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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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Protein analysis of selected mitochondrial proteins in the muscle tissue of porcine model of Huntington's disease
Dosoudilová, Žaneta ; Klempíř, Jiří (advisor) ; Baxa, Monika (referee)
Huntington's disease (HD) is an autosomal dominant hereditary neurodegenerative disease characterized by motor, cognitive and behavioral disorders. HD is caused by expansion of CAG triplet (cytosine-adenosine-guanine) located in a gene on the short arm of the fourth chromosome. This expansion encodes an aberrant polyglutamine chain in the protein huntingtin. Physiological and mutated huntingtin (in case of HD) are expressed in almost all tissues and influences many cellular functions. The prevalence of HD in population is about 1 per 10.000. The disease is currently incurable and its mechanisms are not sufficiently understood. Besides affecting the central nervous system HD also affects peripheral tissues, including skeletal muscles. HD disrupts mitochondrial function and damages oxidative phosphorylation system, which has the task of producing energy in the form of ATP in cells. Research of transgenic minipig model for HD could help elucidate the mechanisms of disease's pathogenesis and potential therapeutic strategy. In this diploma thesis, immunodetection with help of specific antibodies to detect changes in amount of 14 selected mitochondrial proteins in skeletal muscle tissue of three age groups of transgenic HD minipigs - 24, 36 and 48 months old was used. Gradual progression in reduced...
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System of oxidative phosphorylation and methods for its functional analysis
Daňhelovská, Tereza ; Tesařová, Markéta (advisor) ; Míčová, Petra (referee)
The system of oxidative phosphorylation (OXPHOS) is main and essential source of energy (ATP) in eukaryotic cells. It is complex process situated in the inner mitochondrial membrane in which 4 enzymes of the respiratory chain, 2 mobile carriers and the ATP synthase participate. Defect in any part of OXPHOS may lead to the mitochondrial disorders. An incidency of the mitochondrial disorders is estimated to be 1:5000 and mitochondrial disorders are the most common inherent metabolic diseases. Clinically, it is extremely heterogeneous group of disorders affecting primarily tissue with high energy demand, for example brain, hearth and muscle. With regard to broad spectrum of clinical symptoms and relatively poor genotype-phenotype correlation of mitochondrial genetic defects, it is preferred to perform complete laboratory diagnostic tests including several biochemical and molecular genetic approaches. This bachelor thesis summarizes actual information about mechanism of OXPHOS and describes three major approaches to detect its functionality. Three major approaches are: spectrophotometric measurement of individual OXPHOS enzyme activities, measuring capacity of the mitochondrial energy generating system using various radioactive labeled substrates and measuring of oxygen consumption by polarography. To...
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