The thesis presented here describes the steps that were taken in an attempt to solve the protein structure of MenD via molecular replacement and multiple wavelength anomalous dispersion. The introduction provides background on menaquinone biosynthesis and the role of MenD in this metabolic pathway. Also, a detailed discussion of the DC Family of enzymes, a subgroup of ThDP dependent enzymes, which MenD is a part of, is included.<p> Utilizing various software packages a 1.9 Å data set was processed and analyzed in an attempt to provide a molecular replacement result. When molecular replacement was deemed incapable of solving the phase problem of the data set, the production of SeMet protein was attempted to allow for MAD phasing.<p> A homology model of MenD was produced using the program Modeller with benzaldehyde lyase as a template. A structure based sequence alignment was done with all DC Family enzymes with structures published. Then a second structure based sequence alignment was done to compare the same set to the Modeller model. This was done to gain a deeper understanding of MenD and how it interacts with its cofactors ThDP and Mg2+. Furthermore, these results were used to implicate potential active site residues.

MenD, a thiamin diphosphate (ThDP)-dependent enzyme, catalyzes the reaction from isochorismate (ISC) to 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC), and thus is also called SEPHCHC synthase. This conversion is the first committed step in the classical menaquinone (Vitamin K2) biosynthetic pathway, requiring 2-ketoglutarate (2-KG), ThDP and Mg²⁺. Since the biosynthesis of menaquinone is essential in some bacterial pathogens, for example <i>Mycobacterium tuberculosis</i>, MenD or the menaquinone pathway could be a target for drug development.<p> The method for the kinetic assay of the MenD-catalyzed reaction was evaluated by comparing UV spectrophotomeric measurements and HPLC analysis. It was validated that the steady-state kinetics of the MenD-catalyzed reaction can be determined by monitoring UV absorbance of ISC at 278 nm and 300 nm.<p> Phosphonate analogues of 2-KG were synthesized and assayed as inhibitors of the MenD reaction. It was found that the phosphonate analogues of 2-KG are competitive inhibitors with varied affinity for MenD. Of the inhibitors, monomethyl succinyl phosphonate (MMSP) was the most effective, with a <i>K</i>_i of 700 nM. However, the potent MenD inhibitors show no effectiveness against mycobacterial growth.<p> An analogue of isochorismate, trans-(±)-5-carboxymethoxy-6-hydroxy-1,3-cyclohexadiene-1-carboxylate ((±)-CHCD), was synthesized. The (+)-CHCD was found to be an alternative substrate for the MenD-catalyzed reaction. When CHCD was utilized in the MenD reaction, 5-carboxymethoxy-2-(3-carboxy-propionyl)-6-hydroxy-cyclohex-2-enecarboxylate (CCHC) was isolated and characterized, which was believed to be the product of spontaneous isomerization of the SEPHCHC-like analogue. The kinetic study of MenD reaction using (±)-CHCD, in association with the kinetics pattern probed by MMSP, demonstrated for the first time that the MenD-catalyzed reaction has a Ping Pong bi bi kinetic mechanism.<p> The analysis of sequence and structure of MenD from E. coli allowed the investigation of the active site residues and their catalytic functions by mutation of the individual residues. S32A, S32D, R33K, R33Q, E55D, R107K, Q118E, K292Q, R293K, S391A, R395A, R395K, R413K and I418L were prepared and assayed kinetically with respect to 2-KG, ISC, (±)-CHCD, ThDP and Mg²⁺. The values of <i>K</i>_m^a and <i>k</i>_cat^a/<i>K</i>_m^a for the mutants, in comparison with that of wild type MenD, provide valuable insight into the catalytic mechanism of MenD.

MenD, a thiamin diphosphate (ThDP)-dependent enzyme, catalyzes the reaction from isochorismate (ISC) to 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC), and thus is also called SEPHCHC synthase. This conversion is the first committed step in the classical menaquinone (Vitamin K2) biosynthetic pathway, requiring 2-ketoglutarate (2-KG), ThDP and Mg²⁺. Since the biosynthesis of menaquinone is essential in some bacterial pathogens, for example <i>Mycobacterium tuberculosis</i>, MenD or the menaquinone pathway could be a target for drug development.<p> The method for the kinetic assay of the MenD-catalyzed reaction was evaluated by comparing UV spectrophotomeric measurements and HPLC analysis. It was validated that the steady-state kinetics of the MenD-catalyzed reaction can be determined by monitoring UV absorbance of ISC at 278 nm and 300 nm.<p> Phosphonate analogues of 2-KG were synthesized and assayed as inhibitors of the MenD reaction. It was found that the phosphonate analogues of 2-KG are competitive inhibitors with varied affinity for MenD. Of the inhibitors, monomethyl succinyl phosphonate (MMSP) was the most effective, with a <i>K</i>_i of 700 nM. However, the potent MenD inhibitors show no effectiveness against mycobacterial growth.<p> An analogue of isochorismate, trans-(±)-5-carboxymethoxy-6-hydroxy-1,3-cyclohexadiene-1-carboxylate ((±)-CHCD), was synthesized. The (+)-CHCD was found to be an alternative substrate for the MenD-catalyzed reaction. When CHCD was utilized in the MenD reaction, 5-carboxymethoxy-2-(3-carboxy-propionyl)-6-hydroxy-cyclohex-2-enecarboxylate (CCHC) was isolated and characterized, which was believed to be the product of spontaneous isomerization of the SEPHCHC-like analogue. The kinetic study of MenD reaction using (±)-CHCD, in association with the kinetics pattern probed by MMSP, demonstrated for the first time that the MenD-catalyzed reaction has a Ping Pong bi bi kinetic mechanism.<p> The analysis of sequence and structure of MenD from E. coli allowed the investigation of the active site residues and their catalytic functions by mutation of the individual residues. S32A, S32D, R33K, R33Q, E55D, R107K, Q118E, K292Q, R293K, S391A, R395A, R395K, R413K and I418L were prepared and assayed kinetically with respect to 2-KG, ISC, (±)-CHCD, ThDP and Mg²⁺. The values of <i>K</i>_m^a and <i>k</i>_cat^a/<i>K</i>_m^a for the mutants, in comparison with that of wild type MenD, provide valuable insight into the catalytic mechanism of MenD.

The thesis presented here describes the steps that were taken in an attempt to solve the protein structure of MenD via molecular replacement and multiple wavelength anomalous dispersion. The introduction provides background on menaquinone biosynthesis and the role of MenD in this metabolic pathway. Also, a detailed discussion of the DC Family of enzymes, a subgroup of ThDP dependent enzymes, which MenD is a part of, is included.<p> Utilizing various software packages a 1.9 Å data set was processed and analyzed in an attempt to provide a molecular replacement result. When molecular replacement was deemed incapable of solving the phase problem of the data set, the production of SeMet protein was attempted to allow for MAD phasing.<p> A homology model of MenD was produced using the program Modeller with benzaldehyde lyase as a template. A structure based sequence alignment was done with all DC Family enzymes with structures published. Then a second structure based sequence alignment was done to compare the same set to the Modeller model. This was done to gain a deeper understanding of MenD and how it interacts with its cofactors ThDP and Mg2+. Furthermore, these results were used to implicate potential active site residues.

[pt] Constatou-se que os atos piratas no Golfo da Guiné, especialmente em Nigéria, estão fortemente associados ao roubo de cargas de petróleo e à pilhagem das instalações de exploração do produto. As ações dos perpetradores de tais atos, como é o caso da Nigéria que se debruçou mais, visam atingir o Estado Federal e as multinacionais exploradoras do petróleo bruto nacional. Com base na Teoria dos Complexos Regionais de Segurança e nos conceitos teóricos sobre a securitização, procurou-se analisar a pirataria na região estudada como uma ameaça existencial socialmente construída e como um objeto securitizado por uma variedade de atores políticos (regionais e internacionais). O objetivo cabal é mostrar que, no atual contexto da corrida pelo petróleo no Golfo da Guiné, a securitização da pirataria provoca a despolitização de questões econômicas e sociais inerentes aos Estados locais e a ameaça suas soberanias e integridades territoriais. / [en] It was found that pirates acts in the Gulf of Guinea, especially in Nigeria, are strongly associated with the theft of oil cargo and the pillaging of the product exploration installations. The actions of the perpetrators of such acts, as is the case of Nigeria which leaned more, aim to achieve the Federal State and the multinationals exploiting the national crude oil. Based on the Theory of Regional Security Complex and on the theoretical concepts of securitization, we tried to analyze piracy in the region studied as a socially constructed existential threat and a securitized object by a variety of political actors (regional and international). The full goal is to show that, in the current context of the race for oil in the Gulf of Guinea, the securitization of piracy causes the depoliticization of economic and social issues related to local states and threatens their sovereignty and territorial integrity.

Corporations in the resource extraction industry are frequently criticized and their operations opposed by local communities demanding more benefits, compensation for negative consecuences or oppose resource extraction altogether. Research has focused extensively on nonviolent and violent resistance campaigns that target state and quasi-state actors attempting regime change or self-determination. However, campaigns targeting corporations have received little attention so far. This thesis addresses this gap. I argue that nonviolent campaigns have a strategic advantage over violent campaigns in building leverage and forcing corporations to fulfill their demands because they are able to mobilize more numerous and diverse support and have a higher tactical diversity. I test the hypothesis that nonviolent campaigns are more likely to succeed in achieving their objectives and the expected causal mechanism in a qualitative comparative case study using the structured focused comparison method and aspects of process tracing. Applying a most- similar case selection, I select nonviolent and violent resistance campaigns targeting (multinational) corporations in Nigeria and Colombia. I find partial support for the hypothesis. However, limited data availability does not allow for a conclusive evaluation of the theorized causal mechanism. Findings indicate the value of studying resistance campaigns targeting corporations. In particular, future research should use a more fine-grained analysis of causal mechanisms linking the type and outcome of campaigns in this context. Additionally, applying large-n research designs allowing for greater generalizability of findings would be a valuable contribution in the future.