Thesis (doctoral)--Göteborg University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (doctoral)--Göteborg University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Dissertation (Ph.D.) --University of Texas Graduate School of Biomedical Sciences at San Antonio, 2008. / Vita. Includes bibliographical references.

A computational model for mitochondrial function has been developed from oxygen concentration data measured in the Oroboros Oxygraph-2k and oxygen consumption rates measured in the Seahorse XF24 Analyzer. Measurements were acquired using embryonic-cultured cortical neurons and isolated mitochondria from CD1 mice. Based on the biological mechanism of mitochondrial activity, a computational model was developed using biochemical kinetic modelling. To modulate mitochondrial activity, dysfunctions were introduced by injecting the inhibiting reagents oligomycin, rotenone, and antimycin A, and the uncoupling reagent carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) during measurements. To incorporate these changes, model equations were adapted and globally calibrated to experimental data using the genetic algorithm developed by Jason Fiege of the University of Manitoba by fitting oxygen concentration data. The model was coded in MATLAB R2014a along with the development of a graphical user interface for simulating mitochondrial bioenergetics in silico. / October 2016

Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disease characterized by progressive motor neuron degeneration in the brain and spinal cord leading to muscle atrophy, paralysis, and death. Mitochondrial dysfunction is a major contributor to motor neuron degeneration associated with ALS progression. Mitochondrial abnormalities have been determined in spinal cords of animal disease models and ALS patients. However, molecular mechanisms leading to mitochondrial dysfunction in sporadic ALS (sALS) patients remain unclear. Also, segmental or regional variation in mitochondrial activity in the spinal cord has not been extensively examined in ALS. In our study, the activity of mitochondrial electron transport chain complex IV was examined in post-mortem gray and white matter of the cervical and lumbar spinal cords from male and female sALS patients and controls. Mitochondrial distribution and density in spinal cord motor neurons, lateral funiculus, and capillaries in gray and white matter were analyzed by immunohistochemistry. Results showed that complex IV activity was significantly decreased only in gray matter in both cervical and lumbar spinal cords from ALS patients. In ALS cervical and lumbar spinal cords, significantly increased mitochondrial density and altered distribution were observed in motor neurons, lateral funiculus, and cervical white matter capillaries. Discrete decreased complex IV activity in addition to changes in mitochondria distribution and density determined in the spinal cord in sALS patients are novel findings. These explicit mitochondrial defects in the spinal cord may contribute to ALS pathogenesis and should be considered in development of therapeutic approaches for this disease.

Orientador: Gonçalo Amarante Guimarães Pereira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T08:13:02Z (GMT). No. of bitstreams: 1 Bassalo_MarceloColika_M.pdf: 8530833 bytes, checksum: 675d7e6b45b1284552a7e3f9939d929a (MD5) Previous issue date: 2013 / Resumo: A sociedade atual é fundamentalmente dependente do petróleo, recurso natural inserido na grande maioria dos setores da economia. Entretanto, fatores como a limitada disponibilidade deste recurso, sua instabilidade no mercado devido a problemas de natureza geopolítica e a emissão de dióxido de carbono ocasionada pela utilização deste combustível, acentuaram as iniciativas para substituir o petróleo por fontes alternativas e renováveis de matéria prima. A bactéria Propionibacterium acidipropionici surge como uma excelente candidata para a substituição de compostos petroquímicos, através da produção do ácido propiônico. No entanto, antes de transformar esta bactéria em uma plataforma industrial, é necessário aprofundar a compreensão do metabolismo deste microrganismo e desenvolver ferramentas de manipulação genética. No que diz respeito à compreensão do metabolismo, poucos estudos avaliaram o perfil aeróbico desta bactéria, considerada anaeróbica estrita até recentemente. No presente trabalho, foi identificada nesta bactéria a presença de todos os componentes de uma cadeia transportadora de elétrons. No entanto, a citocromo c oxidase identificada apresenta-se mutada e os testes realizados confirmaram a não funcionalidade deste complexo. A existência de uma oxidase alternativa, a citocromo bd oxidase, caracterizada pela alta afinidade ao oxigênio, surge então como uma hipótese promissora acerca da microaerofilia desta bactéria. O trabalho também avaliou o perfil fermentativo dessa bactéria em condições aeróbicas com diferentes fontes de carbono, o que ressaltou a enorme flexibilidade metabólica apresentada por P. acidipropionici, capaz de redirecionar o fluxo de carbono para diferentes produtos finais a depender da necessidade de manutenção do balanço redox. Este estudo também revelou uma propriedade bastante peculiar e industrialmente relevante do xarope de cana-de-açúcar. A fermentação aeróbica com este substrato, ao contrário de todas as outras fontes de carbono, apresentou um crescimento superior ao das condições anaeróbicas e, adicionalmente, exibiu um perfil fermentativo próximo ao observado em ausência de oxigênio. A identificação do composto presente no xarope de cana-de-açúcar, responsável por simular o metabolismo anaeróbico, poderia viabilizar a produção do ácido propiônico em dornas de fermentação aeróbicas, o que traria enormes benefícios para a produção economicamente viável do ácido propiônico e na implementação de P. acidipropionici como uma plataforma industrial / Abstract: The dependence of contemporary society on petroleum is axiomatic, and this natural resource could be found intrinsically embedded in the vast majority of economic sectors. Nonetheless, the limited availability of this natural resource, the instability in the stock market due to geopolitical problems, and also the carbon dioxide emissions associated with the use of fossil fuels have highlighted the need to search for renewable energy sources. The bacteria Propionibacterium acidipropionici arises as an excellent strategy for the substitution of petrochemical compounds, through the production of propionic acid. Before we could implement this bacterium as an industrial platform, however, it becomes necessary to enhance the knowledge regarding the metabolism of P. acidipropionici, and thus create a backbone for the development of genetic manipulation tools. Regarding the metabolism of this bacterium, there aren't comprehensive studies about its aerobic metabolism, thus being considered strict anaerobes until recently. In the present work, it was identified that P. acidipropionici has all required components for a functional electron transport chain. However, the cytochrome c oxidase of this bacterium has a frameshift mutation, and the functional studies proved that this complex is not operative. The presence of an alternative oxidase of high oxygen affinity, called cytochrome bd oxidase, is then suggested as a hypothesis to explain the microaerofilic habit of this bacterium. This work has also shed light into the fermentative profile showed by this bacterium under aerobic cultivation with different carbon sources, bringing attention to the highly flexible metabolism of P. acidipropionici. This bacterium has shown to be capable of completely changing its carbon flux to different end products, as a strategy to maintain the redox balance. In addition, this work has also unveiled an interesting and industrially-relevant property of the sugar cane syrup. It was demonstrated that the aerobic cultivation of P. acidipropionici with sugar cane syrup increased the culture growth, as well as it changed the fermentation end products in a way more similar to the anaerobic cultivation. It was hypothesized that this unusual property found in the sugar cane syrup was due to the presence of a mineral compound that could be used as a final electron acceptor by P. acidipropionici. The identification of this specific compound would allow the aerobic production of propionic acid in industrial conditions, and thus could be a major breakthrough to turn its industrial production into an economically viable process / Mestrado / Genetica de Microorganismos / Mestre em Genética e Biologia Molecular

Sprague-Dawley rats were administered daily, subcutaneous injections of methylmercuric chloride at a dose of 5 mg/Hg/kg beginning on postnatal day 5. By their fourth postnatal week, animals exhibited a constellation of neurological signs of motor impairment which resembled the cerebral palsy syndrome of humans perinatally exposed to methylmercury. Routine histological examination of the brain revealed no gross differences between methylmercury-treated (MeHg), normal control (NC) or weight-matched littermates. The histochemical localization of the mitochondrial enzyme cytochrome oxidase (CO) was utilized in Experiment I to examine possible alterations in the metabolic activity of motor nuclei which might contribute to the observed movement and postural disorders. A population of intensely-staining cytochrome oxidase neurons (ICONs) in the magnocellular portion of the red nucleus (RMC) and interrubral mesencephalon (IRM) were conspicuously present in all MeHg animals at the onset of motor impairment. These morphologically, histochemically, and anatomically distinct neurons did not exhibit intense CO staining in control animals. Conversely, a significant decrease was demonstrated in the oxidative metabolic activity of many neurons in the substantia nigra, zona reticulata of MeHg animals. In Experiment II, the postnatal appearance of ICONs was morphometrically quantified in MeHg animals sacrificed at PND 14, 16, 18, 20, 22, or 25. The histochemically-defined onset of increased metabolic activity in ICONs was first observed on PND 16, at least one week before the onset of clinical signs of neurological impairment. This was the earliest manifestation of methylmercury neurotoxicity yet described in this animal model. A subsequent four-fold increase in the total number of ICONs at PND 18 was followed by a gradual decrease in number to PND 25. Significantly more of the ICONs were found in the IRM than in the RMC at PND 18 & 20. The possibility that the increased activity of ICONs may result from disinhibition of specific afferents to the red nucleus was addressed by introducing either hemidecortication or hemicerebellectomy on PND 10 and then morphometrically determining the deviation from symmetry in the bilateral distribution of the total number of ICONs in the RMC and IRM at PND 22. The distribution of ICONs was symmetrical and not different in either hemidecorticate or unoperated controls. A significant (36%) decrease in the total number of ICONs was observed in both the RMC and IRM contralateral to hemicerebellectomy. The identical ipsilateral regions did not differ from control or hemidecorticate MeHg animals. In Experiment III, the anatomical distribution of major histocompatability complex antigens (MHC) in the brain of MeHg animals was examined using immunohistochemical methods. MHC immunoreactivity was widely distributed throughout the brain of MeHg animals. Areas with low immunoreactivity, or lack of it, stand out and include all of the hippocampus, thalamus, pyriform and entorhinal cortex, and lateral cerebellar hemispheres. Moderate staining intensity was observed in neocortical areas, basal forebrain, caudate-putamen and cerebellar vermis. Strong immunoreactivity was found in red nucleus, substantia nigra, cingulate cortex, retrosplenial cortex, presubiculum, parasubiculum and vestibular nuclei. It was suggested that the increased activity of ICONs likely contributes to the movement and postural disorders resulting from methylmercury intoxication. The increased activity in ICONs was determined to be, at least partially, dependent upon cerebellar input. The results are discussed with reference to the toxic effects of methylmercury and specifically to the susceptibility of GABAergic interneurons in perinatal trauma. Possible analogies are drawn between the mechanisms of methylmercury-induced cerebral palsy syndrome and those of other developmental movement and postural disorders. / Medicine, Faculty of / Graduate

No description available.

Le domaine de la bioénergétique concerne l’étude des échanges et des transformations de l’énergie au sein des organismes vivants. Cette thèse propose une étude électrochimique et spectroscopique de protéines issues de la chaine respiratoire, les oxydases terminales, afin de comprendre l’influence de différentes propriétés de ces enzymes (potentiels des cofacteurs, dépendance pH…) sur leur mécanisme réactionnel. La première partie de ce travail décrit le développement d’une méthode d’immobilisation permettant de conserver l’intégrité et l’activité de ces enzymes. Cette technique a d’abord été utilisée pour étudier l’inhibition de la cytochrome aa3 oxydase de P. denitrificans et a permis de mettre en avant l’importance du transfert de protons sur la réaction de réduction de l’oxygène. Une deuxième étude propose de comparer deux isoformes de la cytochrome cbb3 oxydase dont aucune différence n’a été observée à ce jour. La spectroscopie IRTF couplée à l’électrochimie montre l’implication de résidus acides différents au cours de la réaction d’oxydoréduction suggérant des différences mécanistiques. La dernière partie propose une étude comparative d’oxydases terminales de différents types et met en perspective l’influence des potentiels relatifs des hèmes sur la réaction de réduction de l’oxygène. / The field of bioenergetics concerns the study of exchange and transformation of energy in living organisms. This manuscript proposes an electrochemical and spectroscopic study of the fourth complex of the respiratory chain, the terminal oxidases. The aim of this study was to understand the influence of some properties of these enzymes (potential of the cofactors, pH dependency…) on the catalytic mechanism. The first part describes an immobilization procedure which retains the protein activity and structure. This procedure has been applied for the study the inhibition of the proton pathways of cytochrome aa3 oxidase from P. denitrificans and shows the importance of proton transfer on the oxygen reduction. In a second study, two isoforms of cytochrome cbb3 oxidase were compared. No differences were observed between them until now. Our electrochemically induced FTIR spectroscopy study suggests the implication of different acidic residues during the redox reaction implying differences in the mechanism of these enzymes. The last part deals with the comparison of terminal oxidases of different types and shows the influence of the relative order of the midpoint potentials of the hemes on the oxygen reduction.

Mitochondrial protein homeostasis is crucial for cellular function and integrity. It is ensured by many specific mitochondrial proteases with possible chaperone functions located across the various mitochondrial subcompartments. In the first part, we have focused on characterization of functional overlap and cooperativity of proteolytic subunits AFG3L2 and YME1L of the mitochondrial inner membrane complexes m- and i-AAA in HEK293 cells. The double AFG3L2/YME1L knockdown cells showed severe alteration in OPA1 protein processing, marked elevation in OMA1 protease and severe reduction in SPG7. Our results reveal cooperative and partly redundant involvement of AFG3L2 and YME1L in the maintenance of mitochondrial protein homeostasis and further emphasize their importance for mitochondrial and cellular function and integrity. The aim of the second part was to characterize the cellular function of LACE1 (lactation elevated 1) in mitochondrial protein homeostasis. LACE1 protein is a human homologue of yeast Afg1 (ATPase family gene 1) ATPase. We show that LACE1 is a mitochondrial integral membrane protein that exists as a part of three complexes of approximately 140, 400 and 500 kDa. We demonstrate that LACE1 mediates degradation of nuclear-encoded complex IV subunits COX4, COX5A and COX6A. Using affinity...