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Optimization of real-time PCR method for quantification of human mtDNA in clinical samples
LOSKOT, Martin
This bachelor thesis dealing with measuring mitochondrial DNA (mtDNA) and how is it important to pathological conditions. Mitochondria are one of the cell organelles in eukaryotic cells. They are important to energy metabolism, aging process, and apoptosis. In current studies mitochondrial dysfunctions cause mitochondrial diseases and common illnesses, for example cardiovascular diseases, brain pathology or cancer. Studies show, that copy number of mtDNA correlates with state of health and aging process. Copy number of mtDNA is often measured by ratio of mtDNA to nuclear DNA. Measuring mtDNA of peripheral blood is appropriate indicators for mitochondrial functions because decreased copy number of mtDNA correlates with decreased function of mitochondria. We don't know, how copy number of mtDNA influence on illnesses, because it doesn't still clarify. However, studies show, that copy number of mtDNA can be biomarker of control of state of health, when we take real time measurement. In the practical part of this bachelor thesis describe preparation of samples from buccal swabs and from peripheral blood. It also describes method of measuring mtDNA by real-time PCR. There is calculation of copy number of mtDNA and evaluation of our results too. As part of the measurement optimization are described differences between two methods for diagnostic of relative copy number of mtDNA.
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Molecular basis of deficit of F1Fo-ATP synthase and its impact on energy metabolism of a cell
Štufková, Hana ; Tesařová, Markéta (advisor) ; Kuncová, Jitka (referee) ; Janovská, Petra (referee)
Mitochondria's primary function is to produce energy through the process of oxidative phosphorylation. ATP synthase is a macromolecular rotary machine located in the inner mitochondrial membrane that catalyzes the synthesis of adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and inorganic phosphate (Pi). The mitochondrial disorders due to ATP synthase deficiency represent a heterogeneous group of diseases characterized by variable severity of the phenotype with onset at birth or later in life till adulthood. Mutations in both, mitochondrial or nucelar DNA encoded genes, may result in ATP synthase impairment, either isolated or combined with deficits of other complexes of oxidative phosphorylation. The aims of the thesis were to characterize TMEM70 protein, an ATP synthase assembly factor, and to analyze the impact of novel disease variants leading to ATP synthase deficiency in patients' derived samples. TMEM70 is a 21 kDa hairpin structure protein localized in the inner mitochondrial membrane, with both termini oriented into the matrix, which forms higher oligomer structures. Our results demonstrated that the absence of TMEM70 protein leads to an isolated deficiency of complex V followed in some stage by adaptive/compensatory effect of respiratory chain complexes. Different severities...
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Study of etiopathology of mitochondrial disorders
Rákosníková, Tereza ; Tesařová, Markéta (advisor) ; Pecina, Petr (referee) ; Kalous, Martin (referee)
Mitochondrial disorders are a clinically, biochemically and genetically heterogeneous group of inherited disorders with a prevalence of about 1:5 000 live births. A common sign of those disorders is disruption of mitochondrial energetic metabolism. To this day, more than 400 genes have been associated with mitochondrial disorders, but 45% of patients are still without a genetic diagnosis. Using next-generation sequencing, new candidate genes or variants are found. To confirm the causality of those newly found genes or variants, biochemical characterisation using a plethora of various methods is necessary. The first aim of this thesis was to study the function of ACBD3 protein on mitochondrial energetic metabolism in non-steroidogenic cells HEK293 and HeLa and to confirm the causality of the ACBD3 gene in a patient with combined oxidative phosphorylation (OXPHOS) deficit. The second aim was to confirm the causality of two novel variants in MT-ND1 and MT-ND5 genes, which encode structural subunits of complex I (CI) of the respiratory chain. The third aim of the thesis was to study the formation of supercomplexes (SCs) in patients with rare metabolic diseases. Using functional studies, we showed in this thesis that ACBD3 protein has no essential function in mitochondria but plays an important role in...
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Low-temperature injury in insect tissues and mechanisms of its repair
ŠTĚTINA, Tomáš
While physiological adjustments accompanying insect cold acclimation prior to cold stress have been relatively well explored, processes of recovery after cold stress received much less attention. Using a broad array of physiological, molecular biological and microscopic techniques, I aimed to capture the complexity of processes occurring after the exposure to cold stress in the larvae of two drosophilid species - freeze tolerant Chymomyza costata and freeze sensitive Drosophila melanogaster. Several lines of evidence pointed toward impaired mitochondrial functions in lethally frozen larvae. The freeze-tolerant phenotypes of C. costata retain integrity of mitochondria even after deep freezing.
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Biogenesis, structure and physiological functions of mitochondrial ATP synthase
Eliáš, Jan ; Mráček, Tomáš (advisor) ; Doležal, Pavel (referee)
Mammalian mitochondrial ATP synthase is an enzyme composed of 18 protein subunits, which is localised in the inner mitochondrial membrane. Its main function is to utilise proton gradient, produced by respiratory chain complexes (RCC), for the synthesis of ATP. Aside from the creation of ATP it is known that its dimers contribute to the correct mitochondrial morphology through the formation of cristae apexes. Furthermore, ATP synthase was proposed to have a role in the mitochondrial permeability transition phenomenon, which is important for regulation of programmed cell death. Over the recent years, our understanding of mammalian ATP synthase biogenesis has been tremendously improved. Its assembly process is now clarified, however the knowledge about assembly intermediates of its peripheral stalk and of subunit c are still not sufficient. We focused precisely on those unsolved questions in the fields of ATP synthase biogenesis and its secondary functions, by the production of a KO model of catalytic β subunit of mammalian ATP synthase F1 domain (βKO). This model was successfully prepared on the background of HEK293 cell line. Its characterisation revealed that disruption of the F1 structure resulted in the inability to assemble functional monomer and resulted in a decay of individual subunits. The only...
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Molecular requirements of LACTB-induced tumor suppression
Jakoubě, Pavel ; Kečkéšová, Zuzana (advisor) ; Rohlenová, Kateřina (referee)
LACTB is a recently discovered mitochondrial tumour suppressor protein operating in many different types of tissues. Its mechanism-of-action seems to be context dependent as it has been shown to suppress carcinogenesis through the induction of cell cycle arrest, apoptosis, differentiation and suppression of EMT. These processes can be further dependent on alterations of lipid metabolism and interactions with additional tumour suppressors and signalling pathways. LACTB is derived from bacterial penicillin binding proteins, is localized to the mitochondrial intermembrane space and possesses enzymatic activity. It was shown to form filaments, which consist of two intertwined antiparallel chains, suggesting its role in the organisation of mitochondrial intermembrane space. In the first aim of my thesis, I wanted to examine in more detail the molecular requirement for LACTB's filament formation with the specific focus on the role of disulphide bonds in this process. In the second aim of my thesis, I intended to uncover the binding partners of LACTB, which might have a role in the filament formation. Realizing both aims will uncover important requirements for the proper folding and biological activity of LACTB. Key words: LACTB, tumour suppressor, cancer, structure, disulphide bonds, protein interactions
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Characterization and modulation of MitoTam-induced cell death in breast carcinoma cells
Hrysiuk, Mariia ; Anděra, Ladislav (advisor) ; Dráber, Peter (referee)
Although recent years brought many breakthrough discoveries in anti-cancer research and therapy, malignant diseases such as breast cancer (BC) still present one of the major health threats worldwide. Cancer cells usually gain resistance to the activation of regulated cell death (RCD) modalities such as caspase-dependent apoptosis. Among novel RCD-inducing agents belongs to mitochondria-targeted tamoxifen - MitoTam, which is also the major focus of this Thesis. In a panel of BC cells, we determined the energetic (mitochondrial respiration vs. glycolysis) and major RCD-related proteins (Western blotting) profiles, and using Lumascope LS720-assisted time-lapse monitoring we analyzed their sensitivity to MitoTam-induced RCD. We found out that glycolysis-preferring BC cells as MDA-MB-231 are more resistant to MitoTam treatment than mitochondrial respiration-biased MDA-MB-453 cells. However,the majority of tested BC cells can be sensitized to MitoTam by BH3 mimetics such as BCL-XL targeting A1155463 and some cellular metabolism-modulating compounds such as lactate dehydrogenase inhibitor (R)-GNE-140, especially in the pre-treatment regime. Also, other metabolism-modulating compounds such as Pyruvate Dehydrogenase Kinases inhibitor JX06 potently enhanced the efficacy and kinetics of MitoTam-induced RCD....
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Mitochondrial transfer-mediated modulatory action of stem cells on immune cells
Somova, Veronika ; Krulová, Magdaléna (advisor) ; Balounová, Jana (referee)
Stem cells use different mechanisms of intercellular communication to modulate an immune response. Mitochondrial transfer is one of the mechanisms which induce metabolic changes, support cell survival, and change the phenotype of immune cells. Nevertheless, little is known about the mechanism used for transfer of mitochondria between different cell populations and the faith of mitochondria inside the acceptor cell. This thesis aims to describe the mechanism of transfer and the provided modulation. Factors that could affect mitochondrial transfer including reactive oxygen species production, apoptosis and mitochondria function were analyzed. And the impact of mitochondrial transfer on cell survival and mitophagy was described. The next aim was to compare the ability of mesenchymal stem cells (MSC) and Sertoli cells (SC) to transfer mitochondria, with MSC being more productive in the transfer of mitochondria than SC. Significant differences in the presence of mitochondria from donor MSC or SC in individual populations of immune cells were also detected. To explain these findings, the impact of reactive oxygen species on the transfer of mitochondria was analyzed in detail, although it wasn't confirmed. However, it needs to be highlighted that mitophagy plays an important role before and after...
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Evaluation of antibiotic-induced mitochondrial superoxide production in adherent cells
Ingrová, Kateřina ; Chmelíková, Larisa (referee) ; Zumberg, Inna (advisor)
The theoretical part of this bachelor´s thesis contains a description of the effect of reactive oxygen species on oxidative stress by mitochondria and the consequences of antibiotics use in cell line culturing. The cell line studied in this bachelor´s thesis is the mesenchymal stem cells (MSCs). The practical part describes the procedure of the experiment including cell culturing, passaging and cell labeling. The proposed experiment was repeated with sufficient number of repetitions. Finally, confocal microscopy images were processed in the MATLAB programming environment.
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Mitochondrial production of reactive oxygen species and its role in physiological regulations
Holzerová, Eliška ; Mráček, Tomáš (advisor) ; Kalous, Martin (referee)
The production of mitochondrial reactive oxygen species and the resulting oxidative stress is an important phenomenon driving long-lasting research and intense discussions. Knowledge of exact mechanisms of reactive oxygen species production and pathways leading to their formation could help us to directly affect their production, a task with potential terapeutic implications. The molecular nature of the production of reactive oxygen species by some enzymes has already been well documented, but others still remain controversial and current theories are obviously far from the truth. Much more interesting is the question of physiological importace of this production. The reactive oxygen species were considered harmful factors clearly distorting the integrity of the organism for a long time. However, recent research suggest that their existence can also be beneficial and effective. Evidently they can serve as a signaling molecules in several metabolic and regulatory pathways occurring in the organism. This bachelor thesis offers insight into the current state of knowledge. It focuses on the most detailed description of the reactive oxygen species production by mitochondrial respiratory chain enzymes. Furthermore, it deals with some signaling cascades, where involvement of mitochondrially generated...
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