Beta 2 glycoprotein I (β2GPI) is a major antigenic target in antiphospholipid syndrome (APS). In vitro studies suggest that it may have multifaceted physiological functions, as it displays both anticoagulant and procoagulant properties. Beta 2GPI may bind to FXI and serve as a regulator of FXI activation by thrombin. The possible interaction of β2GPI with thrombin is investigated using enzyme linked immunosorbent assays and surface plasmon resonance based studies. It is demonstrated for the first time that domain V of β2GPI is involved in direct binding to thrombin, and exosites I and II on thrombin take part in this interaction. It is also shown that cleavage of β2GPI at Lys317-Thr318 does not interrupt this binding. A quaternary complex is proposed on the surface of activated platelets in which β2GPI may colocalise with FXI and thrombin to regulate FXIa generation. The effect of anti-β2GPI monoclonal antibodies (mAbs) were investigated on this system using 8 anti-β2GPI mAbs directed against domain I. Anti-β2GPI Abs potentiate the suppressing activity of β2GPI on FXI activation by thrombin. Moreover, they restore the inhibitory effect of clipped β2GPI on this system. The current study demonstrates for the first time a novel biological consequence of thrombin interaction with β2GPI. The effect of β2GPI on thrombin inactivation by the serine protease inhibitor heparin cofactor II (HCII) is investigated using chromogenic assays, platelet aggregation studies, and the platelet release response. The current work shows that β2GPI protects thrombin from inactivation by HCII/Heparin. This ability is modulated by the cleavage of β2GPI. A ternary structure is proposed between β2GPI, thrombin and heparin which may limit the N-terminus of HCII to exosite I therefore inhibit thrombin inactivation by HCII. The effect of anti-β2GPI Abs is examined in this system using patient polyclonal IgGs and a murine anti-β2GPI mAb. Anti-β2GPI Abs potentiate the protective effect of β2GPI on thrombin inhibition by HCII/Heparin. In view of the importance of HCII in regulating thrombin activity within the arterial wall, disruption of this function by β2GPI/anti-β2GPI Ab complexes may be particularly relevant in arterial thrombosis in APS. Beta 2 glycoprotein I (β2GPI) is a major antigenic target in antiphospholipid syndrome (APS). In vitro studies suggest that it may have multifaceted physiological functions, as it displays both anticoagulant and procoagulant properties. Beta 2GPI may bind to FXI and serve as a regulator of FXI activation by thrombin. The possible interaction of β2GPI with thrombin is investigated using enzyme linked immunosorbent assays and surface plasmon resonance based studies. It is demonstrated for the first time that domain V of β2GPI is involved in direct binding to thrombin, and exosites I and II on thrombin take part in this interaction. It is also shown that cleavage of β2GPI at Lys317-Thr318 does not interrupt this binding. A quaternary complex is proposed on the surface of activated platelets in which β2GPI may colocalise with FXI and thrombin to regulate FXIa generation. The effect of anti-β2GPI monoclonal antibodies (mAbs) were investigated on this system using 8 anti-β2GPI mAbs directed against domain I. Anti-β2GPI Abs potentiate the suppressing activity of β2GPI on FXI activation by thrombin. Moreover, they restore the inhibitory effect of clipped β2GPI on this system. The current study demonstrates for the first time a novel biological consequence of thrombin interaction with β2GPI. The effect of β2GPI on thrombin inactivation by the serine protease inhibitor heparin cofactor II (HCII) is investigated using chromogenic assays, platelet aggregation studies, and the platelet release response. The current work shows that β2GPI protects thrombin from inactivation by HCII/Heparin. This ability is modulated by the cleavage of β2GPI. A ternary structure is proposed between β2GPI, thrombin and heparin which may limit the N-terminus of HCII to exosite I therefore inhibit thrombin inactivation by HCII. The effect of anti-β2GPI Abs is examined in this system using patient polyclonal IgGs and a murine anti-β2GPI mAb. Anti-β2GPI Abs potentiate the protective effect of β2GPI on thrombin inhibition by HCII/Heparin. In view of the importance of HCII in regulating thrombin activity within the arterial wall, disruption of this function by β2GPI/anti-β2GPI Ab complexes may be particularly relevant in arterial thrombosis in APS.

Emissions of CO$_2$ largely contribute to global warming. Carbon dioxide can be captured and used to produce value-added chemicals. This thesis focuses on bioelectrocatalysis as a green and sustainable approach. Our aim was to perform conversion of CO$_2$ to methanol via a multi-enzymatic cascade. However, for reactions involving oxidoreductases, ß-NAD is required as a cofactor. Its use in stoichiometric amounts is unprofitable. We address the issue by employing electrochemical regeneration of the cofactor. For the cascade, we expressed and purified formate dehydrogenase, formaldehyde dehydrogenase and alcohol dehydrogenase. Enzymes activity was tested and found to be low for two enzymes. A reliable method to detect methanol via headspace gas chromatography with a flame ionization detector was developed. We tested the cascade with employed in situ electrochemical cofactor regeneration. After two and a half hours of the reaction 4 µmol methanol were detected. Further research is needed to optimize the setup.

Enzymes have found applications in a broad range of industrial production processes. While high catalytic activity, selectivity and mild reaction conditions are attractive advantages of the biocatalysts, particularly costs arising from required cofactors pose a sever limitation. While cofactor-recycling systems are available, their use implies constraints for process set-up and conditions, which are a particular problem e.g. for solid-gas-phase reactions. Several oxidoreductases are able to directly exchange electrons with electrodes. Hence, the co-immobilization of both, an electron-utilizing and an electron-generating oxidoreductase on conductive nanoparticles should facilitate the direct electron flow from an enzymatic oxidation to a reduction reaction circumventing redox-cofactors requirements. In such a set-up, hydrogenases could generate and provide electrons directly form gaseous hydrogen. This thesis describes the co-immobilization of the oxygen tolerant hydrogenases from C. eutropha or C. metallidurans and cytochrome P450BM3 as test system. Conductive material in the form of carbon nanotubes (CNT) serves as a suitable support. A combination of the hydrogenase and the catalytic domain of P450BM3 immobilized on carbon nanotubes were tested for the oxidation of lauric acid in the presence of hydrogen instead of an electron-transport cofactor. The GC-MS analysis reveals the conversion of 4% of lauric acid (LA) into three products, which correspond to the hydroxylated lauric acid in three different positions with a total turnover (TON) of 34. The product distribution is similar to that obtained when using the wildtype P450BM3 with the nicotinamide adenine dinucleotide phosphate (NADPH) cofactor. Such electronic coupling couldn’t be achieved for the conversion of other substrates such as propane and cyclohexane, probably due to the high uncoupling rate within the heme-domain of cytochrome P450BM3 when unnatural substrates are introduced.

No description available.

No description available.

A lesão hepática por isquemia e reperfusão (I/R) ocorre em situações clínicas diversas, como nas grandes hepatectomias e no transplante hepático. A fisiopatologia desta lesão é composta, principalmente, por intensa resposta inflamatória aguda e estresse oxidativo hepático. Em consequência a essa lesão a proliferação pós hepatectomia pode ser prejudicada. Por outro lado, Pirroloquinolina Quinona (PQQ) é um cofator que vem sendo estudado em virtude de suas ações antioxidantes e de estimulação de crescimento. Materiais e métodos: Camundongos balb-c foram submetidos à lesão hepática por I/R normotérmica seguida de hepatectomia e tratados com PQQ (10mg/kg de peso corporal) ou solução salina. Foram avaliados o grau de lesão hepatocelular (nível sérico de aminotransferases e escore histológico de agressão tecidual), a intensidade do estresse oxidativo (dosagem de MDA, GSH, NRF2 e eNOS hepáticos), a intensidade da resposta inflamatória aguda (quantificação de NFkB, Il-1? e TNF-? no fígado) e a proliferação hepatocelular (quantificação hepática de PCNA e ciclina D1). Resultados: PQQ reduziu significativamente os parâmetros de lesão hepática. Adicionalmente, os animais tratados com PQQ exibiram reduzido estresse oxidativo hepático devido à preservação da reserva antioxidante de GSH, à estimulação de expressão de NRF2 e à diminuição dos níveis hepáticos de MDA. Todavia, os níveis de eNOS não sofreram alteração com o tratamento. A atividade inflamatória aguda também foi reduzida com regulação da expressão hepática de NFkB e IL-1?. Entretanto, TNF-?, PCNA e Ciclina D1 não apresentaram diferença estatisticamente significativa entre os grupos. Conclusão: Os resultados obtidos indicam que PQQ é capaz de proteger o fígado contra a lesão por IR normotérmica seguida de hepatectomia e que essa proteção foi relacionada à sua ação antioxidante e anti- inflamatória. Todavia, a capacidade proliferativa hepática não foi alterada pelo tratamento no tempo avaliado. / Liver injury following ischemia and reperfusion (IR) occurs in several clinical situations such as major hepatectomies and liver transplantation. The physiopathology of this damage is composed for hepatic acute inflammatory response and oxidative stress. As a result of this injury the proliferation can be impaired. In other hand, Pyrroloquinoline Quinone (PQQ) is a cofactor that has been studied because has antioxidant activity and stimulates growth. Materials and Methods: Balb-c mice were underwent to hepatic injury by normothermic I/R followed by partial hepatectomy, and treated with PQQ (10mg/kg body weight) or saline solution. We evaluate the degree of hepatocellular damage (Serum level of aminotransferases and histological score of tissue damage), the intensity of liver oxidative stress (measurement of MDA,GSH, NRF2 and eNOS hepatic levels), the intensity of inflammatory response (measurement of NFkB, IL-1? e TNF-? hepatic levels). Results: PQQ significantly reduced the parameters of hepatocellular damage. PQQ-treated animals showed decreased hepatic oxidative stress by preserving antioxidant reserve of GSH, expression stimulation of NRF2 and reduced hepatic level of MDA. However, eNOS levels dind\'t change with the treatment. Inflammatory activity was also reduced by the down- regulation of NF?B and IL1- ?. Nevertheless, TNF-? and PCNA and D1 Cyclin did not show significant differences between the animal groups . Conclusion: These data indicate that PQQ was able to protect the liver against normothermic IR injury followed by hepatectomy and that protection was related with its antioxidant and antiinflammatory capability. Although, the hepatic proliferative capacity was not altered by the treatment in the evaluate time.

<p>In case of the PDE's the concept of solving by separation of variables</p><p>has a well defined meaning. One seeks a solution in a form of a</p><p>product or sum and tries to build the general solution out of these</p><p>particular solutions. There are also known systems of second order</p><p>ODE's describing potential motions and certain rigid bodies that are</p><p>considered to be separable. However, in those cases, the concept of</p><p>separation of variables is more elusive; no general definition is</p><p>given.</p><p>In this thesis we study how these systems of equations separate and find that their separation usually can be reduced to sequential separation of single first order ODE´s. However, it appears that other mechanisms of separability are possible.</p>

In case of the PDE's the concept of solving by separation of variables has a well defined meaning. One seeks a solution in a form of a product or sum and tries to build the general solution out of these particular solutions. There are also known systems of second order ODE's describing potential motions and certain rigid bodies that are considered to be separable. However, in those cases, the concept of separation of variables is more elusive; no general definition is given. In this thesis we study how these systems of equations separate and find that their separation usually can be reduced to sequential separation of single first order ODE´s. However, it appears that other mechanisms of separability are possible.

Stochastic compartment models are widely used in modeling processes for biological populations. The residence time has been especially useful in describing the system dynamics in the models. The direct calculation of the distribution for the residence time of stochastic multi-compartment models is very complicated even with a relatively simple model and often impossible to calculate directly. This dissertation presents an analytical method to obtain the moment generating function for stochastic multi-compartment models and describe the distribution of the residence times, especially systems with nonexponential lifetime distributions. A common method for obtaining moments of the residence time is using the coefficient matrix, however it has a limitation in obtaining high order moments and moments for combined compartments in a system. In this dissertation, we first derive the bivariate moment generating function of the residence time distribution for stochastic two-compartment models with general lifetimes. It provides any order of moments and also enables us to approximate the density of the residence time using the saddlepoint approximation. The approximation method is applied to various situations including the approximation of the bivariate distribution of residence times in two-compartment models or approximations based on the truncated moment generating function. Special attention is given to the distribution of the residence time for multi-compartment semi-Markov models. The cofactor rule and the analytic approach to the two-compartment model facilitate the derivation of the moment generating function. The properties from the embedded Markov chain are also used to extend the application of the approach. This approach provides a complete specification of the residence time distribution based on the moment generating function and thus provides an easier calculation of high-order moments than the approach using the coefficient matrix. Applications to drug kinetics demonstrate the simplicity and usefulness of this approach.

Proteins have evolved over millions of years to serve a plethora of highly specialized functions in biological systems. Given the enormous diversity in structure and function, it is truly surprising that only 20 different amino acids are utilized as the building blocks of proteins. Furthermore, only a small set of metal cations that are biologically available are used as structural or catalytically active cofactors in proteins, whereas rare metal cations such as platinum, ruthenium or rhodium remain absent. In the 20th century myriad catalysts, based on non-biological transition metals, emerged that can facilitate numerous organic transformations. The goal of the thesis was to introduce new functions into proteins by attaching platinum metals and fluorescent metal sensors. Thus, semi-synthetic proteins for catalytic and analytical applications were generated. The replacement of organic solvents by environmentally benign solvents such as water is an imperative step towards achieving "green chemistry". The combination of small molecule catalysts with proteins may introduce new functions and take advantage of the benefits of "both worlds" while avoiding their potential drawbacks. Therefore semi-synthetic catalysts were developed for enantioselective organic reactions in aqueous medium. A suitable reaction, reaction conditions and catalytic system for later utilization in a semi-synthetic protein were designed, developed and characterized. Ruthenium porphyrins catalyzed cyclopropanation reactions with fair yields and high stereoselectivity in aqueous medium. The successful reaction in water was a crucial requirement for a catalytically active semi-synthetic protein. Mechanistic studies did not elucidate the actual catalytic species for the formation of the cyclopropanation product and the side-product diethyl maleate; however, new insights were gained from the analysis of potential reaction pathways. Moreover, studies of the influence of axial ligands, resembling likely residues coordinating to the ruthenium metal center in the active site of a semi-synthetic protein, on the carbene formation of ruthenium porphyrins illustrated that coordination of axial ligands may inhibit the catalytic activity. The generation of ruthenium porphyrin based semi-synthetic proteins and their subsequent catalysis of cyclopropanation reactions was carried out. Myoglobin and myoglobin mutants were successfully reconstituted with a heme-like ruthenium carbonyl porphyrin; however, none of the formed semi-synthetic proteins catalyzed the enantioselective cyclopropanation of styrene. Efforts to determine the reconstitution efficiency of the generated semi-synthetic were hampered by problems to purify the generated semi-synthetic proteins that are probably due to non-specific binding of the ruthenium porphyrin to the protein surface. The exploration of labile metal pools of the biologically relevant transition metals copper, iron and zinc in cells was the goal of developing semi-synthetic proteins for analytical applications. Combining fluorescent proteins with colored or fluorescent metal chelators by forming semi-synthetic proteins allows taking advantage of their beneficial properties while avoiding their downsides. This design offers an attractive platform for in vivo metal sensing. Plasmids encoding fluorescent proteins, targeting sequences and AGT or intein fusion domains (necessary for labeling) for eukaryotic and prokaryotic expression were generated. The targeting of intracellular compartments (mitochondria, nucleus and TGN) was successful (confirmed by light microscopy experiments with transfected mammalian cells). In vitro labeling experiments of expressed and purified fusion proteins with rhodamine derivatives succeeded with AGT based fusion proteins; however, labeling of fusion proteins by trans-splicing with split-inteins failed. A new Zinc(II)-chelator was attached to an AGT based protein and the resulting semi-synthetic protein exhibited strong changes of fluorescence in the presence of zinc(II). This represents an important step towards the goal of in vivo cell imaging of labile zinc(II) pools. Despite extensive efforts, all attempts failed to generate a chelator that forms Cu(I)-complexes with the 1:1 stochiometry (ligand:metal) that is necessary for metal sensing with semi-synthetic proteins.