<p>Advances in modern medicine have helped to prolong human life. These advancements coupled with an ever-increasing population means that diseases associated with aging will become more prevalent in the coming years. As such, it is critical to understand the pathogenesis of disease where aging is the main risk factor. While not widely known, age is in fact a large risk factor in development of obesity and metabolic syndrome. More widely known and discussed are the neurodegenerative diseases that occur late in life. While age as a risk factor is a common point between these types of pathology, there are other similarities, such as the interaction between lipid metabolism and mitochondrial health. </p><p>To study the overlap between obesity and neurodegeneration, we investigated two pathways that regulate both. First, we find that loss of cytoplasmic deacetylase HDAC6 leads to aberrant accumulation of lipid in vitro and in vivo. HDAC6 knock-out (KO) mice gain more weight than WT counterparts after a high-fat diet regimen. Additionally, the intermediary metabolism of cells lacking HDAC6 is disrupted as they increase glucose uptake while downregulating fatty acid oxidation. HDAC6 not only plays a role in lipid metabolism, but regulates mitochondrial dynamics. Upon glucose-withdrawal, HDAC6 KO cells fail to elongate their mitochondria and display increased levels of mitochondrial toxic by-products. Therefore, HDAC6 has critical roles in lipid homeostasis and mitochondrial health. </p><p>The other pathway we investigated is critical in neurodegenerative disease, Parkinson's disease. Parkin, an E3 ubiquitin ligase, flags damaged mitochondria for destruction so they do not poison the other functional organelles. We found that Parkin promotes lipid remodeling at the surface of the mitochondria. Phosphatidic acid (PA) accumulates shortly after mitochondrial damage while diacylglycerol (DAG) appears several hours later. This lipid accumulation is dependent upon Parkin's translocation and E3 ligase activity. Additionally, we found that lipin-1, a PA phosphatase, and endophilin B1 (EndoB1) are critical for DAG accumulation and effective mitochondrial clearance. </p><p>Through this work, we show that two proteins critical in quality control mechanisms also play significant roles in energy homeostasis. We aim to highlight this overlap and posit that common diseases of aging, though presenting differently, might have disruptions in the same basic process.</p> / Dissertation

The presented thesis includes three related projects, that are linked by a common interest in the evolution of eukaryotic organelles and machineries that import proteins into these compartments. The first project considers the possibility of peroxisomes (eukaryotic organelles known in aerobic organisms) being conserved in two related anaerobic protists: a free-living amoeba Mastigamoeba balamuthi and a parasite Entamoeba histolytica. The most important hint for the presence of peroxisomes was the discovery of proteins that are homologous to known components of the peroxisomal protein import machinery. The second project aims to characterize the unknown protein translocase of the inner membrane (TIM) in the mitosomes (extremely reduced mitochondria) of an anaerobic protozoan Giardia intestinalis. We have discovered an important subunit of the mitosomal translocase (Tim44), which usually tethers the Hsp70/PAM (presequence translocase-associated motor) complex to the TIM translocon. The last project shows that the protein translocase of the outer mitochondrial membrane in trypanosomatids is related to a typical eukaryotic channel Tom40. This finding is important because the absence of Tom40 was previously considered an ancestral feature of trypanosomatids.

Charles University in Prague, Faculty of Science Department of Parasitology Ph.D. study program: Parasitology Abstract of the Ph.D. Thesis Reductive Evolution of Mitochondria - Related Organelles in Anaerobic Protist Petr Rada Supervisor: Prof. RNDr. Jan Tachezy,Ph.D. Advisor: Doc. RNDr. Ivan Hrdý, Ph.D. Praha, 2011 1 ABSTRACT Trichomonas vaginalis and Giardia intestinalis are parasitic protists of the Excavata group. Both contain anaerobic forms of mitochondria called hydrogenosomes (Trichomonas) and mitosomes (Giardia). Hydrogenosomes produce hydrogen and ATP by substarte level phosphorylation and mitosomes represent the highly-reduced form of mitochondria that do not participate in cellular energy metabolism and ATP generation. Both types of organelles lost the majority of mitochondrial pathways and their genomes during the mitochondrion to hydrogenosome transition. Consequently, hydrogenosomes and mitosomes facilitate translocation of nuclearly encoded proteins into the matrix of the organelle as well as exchange of metabolites and ions across their membranes. Little is known about the membrane machineries required for the biogenesis of the organelle and metabolite exchange and the limited knowledge of mitosomal proteomes has been mostly gained from genomic analysis and localization studies of a few...

Dans ce travail, nous avons montré qu'un nouveau ligand du TSPO, le TRO40303, possède des propriétés cardioprotectrices, confirmant que le TSPO joue un rôle important dans les effets délétères engendrés par l'ischémie-reperfusion myocardique. Des effets similaires ont déjà été observés avec d'autres ligands du TSPO, notamment le 4'-chlorodiazépam, mais le mécanisme d'action par lequel ces molécules exercent leur effet cardioprotecteur reste pour une large part encore mal connu. Nous avons montré chez le rat que la reperfusion d'un myocarde ischémié s'accompagne d'une augmentation du cholestérol mitochondrial, de la formation d'oxystérols et d'un stress oxydant majeur. Le 4'-chlorodiazépam inhibe ces effets et améliore les fonctions mitochondriales post-ischémiques, révélant que le TSPO est responsable du transport du cholestérol dans la mitochondrie cardiaque et que ce dernier, sous des formes oxydées ou non, pourrait participer aux effets délétères de la reperfusion. La limitation de l'entrée du cholestérol dans la mitochondrie lors de la reperfusion d'un myocarde ischémié est un mécanisme original qui pourrait donc contribuer à l'effet cardioprotecteur des ligands du TSPO. Enfin, nous avons montré que chez des rats génétiquement modifiés et associant hypercholestérolémie, obésité et diabète de type II, le cholestérol mitochondrial est d'une part très élevé, avec ou sans ischémie-reperfusion myocardique et d'autre part que le 4'-chlorodiazépam n'a pas d'effet sur le cholestérol mitochondrial après ischémie-reperfusion. Ces résultats suggèrent que les mécanismes d'action des ligands du TSPO sont probablement différents et que les traitements doivent être adaptés en présence de facteurs de co-morbidité. / In the present work we showed that a new TSPO ligand, TRO40303, has cardioprotective properties confirming that TSPO plays a key role in the deleterious effects of myocardial ischemia-reperfusion. Similar effects have been observed with other TSPO ligands, such as 4'-chlorodiazepam, but the mechanism of action of these molecules is not known. We showed that ischemia-reperfusion in rats increased mitochondrial concentration of cholesterol, oxysterol formation and oxidative stress. 4'-Chlorodiazepam inhibited these effects and improved post-ischemic mitochondrial functions, revealing that TSPO is responsible for cholesterol transport in cardiac mitochondria and that cholesterol, in oxidized or non-oxidized forms, could participate in the deleterious effects of reperfusion. The limitation of the increase in cholesterol in mitochondria during ischemia-reperfusion is an original mechanism which could contribute to the cardioprotective effect of TSPO ligands. We then showed, in genetically modified rats with hypercholesterolemia, obesity and type II diabetes, that the concentration of mitochondrial cholesterol is very high with or without ischemia-reperfusion. We further demonstrated that 4'-chlorodiazepam has no effect on mitochondrial cholesterol after ischemia-reperfusion. These results suggest that the mechanisms of action of TSPO ligands probably differ in these conditions and that treatment must be adapted in accordance with the presence of factors of co-morbidity.

A cisplatina ainda é um dos agentes quimioterápicos mais efetivos. No entanto, em elevadas doses pode ocorrer hepatotoxicidade. Alguns antioxidantes têm sido mostrado amenizar a hepatotoxicidade induzida pela cisplatina mas o mecanismo molecular envolvido não está bem esclarecido.No presente estudo nós investigamos moleculares subjacente ao efeito protetor da dimetiltiuouréia (DMTU), um conhecido eqüestrador de radical hidroxil, contra a lesão oxidativamitocondrial hepática induzida pela cisplatina em ratos. Ratos Wistar machos adultos ( 200 a 220g) foram divididos entre 4 grupos de 8 animais cada. O grupo controle foi tratado apenas com uma injeção intraperitoneal (i.p.) de solução salina (1 ml/ 100g de peso). Ao grupo DMTU foi administrado apenas DMTU (500 mg/kg de peso, i.p., seguido de 125 mg/kg, i.p., duas vezes ao dia até o sacrifício). Ao grupo cisplatina foi administrado uma injeção única de cisplatina (10 mg/kg de peso, i.p.). Ao grupo DMTU + cisplatina foi administrado DMTU (500mg/kg de peso, i.p.), pouco antes da injeção da cisplatina (10 mg/kg de peso, i.p.), seguido por injeções de DMTU (125 mg/kg de peso, i.p.) duas vezes ao dia até o sacrifício ( 72 horas após o tratamento). A hepatotoxicidade foi evidenciada no grupo cisplatina pelo aumento dos níveis séricos de alanina (ALT) e aspartato (AST)aminotransferases. O mecanismo de hepatotoxicidade induzido pela cisplatina mostrou-se envolvido na rigidez de membrana; na redução da razão glutationa reduzida em relação a glutationa oxidada (GSH/GSSG); na redução dos níveis de ATP, GSH e NADPH; na lipoperoxidação; na lesão oxidativa da cardiolipina e de proteínas com grupos fidrílicos. Mais ainda, a morte celular por apoptose foi também demonstrada e os achados fortemente sugerem a participação do xi mecanismo sinalizador mitocondrial neste processo; o DMTU não apresentou nenhum efeito direto sobre a mitocôndria e inibiu substancialmente a lesão mitocondrial induzida pela cisplatina, prevenindo a hepatotoxicidade. Todos os seguintes efeitos induzidos pela cisplatina foram previnidos pelo DMTU: (a) elevação dos níveis séricos de AST e ALT; (b) redução dos níveis de ATP hepático;(c)peroxidação lipídica;(d)oxidação da cardiolipina; (e)oxidação de proteínas sulfidrílicas; (f) rigidez da membrana mitocondrial; (g) oxidação de GSH; (h)oxidação de NADPH e (i) morte celular por apoptose. Os resultados mostraram o papel principal da mitocôndria e dos radicais hidroxilas na proteção do fígado saudável contra a lesão hepática induzida pela cisplatina, delineando um número de etapas que podem ser consideradas no desenvolvimento de futuros agentes citoprotetores / Cisplatin is still one of the most effective chemotherapeutic agents. However, at higher doses hepatotoxicity may occur. Some antioxidants have been shown to ameliorate cisplatin-induced hepatotoxicity but the involved molecular mechanism has not been clarified. In the present study we investigated the molecular mechanism underlying the protective effect of dimethylthiourea (DMTU), a known hydroxyl radical scavenger, against liver mitochondrial oxidative damage induced by cisplatin in rats.Adult male Wistar rats (200 to 220g) were divided into 4 groups of 8 animals each. The control group was treated only with an intraperitoneal (i.p.) injection of saline solution (1ml/100g body weight). The DMTU group was given only DMTU (500 mg/kg body weight, i.p, followed by 125 mg/Kg, i.p., twice a day until sacrifice). The cisplatin group was given a single injection of cisplatin (10 mg/kg body weight, i.p.). The DMTU+cisplatin group was given DMTU (500 mg/kg body weight, i.p.), just before the cisplatin injection (10 mg/kg body weight, i.p.), followed by injections of DMTU (125 mg/kg body weight, i.p.) twice a day until sacrifice (72 hours after the treatment). epatotoxicity was evidenced in the cisplatin group by the increased serum levels of alanine (ALT) and aspartate (AST) aminotransferases. The mechanism of cisplatininduced hepatotoxicity was found to involve membrane rigidification; decreased GSH/GSSG ratio, ATP, GSH and NADPH levels; lipid peroxidation; oxidative damage of cardiolipin and protein sulfhydryl groups. Moreover, cell death by apoptosis was also demonstrated and the findings strongly suggest the participation of the mitochondrial signaling pathway in this process; DMTU did not present any direct effect on mitochondria and substantially inhibited cisplatin-induced mitochondrial injury, therefore preventing the hepatotoxicity. All the following cisplatin-induced xiv effects were prevented by DMTU: (a) elevation of AST and ALT serum levels; (b) decreased hepatic ATP levels; (c) lipid peroxidation; (d)cardiolipin oxidation; (e) sulfhydryl protein oxidation; (f) mitochondrial membrane rigidification; (g) GSH oxidation; (h) NADPH oxidation and (h) apoptotic cell death. Results show the central role of mitochondria and hydroxyl radicals in the protection of healthy liver against cisplatin-induced injury, highlighting a number of steps that might be considered in the development of novel cytoprotective agents.

O fator de transcrição A mitocondrial (TFAM) é um membro da subfamília HMGB que se liga a promotores do DNA mitocondrial (mtDNA). É um gene importante para a manutenção do mtDNA, pois regula o número de cópias e é essencial para inicialização da replicação e transcrição do mtDNA. Recentemente técnicas de edição gênica vêm sendo utilizada como uma ferramenta bastante eficaz na manipulação genômica. A nova tecnologia chamada de CRISPR/ Cas9 (Regulary interspaced clustered short palindromic repeats) utiliza um RNA guia (gRNA) curto que contém 20 nucleotídeos complementares a sequência de DNA. Quando o RNA guia se liga ao local alvo, a proteína Cas9 é recrutada para se ligar no local alvo e induzir a dupla quebra na cadeia de DNA. Neste contexto, este estudo propôs editar o gene TFAM pela tecnologia CRISPR Cas9, com o objetivo de gerar células Rho zero através do knock-out em fibroblastos bovinos. Os fibroblastos bovinos utilizados neste estudo foram derivados de uma biopsia de pele coletada de animais adultos. A sequência do gene foi obtida a partir do banco de dados GenBank (www.ncbi.nlm.nih.gov) e esta foi inserida no site CRISPR direct (crispr.dbcls.jp) e no site rgenome (rgenome.net) a fim de desenhar o gRNA. O gRNA foi desenhado no exon 1 do gene TFAM bovino. Os fibroblastos foram cultivados e após as células atingirem 80% de confluência, estas foram eletrotransfectadas com Cas9 (Addgene 48668), gRNA, GFP e plasmídeo controle. Foi utilizado o kit Primary Mammalian Fibroblasts (VPI-1002) e a transfecção foi realizada no equipamento AMAXA Nucleofector 2B. Após a transfecção foi realizada a citometria de fluxo para avaliar a taxa de transfecção, e as células pós transfectadas foram plaqueadas em placas de 96 poços, pela técnica de sorting. O sorting separarou uma célula por poço de 96. Após 20 dias em cultura essas células foram tripsinizadas em placas de 6 poços e o DNA genômico foi extraído, utilizando o kit Qiamp DNA microkit-Qiagen. Para avaliar a frequência de mutações, foi realizada a digestão com a enzima T7 endonuclease, e após confirmado mutações, os clones foram enviados para analise de sequenciamento. Observamos uma taxa de transfecção eficiente de 51,3%. Obtivemos 40 clones com DNA extraído para analise, no qual 7 destes possuiam mutações no local de inserção da CRISPR Cas 9. Com isso, concluimos uma heterozigose mostrando que o desenho da CRISPR foi eficiente, gerando uma deleção do gene TFAM. / The mitochondrial transcription factor A (TFAM) is a member of HMGB subfamily that binds to promoters of mtDNA. It is a very important gene that maintains mtDNA, regulates the number of copies and is essential for the initiation of transcription mtDNA. Recently, gene edition techniques have been used as a very effective tool in genomic manipulation. The new technology called CRISPR/Cas9 (Regulary interspaced clustered short palindromic repeats) uses a short gRNA containing 20 nucleotides complementary to the DNA sequence. When gRNA binds to the target site, the Cas9 protein is recruited to bind in the chosen location and induce double strands breaks in DNA. In this context, this study proposed to edit the TFAM gene by CRISPR Cas9 technology aiming to generate Rho zero cell through the knock-out in bovine fibroblasts. Bovine fibroblasts used in this study were derived from a skin biopsy collected from an adult. The sequence obtained from the database GenBank (www.ncbi.nlm.nih.gov) was inserted in the CRISPR direct site (crispr.dbcls.jp) and in the rgenome site (rgenome.net) to design the RNA guide. The gRNA was designed in the CRISPR direct site (crispr.dbcls.jp) for the Exon 1 of the gene TFAM bovine and after was performed the CRISPR cloning. The fibroblast were cultured and after reaching 80% of confluence, were electro-transfected with Cas9 (Addgene 48668) and control plasmids using the Nucleofector TM Kit for Primary Mammalian Fibroblasts (VPI-1002) and transfected with Cas 9 (Addgene 48668), GFP and control plasmid. Were used the Primary Mammalian Fibroblasts (VPI-1002) and the transfection was performed on the AMAXA Nucleofector 2B. Post transfected cells were analyzed by flow cytometry to evaluate the rate of transfection. The cells post transfected were further split into 1 cell/well (96- well plates for cell cloning). After days in culture these cells were trypsinized in 6-well plates and the genomic DNA was extracted using the Qiamp DNA microkit- Qiagen. To assess the mutation frequency, T7 endonuclease assay were performed and after confirmed the mutations, the clones were sent for sequencing analysis. We observed that the cells were efficiently transfected since they have a rate of 51,3% transfection. We obtained 40 clones with extracted for analysis, in which 7 of these had mutations at the insertion site of CRISPR/Cas 9. We concluded that until this moment the CRISPR design was efficient and that we obtained a deletion of the TFAM gene.

Os contaminantes emergentes compreendem uma classe de substâncias que apresentam carência de dados toxicológicos, principalmente em relação aos seus efeitos danosos e aos biomarcadores de exposição, além de potencial dano ao ambiente, sendo esta uma preocupação recente da comunidade científica. Dentro dessa classe enquadram-se os praguicidas, mais especificamente os herbicidas, os quais são largamente utilizados na agricultura para prevenir o aparecimento de ervas daninhas, que interferem no rendimento e qualidade da colheita. Esses compostos apresentam potencial contaminação de solo e águas, podendo atingir, dessa forma, organismos não-alvo. Pelo fato da mitocôndria ser reconhecida como a principal organela produtora de energia celular, além de exercer papel fundamental na manutenção de inúmeras funções celulares, ela foi utilizada como modelo experimental para avaliar os efeitos dos herbicidas trifluralina e tebutiurom na faixa de concentração de 1 ?mol/L a 100 ?mol/L. Os resultados obtidos mostram que o tebutiurom não afeta a bioquímica mitocondrial em nenhuma concentração testada. Já a trifluralina, principalmente na concentração mais alta (100 ?mol/L), é capaz de interagir com a membrana mitocondrial, induzir inchamento mitocondrial, dissipar o potencial de membrana, desregular a homeostase cálcica, afetar a respiração celular e alterar os níveis de ATP. Entretanto, não é capaz de induzir estresse oxidativo na mitocôndria. Os resultados indicam possíveis mecanismos de toxicidade do herbicida em organismos não-alvo. / The emerging contaminants comprise a class of substances that exhibit a lack of toxicology data, especially regarding to their harmful effects and biomarkers of exposure, besides the potential damage to the environment, which is a recent concern of the scientific community. Within this class there are the pesticides, more specifically the herbicides, which are widely used in crop production to prevent the appearance of weeds, that can damage the yield and quality of the harvest. These compounds are potential contaminants of soil and water, and may achieve non-target organisms. Because mitochondria are recognized as the main energy-producing cell organelles, and play a vital role in the maintenance of many cellular functions, they were used as an experimental model to evaluate the effects of the herbicides trifluralin and tebuthiuron in concentrations ranging from 1 ?mol/L to 100 ?mol/L. The results show that tebuthiuron does not affect the mitochondrial biochemistry in any concentration tested. Trifluralin, especially at the highest concentration (100 ?mol/L), is capable of interacting with mitochondrial membrane, inducing swelling, dissipating the membrane potential, dysregulating calcium homeostasis, impairing mitochondrial respiration and altering ATP levels. However, it isn\'t able to induce oxidative stress in mitochondria. These events might be involved in the mechanisms of the toxicity of trifluralin in non-target organisms

A Hsp70 mitocondrial humana (mtHsp70 ou mortalina) está envolvida em diversos processos celulares: na matriz mitocondrial atua na importação de proteínas produzidas no citoplasma; no citoplasma, pode atuar sequestrando a p53, estando assim envolvida na proliferação de alguns tipos de câncer. A literatura ainda aponta que a mortalina participa na manutenção de várias doenças causadas pelo envelhecimento, como mal de Parkinson e de Alzheimer. Desse modo, o estudo estrutural e a investigação das principais funções da mortalina in vivo e in vitro, além de sua interação com outras chaperonas e co-chaperonas é de grande relevância científica, podendo proporcionar um maior entendimento de seu papel celular e da maquinaria bioquímica nas doenças onde ela está inserida. Apesar de ser conhecida há bastante tempo, as tentativas de expressão heteróloga da mortalina recombinante resultam na sua produção na forma insolúvel, inviabilizando estudos estruturais e funcionas in vitro. Assim, as informações estruturais e funcionais desta proteína permaneceram limitadas até então. Em 2005, foi descrita uma co-chaperona da mortalina que atua auxiliando o seu enovelamento correto e em sua manutenção na fração solúvel, esta proteína mitocondrial foi denominada de hHep1 (Hsp70-escort protein 1) e por meio de sua co-expressão com a mortalina foi possível obter esta última na sua forma monomérica, solúvel e estável. Isso possibilitou realizar ensaios de caracterização estrutural e funcional da mortalina, sendo o foco principal deste trabalho de doutorado. Os resultados obtidos sugerem que a mortalina se apresenta como um monômero ligeiramente alongado em solução, sendo formada por 2 domínios com estabilidades distintas. Os ensaios funcionais revelaram uma constante de dissociação (KD) para interação com nucleotídeos adenosina da ordem de 1 &micro;M. A mortalina apresenta atividade ATPásica com valores de Vmáx e KM da ordem de 0,21 pmol de ATP por min e 190 ± 20 &micro;M, respectivamente. Este trabalho é pioneiro na caracterização estruturale funcional da mortalina humana e espera-se que estudos posteriores, elucidem mais detalhedamente os mecanismos de interação da mortalina com proteínas clientes nos diversos compartimentos celulares onde ela atua. / The human mitochondrial Hsp70 (mtHsp70 or mortalina) is involved in many cellular processes: in the mitochondrion matrix, mortalin acts in the process of protein importation from cytoplasm; in the cytoplasm may act by sequestering p53, protein involved in the proliferation of some kinds of cancer. The literature also shows that mortalin participates in the maintenance of various diseases caused by aging, such as Parkinson\'s and Alzheimer\'s. Thus, the structural study and research of the main functions of mortalin in vivo and in vitro, and its interaction with other chaperones and co-chaperones is of great scientific importance and may provide a greater understanding of their role and cellular biochemical machinery in diseases where it is inserted. Despite being known for a long time, the expression of heterologous mortalin resulted in an insoluble form of the protein, which precludes its in vitro structural and functional studies. Thus, structural and functional information of this protein, along with its interaction with chaperones, co-chaperones and client proteins, remained unknown. By 2005, it was described co-chaperone that acts on mortalin helping its correct folding and its maintenance in the soluble fraction, this mitochondrial protein was called hHep1 (Hsp70-escort protein 1) and through its co-expression with mortalin it was possible to obtain the recombinant mortalin in its monomeric, soluble and stable. With this protein, it was possible to perform tests of structural and functional characterization of recombinant mortalin, the main focus of this doctoral work. The results suggest that mortalin behaves as a slightly elongated monomer in solution, formed by two domains with different stabilities. Functional assays showed that the dissociation constant for interaction with adenosine nucleotide of the order of 1 &micro;M. Mortalin has ATPase activity with Vmax and KM values of 0.21 pmol ATP per min and 190 ± 20 &micro;M, respectively. It is expected that these results provide information for further studies, such as for elucidating the mechanisms that mortalin interacts with client proteins in various cellular compartments in which it operates.

As peroxirredoxinas (Prxs) são peroxidases dependentes de tiol que catalisam a redução de uma ampla variedade de hidroperóxidos. A atividade catalítica das Prxs é suportada por um resíduo de cisteína catalítico altamente conservado, cuja oxidação pelo hidroperóxido gera o ácido sulfênico (Cys-SOH). Prx1 de Saccharomyces cerevsiae é uma enzima mitocondrial que catalisa a redução do H2O2 gerado no interior da mitocôndria. O mecanismo de redução do ácido sulfênico de Prx1 é uma questão de debate, com a glutarredoxina 2 (Grx2), tiorredoxina 3 (Trx3), tiorredoxina redutase 2 (Trr2) e ascorbato sendo propostos como possíveis redutores. Para avaliar a importância fisiológica de Prx1 na manutenção da homeostase redox mitocondrial, nós investigamos os mecanismos de importação e processamento mitocondrial de Prx1 assim como os de seus possíveis redutores Trr2 e Trx3. Os ensaios de solubilidade e subfracionamento mitocondrial demonstram que Prx1, Trr2 e Trx3 co-localizam na matriz mitocondrial, associadas fracamente com a membrana mitocondrial interna. Além disso, Prx1 apresenta dupla localização, estando presente também no espaço intermembrana mitocondrial possivelmenete na forma solúvel. O mecanismo de importação de Prx1 para o espaço intermembrana envolve a liberação da proteína precursora no interior da bicamada lipídica da membrana interna em decorrência de uma pequena região hidrofóbica localizada imediatamente após a pressequência. Em seguida, a subunidade Imp2 do complexo proteico IMP catalisa a clivagem da região hidrofóbica liberando Prx1 no espaço intermembrana. Durante a importação de Prx1 para a matriz mitocondrial, a enzima é clivada sequencialmente pelas proteases peptidase de processamento mitocondrial (MPP) e octapeptidil aminopeptidase 1 (Oct1). Oct1 catalisa a remoção de oito resíduos de aminoácidos da região N-terminal de Prx1. Esse processamento aumenta a estabilidade de Prx1 no interior da mitocôndria, mas não interfere na sua atividade peroxidásica in vitro. Apesar das enzimas Trr2 e Trx3 não serem clivadas por Oct1, a ausência de Oct1 causa eleavada instabilidade dessas proteínas. O processamento das Prxs por Oct1 parece ser um processo conservado visto que Oct1 de levedura é capaz de clivar a peroxirredoxina mitocondrial humana Prx3 expressa em S. cerevisiae. Estes resultados indicam o envolvimento de Oct1 no processamento das peroxirredoxinas, representando um sistema de controle de qualidade proteico que regula a homeostase das Prxs e, possivelmente, processos redox mitocondriais / Peroxiredoxins (Prxs) are thiol-dependent peroxidases that catalyze the reduction of a wide variety of hydroperoxides. The Prxs catalytic activity is provided by the presence of a highly conserved catalytic cysteine residue whose oxidation by hydroperoxide generates sulfenic acid (Cys-SOH). Saccharomyces cerevsiae Prx1 is a mitochondrial enzyme that catalyzes the reduction of the H2O2 generated endogenously by mitochondria. The mechanism of reduction of Prx harboring Cys-SOH is a matter of debate, with glutaredoxin 2 (GRX2), thioredoxin 3 (Trx3), thioredoxin reductase 2 (Trr2), and ascorbate being proposed as possible reducers. To assess the functional role of Prx1 in maintaining the mitochondrial redox homeostasis, we investigated its mechanisms of import and processing, as well as those ones involved with its possible reducers, Trr2 and Trx3. Assays of solubility and mitochondrial sub-fractionation show that Prx1, Trr2 and Trx3 co-localize in the mitochondrial matrix compartment, being marginally associated with the inner mitochondrial membrane. In addition, Prx1 show dual localization, being also present in the mitochondrial intermembrane space, possibly in their soluble form. The import mechanism of Prx1 to the intermembrane space involves the release of protein\'s precursor within the lipid bilayer of the inner membrane due to a small, hydrophobic region located downstream the presequence. Imp2 subunit of the IMP protein complex then catalyzes the cleavage of the hydrophobic region of Prx1, releasing it to the mitochondrial intermembrane space. During its import into the matrix, Prx1 is sequentially cleaved by the mitochondrial processing-peptidase protease (MPP) and by octapeptidil aminopeptidase 1 (Oct1). Oct1 catalyzes the cleavage of eight amino acid residues from the N-terminal region of Prx1. This process increases stability of Prx1 inside the mitochondria, but does not interfere in its peroxidase activity in vitro. Interestingly, absence of Oct1 causes high instability of Trr2 and Trx3, although these proteins are not cleaved by this protease. Remarkably, the processing of Prxs by Oct1 seems to be a conserved process since yeast Oct1 is able to cleave the human mitochondrial peroxiredoxin Prx3 expressed in S. cerevisiae. Altogether, these results indicate the involvement of Oct1 in the processing of peroxiredoxins, representing a protein quality control system that regulates the homeostasis of Prxs and, possibly, mitochondrial redox processes

αB-crystallin is a small heat-shock chaperone protein (sHSP) required for the homeostasis of multiple tissues including eye lens, retina, heart and brain. Correspondingly, mutation or altered levels of αB-crystallin are associated with multiple degenerative diseases including cataract, retinal degeneration, cardiomyopathy and Lewy body disease. Based on its wide-ranging importance understanding the protective and homeostatic properties of α B-crystallin is critical for understanding degenerative diseases and could lead to the development of therapies to treat these diseases. αB-crystallin is localized to the mitochondria suggesting a direct effect on mitochondrial function. My thesis work has examined those molecular pathways required for translocation of αB-crystallin to the mitochondria and to identify the downstream pathways controlled by mitochondrial translocation of αB-crystallin that could be important for cellular protection and differentiation. My results point to a novel role of αB-crystallin in regulation of key apoptotic pathways that mediate the balance between cell survival and differentiation. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection