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Ketogenic diet as a treatment for inherited disorders of ATP synthase
Kudrnovská, Barbora ; Mráček, Tomáš (advisor) ; Tesařová, Markéta (referee)
The Tmem70 protein plays a significant role in the biogenesis of ATP synthase, mutations in the Tmem70 gene are a common cause of mitochondrial disease and have been associated with neonatal encephalocardiomyopathy. Similar to other mitochondrial disorders, there is currently no available treatment for Tmem70 defects. However, the ketogenic diet (KD) appears to be a potential therapeutic intervention for various mitochondrial disorders, and our preliminary results demonstrate its positive impact in a mouse model of Tmem70 dysfunction. Therefore, using this Tmem70 model, we decided to test a modified KD, which could be a better alternative for a potential application in human patients. The effect of the modified KD was examined in a mouse model of tamoxifen-induced whole-body knockout of Tmem70 (Tmem70 KO). The modified KD led to improved survival, slowed weight loss, and normalization of plasma indicators of liver damage. We detected a positive effect of the modified KD on the assembly of ATP synthase in liver tissue, however, skeletal muscle showed only a minimal response. Based on the results of this study, we infer that although the absence of functional Tmem70 affects both liver and muscle tissues, the greater impact on the phenotypic manifestation in the Tmem70 KO mouse model is due to liver...
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X-ray computed tomography of cartilage tissue of embryos
Tesařová, Markéta ; Křivánek,, Jan (referee) ; Zikmund, Tomáš (advisor)
Research of the cartilage is standardly realized by optical microscopy on histological slices. However, for better understanding of shape-organizing processes, imaging in 3D is required. This thesis deals with X-ray computed tomography of mouse embryos. Specifically, it focuses on visualization of craniofacial cartilage. The thesis summarizes the basic principles of X-ray computed tomography. Short research study dealing with staining of soft tissues for X-ray is also provided. The procedure of implementation of the tomographic measurement of mouse embryos stained by PTA is described. This procedure includes the staining protocol, fixing the sample and setting of the CT machine. Subsequent segmentation and creating of a 3D model of cartilage is also desribed. Results for every step of this procedure are demonstrated at the mouse embryos of developmental stage from 12.5 to 17.5 days. In order to improve data quality and standardization of the whole process, parameters of X-ray tube and temperature conditions during the tomographic measurement are explored. Finally, resulting models of craniofacial cartilage are shown with the possibility of subsequent analysis.
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Implementation of industrial X-ray computed microtomography in developmental biology
Tesařová, Markéta ; Křivánek,, Jan (referee) ; Novotná, Marie (advisor)
Rentgenová počítačová mikrotomografie (CT) je metoda pro trojrozměrné (3D) zobrazování vnitřní struktury objektů. Laboratoř rentgenové počítačové mikro a nanotomografie na CEITEC VUT (Středoevropský technologický institut, Vysoké učení technické v Brně) se zaměřuje zejména na nedestruktivní analýzu v průmyslovém odvětví. Ukázalo se ale, že tato metoda nachází široké uplatnění i v dalších vědeckých oblastech včetně vývojové biologie. Tato práce poskytuje propojení mezi materiálovými a živými vědami. Kompletní proces vedoucí k vytvoření 3D modelu různých anatomických struktur je detailně popsán. Tento postup zahrnuje kontrastování vzorků, samotné CT měření a analýzu dat. Právě zpracování dat je mnohdy považováno za nejdůležitější část v rentgenové počítačové tomografii a proto je této oblasti věnována v práci největší část. Úpravou měřicích parametrů byl dále optimalizován CT přístroj GE v|tome|x L 240 za účelem umožnění automatické segmentace. Navíc je diskutována možnost měření na synchrotronu za účelem zvýšení prostorového rozlišení a diferenciálního kontrastu. Předpokládá se, že v blízké budoucnosti získají průmyslové CT přístroje podobné zobrazovací vlastnosti, a to jak díky nedávnému vývoji v oblasti hardwaru, tak v oblasti zpracování dat.
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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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Quantitative 3D characterization of biological structures by X-ray computed microtomography
Tesařová, Markéta ; Vaňhara,, Petr (referee) ; Oberta,, Peter (referee) ; Zikmund, Tomáš (advisor)
Moderní vývojová biologie využívá široké spektrum metod k analyzování studovaných struktur. Multioborové týmy v poslední době prosazují využívání 3D přístupu k porozumění biologickým procesům. Jedna z hlavních překážek při využívání 3D zobrazovacích technik v biologii je nedostatek metodologie pro kvantifikování pozorovaných jevů, které často bývají příčinou vývojových či genetických onemocnění. V posledních letech se v biologických výzkumech rozšířilo využití rentgenové počítačové mikrotomografie (mikroCT). Technika mikroCT dokáže poskytnout izotropní rozlišení až 1 µm a při správné přípravě vzorku širokou škálu tkáňové diferenciace. Biologické struktury jsou však rozmanitých velikostí a tvarů a každá struktura vyžaduje specifický přístup v oblasti zpracování dat. Tato práce se zabývá využitím mikroCT pro zobrazování, ale především 3D charakterizaci biologických struktur ex-vivo. Zvláštní důraz je kladen na analýzu měkkých tkání v hlavách myších embryí a zobrazování různých druhů mloků se zaměřením na jejich regenerační schopnosti. Tato práce popisuje relevantní informace pro uživatele mikroCT zabývajícími se biologickými projekty: To zahrnuje přípravu vzorku pro měření, výběr vhodných parametrů k samotnému měření, a především analýzu vzniklého 3D datasetu. Nejnovější technologické pokroky ve 3D zobrazovaní byly studovány a testovány na konkrétních projektech vývojové biologie, kde 3D informace získaná pomocí mikroCT hrála klíčovou roli.
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Human F1Fo-ATPsynthase deficiency
Suldovská, Sabina ; Tesařová, Markéta (advisor) ; Černá, Leona (referee)
F1FO-ATPsynthase is a key enzyme in energy metabolism of the cell. Its deficit is caused usually by mutations in two structural genes MT-ATP6 and MT-ATP8 encoded by the mitochondrial DNA or in nuclear genes ATPAF2 and TMEM70 encoding the biogenesis factors and structural gene ATP5E. Deficiency of the F1FO-ATPsynthase leads to progressive and serious phenotype affecting organs with high energy demands. The first symptoms usually occurs in neonatal age and prognosis of the disease is fatal. Mutations in these genes result in both qualitative and quantitative defects of the F1FO-ATPsynthase. The study of molecular bases of mitochondrial disorders including F1FO-ATPsynthase deficiency uses large number of biochemical and molecular-genetic methods to determine a proper diagnosis which is essential for the symptomatic therapy and genetic counselling in affected families. The aim of the diploma thesis was to characterise the F1FO-ATPsynthase deficiency in isolated mitochondria from the lines of cultured cells by the determination oligomycin- sensitive ATP-hydrolytic activity of the F1FO-ATPsynthase, enzymatic activities of the respiratory chain complexes and to analyse changes in the steady-state levels of the representative subunits and whole complex of the F1FO-ATPsynthase in comparison with controls. 3...
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Genetické příčiny deficitu cytochrom c oxidázy u dětí
Vondráčková, Alžběta ; Tesařová, Markéta (advisor) ; Brdička, Radim (referee) ; Procházková, Dagmar (referee)
Mitochondria are the key source of vital ATP molecules, which are largely produced within cells by a system of oxidative phosphorylation (OXPHOS). Genetic defects affecting any of the components of the oxidative phosphorylation system or the structure and function of mitochondria lead to mitochondrial disorders, which occur at an incidence rate of 1 in 5000 live births. Cytochrome c oxidase (COX) is the terminal enzyme and electron acceptor of a respiratory chain that catalyses oxygen to produce a water molecule. In addition to complex I deficiency, isolated or combined COX deficiency is the most common respiratory chain defect in paediatric patients, and it can arise from mutations located either in mitochondrial DNA or in nuclear genes encoding the structural subunits or corresponding assembly factors of the enzyme complex. However, the molecular basis of COX deficiency remains elusive in many patients despite advances in the identification of an increasing number of mutations and genes involved in the disease. This thesis focuses on the identification of the genetic causes of mitochondrial diseases in a cohort of 60 unrelated Czech children with clinically and laboratory confirmed COX-deficiency. With the use of a high-resolution melting analysis mutation screen, four heterozygous sequence...
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