Ozone therapy is an alternative form of therapy that has gained attention in the last couple of years. It is believed that O3 may exert a stimulatory effect on the antioxidant defence and immune systems and may therefore be effective in the treatment of ischemic disorders. diabetes mellitus. AIDS and other diseases. On the other hand. it is well known that O3 is a reactive molecule that is toxic to the pulmonary system. Therefore. there remains scepticism regarding its use as a form of therapy. In order to shed some light on this. the effects of ozone autohemotherapy (O3-AHT) on the energy homeostasis of white blood cells were investigated. The possible protective effects of the plasma antioxidant defence system during O3-AHT, were also investigated. Venous blood from six apparently healthy human donors was collected in heparin. In one aliquot a precise volume of blood was mixed with an equal volume of O2/O3 gas mixture containing 20 or 80 μg/ml O3 for 20 minutes. In the other aliquot, the plasma was washed out and the cells resuspended in a buffered phosphate solution. The buffered blood cells were treated with the same concentrations of O3. Control samples was either not treated or treated with a corresponding volume of O2 . Various biochemical analyses were done on the whole blood and buffered cells to determine the oxidant/antioxidant status, cell viability, apoptosis and mitochondrial function. The higher concentration of O3 increased oxidative stress and caused death of white blood cells. Antioxidant enzyme (catalase, glutathione reductase and glutathione peroxidase) activity and the plasma antioxidant capacity decreased, whereas superoxide dismutase levels increased slightly. Exposure to O3 also increased caspase 3/7 activity. A decrease in mitochondrial function was measured by a decrease in ATP levels and an increase in NADH/NAD+ ratio. Complex IV of the respiratory chain was almost completely inhibited by both O3 concentrations. These results indicated that the death of white blood cells was probably through apoptosis. These effects were more evident in the absence of plasma antioxidants. Therefore. high concentrations of O3 were damaging to the cells, but this effect was lessened by antioxidants present in plasma. In view of the results, the use of O3 as a therapy needs to be reconsidered. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2007.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

The endocannabinoid (EC) system has a role in metabolic homeostasis. The purpose of this study was to determine the effect of ECs and the fatty acids they are derived from on lipid metabolism and mitochondrial function in adipocytes. 3T3-L1 adipocytes on day 8 of differentiation were treated with ECs and fatty acids for 48 hours in the absence or presence of insulin and various inhibitors. Lysates were analyzed via Western immunoblotting, a lipolysis assay and Seahorse XF Analyzer for changes in protein levels, phosphorylation state, lipolysis, and oxygen consumption rate. Results showed that ECs (2-arachidonoyl glycerol) stimulated lipolysis via a novel AMPK-dependent pathway, while fatty acids had varying effects on insulin signaling and mitochondrial function . These data suggest adipose tissue EC receptors may be a suitable target for anti-obesity therapy. Further research is needed to understand how the dietary fatty acid profile may influence synthesis of ECs. / May 2016

PARP-1 has been identified as a major player in apoptotic pathways. Its excessive activation causes mitochondrial dysfunction, permeability, and AIF mitochondrion-to-nucleus translocation. It has been suggested that PARP-1 interacts indirectly with Bnip3, a mitochondrial pro-apoptotic factor. However, the mechanistic linkage is still not well understood. Our lab has shown that cytosolic/nuclear NAD+ depletion is a hallmark for PARP-1 over activation and inhibition of sirtuin activity. Specifically in my project, we think that PARP-1 induced- NAD+ depletion and sirtuin inhibition causes hyperacetylation of the α subunit of the transcription factor HIF-1 allowing increased HIF-1 binding to Bnip3 upstream promoter, and increased Bnip3 expression. Indeed, our PARP-1 Knock out neurons, MNNG and PJ34 treatment, chromatin immunoprecipitation, and HIF-1α loss of function studies strongly confirmed the necessity of HIF-1 to increase Bnip3 expression in hypoxia. Overall, our research suggests a role for HIF-1 in increasing PARP-1 dependent Bnip3 expression in hypoxic models. / May 2016

The national programme of expanded access to antiretroviral therapy in the South African public health sector has resulted in hundreds of thousands of South Africans being subjected to prolonged therapy with the risk of adverse drug effects. Among the most common adverse effects are metabolic disorders one of which is mitochondrial toxicity. Mitochondrial toxicity may manifest as hyperlactatemia. The study was designed to determine the frequency with which hyperlactatemia occurs in HIV – infected adults on long-term antiretroviral therapy (ART). The objective was to determine the proportion of patients with blood lactate levels that exceed a predetermined cut-off level and to attempt to relate hyperlactatemia to a set of factors namely, gender, age, obesity, symptoms, type of ART regime and duration of ART use. The study was conducted at an ART clinic in the provincial state hospital of Bongani in the town of Welkom in Free State. The target population was male and female adult patients (18 years and above) on ART for a duration of 1 year or longer. Participants were selected by a random sampling of hospital case file numbers using random table numbers. The patients answered a set of 7 questions on symptoms, underwent weight and height measurements before having blood drawn for lactate assays Blood specimens for lactate assays were processed at the local National Health laboratory.

As mitocôndrias são os principais locais de geração de espécies reativas de oxigênio (ERO), que são produzidos como subprodutos da cadeia de transporte de elétrons. O ferro livre e as ERO podem se envolver em processos potencialmente nocivos, sendo que a desregulação do metabolismo do ferro nessa organela tem sido associada a várias doenças, como a Ataxia de Friedreich (FA). O transporte seletivo de quelantes de ferro a esta organela é proposto como um meio de melhorar sintomas de FA. A desferrioxamina (DFO) é um sideróforo bacteriano com grande afinidade ao ferro, mas baixa penetração celular. Já os peptídeos altamente catiônicos TAT49-57 (Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg), 1A (Fx-Arg-Fx-Lys-Fx-Arg-Fx -Lys), SS02 (Dmt-(D)-Arg-Phe-Lys) e SS20 (Phe-(D)-Arg-Phe-Lys) são conhecidos por permear as membranas citossólicas e mitocondriais. Nós preparamos conjugados de DFO com peptídeos que penetram as mitocôndrias e estudamos suas características de ligação ao ferro in vitro. Eles foram preparados e conjugados em fase sólida com DFO (produzindo quatro mtDFO), que em seguida foram purificados e caracterizados por meio de LC/MS e análise de aminoácidos. Os mtDFO de alta pureza exibiram capacidade de ligação de ferro idêntica à do quelante livre DFO. Os mtDFO também foram capazes de suprimir a oxidação catalisada pelo sistema ferro-ascorbato. A fim de avaliar a localização intracelular dos mtDFO, estes foram marcados com TAMRA (mtDFO-TAMRA). Frente a uma linhagem de carcinoma de ovario, os mtDFO foram em geral pouco tóxicos e altamente localizados nas mitocôndrias. Não foram observados níveis expressivos de danos a DNA, indução de apoptose, geração de ERO na mitocôndria, arraste de ciclo celular ou de apoptose. Resultados preliminares da aplicação dos mtDFO a cardiomiócitos murínicos com baixo nível de frataxina (modelo de FA) indicam um restabelecimento de aproximadamente 60% na morfologia mitocondrial, o que pode ser interpretado como uma melhora nas funções dessa organela. Estes resultados indicam que os mtDFO produzidos podem ser uma parte importante no arsenal terapêutico para FA. / Mitochondria are the main site for the generation of reactive oxygen species (ROS) as sub products of electron transport chain. Free iron and ROS may interact to generate potentially harmful species, and iron homeostasis in this organelle has been linked to several diseases, such as Friedreich Ataxia (FA). Selective targeting of iron chelators to mitochondria has been proposed as a means of alleviating FA symptoms. Desferrioxamine (DFO) is a bacterial siderophore with high affinity for iron, however low cell penetration. Highly charged peptides TAT49-57 Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg), 1A (Fx-Arg-Fx-Lys-Fx-Arg-Fx -Lys), SS02 (Dmt-(D)-Arg-Phe-Lys) and SS20 (Phe-(D)-Arg-Phe-Lys) are known as both cell- and mitochondria-permeant. We prepared conjugates of DFO with mitochondria-penetrating peptides and studied their iron binding characteristics in vitro. They were prepared in solid phase and conjugated to DFO (generating four mtDFO), which were then purified and characterized by LC/MS and Amino acid analysis. MtDFO exhibited iron binding ability identical to free DFO. The mtDFO of high purity were also able to quench the oxidation catalyzed by the iron-ascorbate system. Cell localization studies were performed by tagging mtDFO with TAMRA. In A2780 cells, mtDFO displayed in general low toxicity and high levels of mitochondrial location. No expressive levels of DNA damage, apoptosis, mitochondrial ERO generation or cell cycle arresting were observed. Preliminary results of the application of mtDFO on mouse cardiomiocytes with low levels of frataxin (animal model of FA) indicate a recovery of ca. 60% of mitochondrial morphology. This is interpreted as an improvement of the functions of the organelle. These results indicate that mtDFO may be important allies in the therapy of FA.

Uma das razões que fazem de Saccharomyces cerevisiae um organismo modelo é o grau de conservação dos mecanismos celulares que existe entre esta levedura e eucariotos superiores. Porém, mesmo após 21 anos do seqüenciamento do seu genoma, ainda existem mais de 600 ORFs com função desconhecida. Neste trabalho, selecionamos quatro delas para o estudo detalhado. MRPL34 (YDR115w) está presente na subunidade maior do ribossomo mitocondrial de levedura e apresenta similaridade com o gene L34 de E. coli e MRP-L34 de humanos. O mutante Δmrpl34 apresenta DNA mitocondrial (mtDNA) instável e para estudá-lo foram gerados alelos sensíveis à temperatura (ts). Com os ensaios de síntese protéica mitocondrial in vivo foi possível identificar clara diminuição da síntese de proteínas do mutante condicional. Mrpl34p foi identificada no extrato ribossomal, conforme esperado. A desestruturação da subunidade maior do ribossomo mitocondrial, utilizando os mutantes ts, nos forneceu indícios sobre intermediários existentes no seu processo de montagem. Verificamos que a porção N-terminal da proteína é responsável pelo endereçamento à mitocôndria. YPR116w também apresenta alta instabilidade do DNA mitocondrial, desta forma, mutantes termossensíveis foram utilizados nos experimentos. Uma das estratégias utilizadas visou a busca de parceiros genéticos. Verificamos que ylr091wp aumenta a estabilidade do mtDNA de ts- ypr116w, sugerindo atividade supressora. Também averiguamos que o alelo ts-ypr16w apresenta menor quantidade de tRNA mitocondrial, através de ensaios de Northen blot. Duas das ORFs escolhidas (YDL119c e YOR022c) tiveram sua caracterização inicial publicada em 2016, refletindo a importância deste tipo de pesquisa. Vimos que a proteína codificada por YDL119c está localizada na membrana interna da mitocôndria e que o mutante Δyor022c apresenta quantidades reduzidas de cardiolipina, quando crescido à 37ºC. / One of the reasons that turn Saccharomyces cerevisiae a model organism is the degree of conservation of cellular mechanisms that exist between this yeast and higher eukaryotes. However, even after 21 years of sequencing their genome, there are still more than 600 ORFs with unknown function. In this work, we selected four of them for the detailed study. MRPL34 (YDR115w) is present in the major subunit of the yeast mitochondrial ribosome and bears similarity to the L34 gene of E. coli and MRP-L34 from humans. The Δ mrpl34 mutant shows unstable mitochondrial DNA (mtDNA) and to study it, temperature sensitive alleles (ts) were generated. With the mitochondrial protein synthesis assays in vivo, it was possible to identify a clear decrease in the protein synthesis of the conditional mutant. Mrpl34p was identified in the ribosomal extract as expected. The disassembly of the major subunit of the mitochondrial ribosome, using the ts mutants, provided us some clues about intermediates in its assembly process. We have verified that the N-terminal portion of the protein is responsible for addressing the mitochondria. YPR116w also shows high mitochondrial DNA instability, in this way, thermosensitive mutants were used in the experiments. One of the strategies used was the search for genetic partners. We verified that ylr091wp increases the stability of ts-ypr116w mtDNA, suggesting suppressor activity. We also found that the ts-ypr16w allele has a smaller amount of mitochondrial tRNA, through Northen blot assays. Two of the chosen ORFs (YDL119c and YOR022c) had their initial characterization published in 2016, reflecting the importance of this type of research. We have seen that the protein encoded by YDL119c is located on the inner membrane of the mitochondria and that the Δyor022c mutant presents reduced amounts of cardiolipin when grown at 37 ºC.

As abelhas da tribo Meliponini (abelhas sem ferrão) estão amplamente distribuídas pelas regiões tropicais do planeta, tendo um importante papel na polinização, sendo o gênero Melipona o que contém o maior número de espécies. A espécie Melipona marginata é uma das menores e mais ancestrais do grupo, e a exemplo de outras espécies nidifica em ocos de árvore. M. marginata, assim como outras espécies do gênero Melipona, vêm sofrendo com a destruição do seu ambiente natural, pelo desmatamento, sendo, aparentemente mais exigente que outras espécies quanto ao tamanho do fragmento florestal para se manter, devido a isso é encontrada apenas em fragmentos maiores, mais antigos e menos perturbados. Tendo em vista a perda de hábitat e o pouco conhecimento da biologia dessa espécie, este trabalho pretende analisar populações de M. marginata, através da técnica PCR+RFLP do DNA mitocondrial e marcadores microssatélites, visando contribuir para entendimento da estrutura populacional de M. marginata. Foram analisados 54 ninhos, provenientes dos estados de MG, SP, PR, SC e RS. Oito regiões do DNA mitocondrial foram amplificadas, e posteriormente digeridas com 12 enzimas de restrição. Foram detectados 14 haplótipos, sendo apenas um compartilhado. A população de SP apresentou o maior número de haplótipos. Os testes estatísticos demonstraram que as populações estão estruturadas e isoladas, não havendo fluxo gênico entre as populações. Já nas análises dos microssatélites foram analisados 4 locos, apresentando alta variabilidade genética, onde também foi verificado que as populações se encontram estruturadas. Os resultados obtidos podem ser explicados principalmente pela redução da área de floresta, mas, podem se dever a eventos antigos em conseqüência de mudanças climáticas ocorridas durante as últimas glaciações. / The tribe Meliponini (stingless bees) is present in all tropical regions of the world and has an important role in pollination. The genus Melipona has the highest number of species in the tribe. The specie Melipona marginata is considered the most ancestral within the genus, and like other species builds the nests in hollow of trees. Unfortunately several bee species have suffered with the devastation of their natural environment. M. marginata seems to be very dependent on the size of the forest fragment, being found only in the biggest, oldest and consequently more preserved ones. In view of the habitat loss and few biological knowledge about this specie, this research intended to analyse M. marginata populations molecularly, through mitochondrial DNA PCR-RFLP and microsatellite data., Fifty four colonies were analyzed, from MG, SP, PR, SC and RS states. Eight mitochondrial DNA regions were amplified and screened with 12 restriction enzymes. Fourteen haplotypes were verified and among them just one was shared. SP population showed the highest number of haplotypes. Statistic tests showed that the 5 populations were genetic structured and isolated, therefore not presenting gene flow. The 4 microsatellites loci analyzed showed high genetic variability. The statistics analysis indicated that the 5 populations are structure and isolated. These results can be explained mainly because the decrease of forest leading to population isolation, however we can not discard the hypothesis that this current scenario is a consequence of climatic changes occurred during the last glaciations.

As mitocôndrias são os principais locais de geração de espécies reativas de oxigênio (ERO), que são produzidos como subprodutos da cadeia de transporte de elétrons. O ferro livre e as ERO podem se envolver em processos potencialmente nocivos, sendo que a desregulação do metabolismo do ferro nessa organela tem sido associada a várias doenças, como a Ataxia de Friedreich (FA). O transporte seletivo de quelantes de ferro a esta organela é proposto como um meio de melhorar sintomas de FA. A desferrioxamina (DFO) é um sideróforo bacteriano com grande afinidade ao ferro, mas baixa penetração celular. Já os peptídeos altamente catiônicos TAT49-57 (Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg), 1A (Fx-Arg-Fx-Lys-Fx-Arg-Fx -Lys), SS02 (Dmt-(D)-Arg-Phe-Lys) e SS20 (Phe-(D)-Arg-Phe-Lys) são conhecidos por permear as membranas citossólicas e mitocondriais. Nós preparamos conjugados de DFO com peptídeos que penetram as mitocôndrias e estudamos suas características de ligação ao ferro in vitro. Eles foram preparados e conjugados em fase sólida com DFO (produzindo quatro mtDFO), que em seguida foram purificados e caracterizados por meio de LC/MS e análise de aminoácidos. Os mtDFO de alta pureza exibiram capacidade de ligação de ferro idêntica à do quelante livre DFO. Os mtDFO também foram capazes de suprimir a oxidação catalisada pelo sistema ferro-ascorbato. A fim de avaliar a localização intracelular dos mtDFO, estes foram marcados com TAMRA (mtDFO-TAMRA). Frente a uma linhagem de carcinoma de ovario, os mtDFO foram em geral pouco tóxicos e altamente localizados nas mitocôndrias. Não foram observados níveis expressivos de danos a DNA, indução de apoptose, geração de ERO na mitocôndria, arraste de ciclo celular ou de apoptose. Resultados preliminares da aplicação dos mtDFO a cardiomiócitos murínicos com baixo nível de frataxina (modelo de FA) indicam um restabelecimento de aproximadamente 60% na morfologia mitocondrial, o que pode ser interpretado como uma melhora nas funções dessa organela. Estes resultados indicam que os mtDFO produzidos podem ser uma parte importante no arsenal terapêutico para FA. / Mitochondria are the main site for the generation of reactive oxygen species (ROS) as sub products of electron transport chain. Free iron and ROS may interact to generate potentially harmful species, and iron homeostasis in this organelle has been linked to several diseases, such as Friedreich Ataxia (FA). Selective targeting of iron chelators to mitochondria has been proposed as a means of alleviating FA symptoms. Desferrioxamine (DFO) is a bacterial siderophore with high affinity for iron, however low cell penetration. Highly charged peptides TAT49-57 Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg), 1A (Fx-Arg-Fx-Lys-Fx-Arg-Fx -Lys), SS02 (Dmt-(D)-Arg-Phe-Lys) and SS20 (Phe-(D)-Arg-Phe-Lys) are known as both cell- and mitochondria-permeant. We prepared conjugates of DFO with mitochondria-penetrating peptides and studied their iron binding characteristics in vitro. They were prepared in solid phase and conjugated to DFO (generating four mtDFO), which were then purified and characterized by LC/MS and Amino acid analysis. MtDFO exhibited iron binding ability identical to free DFO. The mtDFO of high purity were also able to quench the oxidation catalyzed by the iron-ascorbate system. Cell localization studies were performed by tagging mtDFO with TAMRA. In A2780 cells, mtDFO displayed in general low toxicity and high levels of mitochondrial location. No expressive levels of DNA damage, apoptosis, mitochondrial ERO generation or cell cycle arresting were observed. Preliminary results of the application of mtDFO on mouse cardiomiocytes with low levels of frataxin (animal model of FA) indicate a recovery of ca. 60% of mitochondrial morphology. This is interpreted as an improvement of the functions of the organelle. These results indicate that mtDFO may be important allies in the therapy of FA.

Sepsis is defined as potentially fatal systemic inflammation, caused by an infection. It is the leading cause of ICU mortality and the 10th leading cause of death in the United States. Several models exist to mimic this disorder, and have demonstrated differential mortality rates between the models as well as the individual animals. Previous studies have shown that elevated levels of plasma mitochondrial DNA (mtDNA) correlated with mortality in septic patients, and cell-free mitochondrial DNA can elicit toll-like receptor mediated immune responses similar to LPS-mediated septicemia. However, the role of mtDNA in the pathophysiology sepsis is still unknown. The focus of this study was to create sepsis in a mouse model using the murine Cecal Ligation and Puncture (CLP) model, and measure plasma mtDNA levels. After CLP was performed on experimental mice, blood plasma was collected 24 hours later. Elevated amounts of circulating mtDNA were detectable in the plasma using real time PCR and cytochrome B2 as a marker of mitochondria. These data were correlated with plasma IL-6 levels, which were used to predict mortality within 5 days of CLP to stratify mice into two populations of those predicted to live or die following the procedure. We also aimed to investigate the effect of mtDNA and mitochondrial debris on naïve mouse macrophages in an in vitro study of the regulation of inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and interleukin-1 beta (IL-1β). In order to observe the effects of mtDNA on murine macrophages, mitochondria was purified from mouse liver and used to stimulate these cells alongside positive control, LPS. Stimulation with mtDNA and mitochondrial debris resulted in increased levels of TNF-α mRNA in lysed cells as well as their surrounding media as compared to control cells, as well as increased transcript half life as measured over four hours post stimulation with transcription inhibitor actinomycin D. The increases in mRNA half-life elicited by mtDNA were comparable to those observed after LPS addition. Stimulation also caused increased binding of TNF-α mRNA to the RNA binding protein, AUF1, as measured by immunoprecipitation of RNA-protein complexes and assayed for TNF-α binding by PCR. These results demonstrate that mitochondrial damage-associated molecular patterns regulate TNF-α mRNA expression at the post-transcriptional level through AUF1, an mRNA destabilizing factor. This is a novel mechanism that likely contributes to sepsis pathophysiology, and demonstrates the involvement of the mitochondrial fission and fusion balance and its regulation in the sepsis innate immune response.