(EN) Mitochondria are the principal intracellular organelles responsible for fuel generation; however, they are not just cell powerhouses but are involved in a range of other intracellular functions including cell metabolism, proliferation, death, and immune responses. Loss of function in mitochondria will result in oxidative stress, which is one of the underlying causal factors for a variety of diseases including cancer. Cancer cells can predominantly produce energy by glycolysis even in the presence of oxygen. This alternative metabolic behavior is known as the "Warburg Effect." Linked to this, cancer cell mitochondria can switch between glycolysis and oxidative phosphorylation (OXPHOS) for their energy requirements and survival. The electron transport chain (ETC) function is pivotal for mitochondrial respiration, which is also needed for dihydroorotate dehydrogenase (DHODH) activity that is essential for de novo pyrimidine synthesis. In our research, we have used respiration-deficient cancer cells to challenge the dogma that mitochondria with their DNA are constrained within cells in the body. Our results document that mitochondria move from normal cells within the tumor stroma to tumor cells without mitochondrial DNA (mtDNA), resulting in long-lasting recovery of mitochondrial functions and,...

This diploma thesis is focused on topics of and respiratory chain and their processing as educational animations for secondary schools. In the theoretical part of the diploma thesis, supporting terms are defined (visualisations, interdisciplinary relations, animations). Next, themes are included in educational field and thematic unit in RVP G. Two analyses of the topic of respiratory chain are mentioned and evaluated in this part. Firstly, there is an analysis focused on the most used chemical and biological textbooks. The second one is focused on available online animations. For research purpose of the diploma thesis quantitative method was used - a questionnaire survey. Results of this survey are listed in the practical part. Practical part presents educational materials specified for support of education topics of respiration and respiratory chain. Stress on illustrative nature and interdisciplinarity is laid in these materials. The principal materials are educational animations which were made in program Adobe Flash Professional CS6. Study text was also written to these animations. Created educational animations give a complex view on respiratory process. These materials can be used in biology and chemistry subjects or in scientific courses. Animations include two tests giving feedback to...

Espécies reativas de oxigênio (EROs) são normalmente e continuamente geradas em mitocôndrias, majoritariamente na cadeia de transporte de elétrons (CTE). Harman (1956, 1972 e 1992) teorizou que os radicais livres gerados nas mitocôndrias seriam a principal causa do envelhecimento. De fato, durante o envelhecimento é observado um desequilíbrio entre formação e remoção de EROs, que resulta em estresse redox. Essa condição favorece a formação de lesões oxidadas no DNA, acarretando em mutagênese ou morte celular. Diversos mecanismos moleculares cooperam para o reparo de DNA. Duas vias de reparo de DNA lidam com a maioria das lesões: o reparo por excisão de base (BER) e o reparo por excisão de nucleotídeos (NER). A via BER corrige pequenas modificações de bases que surgem de reações de desaminação, alquilação e oxidação. A via NER é mais versátil, reconhecendo lesões que distorcem a dupla hélice de DNA, como danos induzidos por luz UV e adutos volumos. Pacientes xeroderma pigmentoso (XP-A a XP-G) herdam mutações em um de sete genes que codificam proteínas envolvidas na via NER, ou em um gene que codifica uma polimerase translesão (XP-V). A doença é caracterizada por fotosensibilidade e incidência elevada de neoplasias cutâneas. A proteína XPC atua na etapa de reconhecimento da lesão de DNA na subvia de reparo global do genoma (GG-NER), e sua mutação dá origem aos sintomas clássicos de XP. Novas funções de XPC foram recentemente descritas: i) atuando como cofator na via BER auxiliando as DNA glicosilases OGG1, TDG e SMUG; ii) atuando como cofator transcricional de elementos responsivos a Oct4/Sox2, RXR e PPARα; e iii) na adaptação metabólica na transformação de queratinócitos. Então, propusemo-nos a investigar as relações entre XPC e a manutenção da integridade do DNA mitocondrial, a sensibilidade celular a estresse redox mitocondrial e possíveis alterações bioenergéticas e redox. Para tal, padronizamos um ensaio in vitro de cinética de incisão em DNA plasmidial a fim de investigarmos o possível papel de XPC no reparo de lesões oxidadas em mtDNA. Porém, nossos dados revelaram que XPC não se encontra em mitocôndrias. Apesar disso, células XP-C são mais sensíveis ao tratamento com azul de metileno (AM), antimicina A (AA) e rotenona (ROT), que geram estresse redox mitocondrial. A sensibilidade à AA foi completamente revertida em células corrigidas. Células XP-C apresentaram alterações quanto ao uso dos complexos mitocondriais, com diminuição da taxa de consumo de oxigênio (OCR) via complexo I e um aumento da OCR via complexo II, dependente da presença de XPC. Ademais, a linhagem XP-C apresentou um desequilíbrio redox mitocondrial com maior produção de EROs e menor atividade de GPx. O DNA mitocondrial de células XP-C apresentou níveis elevados de lesão e deleção, que no entanto não retornaram aos níveis encontrados em células selvagens na linhagem XP-C corrigida. Observamos uma acentuada diminuição da expressão de PPARGC1A, um importante regulador de biogênese mitocondrial. Contudo, não foi possível determinar o mecanismo de supressão da expressão de PPARGC1A. Por fim, identificamos que o tipo de mutação em XPC pode estar associado a expressão de PPARGC1A. Esse estudo abre novas possibilidade na investigação do papel de proteína XPC, à parte da instabilidade genômica, na adaptação metabólica e desequilíbrio redox em direção da progressão tumoral. / Mitochondria continuously produce reactive oxygen species (ROS), mainly at the electron transport chain. Harman (1956, 1972 e 1992) proposed that normal aging is driven by increased mitochondrially generated free radicals. Indeed, during the course of aging there is an increased imbalance between formation and removal of ROS, leading to redox stress. This condition favours the formation of oxidized DNA lesions, given rise to mutations and cell death. Several molecular mechanisms cooperates to repair the DNA. Two DNA repair pathways deal with the majority of lesions: base excision repair (BER) and nucleotide excision repair (NER). The BER pathway corrects small base modifications that arise from deamination, alkylation and oxidation reactions. The NER pathway is more versitile, recognizing helix-distorting lesions, such as UV-induced damage and bulky adducts. Xeroderma pigmentosum (XP-A to XP-G) patients inherit mutations in one of seven protein-coding genes involved in NER pathway, or in a gene coding a translesion DNA polymerase (XP-V). Photosensitivity and a thousand-fold increased in the risk of developing cutaneous neoplasms are the main clinical features of XP. XPC protein functions in the recognition step of global genome NER (GG-NER) sub-pathway, and mutations in this gene lead to classical XP symptoms. Recently, it has been described that XPC acts: i) as a cofactor in BER pathway through functional interaction with DNA glycosylases OGG1, TDG and SMUG1; ii) as coactivator in transcription at Oct4/Sox2, RXR and PPARα responsive elements; iii) in metabolic shift during keratinocytes transformation. Thus, we sought to investigate a possible role for XPC in the maintenance of mtDNA integrity, cellular sensitivity to mitochondrial redox stress and eventual bioenergetic and redox changes. For this purpose, we established an in vitro plasmid incision assay to investigate the possible role of XPC in the repair of oxidized lesions in mitochondrial DNA. However, our data revealed that XPC did not localized in mitochondria. Nonetheless, XP-C cells are more sensitive to methylene blue, antimycin A (AA) and rotenone treatment, which induce mitochondrial redox stress. The XP-C sensitivity to AA was completely reverted in XPC-corrected cells. XP-C cells presented altered usage of mitochondrial complexes, with decreased oxygen consumption rate (OCR) via complex I and increased OCR through complex II, an XPC-dependent phenomenon. Furthermore, the XP-C cell line showed mitochondrial redox imbalance with increased ROS production and decrease GPx activity. MtDNA from XP-C cells accumulate lesions and deletions, which, however, were found at similar levels in the corrected cell line. We identified a sharp decrease in the expression of PPARGC1A, a master regulator of mitochondrial biogenesis. Nevertheless, it was not possible to determine the mechanism of suppression of PPARGC1A expression. Finally, our results suggest a possible link between the type of XPC mutation and PPARGC1A expression. This study unfolds new possible roles for XPC, aside from its established roles in genomic instability, in metabolic adaptation and redox imbalance towards tumour progression.

Espécies reativas de oxigênio (EROs) são normalmente e continuamente geradas em mitocôndrias, majoritariamente na cadeia de transporte de elétrons (CTE). Harman (1956, 1972 e 1992) teorizou que os radicais livres gerados nas mitocôndrias seriam a principal causa do envelhecimento. De fato, durante o envelhecimento é observado um desequilíbrio entre formação e remoção de EROs, que resulta em estresse redox. Essa condição favorece a formação de lesões oxidadas no DNA, acarretando em mutagênese ou morte celular. Diversos mecanismos moleculares cooperam para o reparo de DNA. Duas vias de reparo de DNA lidam com a maioria das lesões: o reparo por excisão de base (BER) e o reparo por excisão de nucleotídeos (NER). A via BER corrige pequenas modificações de bases que surgem de reações de desaminação, alquilação e oxidação. A via NER é mais versátil, reconhecendo lesões que distorcem a dupla hélice de DNA, como danos induzidos por luz UV e adutos volumos. Pacientes xeroderma pigmentoso (XP-A a XP-G) herdam mutações em um de sete genes que codificam proteínas envolvidas na via NER, ou em um gene que codifica uma polimerase translesão (XP-V). A doença é caracterizada por fotosensibilidade e incidência elevada de neoplasias cutâneas. A proteína XPC atua na etapa de reconhecimento da lesão de DNA na subvia de reparo global do genoma (GG-NER), e sua mutação dá origem aos sintomas clássicos de XP. Novas funções de XPC foram recentemente descritas: i) atuando como cofator na via BER auxiliando as DNA glicosilases OGG1, TDG e SMUG; ii) atuando como cofator transcricional de elementos responsivos a Oct4/Sox2, RXR e PPARα; e iii) na adaptação metabólica na transformação de queratinócitos. Então, propusemo-nos a investigar as relações entre XPC e a manutenção da integridade do DNA mitocondrial, a sensibilidade celular a estresse redox mitocondrial e possíveis alterações bioenergéticas e redox. Para tal, padronizamos um ensaio in vitro de cinética de incisão em DNA plasmidial a fim de investigarmos o possível papel de XPC no reparo de lesões oxidadas em mtDNA. Porém, nossos dados revelaram que XPC não se encontra em mitocôndrias. Apesar disso, células XP-C são mais sensíveis ao tratamento com azul de metileno (AM), antimicina A (AA) e rotenona (ROT), que geram estresse redox mitocondrial. A sensibilidade à AA foi completamente revertida em células corrigidas. Células XP-C apresentaram alterações quanto ao uso dos complexos mitocondriais, com diminuição da taxa de consumo de oxigênio (OCR) via complexo I e um aumento da OCR via complexo II, dependente da presença de XPC. Ademais, a linhagem XP-C apresentou um desequilíbrio redox mitocondrial com maior produção de EROs e menor atividade de GPx. O DNA mitocondrial de células XP-C apresentou níveis elevados de lesão e deleção, que no entanto não retornaram aos níveis encontrados em células selvagens na linhagem XP-C corrigida. Observamos uma acentuada diminuição da expressão de PPARGC1A, um importante regulador de biogênese mitocondrial. Contudo, não foi possível determinar o mecanismo de supressão da expressão de PPARGC1A. Por fim, identificamos que o tipo de mutação em XPC pode estar associado a expressão de PPARGC1A. Esse estudo abre novas possibilidade na investigação do papel de proteína XPC, à parte da instabilidade genômica, na adaptação metabólica e desequilíbrio redox em direção da progressão tumoral. / Mitochondria continuously produce reactive oxygen species (ROS), mainly at the electron transport chain. Harman (1956, 1972 e 1992) proposed that normal aging is driven by increased mitochondrially generated free radicals. Indeed, during the course of aging there is an increased imbalance between formation and removal of ROS, leading to redox stress. This condition favours the formation of oxidized DNA lesions, given rise to mutations and cell death. Several molecular mechanisms cooperates to repair the DNA. Two DNA repair pathways deal with the majority of lesions: base excision repair (BER) and nucleotide excision repair (NER). The BER pathway corrects small base modifications that arise from deamination, alkylation and oxidation reactions. The NER pathway is more versitile, recognizing helix-distorting lesions, such as UV-induced damage and bulky adducts. Xeroderma pigmentosum (XP-A to XP-G) patients inherit mutations in one of seven protein-coding genes involved in NER pathway, or in a gene coding a translesion DNA polymerase (XP-V). Photosensitivity and a thousand-fold increased in the risk of developing cutaneous neoplasms are the main clinical features of XP. XPC protein functions in the recognition step of global genome NER (GG-NER) sub-pathway, and mutations in this gene lead to classical XP symptoms. Recently, it has been described that XPC acts: i) as a cofactor in BER pathway through functional interaction with DNA glycosylases OGG1, TDG and SMUG1; ii) as coactivator in transcription at Oct4/Sox2, RXR and PPARα responsive elements; iii) in metabolic shift during keratinocytes transformation. Thus, we sought to investigate a possible role for XPC in the maintenance of mtDNA integrity, cellular sensitivity to mitochondrial redox stress and eventual bioenergetic and redox changes. For this purpose, we established an in vitro plasmid incision assay to investigate the possible role of XPC in the repair of oxidized lesions in mitochondrial DNA. However, our data revealed that XPC did not localized in mitochondria. Nonetheless, XP-C cells are more sensitive to methylene blue, antimycin A (AA) and rotenone treatment, which induce mitochondrial redox stress. The XP-C sensitivity to AA was completely reverted in XPC-corrected cells. XP-C cells presented altered usage of mitochondrial complexes, with decreased oxygen consumption rate (OCR) via complex I and increased OCR through complex II, an XPC-dependent phenomenon. Furthermore, the XP-C cell line showed mitochondrial redox imbalance with increased ROS production and decrease GPx activity. MtDNA from XP-C cells accumulate lesions and deletions, which, however, were found at similar levels in the corrected cell line. We identified a sharp decrease in the expression of PPARGC1A, a master regulator of mitochondrial biogenesis. Nevertheless, it was not possible to determine the mechanism of suppression of PPARGC1A expression. Finally, our results suggest a possible link between the type of XPC mutation and PPARGC1A expression. This study unfolds new possible roles for XPC, aside from its established roles in genomic instability, in metabolic adaptation and redox imbalance towards tumour progression.

Mood disorders are serious diseases. Nevertheless, their pathophysiology is not sufficiently clarified. Biological markers that would facilitate the diagnosis or successful prediction of pharmacotherapy are still being sought. The aim of the study was to find out whether mitochondrial functions are affected by antidepressants, mood stabilizers and depression. Our research is based on recent hypotheses of mood disorders, the advanced monoamine hypothesis, the neurotrophic hypothesis, and the mitochondrial dysfunction hypothesis. We assume that impaired function of mitochondria leads to neuronal damage and can be related to the origin of mood disorders. Effects of antidepressants and mood stabilizers on mitochondrial functions can be related to their therapeutic or side effects. In vitro effects of pharmacologically different antidepressants and mood stabilizers on the activities of mitochondrial enzymes were measured in mitochondria isolated from pig brains (in vitro model). Activity of monoamine oxidase (MAO) isoforms was determined radiochemically, activities of other mitochondrial enzymes were measured spectrophotometrically. Overall activity of the system of oxidative phosphorylation was measured electrochemically using high- resolution respirometry. Methods were modified to measure the same...

(EN) Tumours are heterogeneous and consist of multiple populations of cells. The population of cells with tumour-initiating capability is known as cancer stem cells (CSC). Cells with increased stemness properties and elevated resistance to anti-cancer treatment have been shown to be highly affected upon decline of mitochondrial respiration, linking the concept of CSCs to deregulated bioenergetics. Consistently, functional electron transport chain (ETC) is crucial in tumorigenesis. Expression of HER2 oncogene, associated with resistance to treatment in breast cancer, has been connected with regulation of mitochondrial function. We therefore investigated the possibility that manipulation of mitochondrial bioenergetics via disruption of ETC eliminates the conventional therapy-resistant populations of tumour, such as CSCs and HER2high cells. We demonstrate that HER2high cells and tumours have increased complex I-driven respiration and increased assembly of respiratory supercomplexes (SC). These cells are highly sensitive to MitoTam, a novel mitochondria-targeted derivative of tamoxifen, acting as a CI inhibitor and SC disruptor. MitoTam was able to overcome resistance to tamoxifen, and to reduce the metastatic potential of HER2high cells. Higher sensitivity of HER2high cells to MitoTam is dependent on...

1 Abstract in English Mitochondrial electron transport chain (ETC) targeting shows a great promise in cancer therapy. However, why modern ETC-targeted compounds are tolerated on the organismal level and what are the molecular reasons for this tolerance remains unclear. Most somatic cells are in a non-proliferative state, and features associated with the ETC in quiescence might therefore contribute to specificity. Thus, we investigated the ETC status and the role of two major consequences of ETC blockade, reactive oxygen species (ROS) generation and inhibition of ATP production, in cell death induction in breast cancer cells and in proliferating and quiescent non-transformed cells. First, we characterised the effect of a newly developed ETC inhibitor mitochondria- targeted tamoxifen (MitoTam) in in vitro and in vivo tumour models of breast cancer with varying status of the Her2 oncogene. We document that Her2high cells and tumours have increased assembly of respiratory supercomplexes (SCs) and increased complex I-driven respiration in vitro and in vivo. They are also highly sensitive to MitoTam. Unlike the parental compound tamoxifen, MitoTam efficiently suppressed experimental Her2high tumours without systemic toxicity. Mechanistically, MitoTam inhibits complex I- driven respiration and disrupts respiratory...

Mood disorders are serious diseases. Nevertheless, their pathophysiology is not sufficiently clarified. Biological markers that would facilitate the diagnosis or successful prediction of pharmacotherapy are still being sought. The aim of the study was to find out whether mitochondrial functions are affected by antidepressants, mood stabilizers and depression. Our research is based on recent hypotheses of mood disorders, the advanced monoamine hypothesis, the neurotrophic hypothesis, and the mitochondrial dysfunction hypothesis. We assume that impaired function of mitochondria leads to neuronal damage and can be related to the origin of mood disorders. Effects of antidepressants and mood stabilizers on mitochondrial functions can be related to their therapeutic or side effects. In vitro effects of pharmacologically different antidepressants and mood stabilizers on the activities of mitochondrial enzymes were measured in mitochondria isolated from pig brains (in vitro model). Activity of monoamine oxidase (MAO) isoforms was determined radiochemically, activities of other mitochondrial enzymes were measured spectrophotometrically. Overall activity of the system of oxidative phosphorylation was measured electrochemically using high- resolution respirometry. Methods were modified to measure the same...

Mitochondria are essential organelles for most eukaryotic cells, containing intricate networks of numerous proteins. These include, among others, complexes I-IV of the electron transport chain. Being at the crossroads of the tricarboxylic acid cycle and the respiratory chain, mitochondrial complex II plays a key role in cellular metabolism. The protein complex, also known as succinate dehydrogenase, is capable of not only succinate oxidation and electron transfer but also contributes to the production of reactive oxygen species. Mitochondrial complex II consists of four subunits, SDHA-D, and four dedicated protein assembly factors SDHAF1-4 that participate in complex II biogenesis. Mutations and epigenetic modulations of genes coding for succinate dehydrogenase subunits or assembly factors are associated with pathological conditions such as neurodegenerative diseases, or may result in tumor formation. However, inborn complex-II-linked mitochondrial pathologies are rather understudied, compared to diseases with causative errors of other mitochondrial complexes, presumably due to the fact that none of complex II subunits is encoded in the mitochondrial genome. Recent studies have shown that impairment of mitochondrial complex II function or assembly leads to accumulation of alternative assembly forms...

No description available.