Ląstelių atsako į nuosaikią hipertermiją molekulinio mechanizmo išaiškinimas yra labai svarbus norint suprasti procesus, kurie vyksta ląstelėse jas kaitinant gydymo tikslais ar organizmui karščiuojant. Šio darbo tikslas buvo nustatyti ir palyginti švelnios (karščiavimo) ir šiurkščios hipertermijos poveikį oksidacinės fosforilinimo sistemos funkcijoms normalių audinių mitochondrijose. Pirmą kartą panaudojome modulių kinetinę analizę hipertermijos poveikiui širdies ir kepenų mitochondrijų oksidacinio fosforilinimo sistemai tirti. Mes nustatėme, kad švelnios hipertermijos (42 ºC) poveikis širdies mitochondrijų funkcijoms yra grįžtamas, bet šiurkštesnė hipertermija (45 ºC) sukelia dalinai negrįžtamą kvėpavimo ir fosforilinimo atskyrimą bei mitochondrijų kvėpavimo greičio trečioje metabolinėje būsenoje slopinimą. Hipertermija didino ROS gamybos greitį ir lipidų peroksidaciją, kurie buvo didžiausi karščiavimo temperatūroje. Nustatėme, kad kepenų mitochondrijų ir hepatocitų atsakas į hipertermiją priklauso nuo žiurkės lyties ir temperatūros. Atlikome palyginamąjį širdies ir kepenų mitochondrijų sandų fazinių virsmų analizę diferencine skenuojamaja kalorimetrija ir nustatėme būdingus skirtumus. / The elucidation of the molecular mechanism of the cell response to moderate heating is of importance for understanding the events that occur in the cell upon use of heating for therapeutic purpose or during illnesses that are associated with fever. The aim of this work was to investigate and to compare the effects of mild (fever) and severe hyperthermia on functional properties of oxidative phosphorylation system in normal tissue mitochondria. Modular kinetic analysis for the first time was applied to evaluate effects of hyperthermia on oxidative phosphorylation in rat heart and liver mitochondria. We demonstrated that changes in mitochondrial functions induced by mild hyperthermia (42 ºC) are reversible but more severe hyperthermia (45 ºC) causes partially irreversible uncoupling and inhibition of mitochondrial respiration in state 3, hyperthermia remarkably (3.6-2.1 fold) activates ROS generation in heart mitochondria and that maximal increase in rate of H2O2 production and lipid peroxidation is observed in the fever temperature range. We show that the response of liver mitochondria and hepatocytes to hyperthermia is to certain extent dependent on gender and temperature. Specific differences of male rat liver and heart mitochondrial components phase transitions have been revealed by DSC analysis.

Perilla L. are significant for multi-pharmacological effect. The aim of this work is to study Perilla L. growth and develop¬ment tendencies, productivity; composition of biologically active compounds and their variations during the vegetation period, and the biological effect of the extracts; to select perspective plants for cultivation in Lithuania and for production of medicinal preparations. The objectives of the Study: To investigate and determine growth dynamics of Perilla L. species and varieties during the vegetation period and to assess the influence of climate conditions on the vegetation rhythmic. To assess the quantity dynamics of Perilla L. species and varieties herbal medicinal raw material and to compare the productivity of Perilla L. species and varieties. To determine the quantity of the essential oil in Perilla L. species and varieties and to identify the chemotypes of the plants. To determine the qualitative composition and variation dynamics of the phenolic acids in Perilla L. species and varieties. To determine the composition and variation dynamics of the flavone complex in Perilla L. species and varieties. To determine the composition and variation dynamics of antho¬cyanidines in Perilla L. species and varieties. To assess the radical scavenging activity of the extracts of Perilla L. species and varieties raw materials. To research the effect of Perilla L. extracts on the oxidative phosphorylation in the rat heart mitochondria. / Perilla L. genties vienmečiai vaistiniai augalai yra augaliniai imunomoduliatoriai, pasižymintys daugeliu farmakologinių poveikių. Darbo tikslas: Ištirti Vidurio Lietuvoje auginamų Perilla L. rūšių ir varietetų augimo ir vystymosi dėsningumus, biologiškai aktyvių junginių sudėtį ir jų įvai¬ravimą vegetacijos metu bei ekstraktų biologinį poveikį; atrinkti perspek¬tyvius augalus auginimui Lietuvoje. Uždaviniai: Ištirti ir nustatyti Perilla L. augimo dinamiką vegetacijos metu ir įvertinti klimatinių veiksnių įtaką augalų vegetacijai. Įvertinti Perilla L. vaistinės augalinės žaliavos kiekio dinamiką vege¬tacijos metu ir palyginti Perilla L. rūšių ir varietetų produk¬tyvumą. Nustatyti Perilla L. rūšių ir varietetų eterinio aliejaus kiekį auga¬luose vegetacijos metu ir identifikuoti augalų chemotipus. Nustatyti Perilla L. rūšių ir varietetų fenolinių rūgščių kokybinę sudėtį ir jų kitimo dėsningumus vegetacijos metu. Nustatyti Perilla L. rūšių ir varietetų flavonų komplekso sudėtį ir kitimo dinamiką vegetacijos metu. Nustatyti Perilla L. rūšių ir varietetų antocianidinų sudėtį bei kitimo dinamiką vegetacijos metu. Įvertinti Perilla L. rūšių ir varietetų žaliavų ekstraktų antiradikalinį aktyvumą. Ištirti Perilla L. rūšių ir varietetų ekstraktų poveikį žiurkės širdies mitochondrijų oksidaciniam fosforilinimui. Tyrimų rezultatai ir poveikiu pasižyminčių junginių identifikavimas atveria perspektyvas ateities tyrimams, kurie reikalingi kuriant preparatus iš perilių augalinių žaliavų.

Vieni populiariausių vaistinių preparatų Europoje yra dviskiaučių ginkmedžių (Ginkgo biloba L.) lapų preparatai. Nors Ginkgo biloba lapų ekstraktai pasižymi plačiu farmakologiniu pritaikymu, tačiau nėra daug duomenų apie jų poveikį mitochondrijoms. Todėl darbo tikslas buvo ištirti dviskiaučių ginkmedžių lapų ekstraktų poveikį žiurkės širdies ir kepenų mitochondrijų oksidacinio fosforilinimo sistemai. Darbo uždaviniai: 1. Ištirti dviskiaučių ginkmedžių lapų ekstraktų poveikį žiurkės širdies permeabilizuotų skaidulų bei izoliuotų širdies ir kepenų mitochondrijų kvėpavimui. 2. Ištirti dviskiaučių ginkmedžių lapų tinktūros poveikio oksidaciniam fosforilinimui širdies mitochondrijose mechanizmą. 3. Įvertinti dviskiaučių ginkmedžių lapų tinktūros poveikį izoliuotos žiurkės širdies elektromechaniniam aktyvumui ir nustatyti kaip greit tinktūros komponentai patenka į ląstelę ir mitochondrijas. 4. Ištirti ar dviskiaučių ginkmedžių lapų tinktūra apsaugo širdies mitochondrijas nuo žalingo išemijos/reperfuzijos poveikio. 5. Ištirti dviskiaučių ginkmedžių lapų tinktūros poveikį mitochondrijų oksidacinio fosforilinimo sistemai in vivo, įvedant ją per os. / Ginkgo biloba-derived preparations have become widely used in medical practice. Thougt extracts of Ginkgo biloba leaves have a wide pharmacological application, little is known about extract effects on mitochondria. Therefore, the aim of this study was to investigate the influence of extracts of Ginkgo biloba leaves on mitochondrial oxidative phosphorylation system. The tasks of the study were following: 1. To investigate the effects of extract of Ginkgo biloba leaves on the respiration of isolated heart and liver mitochondria and permeabilized heart fibers. 2. To analyze the mechanism(s) of extract of Ginkgo biloba leaves on mitochondrial oxidative phosphorylation system. 3. To determine the effect of extract of Ginkgo biloba leaves on perfused rat heart electromechanical activity and to analyze how GBE given to isolated perfused rat hearts readily can penetrate into the heart cells and mitochondria. 4. To test whether perfusion with Ginkgo biloba extract protects heart mitochondria against ischemia/reperfusion damage. 5. To investigate the effect of extract of Ginkgo biloba leaves on mitochondrial oxidative phosphorylation system in vivo.

Mitochondrial mutations and heteroplasmy have been associated with disease states that result from inadequate cellular energy production. As mitochondrial DNA (mtDNA) encodes many of the polypeptides involved in oxidative phosphorylation (OXPHOS), mtDNA mutations may lower energy production which is required for cell division and sustained ATP synthesis. In order to test the relationship between mtDNA mutations and the rate of cell division, a mammary epithelial cancer cell line, MCF-7, is used as a model. Nine proliferate single cell clones have been isolated from MCF-7. Population doubling times of six single cell clones and the MCF-7 stock have been determined. Clones with distinctly different growth rates were selected for mutational analysis. Growth rates of these clones appeared to be different from each other. Using polymerase chain reaction (PCR) and DNA sequencing, three cases of heteroplasmy have been identified in the mitochondrial genes of the MCF-7 stock and four single cell clones (ATPase C9119T, ND6 T14300G, Cytb G15807A). Heteroplasmy present in the Cytb gene is differs between single cell clones. Differences between the growth rates may be indicative of metabolic variations in these single cell clones. The OXPHOS enzymes encoded by the mutated genes were quantified by standard enzymatic assays. The assays demonstrated significant differences in specific activity between the clones, but were not correlated with mitochondrial heteroplasmy. This thesis determines that the differences in specific activity observed between clones is of nuclear origin.

Mitochondrial ATP synthase represents the final complex of oxidative phosphorylation (OXPHOS) system located in the inner mitochondrial membrane. Its primary role is to utilize mitochondrial membrane potential (Δψm) generated by respiratory chain complexes to produce energy in the form of ATP. Mammalian ATP synthase comprises of 17 different subunits organized into membranous Fo and matrix-oriented F1 domains. Defects of complex V and their manifestation have been studied on mitochondrial, cellular, tissue and organism levels using different models, including human cell lines and cell lines derived from patient tissues. In many cases mitochondrial diseases display threshold behaviour, when genetic defect is phenotypically manifested only bellow certain threshold in particular enzyme complex activity and/or content. This work was aimed at elucidation of functional consequences of ATP synthase deficiency in HEK293 cell lines with suppressed gene expression of γ, δ or ε subunits of ATP synthase central stalk. We have analysed range of clones with respective subunits knockdown and found varying decrease in assembled ATP synthase content, which was mirrored by the decrease in individual ATP synthase subunits. The only exception was subunit Fo-c, whose levels remained unchanged or even increased. ATP...

Contusion spinal cord injury (SCI) results in devastating life-long debilitation in which there are currently no effective treatments. The primary injury site presents a complex environment marked by subsequent secondary pathophysiological cascades involving excessive reactive oxygen and nitrogen species (ROS/RNS) production, glutamate-induced excitotoxicity, calcium dysregulation, and delayed neuronal apoptosis. Many of these cascades involve mitochondrial dysfunction, thus a single mitochondrial-centric therapy that targets a variety of these factors could be far reaching in its potential benefits after SCI. As such, this dissertation examines whether transplantation of exogenous mitochondria after SCI can attenuate secondary injury cascades to decrease the spread and severity of the injury. Our first experiment tested the dose-dependent effects of mitochondrial transplantation on the ability to maintain acute overall bioenergetics after SCI. We compared transplantation of mitochondria originating from two different sources-cultured PC12 cells or rat soleus leg muscle. 24 hours after injury, State III oxygen consumption rates were maintained to over 80% of sham levels when 100ug of mitochondria was transplanted, regardless of the origin of the mitochondria. Complex I enzyme activity assays corroborated our findings that the 100ug dosage gave optimal benefits compared to vehicle injection. We also analyzed the rostral-caudal distribution and cell-type colocalization of transplanted transgenically-labeled tGFP mitochondria after SCI. There were greater volumes and rostral-caudal spread of tGFP mitochondria at the 24 hour time point compared to 7 days post injection. tGFP mitochondria had the greatest propensity to colocalize with macrophages and pericytes. Colocalization was evident in endothelial cells, oligodendrocytes and astrocytes, though no such colabeling was present in neurons. Further, colocalization of tGFP was always greater at the 24 hour time compared to 48 hour or 7days post injection time points. These data indicate that there is a cell-type difference in incorporation potential of exogenous mitochondria which changes over time. Finally, we tested the effects of mitochondrial transplantation on long term functional recovery. Animals were injected with either vehicle, 100ug cell-derived mitochondria, or 100ug muscle-derived mitochondria immediately after contusion SCI. Functional analyses including BBB overground locomotor scale and von Frey mechanical sensitivity tests did not show any differences between treatment groups. Likewise, there were no differences in tissue sparing when mitochondria were transplanted compared to vehicle injections, though there were higher neuronal cell counts in tGFP mitochondria injected groups caudal of the injury site. These studies present the potential of mitochondrial transplantation for therapeutic intervention after SCI. While our acute measures do not correspond into long term recovery, we show that at 24 hours transplanted mitochondria do have an effect on bioenergetics and that they are taken into host cells. We believe that further investigation into caveats and technical refinement is necessary at this time to translate the evident acute bioenergetic recovery into long term functional recovery.

Abstract The mitochondrial oxidative phosphorylation system (OXPHOS) generates energy but also deleterious reactive oxygen species (ROS). Changes in the cytoskeleton, composed mainly of microfilaments, microtubules and intermediate filaments, have been observed in OXPHOS deficiency. The 3243A>G point mutation in mitochondrial DNA (mtDNA) leads to mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), which is the most common mitochondrial disease. Interestingly, mitochondrial aberrations have been demonstrated in patients with a mutation in NOTCH3, the genetic cause of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Randomization of vimentin intermediate filament direction and length together with slower population growth was observed in myoblasts with 3243A>G, with no difference in the amount of apoptotic cell death. Upon complex IV inhibition (with or without the microtubule-depolymerizing compound nocodazole) or a lack of mtDNA (ρ0) in osteosarcoma cells the vimentin network collapsed perinuclearly, forming thick bundles, whereas complex I inhibition led to thinner vimentin network bundles. Furthermore, the amount of vimentin was increased in ρ0 cells. Mitochondria accumulated around the nucleus upon complex IV inhibition and in ρ0 cells. Analysis of the total proteome revealed that specific OXPHOS deficiencies led to changes in the expression of cytoskeletal proteins and proteins involved in apoptosis, OXPHOS, glycolysis and oxidative stress response. Muscle histochemical and genetic analysis showed ragged red fibres and cytochrome c oxidase-negative fibres to be associated with 5650G>A in a patient with R133C in NOTCH3 and 5650G>A in MTTA. Immunolabelling of cells with R133C and 5650G>A revealed a sparse tubulin network with asters and less abundant mitochondria by comparison with control cell lines. Comparison of nucleotide diversity between CADASIL pedigrees and controls showed increased mtDNA sequence variation in the CADASIL patients. Also maternal relatives in two CADASIL pedigrees differed from each other in their mtDNA. These findings suggest that defects in OXPHOS lead to selective changes in the vimentin network, which may have a role in the pathophysiology of mitochondrial diseases. They also suggest a relationship between NOTCH3 and mtDNA, and establish the pathogenicity of 5650G>A. The overall results emphasize that a deficiency in the energy converting system together with oxidative stress can lead to cytoskeletal changes.

La mitochondrie est une organelle très importante vu son implication dans plusieurs processus cellulaires. Elle produit notamment la majeure partie de l'énergie qui est consommée par la cellule, grâce aux processus d'oxydation phosphorylante (OXPHOS). La phosphorylation des enzymes impliquées dans les OXPHOS apparait comme une voie de régulation importante de la production énergétique. L'objectif de ce thèse était donc de comprendre comment les phosphorylations, et plus particulièrement, les phosphorylations sur tyrosine induites par la Src kinase influencent les OXPHOS. Il a donc été démontré qu'il existe, à l'intérieur des mitochondries, des voies de régulation de ces processus de phosphorylation induits par la Src kinase. Ces processus pouvant induire la phosphorylation de plusieurs enzymes mitochondriales, notamment plusieurs sous-unités des complexes du système des électrons et ainsi, grandement influencer les OXPHOS. Il a aussi été démontré que la Src kinase semble aussi présente dans les mitochondries de cellules cancéreuses, induisant la phosphorylation d'une sous-unité de la NADH-oxidoréductase et une augmentation du métabolisme énergétique mitochondrial. Cette régulation des OXPHOS dans les cellules cancéreuses par la Src kinase pourrait participer à l'établissement du phénotype hautement prolifératif de ces cellules. / Mitochondria are implicated in several key cellular processes. They are producing most part of the energy that is consumed by the cell via oxidative phosphorylation processes (OXPHOS). Phosphorylation of different components implicated in OXPHOS are known to constitute an important regulation pathway of energetic production. The objective of this thesis was to understand how tyrosine phosphorylation induced by the Src kinase could influence OXPHOS. First, it was shown that Src kinase mediated phosphorylation can be regulated directly in mitochondria, inducing phosphorylation of several mitochondrial proteins and different effects on OXPHOS. I also demonstrated that Src kinase is also present in mitochondria of cancer cells where it can lead to phosphorylation of NADH-oxidoreductase. This phosphorylation site is associated with increase of OXPHOS which could be implicated in the establishment of global phenotype of cancer cells.

Orientador: Roger Frigério Castilho / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T14:26:13Z (GMT). No. of bitstreams: 1 Ruas_JulianaSilveira_M.pdf: 1871713 bytes, checksum: a20e3cd08d0b770aed3b059541e382e3 (MD5) Previous issue date: 2015 / Resumo: A maioria das células tumorais depende da glicólise para a ressíntese de ATP durante um processo de rápida proliferação, mesmo que haja disponibilidade de oxigênio para a transdução de energia mitocondrial (Efeito Warburg). O objetivo do presente estudo foi avaliar o papel do metabolismo oxidativo mitocondrial na proliferação de células de glioblastoma humano U-87MG e T98G. Quando as células foram cultivadas na presença de oligomicina (um inibidor da ATP sintase) ou antimicina A (um inibidor do complexo III da cadeia transportadora de elétrons), observou-se apenas uma inibição parcial da proliferação das células. Notadamente, a incubação dessas células com ambos os inibidores causou uma inibição quase completa na proliferação celular. Resultados semelhantes foram observados em cultura primária de astrócitos, havendo uma queda na proliferação celular somente quando ambos os inibidores mitocondriais estavam presentes. Medidas de consumo de oxigênio indicaram que células de glioblastoma utilizam parcialmente a fosforilação oxidativa para a ressíntese de ATP e apresentam uma respiração bem acoplada. Quando se inibiu, nestas células, a fosforilação oxidativa do ADP com oligomicina ou antimicina A, houve um pequeno aumento no consumo de glicose e na produção de lactato. No entanto, o tratamento com ambos os inibidores mitocondriais promoveu um menor consumo de glicose e produção de lactato, em comparação com os efeitos que a antimicina A promoveu. Isso indica que a cadeia transportadora de elétrons quando inibida pela presença de antimicina A, promove um funcionamento inverso da ATP sintase, promovendo a hidrólise de ATP para que haja um bombeamento de prótons para o espaço intermembranar mitocodrial. De acordo com os resultados acima descritos, uma queda quase completa do potencial de membrana mitocondrial foi observada apenas quando as células de glioblastoma foram incubadas na presença de ambos os inibidores mitocondriais, oligomicina e antimicina A. Quando a análise do ciclo celular foi realizada, observou-se uma diminuição da percentagem das células em G0-G1 e um aumento nas fases S e G2-M quando tratadas com oligomicina. Quando as células foram tratadas com antimicina A e oligomicina mais antimicina A foi constatado uma diminuição significativa nas fases G0-G1 e G2-M, e um aumento na fase S. Em conclusão, estes resultados indicam que a rápida proliferação de células de glioblastoma depende da existência do potencial de membrana mitocondrial, mas não da fosforilação oxidativa ou do transporte de elétrons na cadeia respiratória / Abstract: Most tumor cells rely on glycolysis for ATP resynthesis during rapid proliferation, despite the availability saturating levels of oxygen for mitochondrial energy transduction (Warburg effect). The aim of the present study was to evaluate the role of mitochondrial oxidative metabolism on proliferation of human glioblastoma cells U-87MG and T98G. When cells were cultured in the presence of oligomycin (ATP synthase inhibitor) or antimycin A (inhibitor of complex III of the electron transport chain), we observed only a partial inhibition of cell proliferation. Remarkably, incubation of cells with both inhibitors caused an almost complete inhibition of cell proliferation. Similar results were observed in primary culture of astrocytes, with a decrease in cell proliferation only when both mitochondrial inhibitors were present. Oxygen consumption measurements indicated that glioma cells partially rely on oxidative phosphorylation for ATP turnover and exhibit a well-coupled respiration. In fact, shutting down mitochondrial ADP phosphorylation in these glioma cells with either oligomycin or antimycin inhibitors slightly increased glucose consumption and lactate release. However, the treatment with both mitochondrial inhibitors promoted lower glucose consumption and lactate release as compared with the effects of antimycin alone, which indicates that ATP synthase is operating reversely and thus hydrolyzing ATP and pumping H+ out when the respiratory chain is inhibited by antimycin. In agreement, an almost complete collapse of mitochondrial membrane potential was only observed when the glioma cells were incubated in the presence of both antimycin and oligomycin, but not of only antimycin. When cell cycle analyses were performed in oligomycin-treated cells, a decrease in the percentage of cells in G0-G1 phase and an increase in S and G2-M phases were observed. When cells were treated with antimycin A or oligomycin plus antimycin A, it was observed a significant decrease in G0-G1 and G2-M cell phases and an increase in S phase. Overall, our results suggest that the rapid proliferation of glioblastoma cells is dependent on the mitochondrial membrane potential, but not on oxidative phosphorylation or electron transport in the respiratory chain / Mestrado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Mestra em Ciências

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