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One of the many recorded adaptive responses in respiratory chain complex I deficient cells is the over-expression of the small metal binding proteins, metallothioneins (MTs). The antioxidant properties of MTs putatively protect the deficient cells against oxidative damage, thus limiting further damage and impairment of enzymes involved in energy production. Moreover, the role of metallothioneins in supplying metal cofactors to enzymes and transcription factors in order to promote energy metabolism was previously proposed, which could accompany their role as antioxidants. This view is supported by the observations that MT knockout mice tend to become moderately obese, implying a lower energy metabolic rate. Hence, the involvement of metallothioneins in mitochondrial function and disease cannot be ignored. However, this association is still very vague due to the diversity of their functions and the complexity of the mitochondrion. The use of systems biology technology and more specifically metabolomics technology was thus employed to clarify this association by investigating the metabolic differences between wild type and MT knockout mice in unchallenged conditions as well as when mitochondrial function (energy metabolism) was challenged with exercise and/or a high-fat diet. The metabolic differences between these mice were also studied when complex I of the respiratory chain was inhibited with rotenone. The metabolome content of different tissues and bio-fluids were examined in an untargeted fashion using three standardized analytical platforms and the data mined using modern metabolomics and related statistical methods. Clear metabolic differences were found between the wild type and MT knockout mice during unchallenged conditions. These metabolic differences were persisted and were often amplified when mitochondrial metabolism was specifically challenged through exercise, high-fat intake or complex I inhibition. The data pointed to an overall reduced metabolic rate in the MT knockout mice and possible insulin resistance after the interventions which imply (and confirm) the involvement of MTs in promoting energy metabolism in the wild type mice. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2012

One of the many recorded adaptive responses in respiratory chain complex I deficient cells is the over-expression of the small metal binding proteins, metallothioneins (MTs). The antioxidant properties of MTs putatively protect the deficient cells against oxidative damage, thus limiting further damage and impairment of enzymes involved in energy production. Moreover, the role of metallothioneins in supplying metal cofactors to enzymes and transcription factors in order to promote energy metabolism was previously proposed, which could accompany their role as antioxidants. This view is supported by the observations that MT knockout mice tend to become moderately obese, implying a lower energy metabolic rate. Hence, the involvement of metallothioneins in mitochondrial function and disease cannot be ignored. However, this association is still very vague due to the diversity of their functions and the complexity of the mitochondrion. The use of systems biology technology and more specifically metabolomics technology was thus employed to clarify this association by investigating the metabolic differences between wild type and MT knockout mice in unchallenged conditions as well as when mitochondrial function (energy metabolism) was challenged with exercise and/or a high-fat diet. The metabolic differences between these mice were also studied when complex I of the respiratory chain was inhibited with rotenone. The metabolome content of different tissues and bio-fluids were examined in an untargeted fashion using three standardized analytical platforms and the data mined using modern metabolomics and related statistical methods. Clear metabolic differences were found between the wild type and MT knockout mice during unchallenged conditions. These metabolic differences were persisted and were often amplified when mitochondrial metabolism was specifically challenged through exercise, high-fat intake or complex I inhibition. The data pointed to an overall reduced metabolic rate in the MT knockout mice and possible insulin resistance after the interventions which imply (and confirm) the involvement of MTs in promoting energy metabolism in the wild type mice. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2012

Metalotioneínas (MTs) são proteínas de baixa massa molecular, que tem como principal função a regulação dos níveis de metais nos organismos. A caracterização das MTs da cianobactéria Synechococcus PCC 7942 por eletroforese capilar foi feita em comparação com os padrões comerciais de MTs de rim de cavalo e de fígado de coelho. As MTs de mamíferos apresentam diferentes arranjos moleculares, classificadas em isoformas. Na aplicação da eletroforese capilar como metodologia analítica para a otimização da separação das isoformas existentes, foram investigados a influência da composição da solução eletrolítica, variações da voltagem, comprimento do capilar e diâmetro interno do capilar. Os perfis eletroforéticos das misturas das MTs purificadas a partir de rim de cavalo e fígado de coelho comparados com a de cianobactéria mostraram uma diferenciação no tempo de migração. Para a separação foram testados eletrólitos tais como fosfato, borato e TRIS-HCl, sendo que os melhores resultados foram obtidos com o tampão TRIS-HCl (70 mM, pH 8,2) com adição de 5% de metanol. A separação eletroforética foi testada em capilares de sílica fundida de 75 e 25 m d.i., comprimento de 40, 50 e 60 cm. As soluções das amostras em volume de 327 nL foram introduzidas por injeção hidrodinâmica. As diferenças de potencial testadas foram de 10, 15, 20 e 25 kV. As melhores condições de separação foram atingidas empregado TRIS-HCl com 5% metanol como solução eletrolítica, em capilar de 60 cm e diferença de potencial de 20 kV o que estabilizou a corrente de separação em 42 \'mü\'A. Os resultados mostraram que a eletroforese capilar mostrou-se eficiente para separação das MTs de mamifero e da Synechococcus devido às diferenças de carga e tamanho das moléculas / Metallothioneins (MTs) are low molecular weight proteins, which main functions are the regulation of metals levels in the body and detoxification. The capillary electrophoresis (CE) characterization of MT from Synechococcus cyanobacteria was attained by comparison with commercial standards of horse kidney and rabbit liver MTs. The influence of electrolyte, such as phosphate, borate and TRIS-HCl buffers on the separation performance were tested. Also, parameters such as voltage potential, capillary length and capillary inner diameter were investigated to attain optimized separation of mammal and Synechococcus MTs. The electrophoretic profiles of MTs revealed four abundant metallothionein isoforms for the horse kidney sample, one for rabbit liver MTII and two for cyanobacteria Synechococcus. The separation by CE of horse and cyanobacteria MTs mixtures differentiated two sets of signals, the first with four peaks corresponding to the horse sample and the last to Synechococcus. The mixture of rabbit liver MT and cyanobacteria MTs presented a first peak for rabbit MTII and a second for cyanobacteria. Tests were performed trying phosphate, borate and TRIS-HCl buffers, however the best results were attained with TRIS-HCl buffer (70 mM, pH 8.2) with addition of 5% methanol. Different capillary lengths of 40, 50 and 60 cm and two internal diameters of 75 and 25m were tested. Also, voltages of 10, 15, 20 and 25 kV were studied. The best experimental conditions were attained using a 60 cm long capillary, TRIS-HCl plus 5% methanol as electrolyte, the application of 20 kV which allowed maintaining a separation current of 42 \'mü\'A. Results demonstrated that capillary electrophoresis was efficient for separation of MTs of mammals from that of Synechoccocus due their differences on size to charge

Metalotioneínas (MTs) são proteínas de baixa massa molecular, que tem como principal função a regulação dos níveis de metais nos organismos. A caracterização das MTs da cianobactéria Synechococcus PCC 7942 por eletroforese capilar foi feita em comparação com os padrões comerciais de MTs de rim de cavalo e de fígado de coelho. As MTs de mamíferos apresentam diferentes arranjos moleculares, classificadas em isoformas. Na aplicação da eletroforese capilar como metodologia analítica para a otimização da separação das isoformas existentes, foram investigados a influência da composição da solução eletrolítica, variações da voltagem, comprimento do capilar e diâmetro interno do capilar. Os perfis eletroforéticos das misturas das MTs purificadas a partir de rim de cavalo e fígado de coelho comparados com a de cianobactéria mostraram uma diferenciação no tempo de migração. Para a separação foram testados eletrólitos tais como fosfato, borato e TRIS-HCl, sendo que os melhores resultados foram obtidos com o tampão TRIS-HCl (70 mM, pH 8,2) com adição de 5% de metanol. A separação eletroforética foi testada em capilares de sílica fundida de 75 e 25 m d.i., comprimento de 40, 50 e 60 cm. As soluções das amostras em volume de 327 nL foram introduzidas por injeção hidrodinâmica. As diferenças de potencial testadas foram de 10, 15, 20 e 25 kV. As melhores condições de separação foram atingidas empregado TRIS-HCl com 5% metanol como solução eletrolítica, em capilar de 60 cm e diferença de potencial de 20 kV o que estabilizou a corrente de separação em 42 \'mü\'A. Os resultados mostraram que a eletroforese capilar mostrou-se eficiente para separação das MTs de mamifero e da Synechococcus devido às diferenças de carga e tamanho das moléculas / Metallothioneins (MTs) are low molecular weight proteins, which main functions are the regulation of metals levels in the body and detoxification. The capillary electrophoresis (CE) characterization of MT from Synechococcus cyanobacteria was attained by comparison with commercial standards of horse kidney and rabbit liver MTs. The influence of electrolyte, such as phosphate, borate and TRIS-HCl buffers on the separation performance were tested. Also, parameters such as voltage potential, capillary length and capillary inner diameter were investigated to attain optimized separation of mammal and Synechococcus MTs. The electrophoretic profiles of MTs revealed four abundant metallothionein isoforms for the horse kidney sample, one for rabbit liver MTII and two for cyanobacteria Synechococcus. The separation by CE of horse and cyanobacteria MTs mixtures differentiated two sets of signals, the first with four peaks corresponding to the horse sample and the last to Synechococcus. The mixture of rabbit liver MT and cyanobacteria MTs presented a first peak for rabbit MTII and a second for cyanobacteria. Tests were performed trying phosphate, borate and TRIS-HCl buffers, however the best results were attained with TRIS-HCl buffer (70 mM, pH 8.2) with addition of 5% methanol. Different capillary lengths of 40, 50 and 60 cm and two internal diameters of 75 and 25m were tested. Also, voltages of 10, 15, 20 and 25 kV were studied. The best experimental conditions were attained using a 60 cm long capillary, TRIS-HCl plus 5% methanol as electrolyte, the application of 20 kV which allowed maintaining a separation current of 42 \'mü\'A. Results demonstrated that capillary electrophoresis was efficient for separation of MTs of mammals from that of Synechoccocus due their differences on size to charge

Deficiencies of the oxidative phosphorylation system (OXPHOS) that consists of five enzyme complexes (I-IV) lead to a diversity of cellular consequences. This includes altered Ca2+ homeostasis, reduced ATP production and increased ROS (reactive oxygen species) production. One of the secondary consequences of such deficiencies is the adaptive transcriptional responses of several mitochondrial- and nuclear-encoded genes involved in OXPHOS biogenesis. Additionally, several other genes that are involved in several other functions, such as metallothioneins (MTs), are differentially expressed. In this study we investigated two hypotheses: firstly, that in complex I deficient cells the increased expression of MTs, specifically MT1B and MT2A, has a protective effect against ROS-related consequences of a complex I deficiency. The second hypothesis stated that genes involved in mitochondrial replication and transcription are differentially expressed in OXPHOS deficient cell lines. Firstly, the expression and role of metallothioneins (MTs) in an in vitro complex I deficient model was investigated. The increased expression of different MT isoforms in the presence of the complex I inhibitor rotenone in HeLa cells was confirmed. In this complex I deficient model overexpression of MT1B and especially MT2A isoforms also protected against ROS, mtPTP opening, apoptosis and ROS-induced necrosis. This data supports the hypothesis that increased expression of MT2A has a protective effect against the death-causing cellular consequences of rotenonetreated HeLa cells. Secondly, we investigated the differential expression of selected mitochondrial- and nuclear genes involved in OXPHOS function and regulation. Two experimental in vitro models were developed and utilized in the study. Firstly, a transient siRNA knockdown model of the NDUFS3 subunit of complex I in 143B cells was developed, characterized and introduced. Then the effect of the knockdown on several biochemical parameters (ROS and ATP levels), mtDNA copy number, total mtRNA levels, and RNA levels of several nuclear- and mitochondrial-encoded transcripts encoding structural as well as functional proteins was determined. Additionally, to investigate the effect of stable OXPHOS deficiency, stable shRNA knockdown models of the NDUFS3 subunit of complex I, as well as the Rieske subunit of complex III were introduced and characterized. The second hypothesis about the effect of OXPHOS deficiencies on mtDNA replication and transcription could not, without a doubt, be supported or contradicted by the data. It was determined from the data that an OXPHOS deficiency, which does not result in increased ROS levels, does not significantly affect the regulation of mtDNA replication/transcription or nuclear OXPHOS gene transcription. However, when OXPHOS deficiency was accompanied by increased ROS levels, some structural mitochondrial-encoded transcripts and regulatory nuclear-encoded transcripts were up-regulated, specifically ND6, D-loop, DNApol and TFB2M. Nonetheless, increased ROS production in the presence of OXPHOS deficiency is probably not exclusively responsible for responses of all regulatory proteins involved in mtDNA replication/transcription in vitro. Additionally, this compensatory regulation might be more dependent on mtDNA transcription than mtDNA copy number, and the data showed that TFB2M might be a key regulatory protein involved early in this mechanism before any other regulatory proteins are affected. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2010.

Deficiencies of the oxidative phosphorylation system (OXPHOS) that consists of five enzyme complexes (I-IV) lead to a diversity of cellular consequences. This includes altered Ca2+ homeostasis, reduced ATP production and increased ROS (reactive oxygen species) production. One of the secondary consequences of such deficiencies is the adaptive transcriptional responses of several mitochondrial- and nuclear-encoded genes involved in OXPHOS biogenesis. Additionally, several other genes that are involved in several other functions, such as metallothioneins (MTs), are differentially expressed. In this study we investigated two hypotheses: firstly, that in complex I deficient cells the increased expression of MTs, specifically MT1B and MT2A, has a protective effect against ROS-related consequences of a complex I deficiency. The second hypothesis stated that genes involved in mitochondrial replication and transcription are differentially expressed in OXPHOS deficient cell lines. Firstly, the expression and role of metallothioneins (MTs) in an in vitro complex I deficient model was investigated. The increased expression of different MT isoforms in the presence of the complex I inhibitor rotenone in HeLa cells was confirmed. In this complex I deficient model overexpression of MT1B and especially MT2A isoforms also protected against ROS, mtPTP opening, apoptosis and ROS-induced necrosis. This data supports the hypothesis that increased expression of MT2A has a protective effect against the death-causing cellular consequences of rotenonetreated HeLa cells. Secondly, we investigated the differential expression of selected mitochondrial- and nuclear genes involved in OXPHOS function and regulation. Two experimental in vitro models were developed and utilized in the study. Firstly, a transient siRNA knockdown model of the NDUFS3 subunit of complex I in 143B cells was developed, characterized and introduced. Then the effect of the knockdown on several biochemical parameters (ROS and ATP levels), mtDNA copy number, total mtRNA levels, and RNA levels of several nuclear- and mitochondrial-encoded transcripts encoding structural as well as functional proteins was determined. Additionally, to investigate the effect of stable OXPHOS deficiency, stable shRNA knockdown models of the NDUFS3 subunit of complex I, as well as the Rieske subunit of complex III were introduced and characterized. The second hypothesis about the effect of OXPHOS deficiencies on mtDNA replication and transcription could not, without a doubt, be supported or contradicted by the data. It was determined from the data that an OXPHOS deficiency, which does not result in increased ROS levels, does not significantly affect the regulation of mtDNA replication/transcription or nuclear OXPHOS gene transcription. However, when OXPHOS deficiency was accompanied by increased ROS levels, some structural mitochondrial-encoded transcripts and regulatory nuclear-encoded transcripts were up-regulated, specifically ND6, D-loop, DNApol and TFB2M. Nonetheless, increased ROS production in the presence of OXPHOS deficiency is probably not exclusively responsible for responses of all regulatory proteins involved in mtDNA replication/transcription in vitro. Additionally, this compensatory regulation might be more dependent on mtDNA transcription than mtDNA copy number, and the data showed that TFB2M might be a key regulatory protein involved early in this mechanism before any other regulatory proteins are affected. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2010.

Ischemic injury is a common and debilitating outcome of natural illness and as a complication of commonly performed medical procedures. Whereas naturally occurring ischemic insults are often the result of unpredictable events, such as in the case of stroke or heart attack, the risk of operative and perioperative ischemia is somewhat better characterized in the clinical setting. Given the prevalence and severity of outcomes in ischemic injury, there is significant interest in developing better pharmacological and procedural approaches to improve patient outcomes. One approach that has shown significant promise in the laboratory setting, particularly in the context of planned medical procedures, is the use of delayed anesthetic preconditioning. Delayed anesthetic preconditioning is a phenomenon whereby a prior exposure to clinical concentrations of commonly used inhaled anesthetics, including isoflurane, induces the production of endogenous protective proteins that are able to provide robust protection against subsequent, potentially toxic, ischemic insults. Although many aspects of delayed anesthetic preconditioning have been previously described, a complete understanding of preconditioning mechanism has yet to emerge. The studies described in this dissertation aim to further our understanding of molecular mechanisms involved in delayed anesthetic preconditioning. In the first project, I used DNA microarray to identify genes that were differentially expressed in adult rat liver, kidney and heart following a clinically relevant exposure to the inhaled anesthetic isoflurane. By selecting those genes that were differentially expressed in multiple tissues, I was able to identify a small group of interesting genes for further study. In my second study, I chose from our list two related genes, metallothioneins I + II, to analyze for a role in anesthetic-mediated protection. Using a combination of approaches, I was able to establish that metallotioneins I + II play an essential role in delayed anesthetic preconditioning. In the final study of this dissertation I explore a possible role for metallothioneins I + II as sensor molecules, involved in detecting cellular oxidative stress. Taken together, these three studies represent an important contribution to our understanding of the mechanisms of delayed anesthetic preconditioning and how they might contribute to protecting against ischemic stroke.

Este trabalho apresenta a pesquisa de interação de organismos aquáticos com metais, especificamente cobre e cádmio assim como da combinação deles. Para tanto, descreve-se um protocolo baseado em ensaios de toxicidade com tilápias Oreochromis niloticus, desenvolvido com a finalidade de estabelecer a concentração letal 50 (CL50) 96h. Como recurso adicional, focando a translocação de metais, foram utilizadas soluções enriquecidas isotopicamente de 65Cu (99,7%) e 111Cd (95,5%), as quais foram aplicadas através de injeções intraperitoneais. As concentrações dos metais nos tecidos e nas frações citosólicas do fígado, brânquias e músculo foram determinadas a partir dos extratos preparados em solução tampão tris-HCl 50 mmolL-1, agentes redutores e antiproteolíticos, na proporção 3:1 (m/v). Adicionalmente, foram determinados os teores de proteínas totais seguidos da etapa de isolamento das metalotioneínas. As concentrações letais (CL50) 96h calculadas pelo método Trimmed Spearman-Karber (95% de confiança), foram iguais a 20,13, 3,53 e 1,36 mg L-1 para cádmio, cobre e cobre+cádmio respectivamente. Estes valores indicam uma significativa diferença na sensibilidade dos organismos aos diferentes tratamentos. Observou-se uma redução na bioconcentração dos metais em função da concentração do metal e do tecido analisado, esta é maior para o Cu do que para o Cd e menor para o musculo em relação a brânquia e ao fígado. Os teores de proteínas totais nos tecidos foram determinados e os valores encontrados estiveram entre 3,25 mg/mL para fígados e 11,83 mg/mL para músculo. A presença de MTs nos diferentes tecidos e respectivos tratamentos, foi pesquisada empregando-se eletroforese capilar e separação de proteínas utilizando a eletroforese em gel de poliacrilamida. Por ambas as técnicas eletroforéticas foi possível identificar as metalotioneínas. Contudo, as análises dos extratos dos tecidos por espectrometria de massas, MALDI-TOF/TOF (matrix-assisted laser desorption/ionization) e ESI-MS (electrospray tandem mass spectrometry ) não mostraram-se adequadas para identificação de metalotioneínas / This paper discuss the interactions of Cd, Cu and it mixture upon aquatic organisms. To reach for these goals lethal 50 (LC50) acute toxicity 96h assays were carried out. In order to assess for the metals translocation in fish, isotopically enriched solutions of 65Cu (99,7%) and 111Cd (95,5%), were used, through an intra-peritoneal injections. Metals concentrations in tissue and in the citossolic fractions of liver, and muscular tissue were analysed in the 50 mmol-1 tris-HCl buffer solutions, reducing and un-proteolitic (3:1) solutions. In addition the total protein content were determinate, followed by the metalothyonein isolation. Lethal concentrations were calculated by the Spearman-Karber method at 95% confidence interval, as 20.13, 3.53 and 1.36 mgL-1 for Cd, Cu and Cd+Cu respectively, which denote differences in organisms sensitivity according to the treatment. Metal bio-concentration was reduced depending on the analyte concentration and kind of tissue, being higher to Cu than to Cd. Total protein concentration varied from 3.35 mg L-1 for liver to 11.83 mg L-1 in the muscular tissue. The occurrence of MTs in tissues and in all treatments were investigated by using both, capillary electrophoresis and gel of polyacrilamide. In all situations the occurrence and identification of Mts were verified, but even with MALDI-TOF/TOF (matrix-assisted laser desorption/ionization) and ESI-MS (electronspray tandem mass spectrometry), or actual instrumental facilities no satisfactory results were obtained, being not possible the identification of MTs in the analysed samples

Leishmania are obligate intracellular protozoan parasites that are inoculated into human skin while a sand fly vector takes a blood meal with the resulting disease coined leishmaniasis. The twenty plus species of Leishmania known to cause human disease are found throughout tropical and subtropical regions of the world. Leishmaniasis affects at least eighty-eight countries with three hundred and fifty million people at risk for infection, resulting in an estimated seventy thousand deaths annually. Different species of Leishmania have developed distinct methods for host defense evasion, leading to a wide spectrum of pathologies within humans. Prior studies of macrophage infections with Leishmania have shown global changes in macrophage mRNA expression. We hypothesized miRNAs are important modifiers of mRNA changes during Leishmania infection. Analysis of miRNA expression patterns revealed that changes were detected primarily during macrophage infection with the low virulent logarithmically growing promastigotes. Profiling studies of mRNA and miRNA changes upon infection with promastigotes in logarithmic growth revealed a decrease in miR-200b and increase in miR-744 levels whereas infections with the highly virulent metacyclic promastigotes revealed a decrease in miR-708 levels. Furthermore, microarray studies revealed differences in macrophage mRNA levels between macrophages infected with the low virulent promastigotes verses the highly virulent promastigotes. Correlative studies between miRNA and mRNA changes suggested some of Leishmania induced changes in mRNA levels may be modified by miRNAs. The importance of Toll-like receptors (TLR) in detection of microbial products has been well-documented. Leishmania infection is known to initiate signaling through TLRs 2, 3, 4 and 9, of which TLRs 2, 4 and 9 signal through the adaptor molecule MyD88. We found that miR-200b, a microRNA decreased by infection of macrophages with the low virulent Leishmania promastigotes, regulates signaling through the TLR4 pathway by targeting and repressing MyD88 transcript levels. Furthermore, we have shown that MyD88 repression results in the decreased expression of the downstream effector molecules IL-6, CXCL9 and TNFΑ upon challenge with a TLR4 ligand. The suppression of miR-200b during Leishmania infection could serve to up-regulate inflammatory responses induced through TLR4 and other MyD88 dependent TLRs. This may be responsible, in part, for the decreased virulence of logarithmically growing compared to metacyclic promastigotes. Furthermore, low levels of inflammation may promote parasite survival by promoting the influx of inflammatory phagocytic cells to the site of infection in which the highly virulent parasites can survive. Microarray studies revealed a remarkable increase in expression of metallothionein (MT) transcripts in macrophages infected with low virulent promastigotes but not in macrophages infected with the highly virulent promastigotes. To explore a possible mechanistic role for metallothioneins in leishmaniasis, we used knock-out mice for MT-1 genes. Bone-marrow derived macrophages from MT-1 knock-out mice (MT-KO) generated higher levels of reactive oxygen species upon incubation with Leishmania promastigotes. Consistently, the initial ROS-induced killing of promastigotes, which occurs during the first hours of infection, was greater twenty-four hours after infection of MT-1 KO bone-marrow macrophages than in our wild type controls. Overall, data presented in this thesis documents changes to macrophage mRNA and miRNA expression patterns upon infection with Leishmania promastigotes that correspond to the overall parasite survival in the host macrophage.