Cette thèse présente différents cas d'acclimatation d'une espèce de phytoplancton (Isochrysis sp.) à des modifications de son environnement physico-chimique avec une attention particulière portée sur la composante lipidique de la réponse aux changements. Dans ce contexte nous avons étudié les lipides à la fois comme des produits (triacylglycerol) et des acteurs (glycolipides) de la photosynthèse. Tout d'abord, nous avons étudié l’influence de plusieurs types de limitation par l'azote sur la teneur en lipides neutres des algues. Nous avons montré que la relation entre la disponibilité de l’azote et la teneur en lipide n'était pas monotone et qu'elle présentait un caractère hystérétique. Les cellules d’Isochrysis sp. n’accumulent des lipides neutres que transitoirement lors du passage d'une croissance forte vers une croissance faible. Nous nous sommes ensuite intéressé aux relations qui existent entre le cycle nycthéméral et l'accumulation de réserves énergétiques sous forme de lipides et de sucres. Les réserves sont accumulées pendant le jour en disproportion du reste du carbone et consommées pendant la nuit pour alimenter la division cellulaire et la croissance nocturne. La carence en azote dans ces conditions nycthémérales ne provoque pas d’accumulations significatives de lipides. Les variations diurnes de la teneur en lipides neutres sont plus importantes que les variations obtenues en carence. A travers ces différentes expériences nous avons pu mettre en évidences les relations qui existaient entre l'environnement, la croissance, et l'accumulation de réserves. La compréhension de ces mécanismes a permis de déterminer les conditions environnementales qui favorisent la production d’huile en vue d’une utilisation biocarburant. Nous avons aussi étudié l'influence des conditions de croissance sur la composition des glycolipides des membranes des chloroplastes. Les résultats montrent que les changements de la teneur en MGDG, DGDG et SQDG et de l’insaturation des acides gras de ces lipides accompagnent les modifications d’activité photosynthétique des cellules en limitation azotée et en cycle Jour/Nuit. / This thesis presents various cases of acclimation of a phytoplankton species (Isochrysis sp.) to changes in growth conditions, with special emphasis on the lipid response to those changes. In this context we have studied lipids as both products (triacylglycerol) and actors (glycolipids) of photosynthesis. First, we studied the influence of several types of nitrogen limitation on the neutral lipid content of algae. We showed that the relationship between nitrogen availability and lipid content was not monotonous and presented a hysteretic behaviour. Isochrysis sp. cells accumulated neutral lipids only transiently during the transition from high growth to low growth. We were also interested in the relationship between the circadian cycle and the accumulation of energy reserves as fats and sugars. Reserves were accumulated during the day and consumed during the night to provide energy for cell growth and division. Nitrogen deficiency under alternative light did not lead to significant accumulations of neutral lipids. Through these experiences we could understand the relationship between environment, growth, and accumulation of reserves. These results allowed us to identify the environmental conditions that enhance oil production for biofuel use. We also studied the influence of growth conditions on the composition of glycolipids that comprises chloroplast membranes. Changes in MGDG, DGDG and SQDG contents and in their fatty acids unsaturation were linked to changes in photosynthetic characteristics when growth conditions are modified (L/D cycle, N-limitation). Glycolipids are likely to be important actors of photosynthetic acclimation to environmental changes.

Les vésicules géantes unilamellaires (GUV) permettent d’étudier efficacement les interactions entre les lipides et les peptides. Dans ce manuscrit, il a été montré que les interactions attractives lipides-peptides sont supprimées par l’attachement de polypeptides de type élastine (ELP) sur des peptides riches en arginine et peuvent être modulées par l’auto-assemblage en micelles ainsi que par le nombre de groupements arginine dans la séquence des peptides capables de pénétrer les cellules. De plus, une nouvelle méthode pour former des GUV à partir de systèmes complexes en seulement quelques minutes a été développée. Cette méthode est basée sur le gonflement d’un film de PVA sous une bicouche lipidique. Elle supprime la dégradation des molécules pendant la formation des GUV de lipides synthétiques, tels que des glycolipides et des phospholipides portant des groupements amides, où les méthodes traditionnelles ne réussissent pas à produire des vésicules non endommagées. / Giant Unilamellar Vesicles (GUVs) are a valuable tool to study lipid bilayer-biomolecule interactions in simplified cell-like model systems. In this work, a new method to efficiently form GUVs within minutes from more complex systems was developed. This method is based on swelling of a PVA-film under a lipid bilayer and minimizes damage of involved molecules during GUV formation. It also opens up many interesting perspectives for the formation of GUVs composed from new classes of synthetic lipids, such as glycolipids and amide-bearing phospholipids, where the traditional methods fail to efficiently produce “undamaged” vesicles. GUVs were also used for studying lipid-peptide interactions of a new class of elastin-like polypeptides functionalized with arginine-rich residues. It is shown that attractive interactions between lipids and peptides are suppressed by cargo-attachment and can be tuned by self-assembly into micelles and the arginine-amount of the cell penetrating residue.

Die Struktur der Plasmamembran ist von deren Lipid- und Proteinzusammensetzung abhängig und wird durch die Anbindung an das unterliegende Zytoskelett beeinflusst. Das Ziel der vorliegenden Arbeit war die Untersuchung eines neuen Modellsystems basierend auf po­ren­über­span­nen­den Membranen, welches sowohl die heterogene Lipidzusammensetzung als auch den Einfluss eines unterliegenden Netzwerks berücksichtigt. Lipidmembranen, zusammengesetzt aus der „raft“-ähnlichen Lipidmischung DOPC/Sphingo­myelin/Cho­les­terin (40:40:20), wurden auf porösen, hochgeordneten Siliziumsubstraten mit Po­ren­durch­messern von 0.8, 1.2 und 2.0 µm durch Spreiten und Fusion von Riesenvesikeln (giant unilamellar vesicles, GUVs) präpariert. Die mikroskopische Phasenseparation in koexistierenden flüssig-geordneten (liquid ordered, lo) und flüssig-ungeordneten (liquid disordered, ld) Domänen wurde stark durch das unterliegende poröse Substrat beeinflusst. Die Größe der lo-Domänen konnte durch die Porengröße des Siliziumsubstrats, die Temperatur und den Cholesteringehalt der Membran, welcher durch Zugabe von Methyl-β-Cyclodextrin moduliert wurde, kontrolliert werden. Die Bindung der Shiga Toxin B-Untereinheit (STxB) an po­ren­überspannende Membranen, dotiert mit 5 mol% des Rezeptorlipids Gb3, führte zu einem Anstieg des Anteils der lo-Phase. Außerdem wurde die Bildung von lo-Domänen in nicht-phasenseparierten Membranen, zusammengesetzt aus DOPC/Sphingomyelin/Cholesterin/Gb3 (65:10:20:5), durch die Shiga Toxin-Bindung induziert. Ein Anstieg des Anteils der lo-Phase konnte ebenfalls bei der Bindung der pentameren Cholera Toxin B-Untereinheit (CTxB) an po­ren­überspannende Membranen, dotiert mit 1 mol% des Rezeptorlipids GM1, beobachtet werden. Des Weiteren wurde der Einfluss der chemischen Struktur des Gb3-Moleküls auf die Shiga Toxin-Bindung und die Reorganisation von festkörperunterstützten Membranen (solid supported membranes, SSMs) untersucht. Die STxB-Bindung an α-hydroxyliertes Gb3 erhöhte signifikant den Anteil der lo-Phase, während eine cis-Doppelbindung zur Bildung einer weiteren lo-Phase führte, die vermutlich ungesättigte (Glyko-)Sphingolipide und Cholesterin enthält. Im Falles des ungesättigten Gb3 konnte außerdem eine Kondensation zu größeren Domänen nach der STxB-Bindung beobachtet werden. Die genaue Phasenzuordnung der eingesetzten Glykospingolipide vor der Proteinbindung ist bisher unbekannt. Daher wurde das Phasenverhalten eines fluoreszierenden Polyen-Ga­lac­to­ce­re­bro­sids untersucht, welches bevorzugt in der lo-Phase von GUVs angereichert war. Dieser neue, intrinsische Fluorophor vermag als Grundlage für weitere Studien zum Phasenverhalten von Glykosphingolipiden dienen.

Carbohydrate-protein interactions are responsible for several biological functions. While these interactions maintain high levels of specificities, the binding strength of individual carbohydrate-protein recognitions are weak, with dissociation constants (Kd) ~10-3-10-6 M. In order to increase the binding strengths meaningful to physiological functions, multivalent, clustered patches of carbohydrate ligands are required. Synthetic glycoclusters contribute in a significant manner to understand the fine details of the weak carbohydrate-protein interactions. The extent of clustering of the ligands, spatial, topological orientations and the nature of the scaffolds are prominent issues to address the carbohydrate-protein interactions in general. Chapter 1 of the Thesis presents a summary of the synthetic cluster glycosides, mechanisms and energetics of their interactions with lectins. The presence of several ligands within the molecular scaffold is not sufficient, rather there exists a critical demand on the spatial disposition of the individual ligands in the multivalent ligand system to achieve enhanced binding affinities. In order to assess the multivalent effects, influence of linkers and the spatial disposition of the ligands, a systematic study was undertaken, involving a series of the most minimal of the multivalent sugar ligand system, namely, the bivalent sugar ligands. In a programme, it was desired to study the bivalent and monovalent sugar ligand-lectin interactions in a two-dimensional membrane model system. An appropriate model system was the Langmuir monolayer formations of the sugar ligands and their recognitions of the lectins at the interface. A series of bivalent and monovalent glycolipids were thus designed and synthesized. Molecular structure of the ligands utilized to study the lectins binding behavior at the air-water interface are presented in Figure 1. The sugar density dependent lectin binding at the air-water interface caused by the glycolipids was studied in detail. Prior to lectin binding studies, the monolayer behavior of the glycolipids (GL), non-sugars (NS) and their mixtures were assessed. It was observed that the apparent molecular areas of the mixed monolayers increased with increasing percentage of the glycolipid in the mixed monolayer. Interactions of the glycolipid mixed monolayers with lectin were assessed at a constant surface pressure of 10 mN/m. The adsorption kinetics of the lectin concanavalin A (Con A) with the mixed monolayers was monitored by the surface area variation (ΔA) as a function of time. The detailed studies showed a maximum increase in ΔA of 10% of the bivalent glycolipids in the mixed monolayer and a ΔA of 20% of the monovalent glycolipids (Figure 2). With both bivalent and monovalent glycolipids, change in the area per molecule had decreased progressively with higher percentage of the glycolipids in the monolayers. On the other hand, with ethylene glycol spacers, the relative responses and the amount of bound lectin increased. Figure 2. Ligand-lectin interactions at the air-water interface as a function of the percentage of (a) bivalent glycolipids and (b) monovalent glycolipids in the mixed monolayers. To verify the specificity of these interactions, the mannopyranoside non-specific lectin, namely, wheat germ agglutinin (WGA) was tested and there were no deviations in the ΔA for various ratios of the sugar–non-sugar mixed monolayers. The study established that (i) maximal binding of the lectin to the bivalent glycolipids occurred at lower sugar densities at the interface than that for the monovalent glycolipids and (ii) the surface presenting sparsely populated sugar residues are efficient for a lectin binding. Chapter 2 presents the details of synthesis and ligand-lectin interactions at the air-water interface, relevant in the two-dimensional membrane model system. A study of the multivalent effects originating through glycolipid micelles and their lectin interactions was undertaken in another programme. The kinetic studies of the glycolipid micelles-lectin interactions were conducted with the aid of surface plasmon resonance (SPR) technique. Prior to the SPR studies, the critical micellar concentration (CMC), aggregation number and the hydrodynamic diameter of each glycolipid (GL-1 to GL-6, Figure 1) micelles were determined. The glycolipid micelles were used as the analytes on a Con A immobilized surface. The sensorgrams obtained for the interaction of the various glycolipid micelles with Con A are presented in Figure 3. Figure 3. Sensorgrams obtained for the binding of various glycolipids micelles to a Con A immobilized surface, at a constant glycolipid concentration of 250 µM. The kinetic studies of the interactions were performed and the analysis showed that the bivalent analyte model provided a better fitting for the interaction sensorgrams. The analysis revealed that the ka1/kd1 values remained largely uniform for all the glycolipids, whereas the ka2/kd2 values were about two orders of magnitude larger for the bivalent glycolipid (GL-4 to GL-6) micelle-lectin interactions than for the monovalent series (GL-1 to GL-3) (Table 1). From the SPR studies, it emerged that the additional sugar unit in the bivalent glycolipid micelles provided a favorable complexation between the sugar ligand and the lectin. Further, the glycolipid micelles mediated layer-by-layer Con A multilayer formation was also studied by SPR and atomic force microscopy (AFM) methods. Chapter 3 provides the SPR studies of glycolipid micelles-lectin interactions. A study of the monomolecular recognitions of the mono- and bivalent sugar ligands 1-8 (Figure 4) to a lectin was undertaken subsequently. The kinetic studies of the bivalent vs monovalent ligands during lectin binding were conducted by employing the SPR technique, for which the sugar ligands 1-6 were used as the analytes on a lectin coated sensor surface. Figure 4. Structures of the mono- and bivalent sugar ligands 1-8 and the NS derivative. The following observations were made from the SPR analysis. (i) Within the mono- and bivalent series, the response units increased in the series 1–3 and 4–6; (ii) the equilibrium responses were attained within 105 seconds in the monovalent ligands and (iii) the association response gradually increased for the bivalent ligands 5 and 6 and reached an equilibrium after ~3 min. An important outcome of the kinetic studies was the identification of ka and kd for the monomolecular interactions, that were distinctly different for the bivalent ligands. Specifically, the ka was significantly faster and kd was slower for bivalent sugar ligands, in comparison to the monovalent sugar ligands (Table 2). Table 2. SPR derived kinetic parameters for the interactions of sugar ligand to a Con A immobilized surface at 25 oC. Isothermal titration calorimetry (ITC) studies were also conducted, in order to correlate the functional valencies and the thermodynamic parameters. The studies were conducted at ligand concentrations much below their CMCs. The general observations from the ITC studies were that the binding site saturations were slower for the monovalent sugar ligands, in comparison to the bivalent sugar ligands. It was observed that the binding affinities of bivalent ligands 5 and 6 enhanced ~5 times higher than the monovalent ligands 2 and 3 (Table 3). The effective linker length, which allowed the sugar ligands to be functionally active, was determined to be ~15 Å and this separation was necessary for the intermolecular cross-linking formation. The dynamic light scattering (DLS) study of the bivalent ligands 5 or 6-lectin complexes showed the presence of intermolecular cross-linked complexes that existed in solution from the initial stages of the binding process. Upon realizing the nanometric diameters of the sugar ligand-lectin complex, an attempt was undertaken to visualize the complexes by transmission electron micoscopy (TEM). In TEM, 4-Con A complex exhibited particle sizes in the range of 5-10 nm, matching nearly the size of the lectin alone. On the other hand, 5–Con A and 6–Con A complexes provided sizes varying between 20¬150 nm. These particle sizes corresponded to similar aggregate sizes derived from the DLS studies. Chaper 4 describes the kinetic, thermodynamic, DLS and TEM studies of sugar ligand-lectin intearctions. Table 3. Binding stoichiometries and thermodynamic parameters of the sugar ligand-Con A interactions at 25 oC.a Ligand n Ka (x 10 -4) ΔG ΔH TΔS 1 0.91 9.14 ( ± 0.75) -6.76 -3.39 3.37 2 1.01 5.76 (± 0.80) -6.49 -3.98 2.51 3 1.09 7.06 (± 1.23) -6.61 - 3.01 3.60 4 1.10 5.75 (± 0.27) -6.49 - 6.39 0.10 5 0.50 20.6 (± 1.7) -7.59 - 12.80 -5.21 6 0.47 37. 4 (± 2. 4) -7.61 -11.54 -3.93 7 1.03 0.86 (± 0.06) -5.36 -7.9 -2.62 8 1.05 2.48 (± 0.12) -5.99 -6.3 -0.32 MeαMan 1.04 0.79 (± 0.04) -5.27 -7.83 -2.56 Ka is in the unit of M-1; ΔG, ΔH and TΔS are in the units of kcal mol-1. Errors in ΔG are ~1-4%. Errors in ΔH are in the range of 1-8%. Errors in TΔS are in the range of 1-6 %. A study was undertaken further to assess the kinetic interactions of the tumor-associated T-antigen with a lectin. Synthesis of amine-tethered T-antigen and lactose derivatives (Figure 5) were accomplished and an assessment of their kinetic interactions with lectin peanut agglutinin (PNA) was conducted. Figure 5. Structures of the amine-tethered T-antigen, lactose and mannose derivatives. The lectin PNA was used as the analyte onto the sugar ligand immobilized surfaces. It was found that the interaction with T-antigen showed higher response units than the lactose derivative (Figure 6). The kinetic studies of PNA with immobilized T-antigen and the lactose derivatives demonstrated that the binding followed a bivalent analyte model of the interaction. The T-antigen derivative interacted with the lectin and relatively faster association (ka) and a slower dissociation (kd) were observed, in comparison to the lactose derivative. The ratio of second binding kinetic constants (ka2/kd2) was observed higher than the first binding kinetic constants (ka1/kd1). Further, the ITC studies were conducted, in order to provide the thermodynamic parameters governing the lectin-T-antigen interactions. The combined approach of SPR and ITC studies showed that the T-antigen derivative exhibited a higher binding affinity to PNA than the lactose derivative. Chapter 5 presents synthesis of the T-antigen and lactose derivatives and studies of their lectin interactions. In summary, the thesis provides a detailed insight into the kinetic and thermodynamic parameters of the bivalent sugar ligand-lectin interactions, in comparison to the monovalent sugar ligands. Langmuir monolayer formation of the sugar ligands and the assessment of their lectin binding at the air-water interface demonstrated that the surface presenting sparsely populated sugar residues are efficient for a lectin binding. The kinetic studies of various glycolipid micelles-lectin interactions showed that the additional sugar unit in the bivalent glycolipid micelles provided a favorable complexation between the sugar ligand and the lectin. The detailed monomolecular kinetic studies showed that the bivalent sugar ligands underwent a faster association (kon) and a slower dissociation (koff) of the ligand-lectin complexes. The ITC studies on sugar ligand-lectin interactions led to identify not only the thermodynamic parameters, but also the influence of the hydrophobic alkyl units and the linker moieties. The DLS and TEM characterizations of sugar ligand-lectin complexes showed the status of the complexation, sizes and the morphologies. The studies were extended further to tumor associated T-antigen-lectin interactions. Overall, the Thesis establishes the most minimal multivalent sugar ligands, namely, the bivalent sugar ligand-letin interactions. The studies presented in the Thesis should be useful to design multivalent sugar ligands for highly avid lectin interactions and also to raise possibilities for the construction of defined lectin oligomers, facilitated through the multivalent sugar ligand-lectin cross-linking interactions.

Ces travaux de thèse traitent de l’étude de glycolipides et plus précisément d’esters de trehalose pour la synthèse de nouveaux polymères bio-sourcés. Des monoesters et diesters de trehalose ont ainsi été synthétisés par estérification des alcools primaires du trehalose avec des acides gras selon deux voies de synthèse. La première utilisant un agent de couplage peptidique ne nécessite pas l’utilisation de groupement protecteur pour estérifier sélectivement les alcools primaires. La deuxième est une estérification sélective catalysée par une lipase. L’auto-assemblage des esters de tréhalose a ensuite été étudié. Les monoesters possèdent des propriétés tensio-actives dans l’eau et le trehalose monoeruçate a la capacité de gélifier l’eau. Les diesters, quant à eux sont de bons gélifiants pour les solvants organiques etles huiles végétales. Par conséquent, des gels ont été préparés dans trois huiles végétales, puis leur morphologie et leur propriété rhéologique ont été étudiées. Ensuite, les diesters ont été fonctionnalisé et polymérisés selon différentes stratégies. Ainsi, des polyuréthanes et des poly(hydroxyuréthane)s ont été synthétisés par polycondensation tandis que des glycopolyesters ont été obtenus par polymérisation par métathèse et addition thiol-ène. Finalement,les propriétés d’auto-assemblage de ces polymères ont été étudiées. Ces derniers peuvent former des nanoparticules par la méthode de déplacement de solvants. / The aim of this thesis was to study glycolipids and particularly trehalose esters for the synthesis of new bio-sourced polymers. Trehalose monoesters and diesters were synthesized by two esterification pathways of the primary alcohol of trehalose with different fatty acids. The first synthetic route is a protective group-free esterification using a peptide coupling agent and the second one is a lipase-catalyzed esterification. The self-assembly properties of the trehalose esters were investigated. Trehalose monoesters showed surfactant properties in water and trehalose monoerucate was even able to form gels in water. The trehalose diesters appeared to be good gelators for organic solvent and vegetable oil. Thus, gels in three vegetable oils were prepared and their morphology and rheological properties were studied. Afterwards, trehalose diesters were functionalized and polymerized with different strategies.Thus, polyurethanes and poly(hydroxyurethane)s were obtained by polycondensation where as glyco-polyesters were synthesized by acyclic diene metathesis (ADMET) and thiol-enepolymerization. Finally, the self-assembly properties of these polymers were investigated. The latter were able to form some nanoparticles by solvent displacement method.

Dans le contexte du développement durable, l’'utilisation des ressources renouvelables, biodégradable et peu toxique, est particulièrement recherchée. En particulier, la littérature montre que les molécules amphiphiles biosourcées avec une tête sucre, type glycolipides, sont d'excellents candidats pour substituer les tensioactifs pétrochimiques actuellement utilisés massivement en formulation. Pour limiter le criblage expérimental et orienter le choix des synthèses de telles molécules, une démarche prédictive à partir de l'analyse de la structure moléculaire permettrait d'anticiper les propriétés des molécules amphiphiles et d'identifier celles répondant à des propriétés spécifiques recherchées. L'objectif des travaux de thèse est de développer une méthodologie via la synthèse à façon avec modification graduelle de la structure, la caractérisation et l'analyse systématique de glycolipides, dans le but d'identifier des liens pertinents entre leurs propriétés amphiphiles et leurs caractéristiques structurales. Les données expérimentales générées doivent permettent d'établir une base de données comparables et fiables, nécessaire pour le développement de modèles 1 prédictifs. Les résultats ont mis en évidence l'influence significative de certains paramètres de structure sur j les propriétés physico-chimiques, avec des tendances claires, autrement difficiles à observer à partir des données globales non-comparables de la littérature. Ce travail a également démontré l'importance de prendre en considération le comportement des molécules amphiphiles dans l'eau, via la connaissance de leur diagramme de phase, afin de définir sans ambiguïté certaines propriétés telles que la CMC. / In the context of sustainable development, the use of biodegradable and low toxic renewable resources is particularly important. ln particular, the literature shows that the bio-based amphiphilic molecules with a sugar head, or the glycolipids, are excellent substitutes of the petrochemical surfactants used massively in current formulations. To limit the experimental screening and to orient the choice of the syntheses of such molecules, a predictive approach based on the analysis of the molecular structure would make it possible to anticipate the properties of the amphiphilic molecules and to identify those with specific properties. The objective of this work is to develop a methodology through systematic syntheses, characterizations and analyses of glycolipids with gradual structural modifications, with the aim of identifying relevant links between heir amphiphilic properties and their structural characteristics. The obtained experimental data should make it possible to establish a comparable and reliable database, necessary for the development of predictive models. The results showed the significant influence of some structural parameters on the physico-chemical properties with clear trends, which are otherwise difficult to observe by using the non-comparable data collected from the literature. This work also demonstrated the importance of considering the behavior of amphiphilic molecules in water, through knowledge of their phase diagram, which allows for the unambiguous definition of certain properties like the CMC.

Glycolipids play important roles in the biology of prokaryotes and eukaryotes, including humans, and although theyare found on the cell-membrane surface of all eukaryotic cells, not much is known about their biosynthesis. The aim ofthis project was to characterize two enzymes: glucosylceramide synthase (GCS) which is involved in the biosynthesisof glycolipids such as gangliosides that are abundant in the membranes of nerve cells; and dolicholphosphate mannosesynthase (DPMS), involved in the synthesis precursor for protein glycosylation. Both GCS and DPMS have been shown play a role in cancer as well as in congenital disorders of glycosylation, and are therefore interesting targets tostudy from a therapeutic perspective.With the goal to identify a suitable expression system for GCS, the genes coding for GCS from lancelet (Branchiostoma floridae) and human (Homo sapiens) were cloned and tested for expression in Escherichia coliBL21(DE3)T1 and C41(DE3) using different vectors. Cloning into three different vectors was successful and initial expression testing was performed. SDS-PAGE analysis confirmed initial expression of proteins. Although the correctsize of the protein could be confirmed by Western blot, no fluorescence of the GFP-fusion protein could be detected.DPMS from Thermococcus onnurineus (ToDP) was expressed in E. coli C41(DE3) and purified by immobilized metal ion affinity chromatography and gel filtration. Crystallization optimization was performed for ToDP produced from the vector pNIC28-Bsa4 and plate-like crystals were obtained. X-ray intensity data analysis indicated that thesecrystals contained lipid rather than protein. Crystallization screening for ToDP produced from the vector pNIC-CTHO construct was successful. Crystallization screening using the commercially available MemGold-HT96 crystallization kit resulted in initial crystallization that yielded protein crystals that diffracted to 10 °A resolution. / Glykolipider är viktiga biologiska byggstenar hos prokaryoter och eukaryoter, även människor. Trots att glykolipider finns på cellmembran ytan hos alla eukaryota celler är inte mycket känt kring syntesen av glykolipider. Målet med detta projekt var att karaktärisera två enzym: glukosylceramidsyntas (GCS) som är involverat i biosyntesen av glykolipider som gangliosider vilka förekommer i cellmembranet hos människors nervceller; och dolikolfosfatmannossyntas (DPMS) som är involverat i syntesen av substrat för proteinglykosylering. Både GCS och DPMS harvisat sig spela en roll i cancer och medfödda glykosyleringssjukdomar och är därför intressanta enzym att studera ur ett medicinskt perspektiv.Med målet att identifiera ett lämpligt expressionssystem för GCS, klonades gener från lansett (Branschiostomafloridae) och människa (Homo sapiens) och testades för expression i Escherichia coli BL21(DE3)T1 och C41(DE3)med olika vektorer. Kloning av tre olika vektorer lyckades och expressionstester utfördes. Analys med SDS-PAGE bekräftade expression av protein. Trots att korrekt storlek av proteinet kunde bekräftas med Western blot, detekterades ingen fluorescens från GFP-fusionsproteinet. DPMS från Thermococcus onnurineus (ToDP) i två olika konstrukt uttrycktes i E. coli C41(DE3) och renades med immobiliserad metalljonaffinitetskromatografi och gelfiltrering. Kristalliseringsoptimering utfördes för ToDP uttryckt i vektorn pNIC28-Bsa4 och skivliknande kristaller erhölls. Diffraktionsdata indikerade dock att kristallerna innehöll lipider och inte protein. Kristallisering av ToDP uttryckt i vektorn pNIC-CTHO lyckades och initiala kristallingsförhållanden hittades genom att använda det kommersiellt tillgängliga kristalliseringskitet MemGold-HT96. Diffraktionsdata visade på upplösning ner till 10 Å.

The thesis entitled “Novel Redox Responsive Cationic Lipids, Lipopolymers, Glycolipids and Phospholipid-Cationic Lipid Mixtures: Syntheses, Aggregation and Gene Transfection Properties” elucidates the design, synthesis, aggregation and gene transfection properties of novel cholesterol based cationic lipids with ferrocene as the redox moiety, polyethylenimine based ferrocenylated lipopolymers and cholesterol based non-ionic glycolipids. The thesis also discusses the cationic phospholipid-cationic lipid mixtures as superior gene transfection agents. The work has been divided into six chapters. Chapter 1. Introduction Part A. Various Cholesterol based Systems for Applications as Biomaterials Liposomes composed of cationic lipids have become popular gene delivery vehicles. A great deal of research is being pursued to make efficient vectors by varying their molecular architecture. Cholesterol being ubiquitous component in most of the animal cell membranes is increasingly being used as a hydrophobic segment of synthetic cationic lipids. In this chapter we describe various cholesterol based cationic lipids and focus on the effect of modifying various structural segments like linker and the headgroup of the cationic lipids on gene transfection efficiency with a special emphasis on the importance of ether linkage between cholesteryl backbone and the polar headgroup. Interaction of cationic cholesteryl lipids with dipalmitylphosphatidycholine membranes is also discussed here. Apart from cholesterol being an attractive scaffold in the drug/gene delivery vehicles, certain cholesteryl derivatives have also been shown to be attractive room temperature liquid-crystalline materials. Part B. Diverse Applications of Ferrocene Derivatives This chapter gives a brief overview of ferrocene chemistry followed by description of major applications of ferrocenyl derivatives in a variety of fields like catalysis, materials chemistry, electrochemical sensors, medicinal chemistry etc. We discuss the use of ferrocene as an electrochemical and redox active switch to achieve control over supramolecular aggregation. It also reviews ferrocene based amphiphiles including surfactants, lipids and polymers with an emphasis on the role of ferrocene over aggregate formation and their utilization in biological applications. Chapter 2: Optimization of Redox Active Alkyl-Ferrocene Modified Polyethylenimines for Efficacious Gene Delivery in Serum 1a-c, n = 6, P8-C6-F1, P8-C6-F2, P8-C6-F3 2a-c, n = 11, P8-C11-F1 P8-C11-F2, P8-C11-F3 % ferrocene grafting, F1 = 15%, F2 = 25% and F3 = 50% Figure 1. Structure of the alkyl-ferrocene modified 800 Da Branched Polyethylenimine. In this chapter we present six new lipopolymers based on low molecular weight polyethylenimines (BPEI 800 Da) which are hydrophobically modified using ferrocene terminated alkyl tails of variable lengths. The effects of degree of grafting, spacer length and redox state of ferrocene in the lipopolymer on the self assembly properties were investigated in detail by transmission electron microscopy (TEM), atomic force microscopy (AFM), dynamic light scattering (DLS) and zeta potential measurements. The assemblies displayed a redox induced increase in the size of the aggregates. The coliposomes comprising of the lipopolymer and a helper lipid 1,2-Dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) showed excellent gene delivery capability in serum containing environment in two cancer cell lines (HeLa, U251 cells). Optimized formulations showed remarkably higher transfection activity than BPEI 25 KDa and even better than commercial Lipofectamine 2000 as evidenced from luciferase activity and EGFP expression analysis. Oxidation of ferrocene in lipopolymers led to reduced levels of gene transfection which was also followed by cellular internalization of fluorescently labeled pDNA using confocal microscopy. Cytotoxicity assay revealed no obvious toxicity for the lipopolyplexes in the range of optimized transfection levels. Overall, we have exploited the redox activity of ferrocene in PEI based polymeric gene carriers for trenchant control over gene transfection potential. RLU/mg protein HeLa Cells Figure 2. Maximum transfection efficacies of optimized redox lipopolymer/DOPE formulations by (A) Luciferase Assay and (B) Flow cytometry (GFP expression). Chapter 3. Membranes derived from Redox-active Cholesterol based Cationic Lipids and their Interactions with DNA and Phospholipid Membranes Figure 3. Molecular structures of the electroactive cholesterol based monomeric and gemini lipids. This chapter describes the synthesis and aggregation properties of two series of redox-active ferrocene containing monomeric and gemini cationic lipids with cholesterol as a hydrophobic domain. These cationic lipids are modified at their headgroup region using ferrocene terminated alkyl chains of differing length. All the four cationic lipids formed stable suspensions in water. Aggregation behavior of these cationic lipids in aqueous suspensions in their unoxidized and oxidized state was studied using TEM, DLS, zeta potential measurements and XRD studies. Cationic lipids with ferrocene in natural, reduced state were found form bigger sized vesicles which upon oxidation became smaller aggregates with increased zeta potential. XRD results indicate the existence of nice lamellar arrangements of the lipid bilayers. Thermotropic phase transition behavior of DPPC membranes incorporated with cationic ferrocene lipids was also studied using differential scanning calorimetry. Finally, we assayed pDNA (plasmid DNA) binding ability of all the four cationic lipids using ethidium bromide intercalation assay where all the cationic lipid formulations showed excellent DNA binding capability. In the experiments involving SDS-induced release of DNA, we observed that redox-active monomeric lipids (3a-b) were found to be more efficient in facilitating the release of DNA from the liposome-DNA complex in the presence of negatively charged SDS micelles than their gemini counterparts (4a-b). Chapter 4. Redox-responsive Gene Delivery by Ferrocene containing Cationic Cholesteryl Lipids in Serum This chapter describes the transfection efficacy of redox-active monomeric and gemini cationic lipids with cholesterol backbone. The transfection efficiency of all the lipids could be tuned by changing the oxidation state of the ferrocene moiety. Gene transfection capability was assayed in terms of EGFP expression using pEGFP-C3 plasmid DNA in three cancer cell lines of different origin, namely Caco-2, HEK293T and HeLa in the presence of serum. Figure 4. Effect of oxidation state of ferrocene on maximum transfection efficacies of monomeric and gemini lipids in three different cell lines (Caco-2, HEK 293T and HeLa). Cationic liposomal formulations with ferrocene in its reduced state were observed to be potent transfectants reaching the EGFP expression levels even better than commercial lipofectamine 2000 in the presence of serum as evidenced by flow cytometry. EGFP expression was further substantiated using fluorescence microscopy studies. All liposomal formulations containing oxidized ferrocene displayed diminished levels of gene expression and interestingly, these results were consistent for each formulation in all the three cell lines. Assessment of EGFP expression mediated by both reduced and oxidized ferrocene containing formulations was also undertaken following cellular internalization of labelled pDNA using confocal microscopy and flow cytometry. Lipoplexes derived from different liposomal formulations with reduced and oxidized ferrocene were characterised using TEM, AFM, zeta potential and DLS measurements. Overall, we demonstrate here controlled gene transfection levels using redox driven, transfection efficient cationic monomeric and gemini lipids. Chapter 5: Synthesis of ‘Click Chemistry’ Mediated Glycolipids: Their Aggregation Properties and Interaction with DPPC Membranes This chapter describes the synthesis and aggregation properties of cholesterol based glycolipids along with their interaction with a model phosphatidylcholine membranes. Three series of non-ionic glycolipids with hydrophobic cholesterol backbone and various monosaccharide and disaccharide sugars as the hydrophilic polar domain have been synthesized. These were conjugated to the cholesteryl backbone via oligooxyethylene spacers of different lengths (n = 1, 3 and 4) using Cu (I) catalyzed Huisgen [3+2] cycloaddition, which is popularly known as „Click Chemistry‟. All the synthetic glycolipids (5a-d, 6a-d and 7a-d) formed vesicular aggregates in aqueous medium as confirmed by TEM and DLS. XRD studies with the cast films of lipids revealed that the bilayer width increased with increase in the length of oligoethylene spacer unit that has been incorporated between the hydrophobic and hydrophilic domains. Also, within the same series containing a particular oligoethylene unit, bilayer widths were found to be more for the lipids containing disaccharides as their headgroup than monosaccharides. Figure 5. Molecular structures of various cholesterol-based glycolipids. Calorimetry studies of the coaggregates containing naturally occurring 1, 2-dipalmitoylphosphatidylcholine (DPPC) and various mol-% of each of the glycolipids revealed that more than 30 mol-% of glycolipids are required to completely abolish the phase transition of DPPC membranes. These results were further supported by fluorescence anisotropy measurements of the co-aggregates using 1, 6-diphenylhexatriene (DPH) as a probe. Fluorescence anisotropy of the neat vesicles revealed that 9a and 9c were more rigid than DPPC vesicles in the solid-like gel phase, while the glycolipids with longer oxyethylene spacers (n = 3 and 4) were less rigid than the DPPC vesicles. Chapter 6. Hydrophobic Moiety Decides the Synergistic Increase in Transfection Efficiency in Cationic Phospholipid/Cationic Lipid mixtures This chapter describes the effect of inclusion of cationic lipid/cationic gemini lipids into the membranes of a cationic phospholipid on the gene delivery efficiency across HeLa and HEK293T cell lines. Although all the three cationic lipids have the same quaternary ammonium moiety as their headgroup, they differ from each other in terms of their hydrophobic moiety and in the number of cationic headgroups. Chol-N is a cholesterol based monocationic lipid, while 2C14-N and 2C14N-5-N2C14N are monomeric and gemini cationic lipids respectively with pseudoglycerol backbone consisting of tetradecyl (n-C14H29) chains. Each of the three cationic lipids under the current investigation, namely, Chol-N, 2C14-N and 2C14N-5-N2C14N were added in different ratios to EtDMoPC and the resultant mixed membranes were studied for the biophysical characterization and gene delivery efficacies. Figure 6. Molecular structures of cationic lipids used in this study. All the formulations were characterized using dynamic light scattering and zeta potential measurements to obtain their hydrodynamic diameters and surface charge properties respectively. Their DNA binding ability was also studied by measuring changes in zeta potential and gel electrophoresis of the lipoplexes formed by the coliposomal formulations and pDNA at different Lipid/DNA weight ratios. The gene delivery efficacies of various formulations were studied in terms of EGFP expression using pEGFP-C3 plasmid DNA in two different cell lines, namely HeLa and HEK293T. In the absence of serum we found that the formulation (EtDMoPC+2C14N-5-N2C14N) showed better transfection efficiency than the individual lipids. However, in the case of others, i.e., (EtDMoPC+Chol-N) and (EtDMoPC+2C14-N) formulations, there was a slight decrease in transfection efficiency compared to the individual lipids. In the presence of serum, the formulations (EtDMoPC+2C14-N) and (EtDMoPC+2C14N-5-N2C14N) showed significantly higher transfection efficacies compared to their individual lipids. Fusion assay using labelled cationic lipid formulations and unlabelled anionic liposomes revealed that lipoplexes prepared from EtDMoPC+ 2C14-N and EtDMoPC+ 2C14N-5-N2C14 exhibited much higher fusogenicity as compared to the lipoplexes prepared using EtDMoPC+Chol-N as well as the individual lipids. Thus, the liposome formulations which showed better transfection activity fused more readily with the anionic liposomes than did the formulations with poorer activity. Overall, we found that the hydrophobic domain of the cationic lipid/cationic gemini lipid that is added to cationic phospholipid has an important role on the transfection efficiency of the mixed formulations. Additionally the cytotoxicity studies revealed that each of these formulations was not significantly toxic making them viable for applications in vivo. (For structural formula pl see the abstract pdf file)

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

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