The incidences of obesity and type 2 diabetes and their complications are increasing globally. The presence of elevated circulating free fatty acids has been associated with the initial dysfunction of pancreatic beta cells and microvascular endothelial cells followed later by their demise. The aim of this thesis was to investigate the mechanisms by which demise occurs, and how it may be prevented. Palmitate, a saturated fatty acid, caused cell death in both INS-1 beta cells and HCMec/D3 microvascular cells, whereas the unsaturated fatty acid oleic acid did not cause cell death, and also protected against palmitate-induced toxicity. Etomoxir, the mitochondrial CPT1 inhibitor did not rescue INS-1 or HCMec/D3 cells from palmitate-induced toxicity suggesting that palmitate-induced toxicity does not occur via entry into the mitochondria. Cells were exposed to 2-bromopalmitate, a non-metabolisable fatty acid used to reduce the pool of cytoplasmic CoA, to determine whether palmitate-induced toxicity might be mediated by its ability to be activated. Pre-incubation with 2-bromopalmitate in INS-1 cells significantly prevented palmitate-induced cell death. These data suggest that the activation of palmitate with CoA might mediate cell death. Cell cycle analysis found that neither oleic acid nor palmitate caused an increase or decrease in cell proliferation in both INS-1 and HCMec/D3 cells. The data suggest that the mechanism of oleic acid-induced cytoprotection might not be via a pro-proliferative mechanism. INS-1 cells were imaged using spontaneous Raman microspectroscopy after 24-hour exposure to esterified and non-esterified fatty acids. Uni- and multi-variate analysis and spectral decomposition were carried out using a methodology optimised and validated which is presented in this thesis. The aim was to quantify changes, if any, in lipid disposition: distribution, intensity (as a measure of concentration) and composition after exogenous exposure to these fatty acids. Exposure to 0.125 mM palmitate showed a significant decrease in the percentage of lipid within the cells and a corresponding increase in the intensity of this lipid. This suggests that palmitate, alone, might be shuttled into lipid droplets. This was not observed when the cells were exposed to oleic acid, whereby an increase in the intensity of lipid was observed even though no significant change was observed in the percentage of lipid within the cells. When palmitate and oleic acid were combined, the composition of the lipid droplets changed such that the levels of palmitate decreased and the levels of oleic acid increased. These data suggest that oleic acid does not shuttle palmitate into lipid droplets. These data do not support the hypothesis that oleic acid protects against palmitate-induced cytotoxicity by shuttling palmitate into lipid droplets. The methyl esters of palmitate and oleic acid were employed to determine whether they would affect lipid disposition. No change in lipid distribution or intensity was observed when the cells were exposed to these fatty acids, validating the requirement for the free carboxyl oxygen for the covalent binding to glycerol for the formation of lipid droplets. These data also suggest that INS-1 cells cannot de-esterify esterified fatty acids.

A área das Ciências Farmacêuticas busca constantemente por tratamentos mais eficientes, direcionados para alvos específicos, com diminuição da dose necessária e com a minimização dos efeitos adversos. Neste contexto, a área de Nanotecnologia Farmacêutica apresenta grande potencial de aplicabilidade, com resultados bastante promissores para o tratamento de diversas doenças. Os sistemas nanoestruturados têm sido avaliados para a incorporação de fármacos já utilizados em tratamentos administrados formas farmacêuticas convencionais que apresentam problemas farmacocinéticos ou farmacodinâmicos quando administrados. E, também, para a incorporação de novas moléculas com potencial para o tratamento de determinada doença. Neste trabalho de tese, nanocápsulas de núcleo lipídico versáteis contendo metotrexato na forma ácida e éster, bromelina, etanercept e infliximab foram desenvolvidas buscando contornar as limitações e aumentar a eficácia terapêutica desses fármacos. Inicialmente, as propriedades anti-inflamatórias de nanocápsulas de núcleo lipídico revestidas por micelas de polissorbato 80 contendo metotrexato encapsulado foram avaliadas em experimentos in vitro e in vivo, em células mononucleares obtidas a partir do líquido sinovial de pacientes com artrite reumatoide e em ratos Lewis com artrite induzida por adjuvante completo de Freund, respectivamente. As nanocápsulas de núcleo lipídico demonstraram serem altamente eficazes no controle da inflamação, sendo que os efeitos anti-inflamatórios in vivo foram alcançados em doses 75% menores que o metotrexato em solução. Na sequência, o tratamento in vitro da linhagem de células de carcinoma de mama humano, MCF-7, com nanocápsulas de núcleo lipídico multiparede funcionalizadas com bromelina demonstrou uma redução de 160 vezes na concentração necessária para obter o mesmo efeito quando comparada a uma solução de bromelina. A influência das pseudofases aniônicas e catiônicas no mecanismo de distribuição da indometacina, tacrolimus, aciclovir, metotrexato e éster etílico de metotrexato, foram avaliadas aplicando um algoritmo desenvolvido para nanocápsulas de núcleo lipídico. Verificou-se que somente a indometacina sofreu influência da presença de cargas, aumentando a afinidade pela fase dispersa das formulações. Formulações de nanocápsulas de núcleo lipídico multiparede contendo metotrexato na forma ácida e éster encapsulados e/ou funcionalizando a superfície das nanocápsulas foram desenvolvidas e testadas in vitro em linhagens de células tumorais (MCF-7) e em linhagens de células sadias (HaCaT). Essas formulações demonstraram atividade antiproliferativa maior para as MCF-7 (com redução em mais de 50% na viabilidade celular) em comparação com as soluções de metotrexato e éster etílico de metotrexato e esta atividade foi maior para as formulações em que as moléculas foram funcionalizadas na superfície das nanopartículas. A captação das nanopartículas pelas células também foi maior para as formulações funcionalizadas com metotrexato ou éster etílico de metotrexato em comparação com a formulação em que o éster de metotrexato está encapsulado. As três formulações contendo metotrexato na forma ácida ou éster não demonstraram ação antiproliferativa em linhagens de células sadias (HaCaT). Devido à baixa expressão de receptores de folato nessas células, não houve aumento da captação celular em comparação à formulação sem fármaco. Por último, foram desenvolvidas satisfatoriamente formulações de nanocápsulas de núcleo lipídico multiparede funcionalizadas com os anticorpos monoclonais infliximab e etanercept, e contendo éster etílico de metotrexato encapsulado, demonstrando que são adequadas para futuros estudos visando o tratamento da artrite reumatoide. Esse conjunto de resultados demonstra que as nanocápsulas de núcleo lipídico com funcionalização de superfície versátil, sejam revestidas com polissorbato 80 ou multiparede funcionalizadas são um sistema bastante promissor para a administração de fármacos de modo a aumentar sua especificidade e eficácia. / The Pharmaceutical Sciences field is constantly searching for more effective treatments, aiming specific targets, with dose reduction and minimization of side effects. In this context, the Pharmaceutical Nanotechnology field presents great applicability potential, with highly promising results for the treatment of several diseases. Nanostructured systems have been evaluated for the encapsulation of drugs approved for use in conventional pharmaceutical dosage forms that, however, exhibit pharmacokinetic or pharmacodynamics problems when administered, and for the encapsulation of novel molecules with potential to treat a determined disease. In the present thesis, versatile lipid-core nanocapsules containing methotrexate in the acid and ester forms, bromelain, etanercept and infliximab were developed, seeking to circumvent the limitations and increase the therapeutic efficacy of these drugs. Initially, the anti-inflammatory properties of methotrexate-loaded lipid-core nanocapsules coated with polysorbate 80 micelles were evaluated in in vitro and in vivo experiments, using mononuclear cells obtained from the synovial fluid of rheumatoid arthritis patients and Lewis rats with Freund complete adjuvant-induced arthritis. Lipid-core nanocapsules demonstrated to be highly effective in the control of inflammation, and the in vivo anti-inflammatory effects were reached in a dose 75% lower than the methotrexate in solution. In the sequence, the in vitro treatment of a human breast cancer cell line, MCF-7, with bromelina-functionalized multiple-wall lipid-core nanocapsules demonstrated a 160-fold reduction of the concentration required to obtain the same effect when compared with a bromelain solution. The influence of the anionic and cationic pseudo-phases in the distribution mechanism of indomethacin, tacrolimus, acyclovir, methotrexate and methotrexate ethyl ester was evaluated through an algorithm developed for lipid-core nanocapsules. It was verified that only indomethacin underwent influence in the presence of charge, increasing the affinity by the disperse phase of the formulations. Multiple-wall lipid-core nanocapsules formulations containing methotrexate in the acid and ester forms encapsulated and/or functionalizing the surface of the nanoparticles were developed and tested in vitro in tumour MCF-7 cells and in a healthy cell line (HaCaT). These formulations demonstrated higher anti-proliferative activity for the MCF-7 cells (reduction of over 50 % in cellular viability) in comparison with the methotrexate and methotrexate ethyl ester solutions and this activity was higher for the formulations in which the molecules were functionalized in the surface of the nanoparticles. A higher cellular uptake was observed for the formulations functionalized with methotrexate or methotrexate ethyl ester in comparison with the formulations in which the methotrexate ester is encapsulated. The three formulations containing methotrexate in the acid or ester form did not demonstrate anti-proliferative activity in non-tumour cell lines (HaCaT). Since these cells have a small expression of folate receptors, the uptake was not increased in comparison with the formulation without drug. Lastly, formulations of methotrexate ethyl ester-loaded multiwall lipid core nanocapsules functionalized with monoclonal antibodies infliximab and etanercept were successfully developed demonstrating suitability for future studies aiming the treatment of rheumatoid arthritis. These groups of results demonstrate that versatile lipid core nanocapsules, either coated with polysorbate 80 or multiwalled functionalized are a very promising system for the administration of drugs aiming their specificity and efficacy.

Supported lipid bilayers (SLBs) are unique model systems for biological membranes. SLBs can be formed by fusing liposomes on solid substrates, which can be characterized by a variety of surface analytical techniques, such as Atomic Force Microscopy (AFM), X-ray diffraction, Quartz Crystal Microbalance (QCM), etc. In this study we used AFM to investigate the dynamic process of the formation of SLBs from liposomes in solutions containing metal ions and phase separation between different lipids as a function of temperature. Divalent cations, Ni2+ in particular, was found to be critical to the deposition of bilayers. Lipid rafts are plasma membrane microdomains rich in sphingolipid and cholesterol forming a liquid ordered phase surrounded by a liquid disordered phase. Lipid rafts are insoluble in cold non-ionic detergents, also called Detergent Resistant Membranes (DRMs). The interaction behaviors between detergent (Triton X-100) and mixed bilayers (DOPC/DPPC and DOPC/SpM) were studied by AFM. The way lipid bilayers were solubilized by Triton X-100 was quite different below and above its critical micelle concentration (CMC), and the SpM domains were found to be resistant to detergent extraction in the cold.

The cellular membrane is a complex biological entity, far from being an inert assembly of protein and lipids which separates cells from the surrounding environment. A multitude of biological processes, ranging from active transport of ions into and out of the cell, to the immune response, are regulated at the level of the plasma membrane. The understanding of their molecular basis is among the central goals of modern biological research. In order to dissect the complexity of actual cell membranes, which involves a very complex network of intermolecular interactions, a “divide and conquer” strategy proves very useful. To this end, researchers try to isolate molecules from complex biological contexts to understand their function in simple model systems under controlled conditions. A variety of model membranes have thus been developed in order to gain insight into membrane processes. This approach has resulted in a deeper knowledge on how lipids and proteins interact and how these interactions govern the function of cellular membranes. In the recent past in fact, a connection has been established between the lateral structure of the plasma membrane and its biological function. Furthermore, a large range of biophysical techniques have been used to characterize protein-lipid microdomains. For example, atomic force microscopy (AFM) is a powerful technique which allows a highly detailed topographical characterization of lipid domains in physiological conditions. While AFM imaging offers an extremely high spatial resolution, up to the nanometer scale, the limited image acquisition speed (minutes) can pose a severe drawback in adequately studying fast dynamic processes. On the other hand, fluorescence based imaging techniques are much faster (10-3-100 s), but certainly lack the high spatial resolution that AFM offers. FCS in particular can also provide information about dynamic processes, like diffusion of fluorescent membrane components. For these reasons, implementing a combination of the above mentioned techniques on the same sample (e.g. cell membrane models) would prove extremely beneficial in the complete dynamic and structural characterization of molecular interactions. . The work described in this thesis can be summarized in two main points: i) the development of a novel combined approach of atomic force microscopy (AFM), laser scanning imaging (LSM), and fluorescence correlation spectroscopy (FCS) and ii) the study of the effects of ceramide in the lateral organization of model plasma membranes. We described one of the first simultaneous applications of AFM and FCS on biologically relevant systems. More specifically, model membranes showing complex phase separation were investigated with a combined approach of AFM, confocal fluorescence imaging, force measurements and FCS, based on commercially available instruments. AFM conveys information about the structural and mechanical properties of the different lipid phases. Different membrane domains can be distinguished based on height difference, elastic properties and line tension as measured by the AFM tip. Simultaneous optical measurements offer the correlation of these data in real time with the partition behavior and diffusion of fluorescent lipids and proteins. We established a clear link between the local membrane viscosity, probed by FCS, and the lipid-lipid interactions involved in line tension, probed by AFM force measurements. An example of a significant drawback circumvented by the AFM-FCS approach involves the use of AFM micromanipulation to eliminate unwanted interactions between lipid particles — similar to intra-cellular vesicles found in vivo experiments — and the membrane, which usually result in distorted FCS autocorrelation curves. Finally, the combined application of AFM and FCS on membrane-anchored proteins reconstituted in lipid bilayers has been instrumental in clarifying inconsistencies that arose in work that focused solely on either AFM or fluorescence techniques. We have shown that, in the case of proteins diffusing in the plane of the membrane, AFM can unambiguously detect only a small immobile fraction. Furthermore, since AFM detection of proteins might be facilitated by high local membrane viscosity (e.g. in ordered lipid phases), the measurement of protein partition between disordered and ordered membrane domains might be biased toward the latter. In this case, the use of FCS as a complementary technique allows a more thorough investigation and deeper understanding of the system of interest. The second part of this thesis dealt with the study of complex lipid mixtures which are used to model the putative lipid/proteins domains in cells, called “rafts”. Firstly, we proved how the combined fluorescence imaging/AFM approach is useful in general for studying supported lipid membranes and the role of lipid domains in biological contexts. We investigated the effect of environmental stress on biological membranes and the protective effects of several substances. Our experimental approach was shown to be a new valuable method to visualize the dehydration damage and its effects on the lateral organization of lipid domains. Our results demonstrated that disaccharides like trehalose or sucrose are effective in protecting lipid membranes, not only on a macroscopic scale — preserving the overall integrity of the bilayer — but also on a microscopic scale, preventing the clustering of microdomains. These phenomena are interesting in the context of biological damage to living cells which need to be stored for long time, like organs to be transplanted or blood platelets. Finally, a large section of this thesis focused on the effects of a specific lipid called “ceramide” on the lateral organization of proteins and lipids in the plasma membrane. Ceramide is produced by cells in several situations, like bacterial infections or apoptosis. As consequence of ceramide production in vivo, the local concentration and the dynamic behavior of lipids and membrane receptors are supposed to exhibit strong variations. In order to understand the molecular mechanisms responsible for these effects, we applied a combination of AFM, FCS and fluorescence imaging on simple model membranes containing ceramide. We could show for the first time that, in presence of raft-like Lo/Ld phase separation, physiological quantities of ceramide induced the formation of a highly ordered gel phase, constituted of ceramide and sphingomyelin. The enzymatic production of ceramide was monitored both in supported and in free-standing bilayers. In the second case, ceramide production was connected to selective vesicle budding from the raft-like phase. Since short-chain analogues are often used in both medical applications and biochemical research to mimic the effect of long-chain ceramides, we investigated the effect of chain-length on ceramide-induced membrane reorganization. We could show that only long-chain ceramides (C18 and C16) form highly ordered domains. Interestingly, FCS measurements indicated that the physical properties of the Lo raft-like domains are hardly affected by the presence of ceramide domains. Furthermore, the increased thickness of the Ld phase — as measured by AFM — and its higher viscosity — as measured by FCS — strongly support the hypothesis of ceramide-induced cholesterol displacement from rafts. On the other hand, short-chain ceramides showed completely different biophysical properties that lead to a destabilization of the raft domains, possibly acting as surfactants between the different lipid phases. Our findings contribute to the explanation of in vivo experiments where short-chain ceramides inhibit cell signaling by disrupting the lipid order in the plasma membrane. We have so far demonstrated that ceramide plays a fundamental role in lipid-lipid interactions. In a physiological context, it is also known to produce dramatic effects in living cells. Since a majority of the processes in vivo are thought to be governed by the activity of proteins, it is highly likely that ceramide not only affects lipid organization but also modifies protein-protein and protein-lipid interactions to produce its effects. To test this hypothesis, we reconstituted several membrane proteins in lipid bilayers containing Ld, Lo, and ceramide-rich domains. We were able to show that some membrane proteins are sorted into ceramide-rich domains. More specifically, the raft-associated proteins we tested were enriched in the highly ordered ceramide-rich domains, while the Ld-associated components were excluded from them. Furthermore, the inclusion of any membrane component in ceramide-rich domains is directly connected to a dramatic reduction of its in-plane diffusion. In an in vivo context, such a reorganization of membrane receptors might be used by the cell to alter the signaling process, for example, by i) separating raft receptors from inhibitors with lower raft affinity, ii) bringing both raft-associated receptors and raft-associated signaling molecules into contact, or iii) stabilizing the interactions between a receptor and its ligand by decreasing their diffusion coefficients. In conclusion, this thesis describes a novel combination of AFM, LSM, and FCS for the investigation of the lateral organization of biological membranes. Our results show that this approach applied on model membranes of increasing complexity is an effective tool for understanding the molecular mechanisms behind the organization of biological membranes. This report opens up new possibilities for further investigation in living cell membranes using the same methodology we have described.

Understanding the molecular basis of protein function is a challenging task that lays the foundation for the pharmacological intervention in many diseases originating in altered structural states of the involved proteins. Dissecting a complex functional machinery into modules is a promising approach to protein function. The motivation for this work was to identify minimal requirements for “local” switching processes in the function of multidomain proteins that can adopt a variety of structural substates of different biological activity or representing intermediates of a complex reaction path. For example, modular switches are involved in signal transduction, where receptors respond to ligand-activation by specific conformational changes that are allosterically transmitted to “effector recognition sites” distant from the actual ligand-binding site. Heptahelical receptors have attracted particular attention due to their ubiquitous role in a large variety of pharmacologically relevant processes. Although constituting switches in their own right, it has become clear through mutagenesis and functional studies that receptors exhibit substates of partial active/inactive structure that can explain biological phenotypes of different levels of activity. Here, the notion that microdomains undergo individual switching processes that are integrated in the overall response of structurally regulated proteins is addressed by studies on the molecular basis of proton-dependent (chemical) and force-dependent (mechanical) conformational transitions. A combination of peptide synthesis, biochemical analysis, and secondary structure sensitive spectroscopy (Infrared, Circular dichroism, Fluorescence) was used to prove the switching capability of putative functional modules derived from three selected proteins, in which conformational transitions determine their function in transmembrane signaling (rhodopsin), transmembrane transport (bacteriorhodopsin) and chemical force generation (kinesin-1). The data are then related to the phenotypes of the corresponding full length-systems. In the first two systems the chemical potential of protons is crucial in linking proton exchange reactions to transmembrane protein conformation. This work addresses the hypothesized involvement of lipid protein interactions in this linkage (1). It is shown here that the lipidic phase is a key player in coupling proton uptake at a highly conserved carboxylic acid (DRY motif located at the C-terminus of helix 3) to conformation during activation of class-1 G protein coupled receptors (GPCRs) independently from ligand protein interactions and interhelical contacts. The data rationalize how evolutionary diversity underlying ligand-specifity can be reconciled with the conservation of a cytosolic ‘proton switch’, that is adapted to the general physical constraints of a lipidic bilayer described here for the prototypical class-1 GPCR rhodopsin (2). Whereas the exact sequence of modular switching events is of minor importance for rhodopsin as long as the final overall active conformation is reached, the related heptahelical light-transducing proton pump bacteriorhodopsin (bR), requires the precise relative timing in coupling protonation events to conformationtional switching at the cytosolic, transmembrane, and extracellular domains to guarantee vectorial proton transport. This study has focused on the cytosolic proton uptake site of this retinal protein whose proton exchange reactions at the cytosolic halfchannel resemble that of rhodopsin. It was a prime task in this work to monitor in real time the allosteric coupling between different protein regions. A novel powerful method based on the correlation of simultaneously recorded infrared absorption and fluorescence emission changes during bR function was established here (3), to study the switching kinetics in the cytosolic proton uptake domain relative to internal proton transfer reactions at the retinal and its counter ion. Using an uptake-impaired bR mutant the data proves the modular nature of domain couplings and shows that the energy barrier of the conformational transition in the cytosolic half but not its detailed structure is under the control of proton transfer reactions at the retinal Schiff base and its counter ion Asp85 (4). Despite the different functions of the two studied retinal proteins, the protonation is coupled to local switching mechanisms studied here at two levels of complexity, [a] a single carboxylic acid side chain acting as a lipid-dependent proton switch [b] a full-length system, where concerted modular regions orchestrate the functional coupling of proton translocation reactions. Switching on the level of an individual amino acid is shown to rely on localizable chemical properties (charge state, hydrophobicity, rotamer state). In contrast, switching processes involving longer stretches of amino acids are less understood, less generalizable, and can constitute switches of mechanical, rather than chemical nature. This applies particularly to molecular motors, where local structural switching processes are directly involved in force generation. A controversy exists with respect to the structural requirements for the cooperation of many molecular motors attached to a single cargo. The mechanical properties of the Hinge 1 domain of kinesin-1 linking the “neck” and motor domain to the “tail” were addressed here to complement single molecule data on torsional flexibility with secondary structure analysis and thermal stability of peptides derived from Hinge 1 (5). It is shown that the Hinge 1 exhibits an unexpected helix-forming propensity that resists thermal forces but unfolds under load. The data resolve the paradox that the hinge is required for motor cooperation, whereas it is dispensable for single motor processivity, clearly emphasizing the modular function of the holoprotein. However, the secondary-structural data reveal the functional importance of providing high compliance by force-dependent unfolding, i.e. in a fundamentally different way than disordered domains that are flexible but yet do not support cooperativity.

Metabolic energy is most efficiently stored as triacylglycerol (TAG). This neutral lipid accumulates mainly within adipose tissues, but it can be stored and used in all types of cells. Within cells it is packed in organelles called lipid droplets (LDs). They consist of a core of neutral lipids like TAG and cholesterol esters, which is surrounded by a phospholipid monolayer that mainly consists of phosphatidylcholine (PC). Attached to or inserted into this monolayer are various proteins, mainly LD specific structural proteins or lipid metabolic enzymes. Though excess uptake of nutrition leads to lipid accumulation in all kinds of body tissues, which is accompanied by the augmentation of LDs and results in cellular dysfunction and the development of metabolic diseases, relatively little is known about the biogenesis and growth of LDs. This thesis focuses on diacylglycerol acyltransferase 2 (DGAT2), an enzyme of the TAG biosynthetic pathway, and on lyso-phosphatidylcholine acyltransferases 1 and 2 (LPCAT1 and LPCAT2), both enzymes of one of the PC biosynthetic pathways called Lands cycle. The data presented in this thesis show that these enzymes can localize to LDs and that they actively synthesize TAG and PC at the surface of LDs. While the LPCATs reside on LDs independent from the nutrition status of the cell, DGAT2 accumulates on LDs upon excess availability of oleic acid. DGAT2, LPCAT1 and LPCAT2 differ in their structure from other iso-enzymes that catalyze the same reactions. This thesis shows that they exhibit a monotopic conformation and that they contain a hydrophobic stretch that presumably forms a hairpin. This topology enables them to localize to both a phospholipid bilayer like the membrane of the endoplasmic reticulum and to a phospholipid monolayer like the surface of LDs. The different biophysical properties of the structures of iso-enzymes might be responsible for their subcellular localization and the formation of distinct TAG or PC pools that are destined for different purposes. This would explain, why the iso-enzymes are often not able to replace each other. Knock-down and overexpression experiments performed in this thesis show that the activity of LPCAT1, LPCAT2 and DGAT2 influence the packaging of lipids within LDs. Knock-down of LPCAT1 and LPCAT2 leads to an increase in LD size without concomitant increase in the amount of TAG. Combined with the finding that the profile of the PC species of the LD surface reflects the substrate preferences of LPCAT1 and LPCAT2, the results suggest that these enzymes are responsible for the formation of the LD surface. Therefore, the increase in LD size upon LPCAT1 and LPCAT2 knock-down results from an adjustment of the surface-to-volume ratio in response to reduced availability of surface lipids. The connection between LPCATs and LD size was corroborated in the model organism Drosophila melanogaster. Three different knockout fly strains of the Drosophila homologue of LPCAT1 and LPCAT2, CG32699, exhibit enlarged LDs in the fat body of the L3 larvae. Furthermore, the data presented suggest that the morphology of LDs is important for the secretion of stored lipids. The reduction of LPCAT1 in liver cells leads to a reduction in lipoprotein particle release. This was shown by measuring the amount of released apolipoproteinB with two different methods, by measuring the release of lipids and by quantification of the amount of released hepatitis C virus, which is known to rely on LD interaction for replication and on lipoprotein particles for cellular release. DGAT2 is recruited to LDs upon excess availability of oleic acid and its overexpression leads to the formation of many, but relatively small LDs. Here, it is shown that DGAT2 interacts with acyl-CoA synthetase ligase 1 (ACSL1), an enzyme that catalyzes the activation of free fatty acids with Coenzyme A. This interaction does not influence the stability of DGAT2 nor does it seem to affect lipid synthesis. Nevertheless, it shows an influence on lipid packaging in LDs. While overexpression of DGAT2 results in the appearance of smaller LDs, overexpression of ACSL1 leads to an increase in LD size. Coexpression of ACSL1 and DGAT2 reverses the phenotypes obtained by single overexpression and normalizes the mean LD diameter to values observed at normal conditions. In conclusion, this thesis shows that LDs are able to synthesize the components of their core and their surface, which underlines their independent function in metabolism. Additionally, the results show that LDs can grow by local synthesis and that the responsible enzymes exhibit a monotopic membrane topology, which might be crucial for LD localization. Furthermore, the obtained data suggest that the localization and the ratio between different enzyme activities influence the packaging of lipids and affects lipid secretion and therefore impact the whole body lipid metabolism.

The lymphatic system has fundamental physiological roles in maintaining fluid homeostasis, immune cell trafficking and lipid transport from the small intestine to the venous circulation. Lymphatic vessels are the main functional organ responsible for the diverse transport roles the system plays. Unlike the blood vasculature, the lymphatic system does not have a central pump, such as the heart, and relies on a variety of factors to move lymph through. It was long thought that only external factors, such as skeletal muscle contraction and lymph formation, played a role in the functional transport capacity of these vessels. With the advancement of imaging capabilities (both hardware and software), it has become clear in the past two decades or so that the main factor in driving lymph transport is the ability of these vessels to intrinsically contract whereby each vessel is comprised of a chain of ‘mini pumps’ in series. The functional capacity of these vessels is thus now understood to be primarily determined by this pumping activity that has been shown to be regulated by various mechanical and biochemical cues. Lymphatic vessel dysfunction has been implicated in a variety of diseases including many lipid related pathologies and a neglected tropical disease known as lymphatic filariasis. While it has been possible to study the vessel function in the context of fluid drainage and immune cell trafficking, the capability to understand the role of lymphatic vessels in lipid transport has not been available due to the lack of experimental animal models and acquisition systems. As part of this thesis, we sought to develop an experimental animal model along with hardware and software tools to investigate the interplay between lymphatics and their lipid content. We report the first functional measurements of how vessels respond to elevated lipid loads. We further utilized our engineering expertise to develop an experimental platform allowing us to further understand the parasite known as B. malayi that migrates to and resides in lymphatic vessels.

Die durch den obligat biotrophen Protisten Plasmodiophora brassicae hervorgerufene Pflanzenkrankheit Kohlhernie verursacht weltweit hohe ökonomische Verluste. Bis heute gibt es keine effektiven Möglichkeiten, diese Pflanzenkrankheit zu bekämpfen. Eine Analyse der Genexpression in infizierten Wurzeln im Vergleich zu nicht infizierten Wurzeln ergab, dass die Gene für Lipid-Transfer-Proteine während der gesamten Krankheitsentwicklung differentiell reguliert sind. Über die Funktionen von Lipid-Transfer-Proteinen in Pflanzen wird noch spekuliert. Diskutiert wird dabei eine Funktion bei der Anpassung an verschiedene abiotische Stressfaktoren, bei der Pathogenabwehr sowie bei dem Transfer von Lipiden. In dieser Arbeit wurden transgene Pflanzen generiert, in denen die pathogenbedingte LTP-Genregulation umgekehrt ist. Es wurden transgene A. thaliana Pflanzen erzeugt, die die Gene LTP1, LTP3, LTP4, AT1G12090 sowie AT2G18370 überexprimieren und die Genexpression von AT4G33550 sowie AT1G62510 reprimieren. Die Regulation der LTP-Genexpression erfolgte dabei durch den wurzel- und keimlingsspezifischen Promotor Pyk10. Zusätzlich wurden in dieser Arbeit auch T-DNA-Insertionsmutanten für die Gene AT1G12090, AT2G18370, AT3G22620, AT5G05960, LTP3 sowie LTP4 untersucht. Mittels semiquantitativer Expressionsanalyse konnte die Modulation der LTP-Genexpression in den LTP-Mutanten bestätigt werden. Darüber hinaus konnte gezeigt werden, dass die Modulation der Expression eines LTP-Gens auch die Expression anderer LTP-Gene beeinflusst. Die phytopathologischen Analysen der LTP-Mutanten hinsichtlich der Entwicklung der Pflanzenkrankheit Kohlhernie ergab, dass die Überexpression der Gene LTP1, LTP3 sowie AT2G18370 und die Repression der Expression von AT1G62510 eine verringerte Anfälligkeit für diese Krankheit bewirkt. Die verstärkte Expression der Gene LTP1, LTP3, LTP4, AT1G12090 sowie AT2G18370 resultiert außerdem in einer verringerten Symptomentwicklung infolge einer Pseudomonas syringae-Infektion. Die verringerte Expression des Gens AT4G33550 führt hingegen zu einer größeren Anfälligkeit für eine P. brassicae Infektion; die Infektion mit P. syringae wird dadurch aber nicht beeinflusst. Die physiologische Charakterisierung der LTP-Mutanten umfasste die Analyse des Pflanzenwachstums unter Salzstress bzw. osmotischem Stress sowie die Entwicklung der Seneszenz in abgetrennten Rosettenblättern. Es konnte gezeigt werden, dass die Gene LTP1, LTP3, LTP4, AT4G33550 sowie AT1G62510 bei der Anpassung an Salzstress sowie die Gene LTP3, AT3G22620, AT4G33550 und AT1G62510 bei der Anpassung an osmotischen Stress eine Rolle spielen. Durch die Modulation der Expression der genannten Gene wird das Wachstum unter diesen Stressbedingungen sowohl positiv als auch negativ beeinflusst. Die Entwicklung der Seneszenz wird ebenfalls durch eine veränderte LTP-Genexpression (LTP1, LTP3, LTP4, AT3G22620 sowie AT4G33550) beeinflusst. Für die biochemische Charakterisierung wurden die LTP-Gene aus A. thaliana mit einem Fusionspartner in E. coli exprimiert und die resultierenden Fusionsproteine gereinigt. Diese wurden nach Abspalten des Fusionspartners hinsichtlich ihrer antimikrobiellen Aktivität und auf die Fähigkeit, Calmodulin zu binden, untersucht. Für die gereinigten Lipid-Transfer-Proteine LTP1, LTP3, LTP4, AT2G18370 sowie AT1G62510 konnte unter den bisher getesteten Versuchsbedingungen keine antimikrobielle Aktivität nachgewiesen werden. Für die Proteine LTP1, LTP3 und LTP4 konnte eine calciumunabhängige Calmodulin-Bindung nachgewiesen werden. Die Ergebnisse dieser Versuche ermöglichen keine Aussage bezüglich der genauen Funktion der einzelnen Lipid-Transfer-Proteine, geben aber Hinweise darauf, dass diese bei den entsprechenden Stress-Vorgängen eine Rolle spielen. Welche Funktion sie dabei genau erfüllen, muss in weiterführenden Analysen untersucht werden.

Includes bibliographical references. / 189 leaves, 18, [4] pages : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Physical and Inorganic Chemistry, 1991

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (p. 92-102).