Liver mitochondria play a crucial role in intermediary metabolism and main metabolic pathways. We evaluated the pharmacological effect on liver mitochondria in vitro using two novel anticancer drugs: 3-bromopyruvate and α-tocopheryl succinate. Metabolic influence on liver mitochondria was performed in vivo by high fat and high cholesterol diet. Toxicity of both drugs was evaluated in cell cultures of hepatocytes isolated from rat and mouse liver. The effect of anticancer drugs on liver mitochondrial functions in vitro was studied on suspensions of isolated liver mitochondria, tissue homogenate and permeabilized hepatocytes. Mitochondrial respiration was measured using high-resolution respirometry. 3-bromopyruvate caused morphological and functional damage of primary rat and mouse hepatocytes in cell cultures; this toxic effect was accompanied by an increase of reactive oxygen species production and mitochondrial dysfunction. 3-bromopyruvate decreased the oxygen consumption of mitochondria energized by substrates for complex I and complex II. α-Tocopheryl succinate caused a decrease of succinate-dependent respiration in all experimental models both in coupled and in uncoupled states. The most pronounced effect of α-tocopheryl succinate was apparent in isolated mitochondria and the least pronounced...

In this study, traditional Japanese washi paper was sandwiched with poly(butylene succinate)(PBS) in order to produce a paper-based composite material that can be biologically degraded. Specimens were prepared using a hot press, and the mechanical properties of the washi paperand the new composite material were investigated by tensile tests. The cross-sections of specimens were then observed with a digital microscope as well as by scanning electron microscope (SEM) analysis. The microscope images indicated that therelatively hollow washi provides a structure for the polymer to settle into, which could contribute to the improved strength of the composite material. Specimens made from three layers of washipaper and combined with two layers of PBS film had remarkable mechanical propertiesincluding a UTS of 95 MPa.The composite material was incubated in compost and a MODA C-S (Microbial Oxidative Degradation Analyzer) was used to evaluate its biodegradability. The material showed good biodegradability with significant losses of mass (-32 %) as well as major loss of mechanical strength after four weeks of incubation. The biodegradability of the material will be further investigated.

As nanocomposites provide considerable improvements in material properties, scientists and engineers are focussing on biodegradable nanocomposites having superior material properties as well as degradability. This thesis has investigated the properties of biodegradable nanocomposites of the aliphatic thermoplastic polyester, poly (lactide acid) (PLA) and the synthetic biodegradable polyester, poly (butylene succinate) (PBS). To enhance the properties of this blend, nanometer-sized clay particles, have been added to produce tertiary nanocomposite. High aspect ratio and surface area of clay provide significant improvement in structural, mechanical, thermal and barrier properties in comparison to the base polymer. In this study, a series of PLA/PBS/layered silicate nanocomposites were produced by using a simple twin-screw extruder. PLA/PBS/Cloisite 30BX nanocomposites were prepared containing 1, 3, 5, 7 and 10 wt% of C30BX clay, while PLA and PBS polymers compositions were fixed at a ratio of 80 to 20. This study also included the validation of a gas barrier model for these biodegradable nanocomposites. WAXD indicated an exfoliated structure for nanocomposites having 1 and 3 wt% of clay, while predominantly development of intercalated structures was noticed for nanocomposites higher than 5 wt% of clay. However, TEM images confirmed a mixed morphology of intercalated and exfoliated structure for nanocomposite having 1 wt% of clay, while some clusters or agglomerated tactoids were detected for nanocomposites having more than 3 wt% of clay contents. The percolation threshold region for these nanocomposites lied between 3-5 wt% of clay loadings. Liquid-like behaviour of PLA/PBS blends gradually changed to solid-like behaviour with the increase in concentration of clay. Shear viscosity for the nanocomposites decreased as shear rate increased, exhibiting shear thinning non-Newtonian behaviour. Tensile strength and Young's modulus initially increased for nanocomposites of up to 3 wt% of clay but then decreased with the introduction of more clay. At high clay content (more than 3 wt%), clay particles tend to aggregate which causes microcracks at the interface of clay-polymer by lowering the polymer-clay interaction. Percentage elongation at break did not show any improvement with the addition of clay. PLA/PBS blends were considered as immiscible with each other as two separate glass transition and melting temperatures were observed in modulated differential scanning calorimetry (MDSC) thermograms. MDSC showed that crystallinity of the nanocomposites was not much affected by the addition of clay and hence some compatibilizer is required. Thermogravimetric analysis showed that the nanocomposite containing 3 wt% of clay demonstrated highest thermal stability compared to other nanocomposites. Decrease in thermal stability was noticed above 3 wt% clay; however the initial degradation temperature of nanocomposites with 5, 7 and 10 wt% of clay was higher than that of PLA/PBS blend alone. Gas barrier property measurements were undertaken to investigate the transmission of oxygen gas and water vapours. Oxygen barrier properties showed significant improvement with these nanocomposites, while that for water vapour modest improvement was observed. By comparing the relative permeabilities obtained from the experiments and the model, it was concluded that PLA/PBS/clay nanocomposites validated the Bharadwaj model for up to 3 wt% of clay concentration.

No description available.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Recent studies from our group demonstrated that the (S)-dimethyl 2-(3-(phenyltellanyl) propanamido) succinate compound, a telluroamino acid showed glutathione peroxidase (GPx)-like activity, attesting to its antioxidant potential. However, toxicology and pharmacology from this compound are unknown. Then, the present study aimed to investigate the potencial toxicological properties of this compound in vitro and in vivo. For in vitro analysis were used biochemical tests as: GPx-like activity, tiol oxidase activity and TBARS determination. Futhermore, the in vivo analyses were carried out using swiss albino male adult mice treated and submitted to behavioral tests (exploration and locomotion). For ex vivo studies, the antioxidant enzyme activity, and tests to evaluate citoxicity, genotoxicty and mutagenicity of compound were carried out. In order to verify the potencial mechanisms of action of the compound, we used the GR and TrxR enzymes inhibitors. The results indicated that the compound presented an important antioxidant activity in vitro. However, when the compound was tested in vivo with the highest concentrations, it was shown as a potent genotoxic and mutagenic agent at doses of 175 e 195 μmol/Kg. On the other hand, when the compound was tested at minor doses (1/2 LD50), it did not demonstrate significant signs of toxicity. In relation to treatment with inhibitors of GR and TrxR enzymes, the results showed that the inhibition of these enzymes did not affect the compound action in our model of study. / Estudos recentes de nosso grupo de pesquisa demonstraram que o composto (S) (3-feniltelanil propanamida) succinato de dimetila, um telúrio aminoácido apresentou atividade mimética da enzima Glutationa Peroxidase (GPx), indicando potencial antioxidante. Contudo, a toxicidade e a farmacologia deste composto não são conhecidas. Assim, o presente estudo teve como objetivo investigar as potenciais propriedades toxicológicas deste composto em modelos in vitro e in vivo. Nas análises in vitro foram utilizados testes bioquímicos como: atividade mimética a GPx, atividade tiol oxidase e determinação de substâncias reativas ao ácido tiobarbitúrico (TBARS). Além disso, as análises in vivo foram realizadas com a utilização de camundongos machos adultos, tratados e submetidos a testes comportamentais (exploração e locomoção). Para os estudos ex vivo foram dosados a atividade de enzimas antioxidantes, e testes para avaliar o potencial citotóxico, genotóxico e mutagênico do composto. Com o intuito de verificar potenciais mecanismos de ação do composto, foram utilizados inibidores das enzimas Glutationa Redutase (GR) e Tiorredoxina Redutase (TrxR). Os resultados demonstraram que o composto apresentou importante atividade antioxidante in vitro. No entando, quando testado in vivo em dose maiores, o mesmo demonstrou-se potente agente genotóxico e mutagênico nas doses de 175 e 195 μmol/Kg. Por outro lado, quando testado em doses menores (1/2 DL50) o composto não demostrou significativos sinais de toxicidade. Quanto ao tratamento com os inibidores das enzimas GR e TrxR, os resultados demostraram que a inibição destas enzimas não afeta a ação do composto.

Orientador: Roger Frigerio Castilho / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-11T10:41:34Z (GMT). No. of bitstreams: 1 Mirandola_SandraRegina_D.pdf: 3705031 bytes, checksum: e1e34dd5e1b3949d9c308b3c20a19787 (MD5) Previous issue date: 2008 / Resumo: A acidemia metilmalônica (MMAemia) é uma desordem metabólica hereditária do metabolismo de aminoácidos com cadeia ramificada e de ácidos graxos com cadeia ímpar, envolvendo um defeito na conversão de metilmalonil-CoA a succinil-CoA. Manifestações sistêmicas e neurológicas nesta doença são relacionadas com o acúmulo de metilmalonato (MMA) em tecidos e fluidos biológicos e com o comprometimento do metabolismo energético. Neste trabalho, verificou-se que o MMA inibiu com grande intensidade a conversão de lactato a piruvato catalisada pela enzima lactato desidrogenase (LDH) em homogenatos de fígado e cérebro de rato. A conversão de piruvato a lactato, catalisada pela LDH, foi menos sensível à inibição por MMA. Estudos de cinética enzimática sobre a inibição da LDH de cérebro, utilizando-se lactato como substrato, indicaram que o MMA inibe esta enzima competitivamente (Ki = 3,02 ± 0,59 mM). Propôs-se que a inibição da conversão lactato/piruvato por MMA contribui para a fisiopatologia da MMAemia, resultando, dentre outras alterações, em acúmulo de lactato e acidemia metabólica. Mostrou-se que, em mitocôndrias isoladas de cérebro e músculo de rato, concentrações milimolares de MMA inibiram o consumo de O2 mantido por succinato, enquanto nenhum efeito inibitório foi observado quando substratos para os complexos I ou IV foram utilizados. Notadamente, o efeito inibitório de MMA, mas não de malonato, no consumo mitocondrial de O2 mantido por succinato foi minimizado quando uma permeabilização não-seletiva das mitocôndrias foi induzida por alameticina. Em adição, o MMA apresentou apenas um pequeno efeito inibitório no consumo de O2 por partículas submitocondriais invertidas na presença de succinato. Não se obteve evidência de produção de malonato nas mitocôndrias tratadas com MMA. Conclui-se que o MMA inibe o consumo mitocondrial de O2 na presença de succinato por interferir na captação deste substrato pela mitocôndria. A inibição do transporte mitocondrial de substratos, induzida pelo MMA, através do carreador de dicarboxilatos, pode ter importantes implicações fisiopatológicas na MMAemia. Comparou-se a suscetibilidade de mitocôndrias isoladas de fígado, rim e coração de rato, assim como de diferentes subregiões cerebrais quanto à transição de permeabilidade mitocondrial (MPT) induzida por 3-nitropropionato (3-NP) e Ca2+. A MPT foi estimada pela queda do potencial elétrico transmembrana e inchamento mitocondrial sensíveis à ciclosporina A. Mitocôndrias de cérebro e coração foram mais suscetíveis à MPT induzida por 3-NP e Ca2+ que organelas isoladas de fígado e rim. A comparação de mitocôndrias de diferentes regiões cerebrais indicou que uma inibição parcial da respiração por 3-NP resultou em MPT mais rapidamente em organelas estriatais que corticais ou cerebelares. Em ratos tratados sistemicamente com 3-NP, verificou-se uma inibição de mesma magnitude da succinato desidrogenase em todos os tecidos estudados. Notadamente, mitocôndrias isoladas de cérebro de ratos tratados sistemicamente com 3-NP apresentaram uma maior suscetibilidade à MPT induzida por Ca2+ quanto comparadas a controles. Propôs-se que a maior suscetibilidade do estriado à neurodegeneração induzida por 3-NP pode ser, pelo menos em parte, explicada por uma maior vulnerabilidade desta região cerebral à MPT, juntamente com a vulnerabilidade desta região ao influxo de Ca2+ citosólico mediado pelo estímulo de receptores de glutamato / Abstract: Methylmalonic acidemia (MMAemia) is an inherited metabolic disorder of branched amino acid and odd-chain fatty acid metabolism, involving a defect in the conversion of methylmalonyl-coenzyme A to succinyl-coenzyme A. Systemic and neurological manifestations in this disease are thought to be associated with the accumulation of methylmalonate (MMA) in tissues and biological fluids with consequent impairment of energy metabolism. In the present work it was observed that MMA potently inhibited lactate dehydrogenase (LDH)-catalyzed conversion of lactate to pyruvate in liver and brain homogenates. LDH was about one order of magnitude less sensitive to inhibition by MMA when catalyzing the conversion of pyruvate to lactate. Kinetic studies on the inhibition of brain LDH indicated that MMA inhibits this enzyme competitively with lactate as a substrate (Ki = 3.02 ± ?0.59 mM). We proposed that inhibition of the lactate/pyruvate conversion by MMA contributes to the MMAemia physiophatology, leading to lactate accumulation and metabolic acidemia. While millimolar concentrations of MMA inhibit succinate-supported O2 consumption by isolated rat brain or muscle mitochondria, there is no effect when either a pool of NADH-linked substrates or N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD)/ascorbate were used as electron donors. Interestingly, the inhibitory effect of MMA, but not of malonate, on succinate-supported brain mitochondrial O2 consumption was minimized when nonselective permeabilization of mitochondrial membranes was induced by alamethicin. In addition, only a slight inhibitory effect of MMA was observed on succinate-supported O2 consumption by inside-out submitochondrial particles. Under our experimental conditions, there was no evidence of malonate production in MMA-treated mitochondria. We conclude that MMA inhibits succinate-supported mitochondrial O2 consumption by interfering with the uptake of this substrate. MMA-induced inhibition of substrate transport by the mitochondrial dicarboxylate carrier may have important physiopatological implications. The susceptibility of isolated mitochondria from liver, kidney and heart and different rat brain regions (striatum, cortex and cerebellum) was compared regarding to mitochondrial permeability transition (MPT) evoked by 3-nitropropionate (3-NP) and Ca2+ ions. In general, isolated brain mitochondria from different regions were more sensitive to 3-NP and Ca2+ toxicity than mitochondria from liver and kidney as estimated by decrease in the transmembrane electrical potential and mitochondrial swelling. The comparision of different brain regions revealed that the inhibition of 50% of the mitochondrial succinate-supported respiration elicited by 3-NP resulted in a Ca2+-induced MPT pore opening, inhibited by cyclosporin A, faster in striatal than in cortical and cerebellar mitochondria. It was verified an inhibition of succinate dehydrogenase activity from the same magnitude in all tissues studied after a 3-NP systemic treatment. Interestingly, isolated forebrain mitochondria obtained from rats systemically treated with 3-NP showed a more pronounced susceptibility to Ca2+-induced MPT pore opening when compared to control rats. We proposed that the increased susceptibility of rat striatum to 3-NP-induced neurodegeneration could be in part explain by a region-specific susceptibility to MPT together with increase vulnerability of this brain region to glutamate receptors-mediated cytosolic Ca2+ influx / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutor em Fisiopatologia Medica

Succinate is a dicarboxylic acid that accumulates due to succinate dehydrogenase inhibition. This dicarboxylic acid has a biphasic effect on neural activity in vitro that seems to be mediated by N-methyl-D-aspartate (NMDA) receptors. The NMDA receptor is distributed throughout the central nervous system and mediates synaptic plasticity-related events, such as learning and memory. Although it has been described that succinate modulates NMDA conductance, it is not known if this organic acid modulates learning and me mory. Therefore, in the present study we investigated whether the immediate post-training systemic or intrahippocampal administration of succinate affects the memory of fear conditioning in rats. In addition, we investigated whether the NMDA and β-adrenergic receptors are involved in the facilitatory effect of succinate on memory. Systemic (0,00005, 0,0005, 0,005, 0,05 and 0,5 mg/kg i.p) or bilateral intra-hippocampal (0.21 pmol i.h.) immediate post-training administration of succinate biphasically facilitated contextual fear conditioning and had no effect on conditioning to tone. Systemic or intra-hippocampal administrations of MK-801 (0,001 mg/kg i.p. or 0,21 pmol i.h. respectively), a noncompetitive NMDA receptor antagonist, at a dose that had no effect per se, reversed the facilitatory effect of succinate on contextual fear conditioning. The systemic administration of propranolol (10 mg/kg i.p.), a β-adrenergic antagonist, at a dose that had no effect per se, also reversed the facilitatory effect of succinate on contextual fear conditioning. The administration of succinate (0,005 mg/kg i.p.) immediately after training and 15 minutes before the test did not affect the performance of the animals on fear conditioning. These results suggest that the facilitatory effect of succinate on the memory of fear conditioning involves NMDA and β-adrenergic receptors. The results also indicate that the facilitatory effect of succinate on memory is not related to state-dependence / O sucinato é um intermediário do ciclo de Kreks e também da cadeia respiratória, mas que também parece ter funções não relacionadas ao metabolismo energético. De fato, este ácido dicarboxílico tem efeito bifásico sobre a atividade neural in vitro, que parece ser mediado pelo receptor glutamatérgico N-metil-D-aspartato (NMDA). O receptor NMDA está presente em todo o sistema nervo central (SNC) e os processos mediados por este receptor incluem plasticidade sináptica e formação de circuitos neurais. Embora tenha sido descrito que o sucinato modula a atividade neural in vitro, não se sabe se este ácido orgânico modula processos fisiológicos ligados ao receptor NMDA, como o aprendizado e memória. Consequentemente, neste estudo foi investigado o efeito da administração sistêmica e intrahipocampal de sucinato, MK-801 e propranolol sobre a consolidação da memória em ratos, utilizando a tarefa de medo condicionado clássico. Posteriormente se investigou se os efeitos do sucinato sobre a memória envolvem dependência de estado. O teste do medo condicionado consistiu na apresentação de estímulos pareados, condicionado (tom 2000 Hz 90 dB/10 s) e incondicionado (choque 0.6 mA/1 s). O estado de imobilidade do animal foi utilizado como um indicativo de memória nas sessões de avaliação de memória ao contexto e ao tom. A administração sistêmica de sucinato nas doses de 0,00005, 0,0005, 0,005, 0,05 e 0,5 mg/kg i.p. imediatamente após o treino melhorou a memória. Contudo, a dose de 5 mg/kg de sucinato não alterou a memória dos animais, caracterizando um efeito bifásico sobre a memória. Essa melhora da memória induzida por sucinato (0,005 mg/kg i.p.) foi revertido pela administração pós-treino de um antagonista do receptor NMDA, MK-801 (0,001 mg/kg i.p.) e pela administração imediatamente após o treino de um bloqueador adrenérgico, propranolol (10 mg/kg i.p.). A administração de sucinato (0,005 mg/kg i.p.) imediatamente após o treino e 15 minutos antes do teste não afetou a performance dos animais, não caracterizando dependência de estado. Nos experimentos que visaram identificar se a administração central de sucinato alterava a memória, os animais foram canulados bilateralmente no hipocampo e após a recuperação cirúrgica, recebiam injeções bilaterais imediatamente após o treino, de sucinato (0,21 pmol i.h.) que melhorou a memória. A administração imediatamente após o treino de MK- 801 (0,22 nmol i.h.) foi capaz de reverter esse efeito facilitador do sucinato sobre a memória. Estes resultados sugerem que o efeito facilitador da memória induzida pela administração de sucinato é mediado pelo receptor NMDA e envolve de alguma maneira o sistema adrenérgico

Succinat und ihre Derivate werden in vielfältiger Weise in den Bereichen Tenside, Lebensmittel, Pharmazeutika und Polymere angewendet. Aufgrund der derzeit kostenintensiven petrochemischen Synthese ist die aerobe nicht-konventionelle Hefe Yarrowia (Y.) lipolytica für die biotechnologische Succinatsynthese von großem Interesse. In der vorliegenden Arbeit wurde das Potential dieser Hefe für eine industrielle Succinatproduktion unter Betrachtung des Einflusses der enzymatischen Aktivitäten von Succinyl-CoA Synthetase und Pyruvat-Carboxylase auf die Succinatsynthese untersucht. Es wurde eine Steigerung der Succinatausbeute um 40 % durch die Erhöhung der Pyruvat-Carboxylase Aktivität um den Faktor 7-8 gemeinsam mit der Deletion des Gens der β Untereinheit der Succinyl-CoA Synthetase im genetisch veränderten Y. lipolytica Stamm H222-AK10 (mcPYC Δscs2) erzielt. Unter Verwendung von Glycerol als C-Quelle wurde eine Erhöhung der Succinatbildung der Transformande H222 AK10 im Vergleich zum Wildtyp von 5,1 ± 0,7 g/l auf 8,7 ± 1,6 g/l nachgewiesen. Die Raum-Zeit-Ausbeute dieses Hefestammes verdoppelte sich von 11,9 ± 1,3 mg/l*h auf 21,9 ± 2,5 mg/l*h. Eine Erhöhung der Sekretion organischer Säuren gelang hingegen nicht durch den alleinigen Verlust der Succinyl-CoA Synthetase Aktivität in den Stämmen H222-AK4 (scs1::URA3), H222-AK8 (scs2:.URA3) und H222-AK9 (scs1::URA3 Δscs2) oder durch die alleinige Aktivitätserhöhung der Pyruvat-Carboxylase in H222-AK1 (mcPYC). Des Weiteren wurde ein Y. lipolytica Stamm erzeugt, der durch die Überexpression der für die Succinyl-CoA Synthetase kodierenden Gene SCS1 und SCS2 charakterisiert ist. Die Transformande H222 AK2 (mcSCS1 mcSCS2) bildete unter den gleichen Kultivierungsbedingungen durchschnittlich 2 g/l weniger Succinat als der Wildtyp (5,1 ± 0,7 g/l). Auch die zusätzliche Erhöhung der Pyruvat-Carboxylase Aktivität um den Faktor 4 in der Transformande H222 AK3 (mcPYC mcSCS1 mcSCS2) konnte den negativen Effekt der erhöhten Gen-Dosen von SCS1 und SCS2 auf die Succinatsynthese nicht aufheben. Dementsprechend wurden für H222-AK3 eine Succinatausbeute von 3,1 ± 0,3 g/l bestimmt.:Inhaltsverzeichnis Abbildungsverzeichnis VI Tabellenverzeichnis IX Abkürzungsverzeichnis XI 1. Einleitung 1 1.1. Bernsteinsäure, Succinat 1 1.2. Succinat als Zwischenprodukt des Tricarbonsäurezyklus 3 1.2.1. Die Enzyme des TCC in Saccharomyces cerevisiae 5 1.2.2. Succinatbildung im TCC durch die Succinyl-CoA Synthetase 6 1.2.3. Pyruvat-Carboxylase 7 1.3. Succinat als Endprodukt des Glyoxylatzyklus 9 1.4. Biotechnologische Herstellung von Succinat 10 1.4.1. Succinatproduktion mit Actinobacillus succinogenes und Anaerobiospirillum succiniproducens 11 1.4.2. Succinatproduktion mit Corynebacterium glutamicum 12 1.4.3. Succinatproduktion mit Escherichia coli 12 1.4.4. Yarrowia lipolytica als potentieller Succinatproduzent 15 1.5. Yarrowia lipolytica 15 1.6. Zielstellung 18 2. Material und Methoden 20 2.1. Geräte 20 2.2. Chemikalien, Biochemikalien und Nukleinsäuren 21 2.2.1. Feinchemikalien 21 2.2.2. Enzyme 22 2.2.3. Verbrauchsmaterialien und Kitsysteme 23 2.3. Verwendete Plasmide 23 2.4. Konstruierte Plasmide 24 2.5. Oligonukleotide 25 2.6. Mikroorganismen 26 2.6.1. Escherichia coli 26 2.6.2. Yarrowia lipolytica 27 2.7. Kultivierung 27 2.7.1. Kultivierung von Escherichia coli 27 2.7.2. Kultivierung von Yarrowia lipolytica 28 2.8. Gentechnische Methoden 29 2.8.1. Genomische DNA-Isolierung 29 2.8.2. Agarose-Gelelektrophorese 29 2.8.3. Polymerase Kettenreaktion 30 2.8.4. Verdau der DNA mit Restriktionsendonukleasen 31 2.8.5. DNA-Aufreinigung 31 2.8.6. Plasmidisolierung aus Escherichia coli 31 2.8.7. Dephosphorylierung 31 2.8.8. Ligation 32 2.8.9. Transformation elektrokompetenter Escherichia coli Zellen 32 2.9. Plasmidkonstruktion 33 2.9.1. Konstruktion der Expressionskassette für die Überexpression des Pyruvat-Carboxylase kodierenden Gens 33 2.9.2. Konstruktion der Expressionskassetten für die Überexpression der Gene der α- und β-Untereinheit der Succinyl-CoA Synthetase 34 2.9.3. Konstruktion der Deletionskassette des für die α-Untereinheit der Succinyl-CoA Synthetase kodierenden Gens 35 2.9.4. Konstruktion der Deletionskassette des für die β-Untereinheit der Succinyl CoA Synthetase kodierenden Gens 36 2.9.5. Sequenzierung konstruierter Plasmide 36 2.10. Transformation von Yarrowia lipolytica Zellen 37 2.10.1. Transformation von Yarrowia lipolytica mittels der LiAc-Methode 37 2.10.1.1. Herstellung chemisch kompetenter Yarrowia lipolytica Zellen 37 2.10.1.2. Transformation chemisch kompetenter Hefezellen 37 2.10.2. Herstellung elektrokompetenter Yarrowia lipolytica Zellen 38 2.10.3. Elektrotransformation von Yarrowia lipolytica 38 2.11. Southern Blot 39 2.11.1. Transfer der DNA auf eine Nylonmembran 39 2.11.2. Sondenherstellung 39 2.11.3. Immunologische Detektion 40 2.11.4. Strippen der Southern Blot Membran 40 2.12. Biochemische Methoden 41 2.12.1. Ernte und Aufschluss der Hefezellen 41 2.12.2. Aktivitätsbestimmung der Citrat-Synthase 41 2.12.3. Aktivitätsbestimmung der Aconitase 41 2.12.4. Aktivitätsbestimmung der NADP-abhängige Isocitrat-Dehydrogenase 42 2.12.5. Aktivitätsbestimmung der NAD-abhängige Isocitrat-Dehydrogenase 42 2.12.6. Aktivitätsbestimmung der α-Ketoglutarat-Dehydrogenase 43 2.12.7. Aktivitätsbestimmung der Succinyl-CoA Synthetase 43 2.12.8. Enzymatische Mitochondrienpräparation 46 2.12.9. Aktivitätsbestimmung der Succinat-Dehydrogenase 47 2.12.10. Aktivitätsbestimmung der Fumarase 47 2.12.11. Aktivitätsbestimmung der Malat-Dehydrogenase 48 2.12.12. Aktivitätsbestimmung der Isocitrat-Lyase 48 2.12.13. Aktivitätsbestimmung der Pyruvat-Carboxylase 48 2.12.14. Proteinbestimmung 49 2.13. Mikroskopische Bestimmung der Zellmorphologie 49 2.14. Kultivierung 49 2.14.1. Bestimmung der optischen Dichte 50 2.14.2. Animpfen der Hauptkultur 50 2.14.3. Succinatproduktionsmedium 50 2.14.4. Kultivierung unter Thiaminlimitation 51 2.14.5. Kultivierung unter Stickstofflimitation 52 2.14.6. Bestimmung organischer Säuren mittels Ionenchromatographie 52 2.15. Bioinformatik 53 3. Ergebnisse 54 3.1. Überexpression des Pyruvat-Carboxylase kodierenden Gens 54 3.2. Überexpression der Succinyl-CoA Synthetase kodierenden Gene 58 3.2.1. Bestimmung der Succinyl-CoA Synthetase Enzymaktivität 61 3.2.2. Bestimmung der spezifischen Aktivität weiterer Enzyme 61 3.3. Überexpression der für die Pyruvat-Carboxylase und Succinyl-CoA Synthetase kodierenden Gene 63 3.3.1. Bestimmung der spezifischen Aktivitäten der Enzyme des TCC und der PYC sowie der ICL 66 3.4. Deletion der Succinyl-CoA Synthetase Gene 68 3.4.1. Bestimmung der Succinyl-CoA Synthetase Enzymaktivität 72 3.4.2. Bestimmung weiterer spezifischer Enzymaktivitäten 73 3.5. Überexpression des Gens der Pyruvat-Carboxylase gemeinsam mit der Deletion des Gens für die β Untereinheit der Succinyl-CoA Synthetase 74 3.5.1. Bestimmung der spezifischen Aktivitäten von Enzymen des TCC, sowie der PYC und ICL 76 3.6. Morphologische Veränderungen der Transformanden 78 3.7. Produktion organischer Säuren im Succinatproduktionsmedium 80 3.9.1 Bestimmung der PYC-, ICL- und SDH-Aktivitäten während der Kultivierung im Succinatproduktionsmedium 87 3.8. α-Ketoglutarat-, Pyruvat- und Fumaratproduktion unter Thiaminlimitation 90 3.9. Citrat- und Isocitratproduktion unter Stickstofflimitation 96 4. Diskussion 100 4.1. Auswahl einer geeigneten C-Quelle für die Succinatproduktion 101 4.2. Reduktion der Succinatproduktion von Yarrowia lipolytica 102 4.3. Genetische Veränderungen, für die kein Einfluss auf die Succinatproduktion von Yarrowia lipolytica nachgewiesen wurde 106 4.3.1. Überexpression des Pyruvat-Carboxylase kodierenden Gens 107 4.3.2. Deletion der Succinyl-CoA Synthetase Gene 108 4.4. Erhöhung der Succinatproduktion von Yarrowia lipolytica 112 4.5. Auswirkungen auf die Produktion anderer organischer Säuren 119 4.5.1. α-Ketoglutarat-, Pyruvat- und Fumaratproduktion unter Thiaminlimitation 119 4.5.2. Citrat- und Isocitratproduktion unter Stickstofflimitation 121 4.6. Morphologische Veränderungen 122 4.6.1. Koloniemorphologie 122 4.6.2. Zellmorphologie 124 Literaturverzeichnis 126

A solid phase extraction (SPE) method was developed for simultaneous extraction of dicarboxylic acids and diols formed during hydrolysis of poly(butylene succinate), PBS, and poly(butylene adipate), PBA. The developed SPE method and subsequent GC-MS analysis were used to extract, identify and quantify low molecular weight products migrating from linear and branched poly(butylene adipate) (PBA) and poly(butylene succinate) (PBS) during aging in aqueous media. The combination of SPE and GC-MS proved to be a sensitive tool, able to detect small differences in the degradation rate during early stages of hydrolysis before any significant differences were observed by weight loss and molecular weight measurements. The detected low molecular weight products included monomers i.e. adipic acid and 1,4-butanediol for the PBA polymers and succinic acid and 1,4-butanediol for PBS. Several dimers and trimers i.e. hydroxybutyl adipate, hydroxybutyl succinate, di(hydroxybutyl) adipate, di(hydroxybutyl) succinate and hydroxybutyl disuccinate were also detected. Best extraction efficiency for 1,4-butanediol and succinic acid was achieved with a hydroxylated polystyrene-divinylbenzene resin as solid phase. Linear range for the extracted analytes was 1-500 ng/ml for adipic acid and 2-500 ng/ml for 1,4-butanediol and succinic acid. Detection and quantification limits for all analytes were between 1-2 ng/ml (S/N=3) and 2-7 ng/ml (S/N=10) respectively. Relative standard deviations were between 3 % and 7 %. Comparison of measured weight loss and the amount of monomeric products showed that weight loss during early stages of hydrolysis was mainly caused by the release of water-soluble oligomers that on prolonged ageing were further hydrolyzed to monomeric species. Significant differences in degradation rate could be assigned to degree of branching, molecular weight, aging temperature and degradation medium. Linear and branched PBA was mixed with PVC in solution cast films to study the effects of molecular weight and branching on plasticizer efficiency. Used as polymeric plasticizer, PBA formed a semi-miscible two-phase system with PVC where the amorphous part exhibited one single glass transition temperature and the degree of polyester crystallinity was dependent on molecular weight, degree of branching and blend composition. Plasticizing efficiency was favored by higher degree of branching and a 40 weight-percent polyester composition. / QC 20101209

This research focused on investigating and comparing the cytotoxicity and cellular uptake of RRR-alpha-tocopheryl polyethylene glycol succinate (TPGS, with that of alpha-tocopheryl succinate (α-TS). Both TPGS and α-TS are water-soluble forms of vitamin E with important clinical applications. Cytotoxicity assays with RAW 264.7 and LNCaP cells incubated overnight with TPGS or α-TS at concentrations ≥ 12.4 μM suggest that α-TS is more cytotoxic than TPGS. Macrophages were found to be more sensitive than LNCaP cells when treated with similar concentrations of α-TS. For both cell lines, most of the TPGS or α-TS taken up remained esterified after 24 hours. Our results suggest that cell death was due to TPGS and/or α-TS and not alpha-tocopherol. A para-hydroxyanilide of α-TS (p-HATS) that could be used to distinguish between cellular TPGS and α-TS was studied. It was found that p-HATS can be detected electrochemically and that it is hydrolyzed to α-TOH.