Der Viruspartikelzusammenbau hängt von der Anreicherung viraler Untereinheiten in spezifischen Domänen der PM ab. Es wird vorgeschlagen, dass Membran-Rafts – geordnete, sphingomyelin- und cholesterinreiche Mikrodomänen in der PM – als lokale Rekrutierungsstellen dienen. Hämagglutinin (HA) ist ein homotrimeres Glykoprotein in der Hülle des Influenzavirus. Es dient der Bindung an die Wirtszelle und der Fusion mit dem Endosom. Es wird angenommen, dass HA bei der Abschnürung der Viruspartikel von der Zelle mitwirkt. Zwei Hauptbeobachtungen führten zu der Hypothese, dass sich HA in Lipid-Mikrodomänen einlagert: HA wurde biochemisch in Detergens-resistenten Membranen nachgewiesen und die Virushülle ist mit raftbildenden Lipiden angereichert. Um die Rolle der HA-Transmembrandomäne für die Lipid-Raft-Inkorporation aufzuklären, wurde ein Konstrukt entwickelt, das den C-Terminus von HA mit dem gelb fluoreszierenden Protein YFP fusioniert, und die Transmembrandomäne, nicht aber die N-terminale Ektodomäne von HA enthält. In transfizierten Säugetierzellen wurde der Förster-Resonanz-Energie-Transfer (FRET) zwischen diesem Konstrukt und einem GPI-verankerten cyan fluoreszierenden Protein CFP (Raft-Marker) durch Fluoreszenz-Lebenszeit-Mikroskopie (FLIM) gemessen. Die Ergebnisse zeigen, dass sich HA-Konstrukte in Cholesterin-abhängigen Lipiddomänen anreichern, was durch eine erhöhte FRET-Effizienz nachgewiesen wurde. Zudem führen ein Cholesterinentzug aus der PM und die Deletion hochkonservierter Palmitylierungsstellen zu einer signifikanten Verringerung selbiger; sehr gering war diese zwischen dem HA-Konstrukt und einem Nicht-Raft-Marker. Darüberhinaus konnte durch ortsspezifische Mutagenese gezeigt werden, dass die verwendeten Konstrukte disulfidbrückenverbundene Oligomere bilden, welche Voraussetzung für den Transport der Konstrukte an die PM sind. Zeitaufgelöste Anisotropiemessungen ergaben für diese ein starkes Homo-FRET-Signal, welches die Oligomerisierungshypothese bestätigt. / Numerous enveloped viruses bud from the host cell plasma membrane (PM). Assembly of the new viral particles depends on the accumulation of the viral subunits at specific sites of the cell membrane. Lipid domains or rafts enriched of sphingomyelin and cholesterol were suggested as sites for local recruitment of viral components. Hemagglutinin (HA), a homotrimeric glycoprotein embedded in the envelope of influenza virus, mediates binding of the virus to the host cell and fusion between the viral envelope and the endosomal membrane. HA might play an important role in budding of the viral particles from the host cell. Two observations led to the suggestion that HA entraps in lipid microdomains. First, HA was rescued in DRM fractions, second the viral envelope was found to be enriched in lipids generally forming rafts. To elucidate the role of the HA transmembrane domain in lipid raft localization we expressed constructs harboring the transmembrane domain and the cytoplasmic tail but lacking the N-terminal ectodomain of HA in the PM of mammalian cells. We studied energy transfer (FRET) between these constructs and a GPI anchored CFP as a raft marker by fluorescence lifetime imaging microscopy (FLIM). Our results suggest that HA constructs are indeed sorted into cholesterol-dependent lipid domains since cholesterol depletion of the PM caused a significant decrease of FRET efficiency. Likewise, deletion of the three highly conserved palmitoylation sites of HA is also accompanied by a reduction of FRET efficiency. Site directed mutagenesis demonstrated that TMD-HA constructs form disulfide linked oligomers and that oligomerization is fundamental for the transport to the PM. This result was corroborated by time resolved anisotropy measurements that revealed strong homoFRET between TMD-HA-YFP molecules, thus indicating protein clustering. Accordingly, trimerization of full length HA is fundamental for stability and the subsequent delivery of the protein to the cell surface.

A oxidação de membranas lipídicas pode ser benéfica (p.ex. sinalização celular) ou prejudicial, sendo a permeabilização de membranas uma de suas consequências citotóxicas. A permeabilização fotoinduzida de membranas é parte essencial do mecanismo da terapia fotodinâmica (PDT), uma modalidade clínica em que fotossensibilizadores, luz e oxigênio são combinados para oxidar biomoléculas e consequentemente danificar células indesejadas. Neste trabalho, buscamos entender molecularmente quais fatores levam à permeabilização fotoinduzida de membranas. Enfatizamos os papéis do oxigênio, do status da membrana e de reações específicas do fotossensibilizador em contato com a membrana. Simulações de dinâmica molecular foram usadas para obter a distribuição de oxigênio em membranas em função da temperatura nas fases fluida ou gel. Procedimentos específicos de análise de cinéticas de luminescência de oxigênio singlete foram desenvolvidos para calcular tempos de vida de estado excitado triplete compatíveis com as variações da distribuição de oxigênio em membranas. Caracterizamos um derivado fluorogênico do α-tocoferol como uma sonda para oxigênio singlete em experimentos com lipossomos, possibilitando comparar qualitativamente os níveis de oxigênio singlete atingindo a membrana quando produzido por fotossensibilizadores hidrossolúveis ou lipossolúveis. Experimentos em vesículas unilamelares gigantes (GUVs) nos permitiram comparar a ativação da sonda com o aumento de área superficial da membrana, e estimar a constante de velocidade da reação do oxigênio singlete com lipídeos insaturados como 6 x 104 M-1 s-1. Estreitando nosso foco para a permeabilização fotoinduzida de membranas, inicialmente caracterizamos quatro fotossensibilizadores fenotiazínicos em relação a suas interações com membranas e suas capacidades de promover o vazamento de uma sonda fluorescente. Fotossensibilizadores que se particionaram mais em membranas (e não os geradores de oxigênio singlete mais eficientes) danificaram a membrana de lipossomos mais eficientemente. A ligação à membrana também afetou as vias de decaimento dos estados excitados triplete. Com esse estudo, selecionamos o fotossensibilizador hidrofílico azul de metileno (MB) e o fotossensibilizador mais hidrofóbico DO15 para as investigações subsequentes. Os efeitos de ambos os fotossensibilizadores em GUVs foram caracterizados e observamos que as cinéticas de permeabilização indicaram diferentes taxas de produção de lipídeos formadores de poros para MB e DO15, o que deve depender de interações específicas com a membrana. Para melhor compreender o papel de interações fotossensibilizador/membrana, caracterizamos a oxidação de lipídeos por ambos os fotossensibilizadores, em uma condição em que DO15 permeabilizava membranas 70 vezes mais eficientemente que MB. Observamos principalmente a formação de hidroperóxidos lipídicos para MB, enquanto que para DO15, além desses mesmos produtos, observamos a formação de álcoois, cetonas e aldeídos fosfolipídicos de cadeia truncada, esses últimos tendo sido relacionados a condições em que se observou a permeabilização de membranas. Embora já fosse sabido que aldeídos fosfolipídicos aumentam a permeabilidade da membrana, esse fenômeno nunca havia sido demonstrado para a formação de aldeídos in situ. A fotooxidação lipídica foi acompanhada por aumento do fotobranqueamento de DO15 e pela formação de radicais lipídicos oxigenados, indicando a ocorrência de reações diretas entre lipídeos e fotossensibilizadores. O mapeamento dos fatores que levam à permeabilização fotoinduzida em membranas, focando em reações e interações moleculares, é o maior produto desse trabalho / Oxidation of lipid membranes can be beneficial (e.g., cell signaling) or detrimental, with membrane permeabilization representing one of its cytotoxic outcomes. Photoinduced membrane permeabilization is key to the mechanism of photodynamic therapy (PDT), a clinical modality in which photosensitizers, light and oxygen are combined to oxidize biomolecules and consequently damage diseased cells. In this work, we aimed to understand at the molecular level which factors lead to photoinduced membrane permeabilization. We emphasized the roles of oxygen, membrane status and specific reactions of the photosensitizer in contact with the membrane. Molecular dynamics simulations were used to assess oxygen distribution in membranes as a function of temperature within membranes in gel or liquid phases. Special fitting procedures of singlet oxygen luminescence kinetics were devised to allow the calculation of triplet excited state lifetimes compatible with variable oxygen distributions in membranes. We characterized a fluorogenic α-tocopherol probe as a singlet oxygen trapping molecule in experiments with liposomes, and were able to qualitatively compare the amount of singlet oxygen molecules reaching the membrane after being generated by water soluble or membrane bound photosensitizers. Experiments performed in giant unilamellar vesicles (GUVs) allowed us to compare the activation of the probe with the observed membrane surface area increase and estimate the reaction rate of singlet oxygen with unsaturated lipids to be 6 x 104 M-1 s-1. We then narrowed our focus to photoinduced membrane permeabilization, initially characterizing four phenothiazinium photosensitizers with respect to their interactions with membranes and their capability to promote leakage of a fluorescent probe. Photosensitizers that bound to membranes to a larger extent (and not the most efficient singlet oxygen generators) were the most efficient ones to damage liposomal membranes. Membrane binding also affected triplet excited state deactivation pathways. From this study, we selected the hydrophilic photosensitizer methylene blue (MB) and the more hydrophobic photosensitizer DO15 for subsequent investigations. We characterized the effects of both photosensitizers in GUVs and observed that the kinetics of membrane permeabilization implied different rates of generation of pore-forming lipids for MB and DO15, which should depend on specific interactions with membranes. To further understand the role of photosensitizer/membrane interactions, we characterized the oxidized lipids formed by both photosensitizers in a condition in which the membrane permeabilization efficiency of DO15 was 70 times higher than that of MB. We observed mainly formation of lipid hydroperoxides by MB, while DO15 not only led to these same products, but also to alcohols, ketones and phospholipid truncated aldehydes, the latter being related to conditions in which membrane permeabilization was observed. Although aldehydes were already known to increase membrane permeability, this phenomenon had never before been demonstrated for aldehyde formation in situ. Lipid photooxidation was accompanied by increased photobleaching of DO15 and by formation of lipid oxygenated radicals, indicating the occurrence of direct reactions between lipids and photosensitizers. A roadmap of the factors leading to photoinduced membrane permeabilization focusing on molecular interactions and reactions is the major achievement of this work.

Made available in DSpace on 2017-07-21T14:13:05Z (GMT). No. of bitstreams: 1 Clarissa Elize.pdf: 4457240 bytes, checksum: d8f2520757eb456c2ae4c01f7a1f0343 (MD5) Previous issue date: 2015-12-21 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Skin wounds are interruptions of the normal physiological and anatomical structure of the skin causing damage by loss of barrier function becoming the organism exposed to various types of substances and microorganisms. A fast healing of the wound is essential to avoid the risk of infections and other complications. The aim of this work was to the development of lipid nanoparticles, as solid lipid nanoparticles and lipid-core polymeric nanoparticles containing halcinonide to modulate the inflammatory phase of wound healing, reducing pain, discomfort, edema, exudates and to reduced the drug toxicity. To this end, nanoparticles were obtained and characterized for particle size, physicochemical properties, stability, encapsulation efficiency, scanning electron microscopy by field emission, x-ray diffraction, spectroscopy in the infrared and Raman Fourier Transform, differential scanning calorimetry, thermal gravimetric analysis, study of the in vitro skin permeation and in vivo evaluation of the inflammatory response and drug toxicity. Furthermore, a highperformance liquid chromatography method for quantification of the drug was developed and validated. Nanoparticles had an average diameter ranging from 260-500 nm, polydispersity index below 0.37, zeta potential close to -30 mV, pH between 5.3 and 6.5 and were stability after storage for 60 days. The microscopy images showed spherical shape with smooth surface. The X-ray diffraction analysis showed drug amorphization in the nanostructured systems. By infrared and Raman spectroscopy was identified the characteristic bands of main components of the formulations, indicating no chemical bonding between them. Thermal analysis revealed the melting peaks of the drug and lipids and polymer and the temperature of its degradation. Nanoparticles showed 5.0% of drug permeation in 24 hours. In vivo study showed that the pure halcinonide was toxic; producing systemic adverse effects and nanoparticles containing the drug was able to modulate the inflammation healing, avoiding the edema and exudates formation and however impairing the subsequent stages of the healing process. However, the incorporation of the halcinonide in nanoparticles was able to reduce the drug toxicity due to the control of the drug release. Regarding the samples of lipid nanoparticles, both showed similar results, wherein the lipid-core polymeric nanoparticles showed better retraction of the wound and macroscopic appearance, possibly due to the polymer control associated with the lipid in halcinonide release. / Lesões cutânea são interrupções da sua estrutura anatômica normal e fisiológica que causam danos pela perda da função barreira da pele tornando-a exposta a diversos tipos de substâncias e microrganismos. Dessa forma uma rápida cicatrização desta lesão é fundamental para evitar o risco de infecções e outras complicações. O objetivo deste trabalho foi o desenvolvimento de nanopartículas lipídicas, na forma de nanopartículas lipídicas sólidas e nanopartículas poliméricas de núcleo lipídico, para veiculação de halcinonida para modulação da fase inflamatória da cicatrização, reduzindo dor, desconforto, edema e exsudato, aliado a redução da toxicidade do fármaco. Para tanto, as nanopartículas foram obtidas e caracterizadas quanto ao tamanho de partícula, propriedades físico-químicas, estabilidade, eficiência de encapsulação do fármaco, microscopia eletrônica de varredura por emissão de campo, difração de raios x, espectroscopia na região do infravermelho e Raman com Transformada de Fourier, calorimetria diferencial de varredura, análise termogravimétrica, estudo de permeação cutânea in vitro e avaliação in vivo da resposta inflamatória e da toxicidade do fármaco. Além disso, um método para quantificação do fármaco por cromatografia líquida de alta eficiência foi desenvolvido e validado. As nanopartículas apresentaram diâmetro médio variando entre 260 a 500 nm, índice de polidispersão abaixo de 0,37, potencial zeta próximo a -30 mV, pH entre 5,3 e 6,5 e apresentaram estabilidade após armazenamento de 60 dias. As imagens obtidas por microscopia revelaram formato esférico com superfície lisa. A análise de difração de raios x demonstrou a amorfização do fármaco nos sistemas nanoestruturados. Por espectroscopia na região do infravermelho e Raman puderam-se identificar as bandas características dos principais componentes das formulações, indicando que não houve ligação química entre eles. A análise térmica revelou os picos da fusão do fármaco e dos lipídeos e polímero utilizados e a temperatura da sua degradação. As nanopartículas apresentaram 5,0 % do fármaco permeado em 24 horas de estudo. O estudo in vivo revelou que a halcinonida livre foi tóxica, produzindo efeitos adversos sistêmicos e as nanopartículas contendo o fármaco foram capazes de modular a inflamação da cicatrização, evitando a formação de edema e exsudato, porém prejudicando as fases subsequentes do processo de cicatrização. No entanto, a incorporação da halcinonida nas nanopartículas foi capaz de reduzir a toxicidade do fármaco pelo controle da sua liberação. Em relação às amostras, as duas nanopartículas desenvolvidas apresentaram resultados semelhantes,sendo que as nanopartículas poliméricas de núcleo lipídico mostrou melhor retração da ferida e aspecto macroscópico, possivelmente pelo controle polimérico associado ao do lipídeo para liberação da halcinonida.

Made available in DSpace on 2017-07-21T20:00:00Z (GMT). No. of bitstreams: 1 Luiz Ricardo Olchanheski.pdf: 1069422 bytes, checksum: 4a8faf74d88ea768a689fc8a9a055a56 (MD5) Previous issue date: 2014-02-25 / Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Paraná / The intensive use of agrochemicals has assumed an important role in increasing agricultural production; however, one of the impacts has been changes in population structure of microbiota in the soil, which need to tolerate the xenobiotics that are applied in order to survive. A pertinent question is whether these mechanisms are selected by the presence of different agrochemicals in soils, or if there are constitutive mechanisms of adaptation. The aim of this study was to evaluate the defense system of an un-environmental strain, E. coli DH5-α, in relation to the herbicide mesotrione, which had no prior contact with the soil. The results showed that this strain was able to tolerate higher doses of the herbicide, and that the determination of the herbicide by a method developed and validated by high performance liquid chromatography (HPLC), made it possible to determine the complete disappearance of mesotrione in the sample after 3 h of exposure. Growth rates in the treatment with the herbicide were lower than the control, prior to the period of degradation, showing the toxic effect on the bacterial cells. As regards defense systems, it was noted that changes in the saturation of the membrane lipids reduced the damage caused by reactive oxygen species and possibly hindered the entry of xenobiotics in the cell, as well as activating GST enzyme activity in the antioxidant system and in the metabolizing process of the herbicide. Considering that E. coli DH5-α showed no previous contact with mesotrione, the defense system found in this strain can be considered as general and non-specific. This strategy may be interesting in the adaptation of bacterial strains in agricultural soils, which are subject to changing herbicides in an ever more intense manner. / O uso intensivo de agroquímicos tem assumido um papel relevante no aumento da produção agrícola, mas um dos impactos gerados é a mudança na estrutura populacional de microbiotas de solo, que precisam tolerar os xenobióticos aplicados para sobreviver. Uma pergunta pertinente é se os mecanismos de tolerância são selecionados pela presença de diferentes agroquímicos em solo, ou se há mecanismos constitutivos de adaptação. O objetivo deste trabalho foi avaliar o sistema de defesa de uma linhagem não ambiental, a E. coli DH5-α, em relação ao herbicida mesotrione, com o qual não houve contato prévio em solo. Resultados obtidos mostraram que esta linhagem foi capaz de tolerar elevadas doses do herbicida, e a determinação do herbicida por um método desenvolvido e validado por cromatografia líquida de alta eficiência (CLAE) permitiu determinar que após 3 horas não foi possível detectar o herbicida nas amostras avaliadas. Taxas de crescimento no tratamento com o herbicida foram menores em relação ao controle nos tempos anteriores ao período de degradação, mostrando o efeito tóxico sobre as células bacterianas. Como sistemas de defesa, constataram-se a alteração na saturação dos lipídeos de membrana, diminuindo os danos causados pelas espécies ativas de oxigênio e possivelmente dificultando a entrada do xenobiótico na célula, além da atuação da enzima GST no sistema antioxidante e no processo de metabolização do herbicida. Considerando que a E. coli DH5-α não apresentou um contato prévio com o mesotrione, o sistema de defesa encontrado nesta linhagem pode ser considerado como geral e não específico. Esta estratégia pode ser interessante na adaptação de linhagens bacterianas em solos agrícolas, os quais são submetidos a regimes de trocas de herbicidas de maneira cada vez mais intensa.

Post-translational redox-regulation is a well-known mechanism to regulate enzymes of the Calvin cycle, oxidative pentose phosphate cycle, NADPH export and ATP synthesis in response to light. The aim of the present thesis was to investigate whether a similar mechanism is also regulating carbon storage in leaves. <br><br> Previous studies have shown that the key-regulatory enzyme of starch synthesis, ADPglucose pyrophosphorylase (AGPase) is inactivated by formation of an intermolecular disulfide bridge between the two catalytic subunits (AGPB) of the heterotetrameric holoenzyme in potato tubers, but the relevance of this mechanism to regulate starch synthesis in leaves was not investigated. The work presented in this thesis shows that AGPase is subject to post-translational redox-regulation in leaves of pea, potato and Arabidopsis in response to day night changes. Light was shown to trigger posttranslational redox-regulation of AGPase. AGPB was rapidly converted from a dimer to a monomer when isolated pea chloroplasts were illuminated and from a monomer to a dimer when preilluminated leaves were darkened. Conversion of AGPB from dimer to monomer was accompanied by an increase in activity due to changes in the kinetik properties of the enzyme. Studies with pea chloroplast extracts showed that AGPase redox-activation is mediated by thioredoxins f and m from spinach in-vitro. In a further set of experiments it was shown that sugars provide a second input leading to AGPase redox activation and increased starch synthesis and that they can act as a signal which is independent from light. External feeding of sugars such as sucrose or trehalose to Arabidopsis leaves in the dark led to conversion of AGPB from dimer to monomer and to an increase in the rate of starch synthesis, while there were no significant changes in the level of 3PGA, an allosteric activator of the enyzme, and in the NADPH/NADP+ ratio. Experiments with transgenic Arabidopsis plants with altered levels of trehalose 6-phosphate (T6P), the precursor of trehalose synthesis, provided genetic evidence that T6P rather than trehalose is leading to AGPase redox-activation. Compared to Wt, leaves expressing E.coli trehalose-phosphate synthase (TPS) in the cytosol showed increased activation of AGPase and higher starch level during the day, while trehalose-phosphate phosphatase (TPP) overexpressing leaves showed the opposite. These changes occurred independently of changes in sugar and sugar-phosphate levels and NADPH/NADP+ ratio. External supply of sucrose to Wt and TPS-overexpressing leaves led to monomerisation of AGPB, while this response was attenuated in TPP expressing leaves, indicating that T6P is involved in the sucrose-dependent redox-activation of AGPase. To provide biochemical evidence that T6P promotes redox-activation of AGPase independently of cytosolic elements, T6P was fed to intact isolated chloroplasts for 15 min. incubation with concentrations down to 100 µM of T6P, but not with sucrose 6-phosphate, sucrose, trehalose or Pi as controls, significantly and specifically increased AGPB monomerisation and AGPase activity within 15 minutes, implying T6P as a signal reporting the cytosolic sugar status to the chloroplast. The response to T6P did not involve changes in the NADPH/NADP+ ratio consistent with T6P modulating redox-transfer to AGPase independently of changes in plastidial redox-state. <br><br> Acetyl-CoA carboxylase (ACCase) is known as key-regulatory enzyme of fatty acid and lipid synthesis in plants. At the start of the present thesis there was mainly in vitro evidence in the literature showing redox-regulation of ACCase by DTT, and thioredoxins f and m. In the present thesis the in-vivo relevance of this mechanism to regulate lipid synthesis in leaves was investigated. ACCase activity measurement in leaf tissue collected at the end of the day and night in Arabidopsis leaves revealed a 3-fold higher activation state of the enzyme in the light than in the dark. Redox-activation was accompanied by change in kinetic properties of ACCase, leading to an increase affinity to its substrate acetyl-CoA . In further experiments, DTT as well as sucrose were fed to leaves, and both treatments led to a stimulation in the rate of lipid synthesis accompanied by redox-activation of ACCase and decrease in acetyl-CoA content. <br><br> In a final approach, comparison of metabolic and transcript profiling after DTT feeding and after sucrose feeding to leaves provided evidence that redox-modification is an important regulatory mechanism in central metabolic pathways such as TCA cycle and amino acid synthesis, which acts independently of transcript levels. / Es ist bereits seit längerem bekannt, dass viele Enzyme des Calvinzyklus, des oxidativen Pentosephosphatwegs, des NAD(P)H-Exports und der ATP-Synthese durch post-translationale Redox-Modifikation in Antwort auf Licht reguliert werden. In der vorliegenden Arbeit sollte untersucht werden, ob ein ähnlicher Mechanismus auch die Kohlenstoffspeicherung in Blättern reguliert. <br><br> Vorangegangene Studien mit Kartoffelknollen zeigten, dass das Schlüsselenzym der Stärkesynthese ADP-Glukose-Pyrophosphorylase (AGPase) durch die Bildung einer Disulfidbrücke zwischen den zwei kleinen Untereinheiten (AGPB) des tetrameren Proteins inaktiviert wird, die Bedeutung dieses Mechanismus für die Stärkesynthese in Blättern blieb jedoch bislang ungeklärt. Die vorliegenden Arbeiten zeigen, das AGPase in Erbsen-, Kartoffel- und Arabidopsis-Blättern über post-translationale Redox-Modifikation in Antwort auf Tag-Nacht Änderungen reguliert wird. Dies erfolgt über ein Licht-abhängiges Signal, da, erstens, AGPB in isolierten Chloroplasten durch Belichtung sehr schnell von Dimer zu Monomer umgewandelt wird und, zweitens, ein Abdunkeln der Blätter zu einer schnellen Umwandlung von AGPB von Monomer zu Dimer führt. Die Monomerisierung von AGPB ging mit Änderungen in den kinetischen Eigenschaften des Enzyms einher, die zu einer Aktivierung führten. Studien mit Extrakten aus Erbsenchloroplasten zeigten, dass die AGPase-Redoxaktivierung in-vitro durch die Thioredoxine f und m aus Spinat vermittelt wird. In einem weiteren experimentellen Ansatz konnte gezeigt werden, dass auch Zucker zu Redox-Aktivierung der AGPase und erhöhter Stärkesynthese in Blättern führen, und dass diese unabhängig von Licht wirken. Externe Zugabe von Zuckern wie Saccharose oder Trehalose an Arabidopsis-Blätter im Dunkeln führten zu Monomerisierung von AGPB und einer Erhöhung der Stärkesyntheserate / während die Spiegel des allosterischen Aktivators 3PGA unverändert blieben und keine Änderungen im NADPH/NADP+-Verhältnis auftraten. Experimente mit transgenen Arabidopsis-Pflanzen mit veränderten Spiegeln des Vorläufers der Trehalosesynthese, Trehalose-6-phosphat (T6P), zeigten, dass T6P und nicht Trehalose zu Redox-Aktivierung von AGPase führt. Expression einer E. coli T6P synthase (TPS) im Zytosol führte zu erhöhter Redox-Aktivierung von AGPase und erhöhter Stäreksynthese in Blättern, während die Expression einer T6P-Phosphatase (TPP) gegenteilige Änderungen bewirkte. Diese Auswirkungen erfolgten unabhängig von Änderungen in den Spiegeln von Zuckern und Zuckerphosphaten oder im NADPH/NADP+-Verhältnis. Externe Zugabe von Saccharose führte zu Monomerisierung von AGPB in Wildtyp und TPS exprimierenden Blättern, während diese Antwort in TPP exprimierenden Blättern stark abgeschwächt war. Dies zeigt, dass T6P eine wesentliche Komponente darstellt, die die Redox-Aktivierung der AGPase in Antwort auf Saccharose vermittelt. T6P wurde auch für 15 min direkt an intakte, isolierte Erbsenchloroplasten gefüttert. T6P Konzentrationen im Bereich von 100 µM bis 10 mM führten zu einem signifikanten und spezifischen Anstieg der AGPB-Monomersierung und der AGPase Aktivität. Dies zeigt, dass T6P auch ohne zytosolische Elemente die Redox-Aktivierung der AGPase stimuliert und somit ein Signal zwischen Zytosol und Plastid darstellt. Diese Antwort erfolgte ohne Änderungen im NADPH/NADP+-Verhältnis, was zeigt, dass T6P eher den Redox-Transfer zu AGPase als den Redoxzustand des Chloroplasten moduliert. <br><br> Acetyl-CoA-Carboxylase (ACCase) ist als Schlüsselenzym der Fettsäure- und Lipidsynthese in Pflanzen bekannt. Zu Beginn der vorliegenden Arbeit lagen hauptsächlich in-vitro Befunde vor, die zeigten, dass ACCase durch DTT und thioredoxine f und m über Redox-Modulation reguliert wird. In der Arbeit sollte daher die in-vivo Relevanz dieses Mechanismus für die Regulation der Lipidsynthese in Blättern untersucht werden. ACCase zeigte einen höheren Redox-Aktivierungszustand in Arabidopsis-Blätter, die während des Tages im Vergleich zur Nacht geerntet wurden. Die Redox-Aktivierung der ACCase wurde von Änderungen in den kinetischen Eigenschaften begleitet und führte zu einer erhöhten Affinität des Enzymes gegenüber Acetyl-CoA als Substrat. <br><br> In weiteren Versuchen wurde sowohl DTT als auch Saccharose an Blätter gefüttert, und beide Behandlungen führten zu Redox-Aktivierung von ACCase, was mit erhöhten Lipidsynthesraten und einem Rückgang des Acetyl-CoA-Spiegels einherging.

PEG-lipid stabilized bilayer disks have been found in lipid mixtures containing polyethylene glycol (PEG)-lipids where the combination of a high bending rigidity and low PEG-lipid/lipid miscibility favours disk formation. The disks are planar and circular in shape and their long-term stability is excellent. Theoretical calculations and experimental observations suggest that the micelle forming PEG-lipid are situated at the rim of the aggregate, protecting the hydrophobic lipid chains in the bulk of the aggregate from contact with water. This thesis deals with fundamental aspects concerning the lipid distribution in the disks, as well as with development, optimization, and initial evaluation of the disks as model membranes in partition and interaction studies. Small angle neutron scattering was used to study the partial segregation of components within the bilayer disk. The experiments verified that the PEG-lipids segregate and accumulate at the bilayer disk rim. The proof of component segregation is important from a fundamental point of view and useful, as exemplified in the below-mentioned study of melittin-lipid interaction, when interpreting partition or binding data obtained from studies based on bilayer disks. Today liposomes are often used as model membranes in partition and interaction studies. Using liposomes to predict, e.g., drug partitioning can however have certain drawbacks. In this thesis the disks were proven to be attractive alternatives to liposomes as model membranes in partition studies. The formation of bilayer disks by a technique based on detergent depletion enabled incorporation of a transmembrane protein in the bilayer disks and opened up for the use of disks as model membranes in membrane protein studies. Further, bilayer disks were used in a comparative study focused on the effect of aggregate curvature on the binding of the peptide melittin. Various techniques were used to perform initial evaluations of the bilayer disks as model membranes. Of these, capillary electrophoresis and biosensor-based technology had not been used before in combination with bilayer disks.

Las membranas celulares contienen un canal de Cl- (Maxi-Cl-) que es modulado por estrógenos y antiestrógenos. El trabajo experimental de mi tesis doctoral ha consistido en el estudio de la modulación de este canal y su identidad molecular. El resultado de mi trabajo ha demostrado que la base molecular del canal Maxi-Cl- es una isoforma de la proteína mitocondrial VDAC y que la activación del canal por los antiestrógenos y su inhibición por los estrógenos implica procesos de defosforilación y fosforilación, respectivamente. / Cellular membranes contain a Maxi-Cl- channel that is modulated by oestrogen and antioestrogens. The experimental work of my PhD Thesis has focussed in the study of the molecular identity of the Maxi-Cl- channel and its regulation. My results demonstrated that an isoform of the mitochondrial protein VDAC is the molecular correlate of the Maxi-Cl- channel and that the activation of the channel by antioestrogens and its inhibition by oestrogen requires a dephosphorylation and phosphorylation process, respectively.

Die Degranulation von Mastzellen soll durch Glycosphingolipide, welche mit der Zellmembran wechselwirken inhibiert werden. Der Sphingosingrundkörper wurde in zehn-stufigen Synthese ausgehend von N-Boc-Serin, aufgebaut. Die anschließende Glycosylierung erfolgte nach der Trichloracetimidatmethode in sehr guten Ausbeuten und stellte den Schlüsselschritt dar. Durch die Variation von unter Anderem der Amidseitenkette, der Glycosylkopfgruppe und des Sphingosingrundkörpers wurde eine Vielzahl an Derivaten für das Screening im Degranulationsassay bereitgestellt. / The present dissertation covers the synthesis of glycosphingolipids which interact with the cell membrane in order to inhibit the degranulation of mast cells. The sphingosin body was synthesized in ten steps starting from N-Boc-Serin. The key step, the glycosylation was achieved using the trichloracetimidat method. The variation of the amid sidechain, the gylcosyl headgroup and the sphingosin body created a number of derivatives that were tested in the degranulation assay.

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