Rozprawy doktorskie na temat „Phosphatidic acid”

Phosphatidic Acid (PA) is a natural phospholipid compound derived from lecithin which is commonly found in egg yolk, grains, fish, soybeans, peanuts and yeast. It has been suggested that PA is involved in several intracellular processes associated with muscle hypertrophy. Specifically, PA has been reported to activate protein synthesis through the mammalian target of rapamycin (mTOR) signaling pathway and thereby may enhance the anabolic effects of resistance training. To our knowledge, no one has examined the effect of PA supplementation in humans while undergoing a progressive resistance training program. To examine the effect of PA supplementation on lean soft tissue mass (LM) and strength after 8 weeks of resistance training. Fourteen resistance-trained men (mean [plus or minus] SD; age 22.7 [plus or minus] 3.3 yrs; height: 1.78 [plus or minus] 0.10m; weight: 89.3 [plus or minus] 16.3 kg) volunteered to participate in this randomized, double-blind, placebo-controlled, repeated measures study. The participants were assigned to a PA group (750mg/day; Mediator, ChemiNutra, MN, n=7) or placebo group (PL; rice flower; n=7), delivered in capsule form that was identical in size, shape and color. Participants were tested for 1RM strength in the bench press (BP) and squat (SQ) exercise. LM was measured using dual-energy X-ray absorptiometry. After base line testing, the participants began supplementing PA or PL for 8 weeks during a progressive resistance training program intended for muscular hypertrophy. Data was analyzed using magnitude-based inferences on mean changes for BP, SQ and LM. Furthermore, the magnitudes of the inter-relationships between changes in total training volume and LM were interpreted using Pearson correlation coefficients, which had uncertainty (90% confidence limits) of approximately +0.25. In the PA group, the relationship between changes in training volume and LM was large (r=0.69, [plus or minus] 0.27; 90%CL), however, in the PL group the relationship was small (r=0.21, [plus or minus] 0.44; 90%CL). PA supplementation was determined to be likely beneficial at improving SQ and LM over PL by 26% and 64%, respectively. The strong relationship between changes in total training volume and LM in the PA group suggest that greater training volume most likely lead to the greater changes in LM, however, no such relationship was found with PL group. For the BP data, the PA group resulted in a 42% greater increase in strength over PL, although the effect was considered unclear. While more research is needed to elucidate mechanism of action; the current findings suggest that in experienced resistance trained men supplementing 750mg PA per day for 8 weeks may likely benefit greater changes in muscle mass and strength compared with resistance training only.
M.S.
Masters
Child, Family and Community Sciences
Education and Human Performance
Sport and Exercise Sciences; Applied Exercise Physiology

Thesis (M. S.)--Biology, Georgia Institute of Technology, 2006.
Donald Doyle, Committee Member ; Harish Radhakrishna, Committee Member ; Alfred Merrill, Committee Member ; Marion Sewer, Committee Chair Includes bibliographical references.

The Wingless (Wnt) signaling pathway is one of the critical developmental pathways for control of cell differentiation, proliferation, and cell growth. The DEP domain, located on the C-terminus of Dishevelled (Dvl), plays a role in cytoplasm-membrane association, which branches the canonical and non-canonical Wnt signaling pathway within the cell. It has been suggested that the DEP domain requires the recruitment of ionic lipids, such as phosphatidic acid, to regulate its localization to the plasma membrane and association to the frizzle receptor. However, the physical mechanism for DEP association to the plasma membrane is still unknown. We show that mDvl2-DEP interacts with phosphatidic acid at a distinct patch on the surface formed by a positively charged surface area by NMR spectroscopy. The binding of this interaction was also found at physiologically relevant concentration using fluorescence spectroscopy. We also determined that the interaction is pH-dependent and regulated through a 'histidine switch' mechanism at His464 and His465 where there is increased association of mDvl2-DEP to the plasma membrane at higher pH values (7.5). This association is based on tertiary structure conformational changes with rearrangement of the loop regions by a change in local pH, not its interaction with phosphatidic acid. Overall, our work will contribute to elucidate how cells regulate their developmental pathways through localized molecular interactions.
Master of Science

Emerging evidence suggests that an important mechanism for the negative feedback control of insulin signalling involves the inhibition of tyrosine phosphorylation of IRS-1 by its prior serine/threonine (ser/thr) phosphorylation. IRS-1 ser/thr phosphorylation has been linked to the dissociation of IRS-1 from the insulin receptor and PI3K, and its degradation via a proteasome-dependent pathway. Studies in animal models have shown that increases in plasma free fatty acids (FFAs) are associated with reduced IRS-1-signalling, and so it has been postulated that elevated FFA cause insulin resistance by activating pathways that negatively regulate insulin action, including hyper-phosphorylation of ser/thr residues in IRS-1. We have shown that in the case of linoleate-induced insulin resistance in L6 rat skeletal muscle cells, the inhibition of IRS-1-dependent signalling arises via effects on both the phosphorylation status and degradation of IRS-1, which are mediated, in part, by IKKb. In addition, the reduction of IRS-1 mRNA levels allude to transcriptional effects of linoleate treatment that also contribute to the observed reduction in the total levels of this protein. PtdOH, particularly dilinoleoyl PtdOH, was found to be significantly increased in linoleate treated L6 cells, and sufficient to induce at least some of the effects on insulin-signalling that are observed upon linoleate treatment. It is unlikely, however, that IKKb and PtdOH are components of the same inhibitory pathway, since inhibiting IKKb activity did not alleviate the effects of PtdOH on IRS-1 tyrosine (tyr) phosphorylation. Moreover, although an integral component of the mechanism by which linoleate induces insulin-resistance in L6 cells, it appears that restoring IRS-1 function in linoleate treated cells is not sufficient to reverse insulin resistance. Hence, we hypothesise that linoleate induces multiple inhibitory pathways in L6 cells, with at last two of these involving IKKb- and PtdOH-dependent inhibition of IRS-1 signalling, which act in parallel to reduce glucose disposal and cause insulin resistance in this model.

Thesis (Ph. D.)--University of California, San Diego, 2008.
Title from first page of PDF file (viewed April 7, 2008). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 174-187).

Chez les eucaryotes, les lipides membranaires sont essentiels à la compartimentation et la régulation des voies de sécrétion. Par leurs propriétés physiques, ils sont essentiels aux courbures membranaires et à la régulation de la morphodynamique des endomembranes, de la morphologie des organites et de la formation des vésicules de transport. Chez l’animal, les acide lysophosphatidique acyltransférases (LPAATs) sont impliquées dans la régulation du trafic endomembranaire, mais rien n’est connu sur leur rôle chez la plante. Chez Arabidopsis thaliana, cinq LPAATs ont été identifiées. Nous avons déterminé leur activité enzymatique spécifique pour l'acide lysophosphatidique (LPA) pour produire de l'acide phosphatidique (PA). J'ai ensuite caractérisé leur localisation subcellulaire dans le système endomembranaire de la voie sécrétoire et étudié leur rôle présumé dans cette voie par approche génétique (mutants knock-out), biochimiques (inhibiteurs d'activité, analyses des lipides) et d'imagerie (microscopie confocale). En exploitant les lignées simples, doubles et triples mutantes des gènes LPAAT que j’ai produites, couplées à un traitement au CI-976 qui inhibe l'activité des LPAATs, j’ai montré suite à une analyse lipidique, que la quantité d’acide phosphatidique (PA) dépendante de l’activité LPAAT est essentielle pour l’adressage du transporteur d’auxine PIN2 et de l’aquaporine PIP2,7 vers la membrane plasmique.Ce travail souligne l’importance de la régulation des quantités de lipides dans les endomembranes et l’existence de seuils en-dessous desquels l’homéostasie membranaire peut être finement perturbée au point d’entraîner des disfonctionnements de la voie sécrétoire
In eukaryotic cells, membrane lipids are essential for compartmentalization and regulation of secretory pathways. According to their physical properties, they are essential to membrane curvature and regulation of endomembrane morphodynamics, organelle morphology, and vesicles. In animal cells, lysophosphatidic acid acyltransferases (LPAAT) are involved in the regulation of endomembrane trafficking, but nothing is known about their role in plants. In Arabidopsis thaliana, five LPAATs were identified. We determined their specific enzymatic activity for lysophosphatidic acid (LPA) to produce phosphatidic acid (PA). I then characterized their subcellular localization in the endomembrane system of the secretory pathway and their potential role in this pathway using genetical (knockout mutants), biochemical (activity inhibitors, lipid analyzes) and imaging (confocal microscopy) approaches. Using the single, double and triple mutant lines for LPAAT genes that I produced, in addition to CI-976 treatment that inhibits LPAAT activity, I showed, after lipid analysis, that phosphatidic (PA) dependent on LPAAT activity is essential for the trafficking of the auxin carrier PIN2 and the aquaporin PIP2,7 to the plasma membrane.This work highlights the importance of lipid regulation in endomembranes and thresholds under which membrane homeostasis can be finely disturbed to the point of causing dysfunction of the secretory pathway

Les diacylglycerol kinases catalysent la phosphorylation du diacylglycérol en acide phosphatidique. Nous avons montré que la PLC spécifique des phosphoinositide (PI-PLC) et la diacylglycérol kinase (DGK) régulent négativement l'expression basale de la plupart des gènes DREB2 dans les cellules en suspension d'Arabidopsis thaliana. Les gènes DREB2 codent pour des facteurs de transcription qui se lient aux motifs DRE (Drought Responsive Elements). Ces éléments sont également liés par les facteurs DREB1. Alors que les facteurs DREB2 sont principalement impliqués dans les réponses à la sécheresse et au stress chaud, les DREB1 sont quant à eux induits en réponse au froid. Nous avons également pu montrer que l'inhibition par des agents pharmacologiques des activités PI-PLC ou DGK conduit à l'induction de l'expression basale des gènes DREB1. Cependant, l'induction est beaucoup moins marquée chez les gènes DREB1 que DREB2A, un membre de la famille DREB2. Cela indique que les gènes DREB1 et DREB2, ne sont pas soumis à la même régulation transcriptionnelle et que la signalisation lipidique pourrait en partie expliquer les différences dans la régulation des gènes DREB. Les DGK d'Arabidopsis sont codées par une famille multigénique de 7 gènes. Parmi ces gènes, on retrouve la DGK5 dont les le transcrit peut subir un épissage alternatif, ce qui aboutit à deux transcrits, dont l'un comporte une protéine avec un domaine putatif de liaison à la calmoduline. Le mutant knock-out dgk5.1 à une racine plus courte lorsqu'il est cultivé à 12°C comparé au sauvage. Ce phénotype racinaire est corrélé avec une zone méristématique et des cellules plus petites. La croissance des racines du mutant n'est n'est pas modifiée en présence de la plupart des hormones testées. Pourtant, elle est moins sensible à l'auxine exogène à 12°C par rapport au WT. Le mutant dgk5.1 génère moins de racines secondaires en présence d'auxine exogène que le WT. Le promoteur DR5 n'est pas activé dans le mutant à 12°C par l'IAA exogène dans la zone méristématique, alors qu'il est dans le WT. Nos résultats montrent que le mutant dgk5.1 est altéré dans sa réponse à l'auxine à 12°C, suggérant un rôle de perception/transduction de l’auxine dans les racines courtes
Diacylglycerol kinases catalyse the phosphorylation of diacylglycerol into phosphatidic acid. We show that phosphoinositide dependent-phospholipase C (PI-PLC) and diacylglycerol kinase (DGK) in Arabidopsis thaliana suspension cells negatively regulated the basal expression of most DREB2 genes. DREB2 genes encode transcription factors that bind to Drought Responsive Elements (DRE). Those elements are also bound by DREB1 factors. While DREB2 factors are mostly involved in drought and heat responses, DREB1s are induced in the response to chilling. We show also that the pharmacological inhibition of PI-PLC or DGK leads to the basal induction of DREB1 genes. However, the induction is much less marked for the DREB1 genes than that of DREB2A, a member of the DREB2 family. This illustrates that DREB1 and DREB2 genes, while having the same targets, are not submitted to the same transcription regulation, and that lipid signalling might in part explain these differences in the regulation of the DREB genes. In Arabidopsis, DGKs are encoded by a multigenic family of 7 members. In this thesis, we focus on DGK5. The transcripts can have differential splicing, leading to two mature transcript, one of which leading to a protein with a putative calmodulin binding domain. A dgk5 knocked-out mutant is comparable to the WT, except for shorter root when grown at 12°C. This short root phenotype is correlated with to shorter meristematic zone and smaller cells. The short root phenotype is not altered in presence of most hormones. Yet, the root growth is less sensitive to exogenous auxin at 12°C compared to the WT. Accordingly the mutant produces less secondary roots in presence of exogenous IAA than the WT at 12°C. The DR5 promoter is not activated in the mutant at 12°C by exogenous IAA, in the meristematic zone, while it is in the WT. Our results show that the dgk5.1 mutant is impaired in auxin response at 12°C, suggesting a role of auxin perception /transduction in the short root phenotype

Les cellules eucaryotes possèdent un territoire membranaire dit « électrostatique » qui est définit par la présence de phospholipides négativement chargés sur la face cytosolique des membranes. Cette propriété permet le recrutement de protéine cytosolique contenant des motifs/domaines positivement chargés au niveau des membranes via des interactions électrostatiques. Nous nous sommes demandés si le territoire électrostatique est présent chez les cellules végétales et quel est son organisation ? Quels sont le(s) lipide(s) anionique(s) impliqués dans son maintien ? Et quel est son (ces) rôle(s) dans la signalisation et le développement des plantes ? Premièrement, nous avons mis en avant que la membrane plasmique est le compartiment intracellulaire le plus électronégativement chargé (Simon, Platre et al., 2016 Nature Plants). Ce champ électrostatique est gouverné par trois lipides anioniques différents, l’acide phosphatidique, la phosphatidylserine et le phosphatidylinositol-4-phosphate. Nous avons montré que cette propriété unique de la membrane plasmique permet de réguler des voies de signalisation hormonale, tel que celle de l’auxine et des brassinostéroïdes. Notamment, la phosphatidylserine régule la dynamique spatiotemporelle des petites GTPases de la famille Rho. En réponse à l’auxine, ce lipide permet de regrouper les protéines Rho dans des domaines membranaires. La formation de ces domaines est requise pour l’activité de ces protéines permettant de contrôler l’endocytose, la dynamique du cytosquelette mais également régule la morphogenèse cellulaire ainsi que la réponse gravitropique de la racine
The « electrostatic territory» is part of the eukaryotic membrane organization and is defined by the enrichment of negatively charged phospholipids at the membrane cytosolic face. This feature is involved in the membrane recruitment of cytosolic proteins, which contain positively charged motifs and/or domains. In this work, we used Arabidopsis thaliana as a model and explored the existence of an electrostatic territory in plant cells. We found that the plasma membrane is the most anionic intracellular membrane (Simon, Platre et al., 2016 Nature Plants). This electrostatic field is maintained by lipid cooperation between, phosphatidic acid, phosphatidylserine and phosphatidylinositol-4-phosphate. The cell surface unique feature is involved in the regulation of hormonal signalling such as auxin and brassinosteroids pathways. We found that phosphatidylserine tunes the spatiotemporal dynamics of small GTPases from the Rho family. During auxin response, PS is required to cluster Rho into specialized membrane domains. We show that nanocluster formation is required for Rho-mediated auxin signaling including the regulation of endocytosis, cytoskeleton organization, morphogenesis and the root gravitropic response

L’acide phosphatidique (PA) est un lipide simple qui peut exister sous différentes formes. A partir des sondes que j’ai préparé en se basant sur des domaines de liaison au PA : PDE4A1, Spo20p, et OpiQ2, j’ai pu étudier la localisation subcellulaire du PA dans les cellules PC12 et les macrophages RAW264.7. Ces sondes lient différents formes de PA dans les membranes de différents compartiments subcellulaires. De plus, j’ai pu montrer qu’il y a une néosynthèse de PA et de certaines espèces de PA mono- ou bi-insaturé à la membrane plasmique lors de la stimulation de l’exocytose. Nous avons ainsi observé que la PLD1 semble être la source principale de PA dans les glandes surrénales. D’autre part, mes travaux indiquent une augmentation du niveau global de PA à la membrane plasmique et une diminution importante du PA au niveau du RE dans les macrophages après stimulation de la phagocytose frustrée. Ce qui pourrait ainsi valider le concept d’une fusion d’une partie de la membrane du RE avec la membrane plasmique lors de la phagocytose
The phosphatidic acid (PA) is a simple lipid which may exist in various forms. I have generated probes based on PA binding domains: PDE4A1, Spo20p and OpiQ2 to study the subcellular localization of PA in PC12 cells and RAW264.7 macrophages. These probes bind different form of PA in different subcellular compartments. In addition, I show that PA and certain species mono- or bi-unsaturated of PA are synthesized at the plasma membrane upon stimulation of exocytosis. We observed that the PLD1 seems to be the main source of PA in the adrenal glands. On the other hand, my research indicates an increase in the level of PA at the plasma membrane and a significant decrease in the ER in macrophages after stimulation of phagocytosis frustrated. Thus these results could validate the concept of a fusion of a portion of the ER membrane with the plasma membrane during phagocytosis

Els glicolípids són producte d’alt valor degut a les seves propietats amfipàtiques que els doten d’un ampli rang d’aplicacions en els sectors químic (ex., biosurfactants) o biomèdic (ex., adjuvant de vacunes). Depenent de la unitat lipídica que els forma aquests compostos poden ser classificats en diferents famílies. Si la unitat lpídica és una ceramida o diacilglicerol, el glicolípid resultant es coneixerà com a glicoesfingolípid o glicoglicerolípid (GGL) respectivament. Mentre que els glicoesfingolípids han demostrat jugar un paper clau en diversos processos biològics, els glicoglicerolípids són interessants degut al seu ús potencial per a ser usats com a adjuvants de vacunes o supressors tumorals. Tot i que l’interès per aquests compostos és alt, la seva aplicació es veu obstaculitzada per la seva baixa disponibilitat i alt cost de producció. La síntesi química requereix de complexes passos de protecció i desprotecció per tal d’aconseguir la desitjada regio- i estereoespecificitat de l’enllaç glicosídic que, conseqüentment, comporta una reducció del rendiment i eficiència del procés. Per això, vam considerar l’enginyeria metabòlica com a estratègia potencial per a la producció de glicolípids i ens vam centrar en l’obtenció d’una plataforma d’enginyeria metabòlica en E. coli per tal d’obtenir aquests complexes productes d’interès. En estudis previs, el nostre grup va reportar que la sintasa de glicolípids MG517 de Mycoplasma genitalium era funcional i que s’obtenien glicoglicerolípids a partir de UDP-glucosa (UDP-Glc) i diacilglicerol (DAG). Addicionalment, una primera generació de soques modificades va demostrar que la disponibilitat de DAG era limitant per a la producció de GGL (Mora-Buyé et al., 2012). En el present projecte, cinc estratègies diferents d’enginyeria metabòlica van ser proposades per tal d’augmentar la producció de GGL utilitzant E. coli. Les primeres quatre estratègies tenien com a objectiu incrementar el pool del precursor lipídic, DAG. Sent així, la primera estratègia es basà en augmentar la disponibilitat de DAG a través de l’eliminació de reaccions competitives. Per aconseguir-ho, es van knockejar diferents gens involucrats en la ß-oxidació i l’activació d’àcids grassos (∆tesA i ∆fadE) reportant un increment en la producció de casi el doble. La segona estratègia es va basar en incrementar la disponibilitat d’àcids grassos mitjançant la modulació de factors de transcripció (fabR i fadR). Aquesta estratègia no va reportar un increment de la producció però si un canvi en el perfil lipídic amb un increment d’àcids grassos insaturats. La tercera estratègia es basava en incrementar la conversió dels donadors d’acils a àcid fosfatídic, precursor del DAG, sobreexpressant les aciltransferases PlsC i PlsB. La quarta estratègia es centrà en augmentar la disponibilitat del diacilglicerol per la sobreexpressió de la proteïna de fusió PlsCxPgpB, capaç de redirigir el flux cap a DAG, o CDH promovent la hidròlisi de fosfolípids. D’entre les diferent soques modificades, ∆tesA co-expressant MG517 i la proteïna de fusió PlsCxPgpB va ser la soca més productora, amb un 350% d’increment en la producció de GGL comparant-la amb la soca parental expressant únicament MG517. Especialment interessant és que les soques coexpressant CDH van presentar un canvi en el perfil de GGL cap al lípid diglucosilat (representant al voltant d’un 80% del total de GGLs). Finalment, es va proposar una estratègia metabòlica per incrementar la disponibilitat de l’altre precursor, UDP-Glc. Aquesta cinquena estratègia es va basar en sobre expressar l’enzim GalU, responsable de la biosíntesi d’UDP-Glc, i eliminant l’enzim codificant per la UDP-sucre difosfatasa ushA. No obstant, cap d’aquestes modificacions va aconseguir millorar els nivells de GGLs. Per últim, tal i com va ser reportat pel nostre grup que la fosfatidiletanolamina era intercanviable en les membranes d’E. coli pels nous producte GGL, una llibreria de promotors i RBS va ser dissenyada per tal de disminuir la producció d’aquest fosfolípid, augmentant al mateix temps de la producció de glicolípids.
Los glicolípidos son productos de alto valor debido a sus propiedades amfipáticas, que los dota en un amplio rango de aplicaciones en los sectores químicos (ej., biosurfactantes) o biomédicos (ej., adyuvante de vacunas). Dependiendo de la unidad lipídica que los forma, los glicolípidos son clasificados en diferentes familias. Si la unidad lipídica es una ceramida o diacilglicerol, los glicolípidos son conocidos como glicoesfingolípidos o glicoglicerolípidos (GGL) respectivamente. Mientras que los glicoesfingolípidos han demostrado jugar papeles clave en diversos procesos biológicos, los glicoglicerolípidos son compuestos interesantes debido a su potencial uso como adyuvantes de vacunas o supresores tumorales. Aunque el interés por estos compuestos es muy alto, su aplicación se ve obstaculizada por su baja disponibilidad y altos costes de producción. La síntesis química requiere de complejos pasos de protección y desprotección para conseguir la deseada regio- y estereoespecificidad del enlace glicosídico, que conlleva a una reducción del rendimiento y eficiencia del proceso. Por ello, consideramos la ingeniería metabólica como estrategia potencial para la producción de glicolípidos y nos centramos en construir una plataforma de ingeniería metabólica en E. coli para conseguir estas complejas estructuras de interés. En previos estudios, nuestro grupo reportó que la sintasa de glicolípidos MG517 de Mycoplasma genitalium era funcional y que glicoglicerolípidos podían ser obtenidos a partir de UDP-glucosa (UDP-Glc) y diacilglicerol (DAG). Adicionalmente, la primera generación de cepas modificadas demostró que la disponibilidad de DAG era limitante en la producción de GGL (Mora-Buyé et al., 2012). En el presente proyecto, cinco estrategias diferentes de ingeniería metabólica fueron propuestas para aumentar la producción de GGL en E. Coli. Las primeras cuatro estrategias se centraron en aumentar el pool del precursor lipídico, DAG. Para ello, la primera estrategia se basó en incrementar la disponibilidad de DAG a través de la eliminación de reacciones competitivas. Para lograrlo, se knockearon diferentes genes relacionados con la ß-oxidación y la activación de ácidos grasos (∆tesA y ∆fadE) reportando un incremento de casi el doble. La segunda estrategia se basó en incrementar la disponibilidad de ácidos grasos mediante la modulación de factores de transcripción (fabR y fadR). Aunque estrategia no reportó una mejora en el rendimiento de GGL, sí mostró un cambio en el perfil de los ácidos grasos con un incremento de los ácidos grasos insaturados. La tercera estrategia se basó en incrementar la conversión de los donadores de acilos a ácido fosfatídico, precursor del DAG, mediante la sobreexpresión de las aciltransferasas PlsC y PlsB. La cuarta estrategia se centró en aumentar la disponibilidad de diacilglicerol mediante la sobreexpresión de la proteína de fusión PlsCxPgpB capaz de redirigir el flujo de DAG, o CDH promoviendo la hidrólisis de fosfolípidos. Entre las diferentes cepas modificadas, la cepa ∆tesA coexpresando MG517 y la proteína de fusión PlsCxPgpB fue la mayor productora, con un incremento de los niveles de GGL del 350%, comparándola con la cepa parental expresando únicamente MG517. Interesantemente, las cepas coexpresando CDH mostraron un cambio en el perfil de GGL hacia el lípido diglucosilado (hasta el 80% del total del GGLs). Finalmente, una estrategia metabólica fue propuesta para aumentar la disponibilidad del otro precursor, UDP-Glc. La quinta estrategia se basó en la sobreexpresión de la enzima GalU, responsable de la biosíntesis de UDP-Glc, y la eliminación de la UDP-azúcar difosfatasa codificada por el gen ushA. Sin embargo, ninguna de estas modificaciones mejoró los niveles de GGL. Por último, tal y como reportó nuestro grupo que la fosfatidiletanolamina era intercambiable en las membranas de E. coli por los nuevos compuestos GGL, una librería de promotores y RBS fue diseñada para disminuir la producción de este fosfolípido intentando al mismo tiempo aumentar la producción de glicolípidos.
Glycolipids are products of high-added value due to their amphipathic properties, which endow them with a broad range of applications in the chemical (i.e., biosurfactants) and biomedical sectors (i.e., vaccine adjuvants). Depending on their lipidic moiety, glycolipids are classified in different families. If the lipid moiety is a ceramide or diacylglycerol, the glycolipids are known as glycosphingolipids or glycoglycerolipids respectively. While glycosphingolipids have shown to play essential roles in many biological processes, glycoglycerolipids (GGL) are interesting compounds due to their potential use as vaccine adjuvants or tumor suppressors. Although the interest of these compounds is very high, their applications are hampered by their low availability and high productions costs. Chemical synthesis requires complex protection and deprotection steps to achieve the desired regio- and stereospecificity of the glycosidic linkage, which consequently lower the yield and efficiency of the process. Therefore, we considered metabolic engineering as a potential strategy for the production of glycolipids and we aimed at building up a metabolic engineering platform in E. coli to achieve these complex structures of interest. In previous studies, our group reported that the glycolipid synthase MG517 from Mycoplasma genitalium was functional and glycoglycerolipids were obtained from UDP-glucose (UDP-Glc) and diacylglycerol (DAG). In addition, the first generation of engineered strains demonstrated that the availability of the DAG was the key bottleneck in GGL production (Mora-Buyé et al., 2012). In the present project, five different metabolic strategies were proposed to increase the production of GGL using E. coli. The first four strategies were aimed at increasing the available pool of the lipidic precursor, DAG. Thus, the first strategy was based on increasing DAG availability by removing competing reactions. To achieve so, different genes involved in the ß-oxidation and activation of fatty acids were knocked out (ΔtesA y ΔfadE) reporting an almost 2-fold production increase. The second strategy was based on increasing fatty acids availability by modulating different transcriptional factors (fabR y fadR). Although this strategy did not report an improvement of GGL yield, showed a change in the fatty acid profile with an increase of unsaturated fatty acids. The third strategy was based on increasing the conversion of acyl donors to phosphatidic acid, precursor of DAG, by overexpressing PlsC and PlsB acyltransferases. The fourth strategy was based on increasing diacylglycerol availability by overexpressing the fusion PlsCxPgpB protein that could redirect the flux to DAG or CDH promoting the hydrolysis of phospholipids. Among the different engineered strains, the ∆tesA strain co-expressing MG517 and a fusion PlsCxPgpB protein was the best producer, with a 350% increase of GGL titer compared to the parental strain expressing MG517 alone. Interestingly, the strains co-expressing CDH showed a shift in the GGL profile towards the diglucosylated lipid (up to 80% of total GGLs). Finally, a metabolic strategy was proposed to increase the availability of the other precursor, UDP-Glc. This fifth strategy was based on overexpressing GalU enzyme, which is responsible for the biosynthesis of UDP-Glc, and by removing the UDP-sugar diphosphatase encoding gene ushA. However, none of these modifications further improved the GGL titers. Finally, as it was also reported by our group that phosphatidylethanolamine was exchangeable in the membranes of E. coli by the new GGL compounds, a library of promoters and RBS was designed to decrease the production of this phospholipid trying at the same time to increase the production of glycolipids.

Les diacylglycerol kinases catalysent la phosphorylation du diacylglycérol en acide phosphatidique. Nous avons montré que la PLC spécifique des phosphoinositide (PI-PLC) et la diacylglycérol kinase (DGK) régulent négativement l'expression basale de la plupart des gènes DREB2 dans les cellules en suspension d'Arabidopsis thaliana. Les gènes DREB2 codent pour des facteurs de transcription qui se lient aux motifs DRE (Drought Responsive Elements). Ces éléments sont également liés par les facteurs DREB1. Alors que les facteurs DREB2 sont principalement impliqués dans les réponses à la sécheresse et au stress chaud, les DREB1 sont quant à eux induits en réponse au froid. Nous avons également pu montrer que l'inhibition par des agents pharmacologiques des activités PI-PLC ou DGK conduit à l'induction de l'expression basale des gènes DREB1. Cependant, l'induction est beaucoup moins marquée chez les gènes DREB1 que DREB2A, un membre de la famille DREB2. Cela indique que les gènes DREB1 et DREB2, ne sont pas soumis à la même régulation transcriptionnelle et que la signalisation lipidique pourrait en partie expliquer les différences dans la régulation des gènes DREB. Les DGK d'Arabidopsis sont codées par une famille multigénique de 7 gènes. Parmi ces gènes, on retrouve la DGK5 dont les le transcrit peut subir un épissage alternatif, ce qui aboutit à deux transcrits, dont l'un comporte une protéine avec un domaine putatif de liaison à la calmoduline. Le mutant knock-out dgk5.1 à une racine plus courte lorsqu'il est cultivé à 12°C comparé au sauvage. Ce phénotype racinaire est corrélé avec une zone méristématique et des cellules plus petites. La croissance des racines du mutant n'est n'est pas modifiée en présence de la plupart des hormones testées. Pourtant, elle est moins sensible à l'auxine exogène à 12°C par rapport au WT. Le mutant dgk5.1 génère moins de racines secondaires en présence d'auxine exogène que le WT. Le promoteur DR5 n'est pas activé dans le mutant à 12°C par l'IAA exogène dans la zone méristématique, alors qu'il est dans le WT. Nos résultats montrent que le mutant dgk5.1 est altéré dans sa réponse à l'auxine à 12°C, suggérant un rôle de perception/transduction de l’auxine dans les racines courtes
Diacylglycerol kinases catalyse the phosphorylation of diacylglycerol into phosphatidic acid. We show that phosphoinositide dependent-phospholipase C (PI-PLC) and diacylglycerol kinase (DGK) in Arabidopsis thaliana suspension cells negatively regulated the basal expression of most DREB2 genes. DREB2 genes encode transcription factors that bind to Drought Responsive Elements (DRE). Those elements are also bound by DREB1 factors. While DREB2 factors are mostly involved in drought and heat responses, DREB1s are induced in the response to chilling. We show also that the pharmacological inhibition of PI-PLC or DGK leads to the basal induction of DREB1 genes. However, the induction is much less marked for the DREB1 genes than that of DREB2A, a member of the DREB2 family. This illustrates that DREB1 and DREB2 genes, while having the same targets, are not submitted to the same transcription regulation, and that lipid signalling might in part explain these differences in the regulation of the DREB genes. In Arabidopsis, DGKs are encoded by a multigenic family of 7 members. In this thesis, we focus on DGK5. The transcripts can have differential splicing, leading to two mature transcript, one of which leading to a protein with a putative calmodulin binding domain. A dgk5 knocked-out mutant is comparable to the WT, except for shorter root when grown at 12°C. This short root phenotype is correlated with to shorter meristematic zone and smaller cells. The short root phenotype is not altered in presence of most hormones. Yet, the root growth is less sensitive to exogenous auxin at 12°C compared to the WT. Accordingly the mutant produces less secondary roots in presence of exogenous IAA than the WT at 12°C. The DR5 promoter is not activated in the mutant at 12°C by exogenous IAA, in the meristematic zone, while it is in the WT. Our results show that the dgk5.1 mutant is impaired in auxin response at 12°C, suggesting a role of auxin perception /transduction in the short root phenotype

Le virus de la vaccine (VACV) est l'organisme modèle pour l'étude des Poxviridae. Son cycle de réplication dans le cytoplasme de la cellule hôte a été largement étudié par microscopie optique et microscopie électronique. Grâce à des études génétiques approfondies, le rôle de certaines des 250 protéines du virus a été élucidé. Cependant, les mécanismes d’acquisition de la membrane du virus, notamment le rôle des lipides cellulaires impliqués, restent mal connus. L’étude de la composition des membranes de VACV purifiés par spectrométrie de masse a montré qu’elles présentent un enrichissement en acide phosphatidique (PA) et en dérivés de phosphatidylinositoles (PIPs). De plus, des études in vitro ont permis d’identifier certaines protéines virales capables de se lier aux PIPs in vitro. Le rôle de ces lipides dans le cycle de vie du virus, en particulier, dans la biogenèse de ses membranes n'a pas été identifié. L'objectif de ce projet de thèse est de déterminer l’implication du PA et des PIPs dans la biogenèse des membranes virales. L’expression transitoire de protéines recombinantes contenant des domaines de liaison à ces lipides a permis de déterminer la localisation du PA et des PIPs au cours de la réplication du virus. Afin de compléter ces résultats nous avons également utilisé des anticorps reconnaissant la PI4K et le PI4P. Enfin, l’utilisation d’inhibiteurs des PI3Ks et des PI4Ks a permis d’étudier le rôle de ces kinases durant l’assemblage de la membrane virale. A l'aide de ces outils, j'ai pu montrer que la localisation de ces lipides, à l'exception du PI3P, n'est pas altérée dans les cellules infectées. De plus, aucune co-localisation n’a été observée entre ces lipides et les sites de réplication du virus. Par ailleurs, nous avons observé une co-localisation entre le PI4P et les virus enveloppés ce qui est en accord avec les études précédentes montrant que les membranes du virus mature seraient dérivées de l'appareil de Golgi. Toutefois, des inhibiteurs de la synthèse du PI3P et du PI4P n'ont pas montré d’effets sur la production des membranes virales observables par microscopie optique. En conclusion, ce travail a permis de mieux définir le rôle des lipides durant la réplication de VACV. Ces résultats mettent en lumière un rôle potentiel du PI4P au cours de l’acquisition de l’enveloppe du virus ainsi qu’un rôle PI3P et de protéines reconnaissant spécifiquement le PI3P au cours des phases tardives de la réplication
Vaccinia Virus (VACV) is the model organism for the study of the Poxviridae. Its cytoplasmic life cycle has been studied extensively by light- and electron microscopy. Thanks to a robust genetic system the role of some of its 250 proteins is beginning to be understood. Nevertheless, the acquisition of its membranes is still a matter of debate, in particular the role of cellular lipids. Lipid mass spectrometry of purified VACV previously showed an enrichment of phosphatidic acid (PA) and phosphatidylinositol derivatives (PIPs) in the viral membrane. Although some viral proteins have been shown to bind PIPs in vitro the role of these lipids in the viral life cycle, in particular viral membrane biogenesis, remains elusive.The aim of this work is to determine whether PA and PIPs are relocated in infected cells to the site of viral membrane biogenesis. For both PA and PIPs, I used recombinant proteins containing PA or PIP binding domains fused to eGFP, expressed them by transient transfection to follow their localization during viral replication. In addition, I used antibodies for the recognition of PI4K and PI4P. In order to understand the biochemical role of PIPs, I used pan-PI3K and PI4K inhibitors to study their effect on viral assembly. Using these tools, I could show that the lipids under investigation did not display an altered localization, with the exception of PI3P which showed a different pattern in infected cells. None of the PIPs analyzed co-localized with the sites of primary VACV membrane biogenesis. Consistent with the fact that the mature virus acquires additional membranes derived from the Golgi complex, I could show a co-localization of wrapped virus with PI4P, known to localize to this cellular organelle. However, drugs inhibiting PI3P and PI4P biosynthesis did not show any effect on VACV membrane biogenesis, at least at the light microscopy level. In conclusion, this work sharper defines the role of lipids during VACV replication. In particular, it opens the way to further studies on the putative role of PI4P during wrapping and the fate of PI3P and PI3P binding proteins during late replication

This diploma thesis deals with the preparation and characterization of negatively charged catanionic vesicular systems and their combination with selected polycations. The catanionic vesicular system was prepared by mixing of two oppositely charged surfactants SDS and CTAB. The negative charge as well as the stability of the vesicular system was provided by the incorporation of phosphatidic acid. Polycations, DEAE and TMC, have been selected for use in a pharmaceutical applications. Characterization of the prepared systems was performed by measuring DLS and ELS. The results indicate that we were able to prepare stable negatively charged vesicles that were eligible to non-covalently interact with selected polycations.

La mise en évidence du rôle des lipides dans le trafic membranaire fait partie des avancées majeures de ces dernières années. Mes travaux de thèse se sont focalisés sur le plus simple des phospholipides, l’acide phosphatidique (PA). La production de PA par la phospholipase D (PLD) joue un rôle crucial au cours de la phagocytose et l’exocytose régulée, mais la dynamique de sa formation, tout comme les mécanismes d’action des différentes formes de PA, restent à ce jour totalement inconnus. Durant mon doctorat, j’ai contribué à la caractérisation de trois domaines de liaison peptidique au PA, qui nous ont permis de mieux comprendre les mécanismes d’interaction des protéines avec le PA et de générer des sondes moléculaires pour repérer ce lipide au sein des cellules. Ainsi, j’ai pu visualiser la production de PA au cours de la phagocytose et mis en évidence l’implication de la GTPase Arf6 dans la régulation de la synthèse de PA par la PLD. J’ai également pu montrer que la PLD est impliquée dans plusieurs étapes de l’exocytose dans des cellules neuroendocrines. Des expériences de lipidomique et de restauration ont révélé notamment que des formes mono- et polyinsaturées du PA contrôlent des étapes distinctes de l’exocytose que j’ai pu définir
The discovery of the involvement of lipids in membrane trafficking is one of the major recent progress in cell biology. My thesis work focused on phosphatidic acid (PA), the simplest phospholipid. PA synthesis by phospholipase D (PLD) plays a crucial role during phagocytosis and regulated exocytosis, but its precise dynamics, as well as the mode of action of the different PA species, remain unknown. I characterized three PA binding domains allowing a better understanding of the interaction between proteins and PA and leading to the generation of genetic sensors for PA in cells. Thus I could visualize PA synthesis during phagocytosis and identified that the small GTPase Arf6 regulates PLD activity and consequently PA synthesis. My work also reveals that PLD modulates several steps during exocytosis in neuroendocrine cells. Further lipidomics and rescue experiments allowed me to show that mono- and polyunsaturated forms of PA are involved in distinct steps of exocytosis

Le léiomyome utérin est la pathologie utérine la plus fréquente chez les femmes en âges de procréer. Des résultats précédent obtenus avec les cellules ELT3, une lignée de cellules de léiomyomes de rat, ont montré que l'acide lysophosphatidique (LPA) activait les MAP kinases ERK1/2 via le récepteur LPA1 couplé à la protéine Gi et l'activation de Raf, Ras et de MEK. Durant ce travail, nous avons caractérisé l'activité phosphatase responsable de la dégradation du LPA dans cette lignée de cellules ELT3. Nous avons montré que le LPA était dégradé exclusivement par la lipide-phosphate phosphatase 1 (LPP1), seule isoforme exprimé dans les cellules ELT3. Dans un deuxième temps nous nous somme intéressés aux effets du diacylglycerol pyrophosphate (DGPP). Le DGPP est un médiateur lipidique qui, sous sa forme dioctanoyl (DGPP8:0), est décrit comme un antagoniste des récepteurs LPA1 et LPA3 chez les mammifères. Dans cette étude, nous montrons que le DGPP8:0 n'a pas d'effet antagoniste sur l'activation du module MAP kinase ERK1/2 par le LPA mais qu'il induit une activation de ERK1/2 dans plusieurs lignées de cellules de mammifères. En effet le DGPP active ERK1/2 à travers l'activation des PKC, Raf et MEK. De plus, nous montrons que l'activation induite par le DGPP repose sur sa déphosphorylation catalysée par une LPP. Nous montrons également que l'inhibition de LPP1 par le VPC32183 ou l'utilisation de siRNA dirigé contre la lipide phosphate-phosphatase 1 (LPP1) réduit l'activation ERK1/2 induite par le DGPP. Ceci montre que le DGPP active ERK1/2 via sa déphosphorylation en acide phosphatidique (PA8:0), catalysée par la LPP1. Enfin dans une dernière partie nous montrons que le myomètre sain, contrairement aux cellules ELT3, exprime à la fois la LPP1 et la LPP3. En étudiant l'effet de la surexpression de la LPP3 dans les cellules ELT3, nous avons observé que la LPP3 interagissait avec la LPP1 et qu'elle pourrait la séquestrer dans des compartiments membranaires internes. Cette séquestration entraine une diminution de l'actvité ecto-LPP au profit de l'activité intracellulaire qui pourrait réguler négativement la production de seconds messagers phospholipidiques. Ces résultats montrent l'importance des LPP dans la régulation des effets des phospholipides bioactifs et suggère un lien entre le caractère tumorale des cellules de léiomyomes et l'absence de la LPP3.

This diploma thesis deals with the preparation and characterization of stealth liposomes and their combination with trimethylchitosan (TMC). This complex could find application in the field of inhalation administration. Stealth liposomes were prepared from neutral phophatidylcholine, negatively charged fosfatidic acid and polyethyleneglycol bounded to phosphatidylethanolamine. We have managed to prepare stealth liposomes with suitable properties that should guarantee passive targeting without evocation an immune response, despite the content of the negative component. We also found a suitable method of preparation for stealth liposome–TMC complex, where the change of size and zeta potential confirmed the non–covalent bound between two components despite the content of the polyethyleneglycol.

La croissance neuritique est un mécanisme complexe qui fait toujours l'objet d'intenses investigations. Les donnés actuelles ont permis de mettre en évidence l'implication de trois mécanismes principaux dans la croissance neuritique : i) la dynamique du cytosquelette, ii) le trafic intracellulaire et l'apport membranaire au niveau du cône de croissance et iii) la signalisation cellulaire, principalement via la voie MAPK-ERK1/2, qui abouti à la régulation de la transcription.La PLD1 et son produit l'acide phosphatidique semblent être au centre de voies majeures impliquées dans le développement neuronal. Mes travaux ont permis d'approfondir nos connaissances sur le rôle cellulaire de la PLD1 au cours de la croissance neuritique. J'ai montré que la PLD1 en collaboration avec la kinase RSK2 régule la fusion des vésicules positives pour Ti-VAMP/VAMP7 au cours de la croissance neuritique. D'autre part, j'ai établi que la PLD1 joue un rôle important dans le maintien de la signalisation endosomale de la voie MAPK-ERK1/2-RSK2-CREB induite par les neurotrophines. J'ai également montré que la PLD1 régule l'activation de mTOR/p70S6K en réponse au BDNF. La dérégulation des voies MAPK-ERK1/2 et mTOR/p70-S6K pourraient être à la base de la réduction de l'arborisation dendritique et de la maturation des épines dendritique observée dans les neurones corticaux Pld1-/- en culture. En plus de l'implication de RSK2 dans la régulation de la PLD1, j'ai également montré que la PLD1 régule l'activation de RSK2 en réponse aux neurotrophines, probablement via une boucle de rétrocontrôle. Ainsi les donnés obtenus suggèrent un lien fort entre les deux protéines au cours du développement neuronal. A la lumière de ces donnés, un dysfonctionnement de ce mécanisme pourrait expliquer le retard mental observé chez les patients atteints du syndrome de Coffin-Lowry causé par la perte de l'activité kinase de RSK2. D'autre part, les résultats obtenus suggerent un rôle de la PLD1 dans l'exocytose des vésicules.

Alors que s’accumulent des données épidémiologiques qui suggèrent une importance fondamentale des lipides de l’alimentation dans l’homéostasie cellulaire et le développement de nombreuses pathologies humaines, peu d’informations sur leurs fonctions spécifiques sont disponibles à ce jour. Ceci est particulièrement le cas pour la neurosecrétion qui dépend de la fusion d’organites vésiculaires avec la membrane plasmique. Des études récentes ont montré le rôle clé de la compartimentalisation lipidique au niveau des sites d’exocytose et par ailleurs validées la notion de lipides fusogéniques, comme pour l’acide phosphatidique (PA).La V-ATPase, via ses domaines V0 et V1 est à la fois impliquée dans le remplissage en neurotransmetteurs des vésicules, mais aussi dans leur fusion. Nous montrons ici que V0a1 interagit avec le facteur d’échange pour Arf6 ARNO. En bloquant cette interaction, nous avons observé une réduction de l’activation d’Arf6, de l’activité PLD et de l’exocytose, avec une modification de la cinétique des événements unitaires d’exocytose. Nous proposons que la dissociation de V1 de V0 pourrait représenter un signal permettant l’activation de la voie Arf6-ARNO-PLD1 et ainsi promouvoir la synthèse de PA requise à une exocytose efficace dans les cellules neuroendocrines
Lipids play key cellular functions and are involved in many human diseases and little information is available on their exact function. This is especially the case in neurosecretion that relies on the fusion of specific membrane organelle with the plasma membrane for which relatively little attention has been paid to the necessary role of lipids. Recent studies have established the importance of lipid compartmentalization at the exocytotic sites and validated the contribution of fusogenic lipids such as phosphatidic acid (PA) for membrane fusion. The V-ATPase is involved both in the charging of secretory vesicle and the membrane fusion for secretion of vesicle. Indeed, the V1 and V0 subdomains were shown to dissociate during stimulation allowing subunits of the vesicular V0 to interact with different proteins of the secretory machinery. We show here that V0a1 interacts with the exchange factor ARNO and promotes Arf6 activation during exocytosis in neuroendocrine cells. Interfering with the V0a1-ARNO interaction prevented phospholipase D (PLD) activation, phosphatidic acid synthesis during exocytosis, and altered the kinetic parameters of individual fusion events.We suggest that V1 dissociation from V0 could represent the signal that triggers the activation of the ARNO-Arf6-PLD1 pathway and promotes PA synthesis needed for efficient exocytosis in neuroendocrine cells

El complejo de Golgi participa en el procesamiento, la distribución y el transporte de lípidos y proteínas hacia su destino final dentro de la célula. El transporte desde el Golgi está mediado por intermediarios de transporte (ITs), principalmente vesículas y túbulos. La formación de ITs se inicia con la gemación de la vesícula o el túbulo, a continuación se produce su elongación y finalmente tiene lugar su fisión. Este proceso requiere de una compleja maquinaria molecular en la que intervienen las llamadas proteínas de cubierta, proteínas motoras asociadas al citoesqueleto, y los lípidos de las membranas. Respecto al papel de los lípidos, éstos pueden actuar tanto en el reclutamiento de proteínas citosólicas que participan en la formación de los ITs, como dotando a la membrana de las propiedades físicas óptimas para su deformación en vesículas o túbulos. La curvatura de membrana está facilitada por lípidos con estructura cónica como el ácido lisofosfatídico (LPA), el ácido fosfatídico (PA) y el diacilglicerol (DAG). El DAG cumple una doble función en la formación de ITs desde el Golgi: por una parte actúa en el reclutamiento y la activación de la proteína cinasa D (PKD), necesaria para la correcta fisión de vesículas desde la red trans-Golgi (TGN). Por otra parte ayuda a la formación de curvatura negativa. El DAG en el Golgi está estrechamente regulado por diferentes vías que controlan su formación y metabolismo. Una de estas vías está mediada por las llamadas fosfatasas del ácido fosfatídico (PAPs), que producen DAG a partir de defosforilar el PA. Existen dos familias de proteínas que actúan como fosfatasas del ácido fosfatídico. Las enzimas de la familia PAP1son citosólicas, su actividad catalítica es dependiente de Mg+2, se inhiben por N-etilmaleimida (NSF) y el PA es su único sustrato. Las enzimas PAP2 son proteínas transmembrana, independientes de Mg+2 e insensibles a NSF. Las PAP2 también se conocen como fosfatasas de lípidos fosfato (LPPs), ya que además del PA pueden también catalizar la defosforilación de otros lípidos como la ceramida-1-fosfato (C1P), la esfingosina-1-fosfato (S1P) y el ácido lisofosfatídico (LPA), aunque con diferentes afinidades (PA ~ LPA > C1P > S1P). La inhibición de las PAP por el agente farmacológico propanolol, indica que el DAG que proviene de esta vía es necesario para la formación de ITs desde el Golgi al retículo endoplasmático (RE). En esta tesis hemos demostrado que uno de los miembros de la familia PAP2, la enzima PAP2b, también conocida como LPP3, se localiza en diferentes compartimentos de la vía secretora, desde los sitios de salida del RE (ERES) hasta el complejo de Golgi. El silenciamiento de LPP3: (i) reduce la formación de túbulos desde el compartimento intermedio entre el RE y el Golgi (ERGIC) y el complejo de Golgi, y a su vez incrementa la longitud de aquéllos túbulos formados desde el Golgi; (ii) causa un retraso en el transporte dependiente de Rab6 de la subunidad B de la toxina Shiga desde el Golgi al RE, sin afectar al transporte anterógrado de RE a Golgi; y (iii) a nivel ultraestructural incrementa el número de perfiles vesiculares asociados a las cisternas del Golgi. El silenciamiento de la LPP3 también reduce la síntesis de novo de DAG y los niveles de DAG en el Golgi. Además, también hemos demostrado que la sobreexpresión de un mutante catalíticamente inactivo de la LPP3 reproduce los efectos observados tanto con el silenciamiento de la LPP3, como al inhibir la actividad catalítica de las PAP2 por el propanolol. De forma conjunta, nuestros resultados demuestran que la LPP3, a partir de regular la homeostasis de DAG, participa en la formación de ITs en diferentes compartimentos de la vía secretora, regulando el transporte retrógrado entre el RE y el Golgi.
The Golgi complex is involved in the processing, sorting and transport of membrane components (lipids and proteins) to appropriate subcellular destinations, and is also a membranous platform for signalling, metabolic and cytoskeleton proteins. Transport to and from the Golgi complex is mediated by transport carriers (vesicles and/or tubules), which are generated in sequential stages beginning with the formation of a bud, followed by its elongation, constriction and final fission. Lipids play an essential role in this process through different mechanisms: they can recruit cytosolic proteins, modulate protein functions and modify the architecture and physical properties of the membrane bilayer. Thus, membrane curvature is facilitated by conical lipid molecules such as lysophosphatidic acid (LPA), phosphatidic acid (PA) and diacylglycerol (DAG). In previous results of our group we have demonstrated that the DAG produced by the phosphatidic acid phosphatase type 2 family of proteins (PAP2) is necessary for the retrograde transport from the Golgi to the endoplasmic reticulum (ER). In this thesis we have demonstrated that that the PAP2 family member lipid phosphate phosphatase 3 (LPP3, also known as PAP2b) localizes in compartments of the secretory pathway from ER export sites to the Golgi complex. The depletion of human LPP3: (i) reduces the number of tubules generated from the ER–Golgi intermediate compartment and the Golgi, with those formed from the Golgi being longer in LPP3-silenced cells than in control cells; (ii) impairs the Rab6-dependent retrograde transport of Shiga toxin subunit B from the Golgi to the ER, but not the anterograde transport of VSV-G or ssDsRed; and (iii) induces a high accumulation of Golgi-associated membrane buds. LPP3 depletion also reduces levels of de novo synthesized DAG and the Golgi-associated DAG contents. Remarkably, overexpression of a catalytically inactive form of LPP3 mimics the effects of LPP3 knockdown on Rab6-dependent retrograde transport. We conclude that LPP3 participates in the formation of retrograde transport carriers at the ER–Golgi interface, where it transitorily cycles, and during its route to the plasma membrane.

Epoxy fatty acids (EXA) are valuable to industry as they are used in synthesizing plasticizers such as of poly vinyl chloride, resins, adhesives, coating materials such as paint, lubricant, lubricant additives, insecticides, insect repellants, crop oil concentrates and formulations of carriers for slow release pesticides and herbicides. There is interest in developing commercial oilseeds accumulating epoxy fatty acids to at least 50% of the seed oil. Soybeans are the most widely cultivated oilseed and its oil has high levels of linoleic acid which can be a substrate for epoxygenase enzymes. Cahoon et al., expressed a cytochrome P450 enzyme (CYP726A1) from Euphorbia lagascae in soybean somatic embryos and found that the epoxy fatty acid, vernolic acid, reached ~8% of the total fatty acids in transgenic somatic embryos. Rabbit Livers possess a cytochrome P450, CYP2C2, which catalyzes the same epoxidation reaction as the E. lagascae enzyme but might be less likely to be influenced by regulatory machinery in plant cells. This CYP2C2 gene was placed in a plant expression vector under a seed-specific promoter and used to transform soybean, Glycine max, somatic embryos. The ten putative transgenic clones observed after 4-5 weeks were separated and proliferated under selection. glucuronidase (GUS) assays and PCR analyses performed on selected clones were positive. However vernolic acid in total lipids and specific lipid classes was not detected as analyzed by GC. In vitro enzyme assay performed on microsomes isolated from mature somatic embryos at three weeks of maturation using [14C] 18:2 PC as substrate showed presence of [14C] methyl vernoleate. Preliminary analyses on toxicity of epoxy fatty acids and corresponding diols in bacteria, yeast and caco-2 cells showed that leukotoxin diol (LD) most toxic.

碩士
國立中正大學
化學工程研究所
104
Liposomes containing phosphatidic acid (PA) and with a modified ApoE peptide encapsulate quercetin (QU) and rosmarinic acid (RA) as drug carrier to explore PA on protein phosphorylation targeting treatment. In this study, we used β-Amyloid (Aβ) to induce phosphorylation of protein in SK-N-MC cells for simulating Alzheimer’s disease (AD) in vitro model. In AD vitro model, the experimental results show that Aβ-induced SK-N-MC cells, JNK and ERK and p38 protein phosphorylation (p-JNK: 0.98; p-ERK: 0.89; p-p38: 0.89) was significantly higher than the control group (p-JNK: 0.29; p-ERK: 0.39; p-p38: 0.20). And after containing PA as a target liposomes therapy shown in effect to inhibit JNK, ERK, p38 protein phosphorylation performance (p-JNK: 0.42; p -ERK: 0.45; p-p38: 0.43), slow down cells apoptosis, to make the cell survival effect. Immunofluorescence staining was confirmed by a 10 μM of Aβ disposal of SK-N-MC cells for 24 h, because of the amount of caspase-3 expression was significantly increased, reaching apoptotic effect. Furthermore, Aβ1-42 injected directly into the hippocampus CA1 region of Wistar rats to established in vivo model, after two weeks the formation of Aβ deposition can and plaques, surface ApoE is efficient in transporting PA-liposomes across the BBB and inhibiting the degeneration of SK-N-MC cells with Aβ-induced neurotoxicity. Therefore, the future can be used in clinical treatment of Alzheimer's disease.

碩士
國立中正大學
化學工程研究所
102
Liposomes containing phosphatidic acid and with a modified ApoE peptide encapsulate quercetin and rosmarinic acid (RA) as drug carrier. Comparing the cellular uptake enhance effect of Tween 80 to ApoE. The cell co-culture model was established by consisting of human brain-microvascular endothelial cells (HBMECs), human brain vascular pericytes (HBVPs) and human astrocytes (HAs) to investigate quercetin and RA permeability of the in vitro blood–brain barrier (BBB) model, and then the protective effect of quercetin and RA against the in vitro Alzheimer’s disease (AD) neurodegenerative model was established by SK-N-MC neuroblastoma cell line with Aβ-induced neurotoxicity. The results indicated that an increase in PA mole percentage enhanced the particles size, absolute value of zeta potential, and the entrapment efficiency of quercetin and RA, however, reduced the release rate of quercetin and RA as well as loading efficiency of ApoE. The TEER value was 364 ± 10.4 Ω × cm2 and PI permeability across the BBB without nanocarriers was 2.0 ± 0.5 × 106 cm/s. The permeability of PA-ApoE liposomes was higher than non-modified liposomes. In vitro AD neurodegenerative model indicated that quercetin and RA encapsulated into liposomes can promote the viability and inhibit the apoptosis of SK-N-MC cells. Finally, the results of immunofluorescence demonstrated that conjugation of ApoE on liposomes can target HBMECs via LDL receptor to enhance the permeability against BBB and cellular uptake. Surface ApoE is efficient in transporting PA-liposomes across the BBB and inhibiting the degeneration of SK-N-MC cells with Aβ-induced neurotoxicity. Futhermore, in an AD mouse model by the intracerebroventricular injection of Aβ1–42, PA-ApoE liposomes significantly improved the cognitive ability along with increasing acetylcholinesterase activity and malondialdehyde level. Based on our findings, we conclude that PA-ApoE liposomes can be a promising delivery system for AD treatment in preclinical trials.

碩士
國立中正大學
化學工程研究所
105
Liposomes containing 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, dihexadecyl phosphate (DHDP) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (PA) surface was modified with leptin as drug carriers for hydrophobic, resveratrol (RES) and hydrophilic, epigallocatechin gallate (EGCG) to across the blood‒brain barrier. The cellular uptake of the liposomes has been enhanced by the incorporation of PA and leptin (lep). The protective effect of RES and EGCG against the in vitro Parkinson’s disease (PD) neurodegenerative model was established by SH-SY5Y neuroblastoma cells line with 1-methyl-4-phenylpyridinium (MPP+) neurotoxicity. The results indicated that increasing mole percentage of DHDP and PA increased the particles size, absolute value of zeta potential, and improved the entrapment efficiency of RES and EGCG; however, reduced the releasing rate of RES and EGCG and loading efficiency of leptin.The transendothelial electrical resistance and propidium iodide permeability value of in vitro blood‒brain barrier (BBB) model were obtained as 387 ±9.95 cm2 and 2.47±0.7110-6 cm/s respectively. The permeability of lep-RES-EGCG-PA/liposomes was found to be higher than that of non-modified liposomes. In vitro PD neurodegenerative model indicated that RES and EGCG encapsulated into liposomes promoted the cells viability. Furthermore, incorporation of PA and leptin into the liposomes enhanced the target efficiency and cells viability. The results of immunoflurorescence demonstrated that conjugation of leptin with liposomes targeted HBMECs and SH-SY5Y cells easily via leptin receptor, and enhanced the permeability against BBB and cellular uptake. Immunoflurorescence images also revealed that RES and EGCG encapsulated into the liposomes reduced the apoptosis promotor protein Bcl-2 Assaciated X protein (Bax), and enhanced the apoptosis inhibitor protein B cell lymphoma 2 (Bcl-2), rate-limiting enzyme of dopamine biosynthesis tyrosine hydroxylase (TH), and dopamine transporter (DAT). Liposomes containing PA boost the treatment efficiency of drug, by reducing the Bax and increasing Bcl-2, TH and DAT. Finally, liposomes modified leptin not only enhanced drugs target efficiency, but also protected SH-SY5Y cells against MPP+ induced neuro toxicity. Finally, the results of western blot evidenced that MPP+ enhanced the expression of Bax/Bcl-2 and -synuclein, and reduced the expression of TH and DAT. But, the treatment with different liposomes reduced the expression of Bax/Bcl-2 and -synuclein, and enhanced the expression of TH and DAT. The results of western blot analysis are well concordant with immunoflurorescence images. Hence, leptin-PA liposomes could be a promising delivery system for PD treatment in future.

Protein–lipid binding interactions play crucial roles in various physiological and pathological processes, making it very important to study these interactions at the molecular level. However, investigation of these interactions is complicated by several issues, including the inherent complexity of membranes as well as the diverse mechanisms by which proteins interact with the membrane surfaces. As a result, many of these interactions remain poorly characterized. Synthetic probes are useful tools employed for studying protein–lipid binding interactions. This thesis will detail the design and synthesis of metabolically stabilized analogues of various signaling lipids, which mimic the natural species and are not easily modified by enzymes present in biological systems. A modular approach is employed for synthesizing these lipid probes, giving access to a wide range of derivatized lipid probes that can then be used for several studies. Although a wide variety of metabolically stabilized lipid analogues have been synthesized, their activity has not yet been characterized and quantified in detail. So, there is a great need to synthesize biologically active phosphorothioate and phosphonate analogues of various signaling lipids in order to properly characterize and compare the binding affinities and activity of these analogues. Synthesis of metabolically stabilized lipid analogue would take us one step closer towards understanding protein–lipid interactions in biological systems and in trying to find answers to the myriad of questions pertaining to these systems.

of the thesis The presented work explores the function and regulation of intracellular signaling that utilizes phospholipase D (PLD) and phosphatidic acid (PA), especially in the context of cellular morphogenesis of plants. PLDs cleave membrane phospholipids to phosphatidic acid, which has important biophysical and signaling role in many contexts, such as stress response, regulation of cytoskeletal dynamics and vesicular transport. Vesicular transport is essential in focused tip growth of plant pollen tubes and root hairs. Part of the work deals with NADPH oxidases, that are an emerging counterpart of PLD/PA signaling. Tobacco pollen tubes served as the main experimental model, as it enables assessing of changes in secretory pathway after pharmacological or genetic treatments. A technique utilizing antisense oligonucleotides was used for selective knock-down of PLD isoforms, NADPH oxidase and newly studied family of lipid phosphate phosphatases (LPPs) in pollen tubes. This enabled to assess functions of individual isoforms. For studying of selected gene families, various bioinformatic tool were utilized, such as dendrogram construction, analysis of available expression data and creating of virtual proteome. These tools together enabled to select potentially important genes for further experimental...