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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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"Characterization of Pathogenic Variants of Mitochondrial Carriers and Transporters With the Use of Biochemical Methods: Revealing Novel Mutations and Deletions in Patient Samples, Assumed to be Associated With Rare Diseases."
BEHENSKY, Magdalena Maria
Research concerning orphan diseases pose a challenge to the scientific community, however, they are both of great interest and great necessity, leading to the improvement of treatment opportunities for the 263 to 446 million people affected worldwide and, subsequently, their quality of life. The presence of mitochondria in virtually all kinds of cells, with the exception of mature erythrocytes, make clinical diagnosis of mitochondrial diseases extremely challenging and difficult, as dysfunction in mitochondria can affect any organ, or even whole organ systems. A major challenge for modern medical science, is the identification of the genes affected, which cause specific rare diseases in humans. New technologies, such as next-generation sequencing, and, therefore, the possibility of identifying genetic variations, are rapidly proceeding. Consequently, available treatments, or genetic counseling can be found more often. SLC25A3 is a mitochondrial phosphate-carrier responsible for the transport of inorganic phosphate into the mitochondrial matrix, which is required for the final step of oxidative phosphorylation, and the process of aerobic synthesis of ATP. Alteration in this gene, located on chromosome 12q23.1, is known to cause PiC deficiency (OMIM 610773), which affects the heart-/muscle-specific phosphate-carrier isoform A (PiC-A) [19, 23, 24, 26]. The aim of the Master's Thesis, was to ascertain the impact of the mutation on the activity of the phosphate carrier, and the patient's resulting life expectancies. Previously, three mutations have been described in seven patients, as being homozygous, affecting exon 3A, with the exception of one, which is compound heterozygous and affecting exon 4. The four novel SLC25A3 mutations (p.Gly72Glu, p.Thr81Lys, p.Asp87Tyr, p.Ala124Pro) are homozygous, one (p.Gly72Glu) having been described previously [19,26,30]. In the context of this thesis, four patient samples were used for experimentation, each containing one of these homozygous mutations. SLC19A3 encodes the thiamine transporter 2 (hTHTR2), dysfunctions lead to recurrent encephalopathy, basal ganglia necrosis, generalized dystonia, severe disability and early death. Patients with changes in SLC19A3, located on the second chromosome (q36.3), show a considerable reduction of free-thiamine in cerebrospinal fluid and fibroblasts [34]. The clinical pathology of SLC19A3 is heterogeneous, and, likely, related to the age of onset [49]. Previously, Mayr et al. [27] reported various mutations associated with a thiamine pyrophosphokinase deficiency in encephalopathic children, as well as Kevelam et al. [70], who reported mutated SLC19A3 (p.Ser181Pro, p. Leu385Arg and p.Gly23Val) in patients with an early-infantile, lethal encephalopathy. Furthermore, Schänzer et al. [60], Whitford et al. [71] and Fl?nes et al. [63] have reported bigger partial deletions in SLC19A3. Assuming a partial deletion in the sequence of one of the patient samples, the main task of the second project was the determination of the edges, the localization of the breaking point, and further, the size of the deletion, and, whether this deletion has an impact on gene activity. Additionally, the influence of the location and size of the deletion on the overall condition of the patients was analyzed. Furthermore, a sample from a patient, who was previously identified as having a complete deletion of SLC19A3, was added to the experimentation for verification purposes. Finally, the newly found partial deletion and complete deletion were compared to the previously reported deletions. Large deletions of genes are of particular interest, due to their rarity. Large partial deletions, or even complete deletions are seldomly found and reported, as they are complicated to detect, and generally very rare. As in this case, sometimes, they are only detectable when searching in the non-coding region. Therefore, it is important to search in the right places.
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Three-parent babies - new therapies of mitochondrial disorders
Helebrandtová, Veronika ; Pecinová, Alena (advisor) ; Ješina, Pavel (referee)
Mitochondria are essential parts of living cells, as they play a key role in cellular metabolism, especially in energy production. Due to their unique structure, the energy released during the oxidation of the substrates can be used to form the ATP. Mitochondria also contain their own DNA (mtDNA), which is maternally inherited and encodes catalytic subunits of oxidative phosphorylation complexes. Mitochondrial disorders of nuclear or mitochondrial origin, are common causes of inherited diseases and affect mainly the tissues with high energy requirements, such as heart or brain. Treatment of mitochondrial diseases is usually symptomatic and does not lead to complete recovery of the patient. As a result, new causal therapies, such as a gene therapy, are currently investigated. However, using this approach it is necessary to consider the origin of the mutation. Gene therapy of mitochondrial diseases of mtDNA origin is very complicated, therefore the new treatment strategy, mitochondrial replacement therapy, has been proposed. The principle of this technique is to prevent the transmission of mutated mtDNA from mother to offspring by transferring the nuclear genome of mother with mitochondrial disorder into donor's denucleated oocyte with healthy mitochondria. In this way, the child has genetic...
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Non-pharmacological approaches towards treatments of mitochondrial disorders
Kudrnovská, Barbora ; Mráček, Tomáš (advisor) ; Kalous, Martin (referee)
Mitochondrial diseases are characterized by a high degree of genotypic and phenotypic heterogeneity, but as a whole group, they represent one of the most common forms of inborn errors of metabolism. Treatment of these diseases is still very limited, despite massive body of ongoing research projects, which are focussed on new therapeutic approaches. Besides pharmacological methods, there are also non-pharmacological approaches such as certain diet regimens, physical activity or exposition to hypoxia. Due to their relatively generalized mechanism of action, these methods may offer benefits to a wider range of mitochondrial patients, despite different primary genetic defects. This work summarizes the existing knowledge about the use of exercise, ketogenic diet, and hypoxia application as possible therapeutic approaches towards the treatment of mitochondrial diseases. Keywords: mitochondria, mitochondrial diseases, therapy, exercise, ketogenic diet, hypoxia
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Genetic and functional characterisation of mitochondrial diseases caused by ATP synthase defects
Tauchmannová, Kateřina ; Houštěk, Josef (advisor) ; Flachs, Pavel (referee) ; Kutejová, Eva (referee)
Disorders of ATP synthase, the key enzyme of mitochondrial energy provision belong to the most severe metabolic diseases presenting mostly as early-onset mitochondrial encephalo-cardio-myopathies. Mutations in four nuclear genes can result in isolated deficiency of ATP synthase, all sharing a similar biochemical phenotype - pronounced decrease in the content of fully assembled and functional ATP synthase complex. The thesis summarises studies on two distinct causes of ATP synthase deficiency. First is TMEM70 protein, a novel ancillary factor of ATP synthase, which represents most frequent determinant of severe inborn deficiency of ATP synthase. TMEM70 is a 21 kDa protein of the inner mitochondrial membrane, facilitating the biogenesis of mitochondrial ATP synthase, possibly through TMEM70 protein region exposed to the mitochondrial matrix, but the proper regulatory mechanism remains to be elucidated. In TMEM70-lacking patient fibroblasts the low content of ATP synthase induces compensatory adaptive upregulation of mitochondrial respiratory chain complexes III and IV, interestingly by a posttranscriptional mechanisms. The second type of ATP synthase deficiency studied was mtDNA m.9205delTA mutation affecting maturation of MT-ATP8/MT-ATP6/MT-CO3 mRNA and thus biosynthesis of Atp6 (subunit a) and Cox3...
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Use of Canistherapy for Stimulation of Persons with Rare Diseases
Prokopová, Zuzana ; Hájková, Vanda (advisor) ; Šumníková, Pavlína (referee)
The aim of this thesis was to assess the influence of canistherapeutic intervention on the level of fine motor skills in patients with rare diseases, namely mitochondrial encephalocardiomyopathy caused by TMEM70 gene mutation and Leigh syndrome. Canistherapeutic intervention in these patients is based on activation of motoric functions during programme reasonably designed according to the needs of specific patients. The assistance of the dog is used to achieve this activation. The dog acts as an important motivational factor and the direct contact with it contributes to development of sensomotoric skills of these patients. The thesis summarizes general information about rare diseases and focuses on one of the subgroups of mitochondrial disorders. It presents knowledge about the Leigh syndrome and mitochondrial encephalocardiomyopathy caused by TMEM70 gene mutation. Subsequently the thesis presents information about canistherapy and fine motor skills. The thesis further contains case reports of patients, data from research units and their evaluation. The aim of the thesis was to find out whether patients with mitochondrial disease show improvement of quality and effectivity of selected grasp forms and thus faster and more precise execution assigned tasks focusing on object manipulations. Partial...
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Genetic and functional characterisation of mitochondrial diseases caused by ATP synthase defects
Tauchmannová, Kateřina ; Houštěk, Josef (advisor) ; Flachs, Pavel (referee) ; Kutejová, Eva (referee)
Disorders of ATP synthase, the key enzyme of mitochondrial energy provision belong to the most severe metabolic diseases presenting mostly as early-onset mitochondrial encephalo-cardio-myopathies. Mutations in four nuclear genes can result in isolated deficiency of ATP synthase, all sharing a similar biochemical phenotype - pronounced decrease in the content of fully assembled and functional ATP synthase complex. The thesis summarises studies on two distinct causes of ATP synthase deficiency. First is TMEM70 protein, a novel ancillary factor of ATP synthase, which represents most frequent determinant of severe inborn deficiency of ATP synthase. TMEM70 is a 21 kDa protein of the inner mitochondrial membrane, facilitating the biogenesis of mitochondrial ATP synthase, possibly through TMEM70 protein region exposed to the mitochondrial matrix, but the proper regulatory mechanism remains to be elucidated. In TMEM70-lacking patient fibroblasts the low content of ATP synthase induces compensatory adaptive upregulation of mitochondrial respiratory chain complexes III and IV, interestingly by a posttranscriptional mechanisms. The second type of ATP synthase deficiency studied was mtDNA m.9205delTA mutation affecting maturation of MT-ATP8/MT-ATP6/MT-CO3 mRNA and thus biosynthesis of Atp6 (subunit a) and Cox3...
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