Le développement du parasite Plasmodium falciparum, responsable du paludisme, nécessite la synthèse de phospholipides et plus particulièrement de phosphatidylcholine (PC) et phosphaditylethanolamine (PE) qui représentent environ 85% de la totalité des phospholidipes du parasite. Leur synthèse s'effectue principalement par les voies métaboliques de novo, voies de Kennedy, en trois étapes enzymatiques. Les enzymes CTP: phosphoethanolamine cytidylyltransferase (ECT) et CTP: phosphocholine cytidylyltransferase (CCT) catalysent les étapes limitantes des deux voies de biosynthèse de la PE et de la PC, respectivement. Ces deux enzymes sont essentielles à la survie du parasite murin, P. berghei et représentent ainsi des cibles thérapeutiques potentielles. La PfCCT est constituée de deux domaines cytidylyltranférases (CT) répétés alors que l'enzyme homologue chez l'homme est composée d'un seul domaine. En revanche, pour la ECT, la présence de deux domaines CT est retrouvée chez toutes les espèces mais les analyses de séquences et de structures ont montré que des résidus importants du site catalytique liant le substrat n'étaient pas conservés dans le domaine CT C-terminal de la PfECT. Ce travail a eu pour but de déterminer les propriétés enzymatiques et les caractéristiques cellulaires de la PfECT et de la PfCCT. Les paramètres cinétiques de ces enzymes ont été quantifiés in vitro à l'aide protéines recombinantes ainsi que sur les enzymes endogènes à l'aide d'extraits parasitaires. Grâce à l'utilisation de protéines recombinantes ponctuellement mutées, nous avons montré que seul le domaine CT N-terminal de la PfECT est catalytiquement actif. Chez P. falciparum, la PfECT et la PfCCT sont exprimées tout au long du cycle intra-érythrocytaire du parasite. La PfECT est présente dans la fraction soluble du parasite alors que la PfCCT apparait aussi bien dans la fraction soluble qu'insoluble. Des expériences d'immunofluorescence ont montré que la PfECT est cytosolique. L'ensemble des résultats présentés apportent un éclairage important sur les fonctions et les propriétés de ces deux cibles potentielles et constituent les premières étapes indispensables à l'élaboration d'une approche thérapeutique. / Phospholipids are essential for the growth and development of Plasmodium falciparum malaria parasite. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are its major structural phospholipids. This study focused on CTP: phosphoethanolamine cytidylyltransferase (ECT) and CTP: phosphocholine cytidylyltransferase (CCT) that catalyzes the rate-limiting steps of the de novo Kennedy pathways for PE and PC biosynthesis respectively. Both ECT and CCT are essential in the rodent malaria parasite P. berghei and constitute potential chemotherapeutic targets to fight against malaria. PfCCT consists of two very similar cytidylyltransferase (CT) domains whereas the human enzyme consists of only one CT domain. The presence of two CT domains in ECT seems to be widespread in all the organisms. Sequence and structural analysis showed that the C-terminal CT domain of ECT lacks key residues in the substrate binding motif. This study aimed at unravelling the enzymatic properties and cellular characteristics of PfECT and PfCCT enzymes. In addition, these studies addressed the key question if C-terminal CT domain of PfECT is catalytically active. Kinetic parameters of the enzymes were evaluated in vitro on native proteins as well as on recombinant proteins, the latter being produced in bacterial system. Cellular characterisation studies using polyclonal antisera showed that PfECT and PfCCT are expressed throughout the intra-erythrocytic life cycle of the parasite. PfECT is found mainly in soluble form in the parasite while PfCCT is present in soluble as well as insoluble forms in the parasite. Furthermore, immunofluorescence studies for PfECT revealed that it is mainly cytosolic. To assess the contribution of each CT domain to overall PfECT enzyme activity, recombinant PfECT mutants were generated by site-directed mutagenesis. Kinetic studies on these mutants indicated that the N-terminal CT domain was the only active domain of PfECT. Collectively, these results bring new insights into the kinetic and cellular properties of the enzymes and will pave the way in developing a future pharmacological approach.

Ferroptosis is a form of regulated cell death that results in an accumulation of toxic lipid hydroperoxides. It has been implicated in several human disease models, including cancers, organ failure, and neurodegeneration. Identifying novel regulators and biomarkers for ferroptosis can result in identification of disease states where treatment with ferroptotic inducers or inhibitors can result in a positive outcome for a patient. In this thesis, I will describe several methods that were used to identify novel genetic regulators of ferroptosis. A CRISPR-Cas9-mediated, whole-genome loss of function screen identified several gene knockouts that resulted in a protective phenotype against ferroptosis. These genes were found to play a role in antioxidant response, glutathione bioavailability, and/or labile iron homeostasis consistent with the current model of ferroptotic cell death. Alternatively, a CRISPR-dCas9-mediated whole genome activation screen found Gch1 overexpression to have a potent anti-ferroptotic effect. Gch1 overexpression resulted in an increase to the basal levels of endogenous antioxidants including BH₄ and CoQ₁₀, as well as protection of a specific class of phospholipids containing polyunsaturated fatty acyl (PUFA) chains. This multipronged defense resulted in strong suppression of ferroptosis. Additionally, we were interested in the protection of this unusual class of phospholipids with two PUFA tails, and if their enrichment would result in a change in sensitivity to ferroptosis. We found that the degrees of unsaturation of the acyl chains, as well as differences in head groups of the phospholipids resulted in significant differences in the ability of the phospholipids to induce a strong ferroptotic response. The study of their uptake, subcellular localization, and remodeling pathways can further elucidate biological pathways of ferroptosis.

No description available.

Phospholipid bilayers are the principal component of the cell membrane. Membranes ensure the maintenance of processes required for cells’ survival by regulating the inflow and outflow of nutrients and other molecules using membrane proteins. However, studying the cell membrane is challenging because of its complexity and small size. In-vitro membrane models made of phospholipids are important tools for studying membranes. In this work, we aim to study the fluidity of phospholipid bilayers of different lipids using general polarization (GP) of the fluorescent probe Laurdan as a measure. We will focus on the relative importance of head groups and fatty acids in the phospholipid. For this purpose, we are synthesizing chimeric lipids with the common human head group phosphocholine paired with bacterial fatty acids. We will compare the response of the human and chimeric lipids to temperature and biofuels to ascertain whether improved stress tolerance can be obtained with the chimeras.

(English) Phosphoinositides (PIs) are glycerophospholipids with a negative charge. As components of cell membranes, PIs are involved in membrane and cytoskeletal dynamics, cell movement and signalling, and the modulation of ion channels and transporters. Apart from the cytoplasm, phosphoinositides also localise to the cell nucleus. PIs play a role in crucial nuclear processes, such as DNA transcription, pre-rRNA and pre-mRNA processing, cell differentiation, DNA damage response, or apoptosis. Phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) are the most abundant phosphoinositides in the cell. However, their exact localisation and function in the nucleus are largely unknown. Here, we describe their localisation at super-resolution level and their involvement in some nuclear processes. PI(4)P is present in nuclear lamina, nuclear speckles and nucleoli, and it forms small foci in nucleoplasm. The majority of nuclear PI(4)P localises to the nucleoplasm, whereas almost 16 % is present in nuclear speckles. On the other hand, the majority of nuclear PI(4,5)P2 localises to nuclear speckles, almost 30 % localises to nucleoplasm and the lesser portion to nucleoli. In the nucleoplasm, PI(4,5)P2 forms small foci called nuclear lipid islets (NLIs). Their core is...

Phospholipid molecules self-assemble to form bilayers that are poorly soluble in an aqueous solvent. Phospholipids may, however, be readily dissolved by mixing with a bile salt or amphiphilic drug surfactant that forms mixed surfactant/phospholipid micelles. Mixed bile salt/phospholipid micelles play an important role in the digestion of fats in the gastrointestinal tract as well as solubilizers of water-insoluble drugs and other drug delivery applications. The ability of surfactants to dissolve phospholipids largely depends on the chemical structure of both surfactant and phospholipid. While bile salt and amphiphilic drug surfactants, with a rigid chemical structure, are good solubilizers of phospholipids, conventional surfactants, with a flexible aliphatic hydrocarbon tail, are poor solubilizers. In addition, the chemical structure of phospholipids, such as tail lengths and charge number, or the fraction of a cosurfactant, for instance cholesterol, is expected to influence the ability to form mixed micelles. In this paper, the aggregation behaviour and mixed micelle formation of the phospholipid dimyristoyl phosphatidylglycerol (DMPC) and two different surfactants: the anionic surfactant sodium dodecyl sulfate (SDS) and the amphiphilic drug surfactant Sodium fusidate (SF, similar structure to that of bile salt), have been studied, and the transition from micelles to bilayers has  been determined for the different surfactants, as well as the size and structure of micelles and bilayers close to the points of transition. The self-assembly of the mixed micelles of surfactants/phospholipids have been investigated using surface tension measurements, refractive index increment and static and dynamic light scattering (SLS and DLS). The results suggest that the transition from micelles to bilayers are found to exist in the following range of bile salt/phospholipid compositions: For SF, 70-75 mol % phospholipid in the micelle was determined to be the point of transition, whilst 20-30 mol % for SDS. As the mole fractions of DMPC increased for both mixtures, the samples became turbid, which indicates the transition of micelles to bilayers. An exact value for molar ratio of transition might not be possible to determine from this study, but instead a, somewhat wider, range of values. In spite of this, a clear trend and difference between the two surfactants was observed.

The influence of the membrane environment on the α1-adrenoceptor has been investigated by examining the effect of phospholipase digestion on the binding of [3H]prazosin to aortic and hepatic membranes. Membrane digestion by phospholipase A2 and phospholipase C was found to markedly reduce prazosin binding to the α1-adrenoceptor whereas phospholipase D had comparatively little effect. In addition, there were differences between membrane preparations since the aortic α1-adrenoceptor was less sensitive to phospholipase A2 and phospholipase C than the hepatic receptor. The results support a major role for hydrophobic groups and the negatively charged, hydrophilic phosphate moiety of phospholipids in the interaction between prazosin and the α1-adrenoceptor.

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No description available.

Plant autophagy is a crucial evolutionary conserved process for recycling cytoplasmic material under stress conditions or during development. The autophagic pathway is negatively regulated by TOR kinase, a versatile molecule modulating a wide range of cellular processes. In mammals, TOR kinase may be activated by phosphatidic acid, a vital signalling lipid. This thesis aims to prove the possible involvement of phospholipids in plant autophagy. I analysed the rate of primary root inhibition in knock-out mutants coding phospholipases in A. thaliana with induced autophagy, measured activity of lipid metabolising enzymes in wild type and atg10 mutant and observed autophagosome formation in selected mutants. Autophagosomes were labelled by fluorescent protein in vivo and by indirect immunolabelling in fixed samples. Using advanced stereological approach, I optimized a method for obtaining an unbiased estimate of autophagosome number in plant root cells.