis of Hep2 cells was stimulated as well. Inversely, addition of phospholipids, which remain on the outer layer, reduced bulk flow or clathrin-mediated endocytosis in various cell lines. Notably, also clathrin-mediated endocytosis was influenced by the addition of lipids, although many proteins noted for their ability to induce membrane curvature are known to be implicated in this pathway. This corroborates a recent model how aminophospholipid translocases are implicated in endocytosis. Upon translocating lipids and additionally interacting with endocytic accessory proteins, lipid translocators could integrate two processes to generate curvature: membrane bending based on monolayer area asymmetry and protein-related mechanisms. Collectively, findings in the present study suggest that curvature generation in endocytosis is supported by the induction of monolayer area asymmetry mediated by the translocation of lipids.
33
Karlsson, Emma. "Compositional clues to sources and sinks of terrestrial organic matter transported to the Eurasian Arctic shelf". Doctoral thesis, Stockholms universitet, Institutionen för miljövetenskap och analytisk kemi, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-116876.
Resumen
The amount of organic carbon (OC) present in Siberian Arctic permafrost soils is estimated at twice the amount of carbon currently in the atmosphere. The shelf seas of the Arctic Ocean receive large amounts of this terrestrial OC from Eurasian Arctic rivers and from coastal erosion. Degradation of this land-derived material in the sea would result in the production of dissolved carbon dioxide and may then add to the atmospheric carbon dioxide reservoir. Observations from the Siberian Arctic suggest that transfer of carbon from land to the marine environment is accelerating. However, it is not clear how much of the transported OC is degraded and oxidized, nor how much is removed from the active carbon cycle by burial in marine sediment. Using bulk geochemical parameters, total OC, d13C and D14C isotope composition, and specific molecular markers of plant wax lipids and lignin phenols, the abundance and composition of OC was determined in both dissolved and particulate carrier phases: the colloidal OC (COC; part of the dissolved OC), particulate OC (POC), and sedimentary OC (SOC). Statistical modelling was used to quantify the relative contribution of OC sources to these phases. Terrestrial OC is derived from the seasonally thawing top layer of permafrost soil (topsoil OC) and frozen OC derived from beneath the active layer eroded at the coast, commonly identified as yedoma ice complex deposit OC (yedoma ICD-OC). These carbon pools are transported differently in the aquatic conduits. Topsoil OC was found in young DOC and POC, in the river water, and the shelf water column, suggesting long-distance transport of this fraction. The yedoma ICD-OC was found as old particulate OC that settles out rapidly to the underlying sediment and is laterally transported across the shelf, likely dispersed by bottom nepheloid layer transport or via ice rafting. These two modes of OC transport resulted in different degradation states of topsoil OC and yedoma ICD-OC. Terrestrial CuO oxidation derived biomarkers indicated a highly degraded component in the COC. In contrast, the terrestrial component of the SOC was much less degraded. In line with earlier suggestions the mineral component in yedoma ICD functions as weight and surface protection of the associated OC, which led to burial in the sediment, and limited OC degradation. The degradability of the terrestrial OC in shelf sediment was also addressed in direct incubation studies. Molecular markers indicate marine OC (from primary production) was more readily degraded than terrestrial OC. Degradation was also faster in sediment from the East Siberian Sea, where the marine contribution was higher compared to the Laptev Sea. Although terrestrial carbon in the sediment was degraded slower, the terrestrial component also contributed to carbon dioxide formation in the incubations of marine sediment. These results contribute to our understanding of the marine fate of land-derived OC from the Siberian Arctic. The mobilization of topsoil OC is expected to grow in magnitude with climate warming and associated active layer deepening. This translocated topsoil OC component was found to be highly degraded, which suggests degradation during transport and a possible contribution to atmospheric carbon dioxide. Similarly, the yedoma ICD-OC (and or old mineral soil carbon) may become a stronger source with accelerated warming, but slow degradation may limit its impact on active carbon cycling in the Siberian Shelf Seas.At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.
34
Jacquot, Aurore. "Co-expression et caractérisation fonctionnelle d’un transporteur de lipides (une « flippase ») de la levure S. cerevisiae : l’ATPase P4 Drs2p, en complexe avec sa sous-unité associée Cdc50p". Thesis, Paris 11, 2012. http://www.theses.fr/2012PA11T081/document.
Resumen
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 membranaireTrans-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
35
Trotier-Faurion, Alexandra. "Optimisation pharmacologique des dérivés de la créatine pour le traitement du déficit en transporteur de la créatine". Phd thesis, Université Paris Sud - Paris XI, 2013. http://tel.archives-ouvertes.fr/tel-00806976.
Resumen
Le déficit en transporteur de la créatine est une maladie rare neurologique dans laquelle la perte de fonctionnalité du transporteur de la créatine (SLC6A8) conduit à une absence de créatine au niveau cérébral et à des retards de développement majeurs chez les enfants. A l'heure actuelle, aucune thérapie efficace n'est disponible.Une approche thérapeutique potentielle est le développement de molécules prodrogues de la créatine plus lipophiles qui franchiront les membranes cellulaires de façon passive et la recherche d'une formulation galénique susceptible d'emmener la prodrogue vers les cellules cibles d'intérêt, les neurones. Ainsi, dans cette thèse, nous proposons une nouvelle voie de synthèse originale d'esters de la créatine à longue chaîne aliphatique. Ces composés présentent des propriétés pharmacologiques intéressantes : nous montrons qu'il existe une relation de structure-activité entre la taille de la chaîne aliphatique (et donc la lipophilie) et la capacité de la molécule à être internalisée dans les cellules endothéliales cérébrales, astrocytaires et neuronales, constituant l'unité neurovasculaire. Il ressort de nos observations expérimentales que l'ester dodécylique de créatine est le meilleur candidat médicament. De plus, après avoir été internalisé dans les fibroblastes des patients présentant un déficit fonctionnel du transporteur de la créatine, l'ester dodécylique subit une conversion par les estérases cellulaires, libérant ainsi la créatine dans le compartiment intracellulaire.La formulation galénique permettant de protéger ces esters de créatine jusqu'au cerveau repose, elle, sur la nanovectorisation, par encapsulation de l'ester dodécylique de créatine dans des NanoCapsules Lipidiques. L'avantage de cette formulation est de permettre également un ciblage actif vers la Barrière Hémato-Encéphalique, obstacle majeur dans le développement de thérapies ciblant le Système Nerveux Central. Nos observations expérimentales mettent en exergue cette double stratégie thérapeutique pour le traitement du déficit en transporteur de la créatine.Ce travail a été soutenu financièrement par la Fondation Lejeune.
36
Carswell, Casey. "The Structural Characterization of Two Prokaryotic Membrane Proteins: CfrA and ELIC". Thesis, Université d'Ottawa / University of Ottawa, 2014. http://hdl.handle.net/10393/31214.
Resumen
This thesis focuses on the structural and functional characterization of two integral membrane proteins; CfrA, an outer membrane TonB-dependent transporter (TBDT) from Campylobacter jejuni, and ELIC, a pentameric ligand-gated ion channel (pLGIC) from Erwinia Chrysanthemi. The spectroscopic characterization of CfrA revealed a fold consistent with the structural and biophysical properties observed for other TBDT. Both a homology model of CfrA and sequence alignments of CfrA with other ferric-enterobactin transporters suggested a unique mode of ligand binding, thus raising the possibility that C. jejuni can be specifically inhibited. To investigate the molecular determinates of binding to CfrA, I set out to crystallize CfrA. Hundreds of crystal trials led to crystals diffracting to 3.6 Å resolution, with a complete data set acquired at 5 Å resolution that led to a structural model of the CfrA β-barrel.
In the second part of this thesis, I reconstituted ELIC into model membranes in order to test the role of intramembrane aromatic interactions in ELIC gating and lipid sensing. ELIC was reconstituted into both asolectin (aso-ELIC) and 1-palmitoyl-2-oleoyl phosphatidylcholine (PC-ELIC), membranes that stabilize the homologous nicotinic acetylcholine receptor (nAChR) in functional coupled versus non-functional uncoupled conformations, respectively. In both membrane environments, ELIC exhibits a mixed α-helical and β-sheet secondary structure, with a thermal denaturation intermediate between those of the nAChR and the close prokaryotic homolog, GLIC, in similar membranes. The data suggest that although ELIC has a decreased propensity to adopt an uncoupled conformation relative to the nAChR, its ability to undergo cysteamine-induced channel gating is sensitive to its lipid environment. The decreased propensity to uncouple may reflect an increased level of aromatics at the interface between the transmembrane α-helices, M1, M3, and M4. To test this hypothesis further, the level or aromatic residues at the M1, M3, and M4 interface in both GLIC and ELIC were varied, and in both cases the levels of intramembrane aromatic interactions correlated with the efficiency of coupling binding to gating. The data provide further evidence for a role of intramembrane aromatics in channel gating and in dictating the propensity of pentameric ligand-gated ion channels to adopt an uncoupled conformation.
37
Hardy, Lise. "Identification de nouveaux acteurs du métabolisme des HDL : impact sur les maladies cardiovasculaires Critical role of the human ATP-binding cassette G1 transporter in cardiometabolic diseases A Genome Wide Association Study on plasma FV levels identified PLXDC2 as a new modifier of the coagulation process". Thesis, Sorbonne université, 2019. http://www.theses.fr/2019SORUS546.
Resumen
De faibles concentrations de cholestérol associé aux HDL (HDL-C) est un facteur de risque indépendant des maladies cardiovasculaires (MCV). Les HDL sont capables de réaliser de l’efflux de lipides des tissus périphériques pour assurer son retour vers le foie, et ont des rôles athéroprotecteurs. Le travail mené ici vise à identifier de nouveaux acteurs impliqués dans la détermination des fonctions des HDL. Le transporteur ABCG1 réalise de l’efflux de cholestérol, de phospholipides (PL), ou encore de vitamines à partir des macrophages périphériques vers les HDL. Nous avons montré que la stimulation de l’expression d’ABCG1 dans l’hépatocyte favorisait un réarrangement du contenu des HDL en PL. Ce remodelage est associé à une amélioration de la capacité d’efflux de cholestérol des HDL et de leur fonction anti-inflammatoire. En parallèle, des études épidémiologiques nous ont permis d’identifier ZNF471, nouveau facteur de transcription. Il semble capable de moduler l’expression et l’activité de protéines clés du métabolisme des lipides, via une régulation épigénétique impactant la méthylation de l’ADN : ZNF471 dans les hépatocytes augmente la méthylation de l’ADN sur la région promotrice du gène codant la CETP. En conséquence, l’expression du gène et l’activité protéique de la CETP sont réduits, ce qui favorise l’accumulation de HDL-C. De plus, les capacités d’efflux de cholestérol des HDL sont également stimulées par ZNF471. Ces travaux de recherche permettent l’identification de nouveaux acteurs du métabolisme des lipoprotéines HDL. Ils ouvrent ainsi la voie à de nouvelles explorations thérapeutique et mécanistique sur les rôles des HDL dans les MCV liées à l’athéroscléroseSince low concentrations of High-Density Lipoproteins-cholesterol (HDL-C) are associated with increased cardiovascular disease (CVD) risk, HDL are recognized as protective in atherosclerotic CVD (ASCVD). Indeed, HDL promote lipid efflux from macrophages and other atheroprotective activities. Here, we aimed to identify new factors implicated in the determination of atheroprotective functions of HDL. ATP-Binding Cassette G1 (ABCG1) transporter perform cholesterol, phospholipids or vitamin efflux from peripheral macrophages to HDL. We showed that ABCG1 expression in hepatocytes promoted HDL phospholipid content rearrangement. This HDL remodeling is associated with a better cholesterol efflux capacity and an improvement of their anti-inflammatory properties. Simultaneously, epidemiological studies allow us to identify a novel transcription factor, ZNF471 (Zinc Finger Protein 471). ZNF471 seems to modulate expression and activity of key proteins implicated in lipid metabolism, through epigenetic DNA methylation regulation. We highlighted that ZNF471 expression in hepatocytes increased DNA methylation in CETP (Cholesterol Ester Transfer Protein) gene promoter region. As a consequence, CETP gene expression and protein activity were diminished, which raised HDL-C circulating concentrations. We also pointed out that ZNF471 expression stimulated HDL cholesterol efflux capacities. This work allows the identification of novels genetic and epigenetic actors in determining HDL lipoproteins activities. It paves the way for new therapeutic and mechanistic insights on the roles of HDL in ASCVD
38
Gumí, Audenis Berta. "Structural organization of model membranes: a complementary approach combining atomic force microscopy and X-ray techniques". Doctoral thesis, Universitat de Barcelona, 2018. http://hdl.handle.net/10803/550973.
Resumen
Biological membranes (BMs) are self-sealing boundaries, which confine the permeability barriers of cells and organelles and provide the means to compartmentalize functions. Apart from being crucial for the cell structure, they provide a support matrix for all the proteins inserted in the cell, acting as channels to exchange mass, energy and information with the environment. BMs mediate several biological functions, such as trafficking, cell division, endocytosis and exocytosis, demanding strong conformational changes of the lipid membrane like fusion, fission or tubes growth. These mechanical requirements are only possible due to the organization of the chemical composition of the lipids into the membrane of each organelle, which is directly linked to the organelle function. Thanks to the dynamic behavior of the membrane, lateral and transverse forces within the membrane are significant and change rapidly as the membrane is bent or stretched, and as new constituents are added, removed or chemically modified. Differences in structure between the two leaflets and between different areas of the bilayer can be associate to membrane deformation to alter the activities of membrane binding proteins. It is then the correlation between the composition and the packing of the lipids what essentially governs the membrane physicochemical and mechanical properties.
Considering the complex chemical diversity of BMs, model bilayers systems are frequently used to study membrane properties and biological processes. Because of the micro and nanoscale range of domains in BMs, and the consequent need of local techniques to explore BMs at the nanometric level, supported bilayer systems are very manageable platforms, since they retain two-dimensional order and lateral mobility and offer excellent environments for the insertion of membrane proteins. In particular, supported lipid bilayers (SLBs) facilitate the use of surface analytical techniques, being ideal models to study the lipid lateral interactions, the growth of lipid domains, as well as interactions between the lipid membrane and proteins, peptides and drugs, cell signaling, etc.
Several reports demonstrate the wide variety of useful techniques to study supported and non-supported lipid membranes. Thanks to the possibility of working under controlled environment and with distance and force resolution at the nanoscale, atomic force microscopy (AFM) is nowadays a well-established technique for both imaging the morphology and probing the local physical and mechanical properties of SLBs by means of force spectroscopy. However, the resolution given by AFM might be inferior to the one achievable with X-ray (XR) and neutron techniques. In particular, XR techniques such as XR reflectivity (XRR) and grazing incidence XR diffraction (GIXD) are powerful tools to characterize surfaces below the nanoscale, providing structural information in the reciprocal space through the interaction between XR and the sample electronic structure. Still, since these techniques do not involve any mechanical interaction with the specimen, mechanical properties cannot be evaluated with XR.
The general objective of this thesis is to investigate the physicochemical and structural properties of model lipid membranes combining atomic force microscopy (AFM) and spectroscopy (AFM-FS) and X-Ray techniques. The AFM provides the morphological and mechanical information of the SLBs, whereas the XR gives more understandings on the electronic structure of the bilayers. We also propose advanced methodologies based on AFM and XR as well as the coupling of both techniques for local in situ experiments. These technical progresses allow us to study not only the diversity on the chemical composition of the bilayers, but also the effect of small molecules or peptides to the membrane physical and structural properties. In addition, by means of AFM and AFM-FS we also characterize vesicular systems that are not composed by phospholipid molecules, which have a technological application: to act as nanocarriers for drug delivery.Les membranes biològiques (BMs) són fronteres autosegellants, que limiten les barreres permeables de les cèl·lules i els orgànuls i proporcionen els mitjans necessaris per compartir funcions. A part de ser crucials per l’estructura cel·lular, proporcionen una matriu de suport per a totes les proteïnes que es troben inserides a la cèl·lula, actuant com canals per l’intercanvi de massa, energia i informació amb l’exterior. Les BMs intervenen en moltes funcions biològiques, com el tràfic, la divisió cel·lular, l’endocitosi i l’exocitosi, que exigeixen canvis conformacionals durs en la membrana lipídica com la fusió, la fissió o el creixement de tubs. La correlació entre la composició i l’empaquetament dels lípid regeix les propietats fisicoquímiques de la membrana i la seva estructura mecànica Considerant la complexa diversitat química de les BMs, sistemes de membranes model són utilitzats sovint per estudiar propietats de membrana. Degut a la heterogeneïtat de les BMs i la conseqüent necessitat de tècniques locals per a explorar BMs a escala nanomètrica, sistemes de bicapes suportades, com les bicapes de lípids suportades (SLBs), s’han proposat com models, ja que conserven l’ordre bidimensional i la mobilitat lateral, oferint ambients excel·lents per a la inserció de proteïnes de membrana. Diversos informes demostren la gran varietat de tècniques útils per estudiar membranes lipídiques suportades i sense suport. Gràcies a la possibilitat de treballar sota un ambient controlat i amb una resolució nanomètrica en distància i força, la microscòpia de forces atòmiques (AFM) és, avui en dia, una tècnica ben establerta tant per a obtenir una imatge de la morfologia com per mesurar les propietats locals físiques i mecàniques de les SLBs mitjançant modes d’espectroscòpia de forces (AFM-FS). De totes formes, la resolució que s’obté amb l’AFM és inferior a la que es pot obtenir amb tècniques de raigs X (XR) i neutrons. L’objectiu general d’aquesta tesis és investigar les propietats fisicoquímiques i estructurals de membranes lipídiques model combinant tècniques d’AFM, d’AFM-FS i de XR. També proposem metodologies avançades basades en AFM-FS i XR, així com l’acoblament de les dues tècniques per dur a terme experiments locals in situ. A més, amb AFM i AFM-FS també hem caracteritzat sistemes vesiculars que no contenen fosfolípids, els quals tenen una aplicació tecnològica: actuar com a nanotransportadors per al lliurament de fàrmacs.
39
Brown, Dustin Paul. "TARGET-DIRECTED BIOSYNTHETIC EVOLUTION: REDIRECTING PLANT EVOLUTION TO GENOMICALLY OPTIMIZE A PLANT’S PHARMACOLOGICAL PROFILE". UKnowledge, 2015. http://uknowledge.uky.edu/neurobio_etds/13.
Resumen
The dissertation describes a novel method for plant drug discovery based on mutation and selection of plant cells. Despite the industry focus on chemical synthesis, plants remain a source of potent and complex bioactive metabolites. Many of these have evolved as defensive compounds targeted on key proteins in the CNS of herbivorous insects, for example the insect dopamine transporter (DAT). Because of homology with the human DAT protein some of these metabolites have high abuse potential, but others may be valuable in treating drug dependence. This dissertation redirects the evolution of a native Lobelia species toward metabolites with greater activity at this therapeutic target, i.e. the human DAT. This was achieved by expressing the human DAT protein in transgenic plant cells and selecting gain-of-function mutants for survival on medium containing a neurotoxin that is accumulated by the human DAT. This created a sub-population of mutants with increased DAT inhibitory activity. Some of the active metabolites in these mutants are novel (i.e. not detectable in wild-type cells). Others are cytoprotective, and also protect DAergic neurons against the neurotoxin. This provides proof-of-concept for a novel plant drug discovery platform, which is applicable to many different therapeutic target proteins and plant species.
40
Sigrüner, Alexander [Verfasser]. "ABCA transporters and associated genes in lipid metabolism / vorgelegt von Alexander Sigrüner". 2007. http://d-nb.info/986961256/34.
41
Hsiao, Man-Yun y 蕭蔓妘. "Extraction of membrane lipid rafts from gills of euryhaline fish and salinity effects on abundance of ion transporters in branchial lipid rafts". Thesis, 2018. http://ndltd.ncl.edu.tw/handle/bfwwyh.
Resumen
碩士國立中興大學
生命科學系所
106
Lipid rafts are microdomains of the plasma membrane enriched in cholesterol, sphingolipids, glycosphingolipids and proteins (e.g. Flotillin). Lipids in the lipid raft are composed of long-chain saturated fatty acids, which make it insoluble in nonionic detergents. In previous studies on mammalian cells, ion exchanges mainly occurred in lipid rafts. However, in fish, there are few studies on lipid rafts. Therefore, the aim of the present study is to establish the extraction method of lipid rafts in fish gills, and to investigate the localization and protein expression of the ion transporters in the lipid rafts of fish gills. In the first part of this study, the amino acid sequence of the Na+, K+-ATPase (NKA) was predicted to possess more N-Myristoylation and Palmitoylation sites than that of the vacuolar-type H+-ATPase (V-HA). Thus, the NKA might have a higher tendency to locate on lipid rafts, compared to the V-HA. Salinity effects on distribution of NKA and Flotillin were found in lipid rafts of medaka gills, but the relations between NKA and Flotillin were not clear yet. Meanwhile, 0.1% Triton X-100 and 0.05% Tween 20 were found to successfully extract lipid rafts from milkfish and tilapia gills, respectively. In the second part of this study, the distribution of NKA was found only in lipid rafts of milkfish and tilapia gills, and distribution of NKA in the freshwater (FW) group was significantly higher than the seawater (SW) group. However, expression of NKA was not affected by lipid raft contents. On the other hand, V-HA isoform1 and isoform2 of milkfish gills as well as V-HA of tilapia gills appeared in the lipid rafts and non-lipid rafts of cell membrane. Distribution of V-HA isoform1 and isoform2 in non-lipid rafts was significantly higher of the FW milkfish gills than the SW group. Most V-HA distribution was found in non-lipid rafts of tilapia gills, significantly higher in the FW group than the SW group. In summary, this study has established the extraction method of lipid rafts on milkfish and tilapia gills. The NKA of milkfish and tilapia gills was located only in lipid rafts of cell membrane, and its expression was not affected by contents of the lipid rafts. V-HA was mainly localized in non-lipid rafts of both milkfish and tilapia gills with significant differences in expression between the SW and FW groups.
42
Gulati, Sonia. "Characterizing the Interaction of the ATP Binding Cassette Transporters (G subfamily) with the Intracellular Protein Lipid Environment". Thesis, 2011. https://doi.org/10.7916/D8ZW1SW7.
Resumen
Cholesterol is an essential molecule that mediates a myriad of critical cellular processes, such as signal transduction in eukaryotes, membrane fluidity, and steroidogenesis. As such it is not surprising that cholesterol homeostasis is tightly regulated, striking a precise balance between endogenous synthesis and regulated uptake/efflux to and from extracellular acceptors. In mammalian cells, sterol efflux is a key component of the homeostatic equation and is mediated by members of the ATP binding cassette (ABC) transporter superfamily. ATP-binding cassette (ABC) transporters represent a group of evolutionarily highly conserved cellular transmembrane proteins that mediate the ATP-dependent translocation of substrates across membranes. Members of this superfamily, ABCA1 and ABCG1, are key components of the reverse cholesterol transport pathway. ABCG1 acts in concert with ABCA1 to maximize the removal of excess cholesterol from cells by promoting cholesterol efflux onto mature and nascent HDL particles, respectively. To date, mammalian ABC transporters are exclusively associated with efflux of cholesterol. In Saccharomyces cerevisiae, we have demonstrated that the opposite (i.e inward) transport of sterol in yeast is also dependent on two ABC transporters (Aus1p and Pdr11p). This prompts the question what dictates directionality of sterol transport by ABC transporters. The main focus of this study is to define the parameters that result in sterol movement across membranes. The comparison between these contrasting states (outward v. inward transport of the same substrate) will allow us to dissect whether sterol transport across the plasma membrane is defined by the molecule (i.e. the ABC transporter) or by microenvironment (i.e. the status of other proteins and lipids) in which it resides. We have developed the model eukaryote Saccharomyces cerevisiae as a tool to understand the mechanisms that influence ABC-transporter mediated movement of sterols. Specifically, we expressed murine ABCG1 (mABCG1) in yeast and assessed how changes in the intracellular sterol environment affect movement of sterols by this transporter. We found that expression of mABCG1 is able to vary (both increase and decrease) the concentration of exogenous sterols in the cell in response to intracellular sterol changes. We also found that yeast members of the ABCG subfamily, Aus1p and Pdr11p are able to promote either influx of cholesterol or efflux of a cholesterol derivative depending on the sterol context of the cell. This is the first example of an ABC transporter mediating bi-directional transport. These data suggest that direction of transport is not a static property of the transporter but rather can adapt in response to changes in the intracellular microenvironment. In addition to sterols we also found that proteins in the microenvironment may also influence direction of transport. Specifically, we found that the yeast sterol esterifying enzyme Are2p, physically interacts with the ABC transporters Aus1p and Pdr11p. Furthermore, all three proteins were found to co-localize to detergent resistant membrane microdomains. Deletion of either ABC transporter resulted in Are2p re-localization from DRMs to a detergent soluble fraction as well as a significant decrease in the percent of sterol esterified. This phenomenon is evolutionarily conserved in the murine lung where ABCG1 and ACAT1 were observed to co-localize with flotillin-1, a marker of DRMs. We propose that co-localization and complex formation of sterol esterification enzymes and ABC transporters in DRMs reflects a novel mechanism that directs membrane sterols to the esterification reaction. The studies presented in this thesis provide evidence that direction of transport is not a static inherent property of the transporter, but rather that it is mutable and influenced by surrounding sterols and proteins. The data provided here offers further insight as to how ABC transporters move cholesterol from the membrane and therefore may provide a platform for innovative strategies to combat atherosclerosis.
43
Tan, Kah Poh. "Nuclear Factor (Erythroid 2-like) Factor 2 (Nrf2) as Cellular Protector in Bile Acid and Retinoid Toxicities". Thesis, 2008. http://hdl.handle.net/1807/17287.
Resumen
Exposure to toxic bile acids (BA) and retinoic acids (RA) is implicated in toxicities related to excessive oxidative stress. This thesis examined roles and mechanisms of the oxidative stress-responsive nuclear factor (erythroid 2-like) factor 2 (Nrf2) in adaptive cell defense against BA and RA toxicities. Using liver cells and mouse models, many antioxidant proteins known to be Nrf2 target genes, particularly the rate-limiting enzyme for glutathione (GSH) biosynthesis, i.e., glutamate-cysteine ligase subunits (GCLM/GCLC), were induced by BA [lithocholic acid (LCA)] or RA (all-trans, 9-cis and 13-cis) treatment. Evidence for increased Nrf2 transactivation by LCA and all-trans-RA was exemplified in HepG2 by: (1) reduced constitutive and inducible expression of GCLM/GCLC upon Nrf2 silencing via small-interfering RNA; (2) increased inducible expression of GCLM/GCLC genes by Nrf2 overexpression, but overexpression of dominant-negative Nrf2 decreased it; (3) increased nuclear accumulation of Nrf2 as signature event of receptor activation; (4) enhanced Nrf2-dependent antioxidant-response-element (ARE) reporter activity as indicative of increased Nrf2 transactivation; and (5) increased Nrf2 occupancy to AREs of GCLM and GCLC. Additionally, in BA-treated HepG2 cells, we observed concomitant increases of many ATP-binding cassette (ABC) transporters (MRPs 1-5, MDR1 and BCRP) in parallel with increased cellular efflux. Nrf2 silencing in HepG2 cells decreased constitutive and inducible expression of MRP2, MRP3 and ABCG2. However, Nrf2-silenced mouse hepatoma cells, Hepa1c1c7, and Nrf2-/- mice had decreased constitutive and/or inducible expression of Mrps 1-4, suggesting species differences in Nrf2-dependent regulation of hepatic ABC transporters. Protection by Nrf2 against BA and RA toxicities was confirmed by observations that Nrf2 silencing increased cell susceptibility to BA- and RA-induced cell death. Moreover, Nrf2-/- mice suffered more severe liver injury than the wildtype. Increased GSH and efflux activity following increased GCLM/GCLC and ABC transporters, respectively, can mitigate LCA toxicity. Activation of MEK1-ERK1/2 MAPK was shown to primarily mediate Nrf2 transactivation and LCA-induced expression of antioxidant proteins and Nrf2-dependent and -independent ABC transporters. In conclusion, Nrf2 activation by BA and RA led to coordinated induction of antioxidant and ABC proteins, thereby counteracting resultant oxidative cytotoxicity. The potential of targeting Nrf2 in management of BA and RA toxicities merits further investigation.
44
Mendes, Maria Manuel Feliciano da Costa. "Lipid nanoparticles as a versatile system for drug administration". Master's thesis, 2016. http://hdl.handle.net/10316/36494.
Resumen
Dissertação de mestrado em Tecnologias do Medicamento, apresentada à Faculdade de Farmácia da Universidade de CoimbraA nanotecnologia é uma ciência emergente com aplicação em diferentes áreas, com especial ênfase nas ciências da saúde. As nanopartículas de lipídicas (NL) são um nanosistema promissor para a entrega de fármacos, que suscita interesse particular em comparação com os sistemas coloidais convencionais. A sua natureza biocompatível e biodegradável, a estabilidade físico-química, o controle sobre a libertação do fármaco, relação custo-eficácia e fácil transposição de escala são algumas das suas vantagens. As NL tem sido avaliadas para administração por várias vias, superando, assim, algumas limitações associadas às formas farmacêuticas de dosagem convencionais. Neste trabalho, o objetivo principal foi o desenvolvimento e caracterização de formulações orais e transdérmica baseadas em nanopartículas lipídicas para co-encapsulação de olanzapina (OL) e de sinvastatina (SV). A aplicação particular das NL para administração oral, é vantajosa, devido ao efeito potenciador dos lípidos na absorção do fármaco e a na sua absorção preferencial pelo sistema linfático, evitando o efeito de primeira passagem hepática e assim, aumentar a biodisponibilidade oral. Por sua vez, a sua utilização na administração transdérmica pode aumentar a penetração dos fármacos, como resultado do efeito oclusivo promovido pelo pequeno tamanho das partículas e a pela sua adesividade. Na administração oral, foi realizada uma dupla abordagem de desenvolvimento. Em primeiro lugar, pretendeu-se avaliar a exequibilidade de transposição de escala das nanopartículas lipídicas e, subsequentemente, da conversão de NLC numa forma de dosagem sólida e flexível, em termos de modulação de libertação dos fármacos. Para isso, as dispersões de Combo-NLC foram produzidas por homogeneização a alta pressão, e avaliada em termos de estabilidade, processo de secagem, desempenho dos comprimidos na libertação in vitro e na permeabilidade intestinal. As NLC revestidas com polímeros permitiram obtenção de mecanismos de liberação distintos, incluindo libertação imediata, retardada e prolongada. Os estudos de citotoxicidade e de permeabilidade foram realizados para todas as formulações. A melhor formulação foi associada à baixa toxicidade e alta permeabilidade intestinal, o que pode ainda resultar num melhoramento permeabilidade oral. Tais resultados poderiam ser atribuídos à proteção dos fármacos e controle sobre liberação promovido pela NLC. Na administração transdérmica, como segunda abordagem, um sistema monolítico de fármaco-nas-NLC-no-adesivo foi desenvolvido, formulado e caracterizado por metodologias 10 in vitro, in silico e viabilidade celular. Foram aplicadas estratégias passivas para aumentar a permeação dos fármacos, como as nanopartículas lipídicas juntamente com promotores químicos de permeação, e microagulhas usadas como método activo. Em primeiro lugar, foi avaliada a combinação de NL com diferentes promotores em termos de comportamento de permeação. O propileno glicol revelou o maior valor de permeação para os dois fármacos. Estes resultados foram complementados por estudos de dinâmica molecular. Depois, diferentes adesivos foram formulados e convenientemente avaliados. Entre todos os adesivos testados, a combinação entre PVP K30 e PEG 400 resultou na permeação do fármaco mais elevada através da pele, simultaneamente revelou as melhores propriedades adesivas in vitro-in vivo. A influência do promotor químico de permeação e o adesivo sobre o desempenho formulação foi racionalizada usando um planeamento factorial. Os resultados mostraram que a presença do adesivo é importante para promover a permeação através da pele, enquanto que a permeação de SV é influenciado principalmente pela presença do promotor químico. Finalmente, avaliou-se a influência das micro-agulha. Esta abordagem combinada não demonstrou uma melhoria significativa sobre a permeação dos fármacos, o que pode indicar que a própria formulação proporciona a principal força motriz para a permeação dos fármacos na pele. Os estudos de citotoxicidade revelaram que as nanopartículas de lipídicas são capazes de reduzir a citotoxicidade associada à SV em solução a concentrações inferiores a 90 μM e que o efeito isolado de adesivo em termos de citotoxicidade foi mais elevado do que o induzido pelo promotor químico. Utilizando esta abordagem combinatória, o projecto demonstrou a versatilidade das nanopartículas de lípidos como sistemas para administração de fármacos coloidais.
Nanotechnology is an emerging science with application in different areas, with special emphasis on health sciences. Lipid nanoparticles (LN) are a promising nanosystem for drug delivery, which has raised particular interest in comparison to conventional colloidal systems. Their biocompatible and biodegradable nature, physicochemical stability, control over drug release, cost-effectiveness and easy scaling-up are some of their advantages. LN have been assessed for administration by several routes, thereby overcoming some limitations associated to conventional dosage forms. In this work, the main aim was the development and characterization of oral and transdermal formulations based on lipid nanoparticles for co-delivery of olanzapine (OL) and simvastatin (SV). The particular application of LN for oral administration is advantageous, due to the enhancer effect of lipids on drug absorption and their preferential uptake by the lymphatic system, thus avoiding liver first pass and increasing bioavailability. In turn, their use in the transdermal administration can increase drug penetration, as a result of the occlusive effect promoted by the small particle size and adhesiveness. In oral administration, a dual step development approach was carried out, firstly, focused on the assessment of the NLC scale-up feasibility, and subsequently, on the conversion of NLC into a solid and flexible dosage form, in terms of drug release modulation. For that, Combo-NLC dispersions were produced by high pressure homogenization, and evaluated in terms of scalability, drying procedure, tableting and performance from an in vitro release and permeability perspective. Modified NLC formulations with the polymers allowed to obtain distinct release mechanisms, including immediate, delayed and prolonged release. Cytotoxicity and permeability studies were performed in order to assess all formulations. The better formulation was associated to low cytotoxicity and high intestinal permeability, which can further result in enhanced oral permeability. Such findings could be ascribed to drug protection and control over release promoted by the NLC. In transdermal administration, as second approach, a transdermal drug delivery system based on an innovative drug-in-LN-in-adhesive design and adhesive composition was formulated and characterized by in vitro, in silico and cellular viability methodologies. As enhancement permeation strategies, passive, including LN coupled with chemical penetration enhancers, CPEs, and microneedles as active method were investigated. Firstly, it was assessed the combination of LN with different enhancers in terms of permeation behaviour. 12 Propylene glycol revealed the higher permeation of both drugs. These results were supported by molecular dynamics studies. After that, different adhesives were formulated and suitably evaluated. Among all adhesives tested, the combination between PVP k30 and PEG 400 resulted in the higher drug permeation across the skin, along with the best in vitroin vivo adhesive properties. The influence of the enhancer and the adhesive on the formulation performance was rationalized using a factorial design. Results showed that the presence of adhesive is crucial to promote of permeation through the skin, while permeation of SV is mainly influenced by the presence of enhancer. Finally, the pre-application of a microneedle device followed by the optimal patch was evaluated. This combined approach did not a show a significant improvement over the drug permeation, which may indicate that the formulation itself provides the main driving force for skin permeation. Cytotoxicity studies revealed that lipid nanoparticles are able to reduce cytotoxicity associated to the SV in solution at concentrations lower than 90 !M!! and it can be visualized that the isolated effect of adhesive in terms of cytotoxicity was higher than the one induced by the enhancer. Using this combinational approach, the project demonstrated the versatility of the lipid nanoparticles as colloidal systems for drug administration.
45
Pandey, Aparamita. "Glucose and Lipid Metabolism during Pregnancy and Lactation in Rats : Role of Undercarboxylated Osteocalcin". Thesis, 2016. http://etd.iisc.ac.in/handle/2005/3074.
Resumen
Energy homeostasis is an important physiological mechanism essential for balancingenergy flow through the living systems by managing overall metabolism in the body.
Thus, energy homeostasis is under a tight control by means of extremely well-regulated energy metabolism. One of the most common metabolic disorders that occur following disruption in energy homeostasis mechanisms is obesity. Obese individuals develop insulin resistance in the peripheral tissues (fat and muscle) and may also include non-alcoholic fatty liver disease. Insulin resistance is the primary factor responsible for the development of type 2 diabetes mellitus (T2D). Towards control and management of T2D condition, insulin, drugs that regulate the insulin sensitivity and drugs that regulate glucose metabolism are widely used. Repeated insulin administration is painful, expensive and requires constant glucose monitoring while other drugs have various limitations and side effects. Therefore, there is wide scope development of new anti-diabetic molecules for effective management of T2D. Studies related to energy metabolism are necessary to understand the cause of such disorders and improve existing methods to manage metabolic abnormalities. Animal models to understand such metabolic disorders have been developed by chemical treatments and genetic modifications, but diet-induced obese (DIO) animal models appear to be the better among all the models reported. DIO animal models are known to most closely mimic the physiological situation. Apart from the experimental model system studies have been conducted under physiological conditions to gain knowledge on possible mechanisms behind energy balance maintained and established during extreme situations such as pregnancy and lactation. To support fetal growth and milk synthesis several metabolic adjustments occur during pregnancy and lactation without the major disruption in the maternal energy homeostasis.
In the present study, to gain knowledge on the mother’s body glucose, lipid management and insulin responses throughout the gestation and lactation periods analyses were carried out during at different stages of pregnancy and lactation in rats. It was observed that during pregnancy, the dam developed insulin resistance in peripheral tissues with decreased activation of insulin pathway and reduced glucose utilization while the liver remained unaffected. Although, as soon as the lactation began, peripheral tissue such as muscle developed increased insulin sensitivity associated with increased expression of glucose transporter gene and higher glucose metabolism. The reversal of insulin response in the muscle tissue observed during lactation appears to be a suitable model system for understanding the process by which the body undergoes a transition from insulin resistant state to sensitive state under a physiological condition. Interestingly, early lactation period is known to have much lower levels of insulin available to act upon peripheral tissues. Factors involved in this transition could be potential therapeutic agents for control of T2D, since during early stages of T2D muscle appears to be the first metabolic organ to exhibit resistance to insulin. The undercarboxylated osteocalcin (UNOC) has been reported to function as anti-diabetic molecule. UNOC is released from skeletal system during bone turnover, especially due to resorption process. Experiments were carried out to examine the role of UNOC during the transition from insulin resistant state of pregnancy to sensitive state of lactation period. It was observed that UNOC levels were lower during pregnancy, but increased during early lactation (day 3 to 6 of lactation). The increased UNOC levels seen during early lactation was higher than the levels observed in non-pregnant, non-lactating (NPNL) rats and the UNOC levels decreased following removal of pups immediately after parturition. It was noted that altering UNOC levels during early lactation altered the insulin response of the whole body and muscle transporter-4 expression (glut4) of lactating rats. A significant increase in bone turnover was also observed during lactation compared to NPNL and pregnant rats. The data suggest that increased bone turnover leads to increased UNOC levels in blood during lactation. Estrogen is known as bone protector molecule which acts via its receptors, estrogen receptor α and β (ERα and β). It was reported that ERβ is a dominant regulator of estrogen signaling when both the receptors of estrogen i.e. ERα and ERβ coexist in the target tissue and estrogen levels are relatively higher. Compared to NPNL rats estrogen levels have shown to be higher during late pregnancy and lower during early lactation. It was observed that liver and adipose tissues largely express ERα, but the muscle showed expression of both the receptors in NPNL rats indicating that muscle is the metabolic tissue that may be modulated by both the receptors. It has been reported that ERβ suppresses ERα action on glut4 transcription in the myocytes. It is possible that the altered ERs ratio modulates glut4 expression during late pregnancy and early lactation. The receptor expression ratio data indicated that muscle is an ERβ dominant during late pregnancy, while it is ERα dominant during early lactation. Further, alteration in UNOC levels during early lactation changed ERs ratio but not sufficient enough to alter the ER dominance, indicating lack of effect of UNOC on ER dominance during early lactation. Experiments were conducted to alter insulin sensitivity during early lactation to extrapolate physiological findings to a pathological condition of the DIO model by feeding rats with high-fat diet (HFD). During early lactation, HFD dams had lower insulin response, lower circulatory UNOC level and lower UNOC receptor (GPRC6A) expression in the muscle. Gene expression of muscle glut4 was lower in HFD rats and the tissue remained ERα dominant indicating no role of HFD on ERs ratio in muscle during early lactation.
UNOC has been found to have negative effect on lipid accumulation. During pregnancy, lipid accumulation is one of the first events essential for proper fetal development. Since UNOC levels were suppressed during pregnancy, experiments were carried out to examine relevance of UNOC suppression on lipid accumulation during early pregnancy. For this purpose, pharmacological approaches were utilized to alter UNOC levels during early pregnancy. It was observed that the transient elevation of UNOC levels caused decrease in maternal fat depots without changing circulatory triacylglyceride (TAG) levels. In experiments that decreased UNOC levels in NPNL state to mimic lower levels of UNOC present during early pregnancy, it was found fat storage was higher and TG was found to be lowered in the circulation. These results indicate that UNOC can cause a reduction in fat accumulation and TG levels but UNOC effects on TG levels, was not observed during pregnancy. The data taken together suggest that suppression of UNOC is required for better fat deposition in the mother’s body. Although, some studies have indicated an insulin response transition occurring during pregnancy to lactation, but the factors involved in this transition have not been reported. This report discusses about the factors such as UNOC and ERs and their involvement in the transition process. UNOC role has been studied in genetically modified models and in metabolic disorders such as obesity model system and evidence for physiological role of UNOC would further support its candidature as anti-diabetic molecule. The present research work is the first report to detail relevance of UNOC in physiological conditions such as pregnancy and lactation for glucose and lipid management.
46
Pandey, Aparamita. "Glucose and Lipid Metabolism during Pregnancy and Lactation in Rats : Role of Undercarboxylated Osteocalcin". Thesis, 2016. http://hdl.handle.net/2005/3074.
Resumen
Energy homeostasis is an important physiological mechanism essential for balancingenergy flow through the living systems by managing overall metabolism in the body.
Thus, energy homeostasis is under a tight control by means of extremely well-regulated energy metabolism. One of the most common metabolic disorders that occur following disruption in energy homeostasis mechanisms is obesity. Obese individuals develop insulin resistance in the peripheral tissues (fat and muscle) and may also include non-alcoholic fatty liver disease. Insulin resistance is the primary factor responsible for the development of type 2 diabetes mellitus (T2D). Towards control and management of T2D condition, insulin, drugs that regulate the insulin sensitivity and drugs that regulate glucose metabolism are widely used. Repeated insulin administration is painful, expensive and requires constant glucose monitoring while other drugs have various limitations and side effects. Therefore, there is wide scope development of new anti-diabetic molecules for effective management of T2D. Studies related to energy metabolism are necessary to understand the cause of such disorders and improve existing methods to manage metabolic abnormalities. Animal models to understand such metabolic disorders have been developed by chemical treatments and genetic modifications, but diet-induced obese (DIO) animal models appear to be the better among all the models reported. DIO animal models are known to most closely mimic the physiological situation. Apart from the experimental model system studies have been conducted under physiological conditions to gain knowledge on possible mechanisms behind energy balance maintained and established during extreme situations such as pregnancy and lactation. To support fetal growth and milk synthesis several metabolic adjustments occur during pregnancy and lactation without the major disruption in the maternal energy homeostasis.
In the present study, to gain knowledge on the mother’s body glucose, lipid management and insulin responses throughout the gestation and lactation periods analyses were carried out during at different stages of pregnancy and lactation in rats. It was observed that during pregnancy, the dam developed insulin resistance in peripheral tissues with decreased activation of insulin pathway and reduced glucose utilization while the liver remained unaffected. Although, as soon as the lactation began, peripheral tissue such as muscle developed increased insulin sensitivity associated with increased expression of glucose transporter gene and higher glucose metabolism. The reversal of insulin response in the muscle tissue observed during lactation appears to be a suitable model system for understanding the process by which the body undergoes a transition from insulin resistant state to sensitive state under a physiological condition. Interestingly, early lactation period is known to have much lower levels of insulin available to act upon peripheral tissues. Factors involved in this transition could be potential therapeutic agents for control of T2D, since during early stages of T2D muscle appears to be the first metabolic organ to exhibit resistance to insulin. The undercarboxylated osteocalcin (UNOC) has been reported to function as anti-diabetic molecule. UNOC is released from skeletal system during bone turnover, especially due to resorption process. Experiments were carried out to examine the role of UNOC during the transition from insulin resistant state of pregnancy to sensitive state of lactation period. It was observed that UNOC levels were lower during pregnancy, but increased during early lactation (day 3 to 6 of lactation). The increased UNOC levels seen during early lactation was higher than the levels observed in non-pregnant, non-lactating (NPNL) rats and the UNOC levels decreased following removal of pups immediately after parturition. It was noted that altering UNOC levels during early lactation altered the insulin response of the whole body and muscle transporter-4 expression (glut4) of lactating rats. A significant increase in bone turnover was also observed during lactation compared to NPNL and pregnant rats. The data suggest that increased bone turnover leads to increased UNOC levels in blood during lactation. Estrogen is known as bone protector molecule which acts via its receptors, estrogen receptor α and β (ERα and β). It was reported that ERβ is a dominant regulator of estrogen signaling when both the receptors of estrogen i.e. ERα and ERβ coexist in the target tissue and estrogen levels are relatively higher. Compared to NPNL rats estrogen levels have shown to be higher during late pregnancy and lower during early lactation. It was observed that liver and adipose tissues largely express ERα, but the muscle showed expression of both the receptors in NPNL rats indicating that muscle is the metabolic tissue that may be modulated by both the receptors. It has been reported that ERβ suppresses ERα action on glut4 transcription in the myocytes. It is possible that the altered ERs ratio modulates glut4 expression during late pregnancy and early lactation. The receptor expression ratio data indicated that muscle is an ERβ dominant during late pregnancy, while it is ERα dominant during early lactation. Further, alteration in UNOC levels during early lactation changed ERs ratio but not sufficient enough to alter the ER dominance, indicating lack of effect of UNOC on ER dominance during early lactation. Experiments were conducted to alter insulin sensitivity during early lactation to extrapolate physiological findings to a pathological condition of the DIO model by feeding rats with high-fat diet (HFD). During early lactation, HFD dams had lower insulin response, lower circulatory UNOC level and lower UNOC receptor (GPRC6A) expression in the muscle. Gene expression of muscle glut4 was lower in HFD rats and the tissue remained ERα dominant indicating no role of HFD on ERs ratio in muscle during early lactation.
UNOC has been found to have negative effect on lipid accumulation. During pregnancy, lipid accumulation is one of the first events essential for proper fetal development. Since UNOC levels were suppressed during pregnancy, experiments were carried out to examine relevance of UNOC suppression on lipid accumulation during early pregnancy. For this purpose, pharmacological approaches were utilized to alter UNOC levels during early pregnancy. It was observed that the transient elevation of UNOC levels caused decrease in maternal fat depots without changing circulatory triacylglyceride (TAG) levels. In experiments that decreased UNOC levels in NPNL state to mimic lower levels of UNOC present during early pregnancy, it was found fat storage was higher and TG was found to be lowered in the circulation. These results indicate that UNOC can cause a reduction in fat accumulation and TG levels but UNOC effects on TG levels, was not observed during pregnancy. The data taken together suggest that suppression of UNOC is required for better fat deposition in the mother’s body. Although, some studies have indicated an insulin response transition occurring during pregnancy to lactation, but the factors involved in this transition have not been reported. This report discusses about the factors such as UNOC and ERs and their involvement in the transition process. UNOC role has been studied in genetically modified models and in metabolic disorders such as obesity model system and evidence for physiological role of UNOC would further support its candidature as anti-diabetic molecule. The present research work is the first report to detail relevance of UNOC in physiological conditions such as pregnancy and lactation for glucose and lipid management.
47
Chang, Shu-Wen y 張書聞. "Effects of Collapsing Response Mediator Protein 2 on Glucose Transporter Type 4 Translocation and Lipid Metabolism in Adipocyte Differentiation via Cytoskeleton Dynamics". Thesis, 2016. http://ndltd.ncl.edu.tw/handle/27002176841105689762.
Resumen
碩士國立陽明大學
醫學生物技術暨檢驗學系
104
Diabetes mellitus is a metabolic abnormality defined by hyperglycemia resulting from defects in insulin action and/or insulin secretion. Diabetic patients with long-term hyperglycemia are susceptible to develop multiple complications and neurodegenerative diseases due to hyperglycemia-resulted oxidative stress and damage. Cytoskeleton is abnormally assembled and neurotransmission is impaired in neurodegenerative diseases. Moreover, alterations in cytoskeleton dynamics have been implicated in insulin action, glucose transporter type 4 (GLUT4) translocation and trafficking of lipid droplets in adipocytes. Collapsin response mediator protein 2 (CRMP2) regulates neurite outgrowth through mediating microtubules dynamics, which is regulated by glycogen synthase kinase-3β (GSK-3β), the important mediator in the signaling of insulin and GLUT4 translocation. Accordingly, we hypothesized that CRMP2 may be involved in energy homeostasis and thus adipocyte differentiation (adipogenesis). The aim of this study is to investigate expression profile and effects of CRMP2 on glucose and lipid metabolism in adipogenic process of 3T3-L1 pre-adipocytes under high (hyperglycemia) or low (normoglycemia) glucose environment. Our results showed that CRMP2 overexpression in mature adipocytes led to reduced lipids accumulation and decreased expression of fatty acid synthesis enzyme (acetyl CoA carboxylase, ACC and fatty acid synthase, FAS) and adipogenesis marker peroxisome proliferator-activated receptor gamma (PPAR-γ), CCAAT/enhancer binding protein α (C/EBP-α) and fatty acid binding protein 4 (FABP4), while actin filament polymerization is increased. In contrast, knockdown of CRMP2 expression attenuated the above effects. Besides, CRMP2 expression and basal glucose uptake activity were significantly increased in cells under normoglycemic (100 mg/dL) condition. Although lipids contents were reduced in cells exposed to low glucose, expression of PPAR-γ, FABP4 and GLUT4 were elevated. Nevertheless, glucose levels did not affect CRMP2 mRNA levels and proteasomal degradation in adipogenic process. The above results suggest that under physiological glucose environment, the increased CRMP2 expression may result in upregulation of FABP4, PPAR-γ and GLUT4 that facilitates adipocyte differentiation as well as glucose uptake. In addition, the CRMP2-mediated decrease of fatty acid synthesis enzymes and adipogenesis markers and increase of cytoskeleton polymerization result in the reduced lipid accumulation. The above results suggest that CRMP2 may participate in diabetic pathogenesis and diabetic neuropathy through mediating lipid and glucose metabolism in response to external nutrient condition.
48
Dagenais, Bellefeuille Steve DB. "Nitrate metabolism in the dinoflagellate Lingulodinium polyedrum". Thèse, 2015. http://hdl.handle.net/1866/15897.
Resumen
Les dinoflagellés sont des eucaryotes unicellulaires retrouvés dans la plupart des
écosystèmes aquatiques du globe. Ces organismes amènent une contribution substantielle à la
production primaire des océans, soit en tant que membre du phytoplancton, soit en tant que
symbiontes des anthozoaires formant les récifs coralliens. Malheureusement, ce rôle
écologique majeur est souvent négligé face à la capacité de certaines espèces de dinoflagellés
à former des fleurs d'eau, parfois d'étendue et de durée spectaculaires. Ces floraisons d'algues,
communément appelées "marées rouges", peuvent avoir de graves conséquences sur les
écosystèmes côtiers, sur les industries de la pêche et du tourisme, ainsi que sur la santé
humaine. Un des facteurs souvent corrélé avec la formation des fleurs d'eau est une
augmentation dans la concentration de nutriments, notamment l’azote et le phosphore. Le
nitrate est un des composants principaux retrouvés dans les eaux de ruissellement agricoles,
mais également la forme d'azote bioaccessible la plus abondante dans les écosystèmes marins.
Ainsi, l'agriculture humaine a contribué à magnifier significativement les problèmes associés
aux marées rouges au niveau mondial. Cependant, la pollution ne peut pas expliquer à elle
seule la formation et la persistance des fleurs d'eau, qui impliquent plusieurs facteurs biotiques
et abiotiques. Il est particulièrement difficile d'évaluer l'importance relative qu'ont les ajouts de
nitrate par rapport à ces autres facteurs, parce que le métabolisme du nitrate chez les
dinoflagellés est largement méconnu. Le but principal de cette thèse vise à remédier à cette
lacune. J'ai choisi Lingulodinium polyedrum comme modèle pour l'étude du métabolisme du
nitrate, parce que ce dinoflagellé est facilement cultivable en laboratoire et qu'une étude
transcriptomique a récemment fourni une liste de gènes pratiquement complète pour cette
espèce. Il est également intéressant que certaines composantes moléculaires de la voie du
nitrate chez cet organisme soient sous contrôle circadien. Ainsi, dans ce projet, j'ai utilisé des
analyses physiologiques, biochimiques, transcriptomiques et bioinformatiques pour enrichir
nos connaissances sur le métabolisme du nitrate des dinoflagellés et nous permettre de mieux
apprécier le rôle de l'horloge circadienne dans la régulation de cette importante voie
métabolique primaire.
Je me suis tout d'abord penché sur les cas particuliers où des floraisons de dinoflagellés
sont observées dans des conditions de carence en azote. Cette idée peut sembler contreintuitive,
parce que l'ajout de nitrate plutôt que son épuisement dans le milieu est généralement
associé aux floraisons d'algues. Cependant, j’ai découvert que lorsque du nitrate était ajouté à
des cultures initialement carencées ou enrichies en azote, celles qui s'étaient acclimatées au
stress d'azote arrivaient à survivre près de deux mois à haute densité cellulaire, alors que les
cellules qui n'étaient pas acclimatées mourraient après deux semaines. En condition de carence
d'azote sévère, les cellules arrivaient à survivre un peu plus de deux semaines et ce, en arrêtant
leur cycle cellulaire et en diminuant leur activité photosynthétique. L’incapacité pour ces
cellules carencées à synthétiser de nouveaux acides aminés dans un contexte où la
photosynthèse était toujours active a mené à l’accumulation de carbone réduit sous forme de
granules d’amidon et corps lipidiques. Curieusement, ces deux réserves de carbone se
trouvaient à des pôles opposés de la cellule, suggérant un rôle fonctionnel à cette polarisation.
La deuxième contribution de ma thèse fut d’identifier et de caractériser les premiers
transporteurs de nitrate chez les dinoflagellés. J'ai découvert que Lingulodinium ne possédait
que très peu de transporteurs comparativement à ce qui est observé chez les plantes et j'ai
suggéré que seuls les membres de la famille des transporteurs de nitrate de haute affinité 2
(NRT2) étaient réellement impliqués dans le transport du nitrate. Le principal transporteur
chez Lingulodinium était exprimé constitutivement, suggérant que l’acquisition du nitrate chez
ce dinoflagellé se fondait majoritairement sur un système constitutif plutôt qu’inductible.
Enfin, j'ai démontré que l'acquisition du nitrate chez Lingulodinium était régulée par la lumière
et non par l'horloge circadienne, tel qu'il avait été proposé dans une étude antérieure.
Finalement, j’ai utilisé une approche RNA-seq pour vérifier si certains transcrits de
composantes impliquées dans le métabolisme du nitrate de Lingulodinium étaient sous
contrôle circadien. Non seulement ai-je découvert qu’il n’y avait aucune variation journalière
dans les niveaux des transcrits impliqués dans le métabolisme du nitrate, j’ai aussi constaté
qu’il n’y avait aucune variation journalière pour n’importe quel ARN du transcriptome de
Lingulodinium. Cette découverte a démontré que l’horloge de ce dinoflagellé n'avait pas
besoin de transcription rythmique pour générer des rythmes physiologiques comme observé
chez les autres eukaryotes.Dinoflagellates are unicellular eukaryotes found in most aquatic ecosystems of the world. They are major contributors to carbon fixation in the oceans, either as free-living phytoplankton or as symbionts to corals. Dinoflagellates are also infamous because some species can form spectacular blooms called red tides, which can cause serious damage to ecosystems, human health, fisheries and tourism. One of the factors often correlated with algal blooms are increases in nutrients, particularly nitrogen and phosphorus. Nitrate is one of the main components of agricultural runoffs, but also the most abundant bioavailable form of nitrogen in marine environments. Thus, agricultural activities have globally contributed to the magnification of the problems associated with red tides. However, bloom formation and persistence cannot be ascribed to human pollution alone, because other biotic and abiotic factors are at play. Particularly, it is difficult to assess the relative importance of nitrate addition over these other factors, because nitrate metabolism in dinoflagellate is mostly unknown. Filling part of this gap was the main goal of this thesis. I selected Lingulodinium polyedrum as a model for studying nitrate metabolism, because this dinoflagellate can easily be cultured in the lab and a recent transcriptomic survey has provided an almost complete gene catalogue for this species. It is also interesting that some molecular components of the nitrate pathway in this organism have been reported to be under circadian control. Thus, in this project, I used physiological, biochemical, transcriptomic and bioinformatic approaches to enrich our understanding of dinoflagellate nitrate metabolism and to increase our appreciation of the role of the circadian clock in regulating this important primary metabolic pathway. I first studied the particular case of dinoflagellate blooms that occur and persist in conditions of nitrogen depletion. This idea may seems counterintuitive, because nitrogen addition rather than depletion, is generally associated with algal blooms. However, I discovered that when nitrate was added to nitrogen-deficient or nitrogen-sufficient cultures, those that had been acclimated to nitrogen stress were able to survive for about two months at high cell densities, while non-acclimated cells died after two weeks. In conditions of severe nitrogen limitation, cells could survive a little bit more than two weeks by arresting cell division and reducing photosynthetic rates. The incapacity to synthesize new amino acids for these deprived cells in a context of on-going photosynthesis led to the accumulation of reduced carbon in the form of starch granules and lipid bodies. Interestingly, both of these carbon storage compounds were polarized in Lingulodinium cells, suggesting a functional role. The second contribution of my thesis was to identify and characterize the first nitrate transporters in dinoflagellates. I found that in contrast to plants, Lingulodinium had a reduced suite of nitrate transporters and only members of the high-affinity nitrate transporter 2 (NRT2) family were predicted to be functionally relevant in the transport of nitrate. The main transporter was constitutively expressed, which suggested that nitrate uptake in Lingulodinium was mostly a constitutive process rather than an inducible one. I also discovered that nitrate uptake in this organism was light-dependent and not a circadian-regulated process, as previously suggested. Finally, I used RNA-seq to verify if any transcripts involved in the nitrate metabolism of Lingulodinium were under circadian control. Not only did I discovered that there were no daily variations in the level of transcripts involved in nitrate metabolism, but also that there were no changes for any transcripts present in the whole transcriptome of Lingulodinium. This discovery showed that the circadian timer in this species did not require rhythmic transcription to generate biological rhythms, as observed in other eukaryotes.
49
Mok, Leo. "In Vitro Characterization of the Function of ABCA1: Effects of Naturally Occurring Mutations". Thesis, 2010. http://hdl.handle.net/1974/5434.
Resumen
The ATP-binding cassette (ABC) transporter, ABCA1, plays a pivotal role in reverse cholesterol transport, which is the elimination of excess sterols from peripheral cells and their transport to the liver for elimination. Early studies failed to detect significant ATPase activity, prompting the suggestion that ABCA1 was an ATP-regulated receptor, rather than an active transporter. We have provided evidence that ABCA1 can bind ATP and trap its hydrolysis product, ADP, in the presence of either ortho-vanadate or beryllium fluoride and Mg2+ or Mn2+. We have also shown that both nucleotide-binding domains (NBDs) trap nucleotide comparably, suggesting that ABCA1 is a functional ATPase. In addition, we have shown that ABCA1 can directly transport 25-hydroxycholesterol (25-OHC) in an ATP-dependent manner using a membrane vesicle uptake assay, and can do so when the physiological substrate acceptor apoA-I is replaced with BSA as a non-specific binding protein.
Although more than 50 naturally occurring missense mutations and polymorphisms in ABCA1 have been identified in individuals with HDL-C levels within the lowest 5th percentile of the general population, the extent to which many of these mutations affect ABCA1 function is not known and cannot be predicted. Naturally occurring extracellular loop (ECL) mutations W590S and C1477R have both been shown to effectively eliminate the ability to mediate lipid efflux, despite the fact that the W590S mutant protein retains the ability to bind apoA-I. We show that neither mutant can transport nor efflux 25-OHC, whether in the presence of apoA-I or BSA, despite apparently full retention of the ability to bind and trap nucleotide. This suggests that these two ECL mutations inhibit transport by a mechanism that is independent of their effect on apoA-I binding. By introduction of naturally occurring mutations in the NBDs, we show that although some mutations associated with Tangier Disease, such as N935S, essentially eliminate nucleotide trapping and substrate translocation, other polymorphisms such as L1026P and T2073A associated with low HDL-C, appear to be fully functional. Lastly, we observed differences in the behaviour of both wild-type and mutant forms of ABCA1-GFP depending on whether they were expressed in insect or mammalian cell lines.Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2010-02-12 11:14:11.381