Mitochondria are essential for the viability of mammalian cells and provide a compartment for specific chemical reactions. Cellular respiration -- the main mitochondrial function -- is tightly connected with ROS production: the mitochondrial electron transport chain complexes I and III are the main ROS sources in mammalian cells. It has been reported that complex I and complex III activities are essential for cell cycle, apoptosis and stem cell differentiation (Spitkovsky et al., 2004; Varum et al., 2009; Lee et al., 2011; Ma et al., 2011; Tormos et al., 2012). In our work, we aimed to investigate the role of mitochondrial electron transport chain activity in the regulation of the differentiation potential and to unravel signaling pathways that could participate in this regulation. As a model, we used the P19 pluripotent stem cell line that can be easily differentiated into trophoblasts, expressing intermediate filaments cytokeratin 8/18, and neurons, which express cytoskeleton protein beta-III-tubulin. We first showed that both trophoblast and neural differentiation of P19 cells were accompanied by activation of cellular respiration. The analysis of respiratory chain complexes and supercomplexes, however, showed that undifferentiated P19 cells, as well as their differentiated derivatives did not differ in their respiratory machinery, including functional respirasomes. While undifferentiated cells did not use respiration as the main energy source, cellular respiration was activated during differentiation, indicating that oxidative metabolism was important for efficient differentiation. To investigate the potential role of mitochondrial electron transport chain activity we monitored the influence of a disrupted electron flow on the differentiation of P19 cells. We found that the activity of complex I and complex III influenced the differentiation potential of the pluripotent P19 cell line: the presence of complex I and complex III inhibitors rotenone, antimycin A, or myxothiazol increased the amount of cytokeratin 8/18+ cells during trophoblast differentiation, but almost completely prevented the formation of neuron-like beta-III-tubulin+ cells during neuron differentiation. Moreover, a low oxygen level (1 % O2 vs 21 % O2 in atmosphere) - the final electron acceptor - had the same effect on differentiation. These data suggest that mitochondrial electron transport chain activity contributes to the regulation of differentiation. The presence of complex I and complex III inhibitors, as well as oxygen scarcity, increase ROS production. We suggested that increased ROS level could explain the observed effects. By visualizing mitochondrial superoxide production with a specific dye – MitoSox - we confirmed that rotenone, antimycin A, myxothiazol, as well as low oxygen conditions, increased the superoxide level. These results suggest that the observed changes of the differentiation potential of P19 cells are associated with ROS production. To prove this idea, we differentiated P19 cells in presence of paraquat – a known ROS inducer. In line with our hypothesis paraquat promoted trophoblast differentiation. The received results suggest that the mitochondrial electron transport chain activity regulates differentiation through the ROS level. ROS are secondary messengers that participate in numerous processes including cell proliferation and differentiation. We aimed to predict the signal pathway that connects ROS level in stem cells and their differentiation potential. For this purpose, we performed a microarray analysis and compared the gene expression profiles of cells grown under hypoxia or in the presence of the complex III inhibitor myxothiazol with untreated control cells. The expression analysis revealed p53 as a transcriptional factor that impacts the differentiation potential in treated cells. p53 is a known redox-sensing molecule (Bigarella et al., 2014) that influences the differentiation potential through cell cycle control (Maimets et al., 2008). This observation is in line with our results and suggests that p53 may regulate the differentiation potential of P19 cells. We are planning to investigate the role of p53 signaling in the regulation of cell cycle and differentiation potential of P19 cell line.

Increasing freight transportation volumes continue to increase problems related to human health, congestion on the transport infrastructure, noise, climate changes, etc. Governments often want to minimize these negative effects, and this wish is expressed in societal goals, e.g., to reach environmental targets. An important instrument for reaching societal goals is governmental control policies, e.g., regulations, taxes and fees, which can influence the behaviour of the actors in a transport chain. Before implementing such control policies, it is crucial to predict their effects in order to make probable that it is a good measure. A review of models that consider the effects of governmental control policies on transportation has been performed which shows that macro-level models are mainly used for this purpose. However, the behaviour of the individual transport chain actors can hardly be captured in such models since the decision making actors are not modelled explicitly. Consequently, the negative effects caused by the decisions taken by the individual transport chain actors are not fully captured in these models. We believe that micro-level models have the potential to bridge the gap between governmental policy-making and the behaviour of transport chain actors. A micro-level model based on agent-technology has been developed which captures the environmental, quality and economical performance in transport chains, given different governmental control policies. The transport chain actors are represented by decision-making agents in the model. Logistical factors for characterizing transport chains have been identified and described according to degree of influence. To illustrate the usage of the micro-level model, simulation experiments based on a real world case have been performed where different levels of governmental control policies are introduced. The simulation results so far have showed that the model seems to simulate the behaviour of the transport chain actors correctly in the studied scenarios. The simulation tool can then be used as a decision support for policy-makers and serve as a complement to existing tools based on macro-level models.

The isolation and antimycobacterial activity of several naphthoquinones from Euclea natalensis were previously reported and initiated this study into the occurrence, chemistry and biological activity of this class of compounds. The structure activity relationship of the isolated naphthoquinones, and commercially available derivatives were also studied. Several plant species were investigated to establish a possible link between their traditional use for chest related symptoms (including tuberculosis infection) and the occurrence of 7-methyljuglone in these plants. The plants were extracted and tested qualitatively with the use of three analytical tools for the presence of 7-methyljuglone or related naphthoquinones. Due to its commercial unavailability, the chemical synthesis of two of these naphthoquinones, 7-methyljuglone and diospyrin, was attempted with varying degrees of success. The Friedel-Crafts acylation method was used to synthesise 7-methyljuglone from m-cresol and maleic anhydride as starting material. The optimisation of the synthesis was also investigated. Through a two-step pathway of epoxidation and steam distillation, diospyrin was successfully synthesised albeit in small quantities. During the attempts to synthesise diospyrin, two other related compounds were also synthesised. These compounds, neodiospyrin and mamegakinone, are structural isomers of diospyrin. The stability of some of the naphthoquinones was tested in various carriers in an attempt to explain the influence this will have on the obtained antituberculosis and toxicity data. The BACTEC vial solution, which is widely used to determine potency against Mycobacterium tuberculosis, was analysed with HPLC to determine the stability of these compounds in it. In addition the stability in organic solvents especially DMSO, was also tested as this is the solvent of choice for hydrophobic compounds in almost all bioassays. The antituberculosis activity and/or toxicity of 7-methyljuglone was investigated with three bioassays, to broaden our knowledge on the mechanism of action of naphthoquinones. Vero cells were employed to determine the inhibitory concentration (IC50) of most of the naphthoquinones. Mice experiments were carried out to determine the toxicity of 7-methyljuglone and diospyrin in vivo. In addition the lead compound, 7-methyljuglone, was tested on Musca domestica (house fly) to establish its toxicity on this organism. In order to find the pharmacophore of this class of compounds, a preliminary structure-activity relationship was conducted. During this study the active site in the compounds which confers potency and toxicity was partly established. The mode of action of some of the naphthoquinones was investigated and it was established that the compounds might interfere with the mycobacterial electron transport chain. A fluorinated 7-methyljuglone stops the production of menaquinone which transports electrons from the NADH dehydrogenase complex to the cytochrome bc complex and effectively kills the mycobacterium. / Thesis (PhD (Botany))--University of Pretoria, 2007. / Plant Science / unrestricted

Freight demand models have not been developed that much as passenger demand models. The reason is existence of too many complexities in this area. To estimate a disaggregate freight transport models large input data is required. The Swedish Flow Commodity survey 2004/2005 (CFS) which is a unique data source at the level of individual firms made it possible to estimate a disaggregate model to analyze the choice of transport chains and shipment size for the domestic metal products. The output of logistics module of the Swedish national freight transport (SAMGODS) is used as an auxiliary database to incorporate logistics decisions which CFS lacks in the model. The model comprises logistics perspective by considering both shipment size and transport chains as endogenous choices. Characteristics of shippers, shipments and transport chains are included in the model to analyze the choice of transport chain and shipment size. It has been tried to include as many transport chains as possible in the choice sets to consider their effects on decision making. Transport costs have been included in the model as shipment size specific to incorporate the concept of logistics more precisely in the model. From the results it can be seen that the freight transport demand is almost inelastic to the cost. The model gives a positive sign for the coefficient of the transport time which can be explained as the storage cost is so high that shippers prefer to use transport modes as the moving inventories instead. Finally, it is suggested to estimate panel discrete choice models on this dataset.

The cardiac toxicity of doxorubicin (DOX), a potent anticancer anthracycline antibiotic, is believed to be mediated through the generation of reactive oxygen species (ROS) in cardiomyocytes. This study aims to determine the function of cellular glutathione peroxidase (Gpx1), which is located in both mitochondria and cytosol, in defense against DOX-induced cardiomyopathy using a line of transgenic mice with cardiac overexpression of Gpx1. The Gpx1-overexpressing hearts were markedly more resistant than nontransgenic hearts to DOX-induced acute functional derangements, including impaired contractility and diastolic properties, decreased coronary flow rate, and reduced heart rate. In addition, DOX treatment impairs mitochondrial function of nontransgenic hearts as evident in a decreased rate of NAD-linked State 3 respiration, presumably a result of inactivation of complex I activity. This is associated with increases in the rates of NAD- and FAD-linked State 4 respiration and declines in P/O ratio, suggesting that the electron transfer and oxidative phosphorylation are uncoupled in these mitochondrial samples. These functional deficits of mitochondria could be largely prevented by Gpx1 overexpression. Taken together, these studies provide new evidence to further support the role of ROS, particularly H2O2 and/or fatty acid hydroperoxides, in causing contractile and mitochondrial dysfunction in mouse hearts acutely exposed to DOX.

abstract: This study aims to address the deficiencies of the Marcus model of electron transfer (ET) and then provide modifications to the model. A confirmation of the inverted energy gap law, which is the cleanest verification so far, is presented for donor-acceptor complexes. In addition to the macroscopic properties of the solvent, the physical properties of the solvent are incorporated in the model via the microscopic solvation model. For the molecules studied in this dissertation, the rate constant first increases with cooling, in contrast to the prediction of the Arrhenius law, and then decreases at lower temperatures. Additionally, the polarizability of solute, which was not considered in the original Marcus theory, is included by the Q-model of ET. Through accounting for the polarizability of the reactants, the Q-model offers an important design principle for achieving high performance solar energy conversion materials. By means of the analytical Q-model of ET, it is shown that including molecular polarizability of C60 affects the reorganization energy and the activation barrier of ET reaction. The theory and Electrochemistry of Ferredoxin and Cytochrome c are also investigated. By providing a new formulation for reaction reorganization energy, a long-standing disconnect between the results of atomistic simulations and cyclic voltametery experiments is resolved. The significant role of polarizability of enzymes in reducing the activation energy of ET is discussed. The binding/unbinding of waters to the active site of Ferredoxin leads to non-Gaussian statistics of energy gap and result in a smaller activation energy of ET. Furthermore, the dielectric constant of water at the interface of neutral and charged C60 is studied. The dielectric constant is found to be in the range of 10 to 22 which is remarkably smaller compared to bulk water( 80). Moreover, the interfacial structural crossover and hydration thermodynamic of charged C60 in water is studied. Increasing the charge of the C60 molecule result in a dramatic structural transition in the hydration shell, which lead to increase in the population of dangling O-H bonds at the interface. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2019

La bactérie Salmonella enterica sérovar Typhi est l’agent responsable de la fièvre typhoïde qui cause 20 millions de cas et plus de 200 000 morts annuellement. Elle possède plusieurs facteurs de virulence y compris 14 fimbriae, des structures protéiques que l’on retrouve à la surface de certaines bactéries. Plusieurs facteurs impliqués dans la chaîne de transport d'électrons (CTE) influencent l’expression du fimbriae fim, tel que Ndh, une NADH déshydrogénase (NADH-2), qui accepte et transmet les électrons du complexe I au pool de quinones entre le complexe II et III, et est un facteur majeur de la respiration aérobie chez les bactéries qui régit l'équilibre entrele NADH et NAD, et YqiC, une protéine multifactorielle et un facteur accessoire des ubiquinones, ont été identifiés comme activateur du fimbriae Fim. Notre hypothèse est que la CTE est impliquée dans la régulation du fimbriae fim. Nous voulions déterminer si la CTE régule l’expression du fimbriae fim de façon directe ou indirecte. Pour ce faire, nous avons déterminé le rôle du complexe I ; l’effet du stress oxydatif; et le rôle de l’accepteur final d’électron. Plusieurs mutants ont été créés par échange allélique et l’expression de fim chez ces mutants est quantifiée à l’aide d’un gène rapporteur, soit une fusion du promoteur fim avec le gène rapporteur lacZ. Pour déterminer le rôle du complexe I de la CTE, nous avons testé le mutant NDH-1 et NDH-2, qui s’avère à jouer un rôle important essentiel pour la régulation de fim. Pour évaluer l’effet du stress oxydatif, nous avons testé les mutants OxyR, SodB, la catalase KatG et les 3 peroxydases (Tpx, AhpC et TsaA) qui détoxifient les espèces réactives d’oxygènes (ROS) produites lors de la respiration. L’absence combinée de la catalase KatG et de la peroxydase AhpC empêche totalement l’expression de fim. Le rôle de l’accepteur final d’électron a été évalué par la présence ou absence d’oxygène, avec ou sans ajout de nitrate, nous suggérant un rôle important de YqiC pour la synthèse des transporteurs d’électrons, la génération d’ATP et le maintien du métabolisme bactérien en cas de stress oxydatif. Cette étude a permis d’identifier Ndh (NDH-2) comme une cible thérapeutique potentielle puisqu’elle ne se retrouve pas dans la chaîne d’électron de la mitochondrie chez les mammifères, donc chez les humains. Aussi, cette étude a permis de cibler les gènes détoxifiant essentiels à la régulation de fim, devenant eux aussi des cibles thérapeutiques potentielles. / The bacterium Salmonella enterica serovar Typhi is the causative agent of typhoid fever, which causes 20 million cases and more than 200,000 deaths annually. It has several virulence factors including 14 fimbriae, protein structures found on the bacterial surface. Several factors involved in the electron transport chain (ETC) influence expression of the fim fimbriae, such as Ndh, an NADH dehydrogenase (NDH-2), which accepts and transports electrons from complex I to the quinone pool between the complex II and III, and is a major factor in aerobic respiration in bacteria that governs the balance between NADH and NAD, and YqiC, a multifactorial protein and an ubiquinone biosynthesis accessory factor, have been identified as an activator of Fim fimbriae . Our hypothesis is that ETC is involved in the regulation of the fim fimbriae. Our aim was to determine whether ETC regulates the expression of the fim fimbriae directly or indirectly. To do this, we determined the role of complex I; the effect of oxidative stress; and the role of the final electron acceptor. Several mutants were created by allelic exchange and fim expression in these mutants was quantified using a reporter gene, i.e. a fusion of the fim promoter with the lacZ reporter gene. To determine the role of ETC complex I, we tested the NDH-1 and NDH-2 mutants, which appears to play an important role essential for fim regulation. To assess the effect of oxidative stress, we tested mutants of OxyR, SodB, catalase KatG and the 3 peroxidases (Tpx, AhpC and TsaA) which detoxify reactive oxygen species (ROS) produced during respiration. When both the catalase KatG and the peroxidase AhpC were mutated, the fim expression was totally turned off. The role of the final electron acceptor was evaluated by the presence or absence of oxygen, with or without addition of nitrate, suggesting an important role of YqiC for electron carrier synthesis, the generation of ATP and the maintain bacterial metabolism in the event of oxidative stress. This study made it possible to identify Ndh (NDH-2) as a potential therapeutic target since it is not found in the electron chain of the mitochondria in mammals, therefore in humans. Also, this study made it possible to target the detoxifying genes essential to fim regulation, also becoming potential therapeutics targets.

Mitochondrial ATP production is achieved using the electron transport chain (ETC), whereby the controlled oxidation of biomolecules is coupled to the activity of ATP synthase. ETC complexes organize into supramolecular structures called supercomplexes (ETC SCs). Protein factors regulating ETC SCs remain largely unknown despite their fundamental implications to mitochondrial respiratory function. Recent knock-out studies have delineated external ETC proteins HIGD1A and HIGD2A as assembly factors of ETC complexes III and IV, and their incorporation into SCs. In order to clarify the primary functions of HIGD1A and HIGD2A, as well as other previously uncharacterized HIG1 protein family members, stable overexpression (OE) models of each HIG1 protein were generated in HEK293t cells to preform comparative studies. We uncover a general dichotomy in the effects observed from HIGD2A vs. HIGD1A/1B/1C OE. Furthermore, we demonstrate that the previously unstudied protein family member HIGD1C is a negative regulator of complex IV SCs. A very limited number of protein factors specifically regulating the I1III2IV1 “respirasome” ETC SC have been identified. We propose a new framework where select complex I accessory subunits regulate respirasome assembly through protein-protein interactions between ETC complexes. Through specific point mutations to one such subunit, we generate a novel cell model with selective disassembly of the respirasome but otherwise functional individual ETC complexes. We demonstrate that respirasome disassembly limits respiration and modifies electron transfer pathways within the ETC. These findings to respirasome assembly and function may represent just a portion of higher order regulation that we are beginning to describe within eukaryotic metabolism.

No description available.

Aspergillus fumigatus é um fungo oportunista, sendo uma importante causa de morbidade e mortalidade entre os pacientes imunossuprimidos, acometidos com aspergilose invasiva. O sistema de defesa antioxidante do fungo atua como fator primordial de sua patogenicidade, mitigando os efeitos deletérios de espécies reativas produzidas pelo hospedeiro. Este trabalho teve o objetivo de avaliar o nível de transcrição relativo dos genes associados às proteínas mitocondriais e antioxidantes na presença de diferentes agentes pró-oxidantes para hifas e germinantes de cepas controle (CEA17) e nocaute para oxidase alternativa (?aoxA) de A. fumigatus. Utilizando qPCR, foram analisados 23 genes para as cepas CEA17 e mutante, dentre os quais, 5 associados à cadeia de transporte de elétrons (CTE), 4 relacionados aos componentes alternativos mitocondriais e 14 associados às proteínas antioxidantes. Os resultados foram obtidos através da normalização com o gene constitutivo gapdh utilizando a cepa CEA17 sem tratamento de cada fase de desenvolvimento celular como calibrador. As diferenças de transcrição da maioria dos genes estudados variaram com o pró-oxidante utilizado e com a fase de desenvolvimento celular (germinantes e hifas). A CTE em germinantes apresentou maiores níveis de transcritos dos complexos II e III em CEA17 do que em ?aoxA tratadas com menadiona. Com os demais complexos e atpase não foram observadas diferenças significativas quando comparados os resultados de ambas as cepas. Entretanto, os níveis de transcritos de atpase não foram superiores ao controle em nenhuma das cepas, sugerindo sua regulação na presença de ucp. ucp-like teve seus níveis de transcritos aumentados no mutante, sugerindo o desacoplamento da CTE em relação ao controle e à CEA17, que apresentou alto nível de transcritos de nde. Nesta cepa, os níveis de aoxA e ucp não foram significativamente diferentes. Esse fato, associado a níveis menores de ndiA e transcritos dos complexos I e II, sugerem manutenção do fluxo de elétrons na mitocôndria. Em relação às enzimas antioxidantes, houve aumento significativo nos níveis de cat2, catA, prx1, trx1 e grx1, indicando provável resposta à ausência de AOX. Os transcritos relacionados à CTE em hifas não apresentou diferença significativa nos níveis de complexo III e atpase em CEA17 e ?aoxA. No entanto, complexo IV se mostrou superior no mutante. Nesta fase de desenvolvimento, o aumento dos níveis de ucp-like está relacionado ao tratamento com menadiona e não com a presença de aoxA, explicando os mesmos níveis de transcritos em CEA17 e ?aoxA. Os níveis de nde sugerem atividade coordenada de sua proteína com AOX, por seus níveis estarem modulados pela presença de aoxA. A participação da Nde e da AOX na detoxificação das espécies reativas de oxigênio é sugerida ao se obter altos níveis de seus transcritos em hifas tratadas com menadiona. Diferentemente de germinantes, transcritos de sod3 foram predominantes em hifas de CEA17, enquanto transcritos de sod2 foram maiores em ?aoxA. Entre as catalases, os níveis de catA foram maiores no mutante, bem como a atividade das catalases como um todo. prx1 e trx1 foram maiores em CEA17 e ii grx1 em ?aoxA. Porém, trx1 atingiu os mais altos níveis de transcritos em germinantes, indicando possível redução de proteínas tiólicas e ribonucleotídeos por diferentes vias, de acordo com a fase de desenvolvimento do fungo. As enzimas antioxidantes clássicas, como superóxido dismutase, catalase e enzimas tiólicas, apresentaram aumento de transcrição na maioria das condições estudadas. No entanto, menadiona e paraquat promoveram maiores variações nos transcritos do que o observado com peróxido de hidrogênio gerado pelo sistema glicose/glicose oxidase. / Aspergillus fumigatus is an opportunistic fungus which has been referred to as a major cause of morbidity and mortality in imunosupressed patients affected with invasive aspergillosis. The antioxidant defense system of the fungus plays an important role in its pathogenicity by overcoming the negative effects of the oxygen reactive species produced by host. This work aims evaluating the transcription of genes associated to mitochondrial and antioxidant proteins in the presence of different prooxidants for hyphae and germinants of control (CEA17) and alternative oxidase knockout (?aoxA) strains. Using qPCR, 23 genes have been analyzed, 5 of which are electron transport chain (ETC), 4 are related to alternative components of the mitochondria, and 14 are associated to antioxidant proteins. The results were obtained via normalization with the constitutive gene gapdh using untreated CEA17 strain in each developmental phase as calibrator. The differences in the transcription of most analyzed genes varied with the prooxidant and the developmental phase (germinants and hyphae). The ETC in germinants presented higher amounts of transcripts of the complex II and III in CEA17 than in ?aoxA treated with menadione. With other complexes and the atpase no significant difference has been noted between both strains. However, the transcript levels of ATP synthase were not higher than control in any strain, which is related to its regulation in the presence of UCP. UCP-like had increased transcription in mutant, suggesting uncoupling from the ETC, relative to control and CEA17, which showed high levels of nde. In this strain, aoxA and ucp-like transcripts were not significantly different. This fact associated the lower levels of ndiA and transcripts of the complexes I and II suggest the maintenance of the electron flow in the mitochondria. Concerning the antioxidant enzymes, there was significant increase in the transcript production of cat2, catA, prx1, Ttrx1 and Ggrx1, suggesting a response to the absence of AOX. The ETC transcripts in hyphae did not present significant difference in the levels of complex III and atpase in CEA17 and ?aoxA. Yet the complex IV has shown higher in mutant than in CEA17. In this developmental phase, the increase of ucp-like is related to the treatment with menadione and not with the presence of aoxA, what explains the equivalent amount of transcripts in CEA17 and ?aoxA. The levels of nde suggest coordinated action of the protein with AOX, because it is modulated by the presence of aoxA. The role of Nde and AOX in the detoxification of reactive oxygen species is suggested by their high levels of transcripts in hyphae treated with menadione. Different from germinants, transcripts of sod3 was predominant in CEA17 hyphae, whereas transcripts of sod2 were higher for ?aoxA. Among catalases, levels of catA were higher in mutant, as was the activity of catalase as a whole. prx1 and trx1 transcripts were higher in CEA17 and grx1 in ?aoxA. Yet, trx1 reached the highest levels of transcripts in germinants, indicating a possible reduction of thiol proteins and ribonucleotides through different ways, according to the developmental phase of the fungus. The classical antioxidant enzymes, as superoxide dismutase, catalase and thiol enzymes showed increase in the transcripts in most of the analyzed situations. iv However, menadione and paraquat promoted higher variation in the transcripts than that observed with hydrogen peroxide generated by the system glucose/glucose oxidase.