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Mitochondrial structure and energetic metabolism changes in patients with Huntington's disease and in transgenic minipig model
Vanišová, Marie ; Hansíková, Hana (advisor) ; Kalous, Martin (referee) ; Mühlbäck, Alžbeta (referee)
Huntington's disease (HD) is a severe neurodegenerative disease with autosomal dominant inheritance. HD is caused by the expansion of the CAG triplet in the gene for the huntingtin protein (Htt), which leads to damage and loss of its functions. Htt is essential in the development of the nervous system, it is involved in axonal transport, regulation of mitochondrial metabolism gene expression or spermiogenesis. In HD, the nerve tissue is most significantly damaged, but pathological changes associated with the disease are detected throughout the organism. There is currently no satisfactory treatment. Mitochondrial damage has been shown to significantly affect the progression of HD in patients with HD, but the mechanisms of mitopathy and its development with all the effects on tissue physiology in HD are still not fully understood. The aim of the dissertation theses was to study mitochondrial energy metabolism impairment, mitochondrial network organization and mitochondrial ultrastructure in HD in selected tissues of patients with HD and in a minipig model transgenic for HD (TgHD). Furthermore, the effort was to find and characterize a mitochondrial biomarker of HD, which would well reflect the patient's current clinical phenotype state and it would be possible to monitor changes in its parameters...
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The study of energetic metabolism in patients with mitochondrial translation defects
Hýbl, Martin ; Hansíková, Hana (advisor) ; Čermáková, Michaela (referee)
Mitochondria are semi-autonomous organelles that contain their own DNA. Human mitochondrial DNA (mtDNA) encodes a total of 37 genes: 13 subunits of oxidative phosphorylation complexes (OXPHOS), 22 transfer RNA (tRNA) molecules and 2 ribosomal RNA (rRNA) molecules. Pathogenic mutations in genes associated with mitochondrial translation are a common cause of mitochondrial disease. These mutations can be found in mtDNA or in nuclear genes encoding ribosomal proteins, initiation, elongation and termination factors, mitochondrial tRNA-modifying enzymes and aminoacyl-tRNA synthetases. Mitochondrial aminoacyl-tRNA synthetases (mt-aaRS) are enzymes that catalyse the addition of single amino acids to specific tRNAs. The aim of the bachelor thesis was an introduction to the work in the tissue culture laboratory. To prepare samples for the following experiments, skin fibroblasts from five patients with mt-aaRS disorders (AARS2, DARS2, NARS2, SARS2) and control lines were cultured in glucose and galactose media. Subsequently, the procedure for determining the equilibrium amount of selected subunits of the OXPHOS complexes was optimized and applied to the analysis of fibroblasts from five patients with mt-aaRS disorder. When the cells were cultured in glucose medium, decreased levels of some subunits of complex...
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Modulation of Mitophagy in Huntington Disease
Šonský, Ivan ; Hansíková, Hana (advisor) ; Kalous, Martin (referee)
Huntington diseases (HD) is a hereditary neurodegenerative disorder characterized by the presence of the aggregation prone mutated version of protein huntingtin (HTT). Mutation in huntingtin (mHTT) results in an aberrant expansion of the polyglutamine tract, thereby gaining toxic properties, which causes progressive loss of striatal medium spiny neurons. Neurons heavily rely on a healthy mitochondrial pool. Thereby, it is crucial to preserve biological mechanisms maintaining its turnover and quality control, such as mitophagy. However, mHTT impairs mitophagy, therefore preventing autophagosomes from engulfing mitochondria and resulting in an accumulation of dysfunctional mitochondria. Our recent results showed that mHTT-caused mitochondrial impairments can be observed in more easily accessible extraneuronal cells such as skin fibroblasts. While mitophagy is considered a fundamental cellular process, there is a lack of compounds selectively modulating mitophagy. Thereby, the aim of this diploma thesis was to introduce a small-molecule compound, MIND4-17, which showed neuroprotective effects in HD, and to study its selective effect on mitophagy in cultivated fibroblasts from HD patients and controls. Here we report that MIND4-17 increased the expression of specific autophagy markers in fibroblasts...
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Complex I of mitochondrial respiratory chain a its disorders.
Rodinová, Marie ; Hansíková, Hana (advisor) ; Kalous, Martin (referee)
NADH: ubiquinone oxidoreductase (Complex I) is a multisubunit protein complex of inner mitochondrial membrane. Complex I is the biggest and most complicated part of oxidative phosphorylation system, which is responsible for the cell ATP production. It consists of 45 subunits. 7 subunits are mitochondrial encoded, remainder 38 are nuclear encoded. NADH: ubiquinone oxidoreductase has L-shaped structure, which is built of two arms: membrane arm and matrix located peripheral arm. Complex I oxidize the NADH molecule. The electron transport is coupled with proton pumping across the inner mitochondrial membrane to intermembrane space, where proton gradient developed and which is used by ATP synthase to ATP synthesis. Deficiencies of NADH: ubiquinone oxidoreductase represent extensive, clinically and genetic heterogeneous group of mitochondrial diseases. Decrease of activity and amount of complex I, decrease of ATP production, changes of membrane potential, mitochondrial morphology and mitochondrial network and increasing of production of reactive oxygen species are found in cells with defects of NADH: ubiquinone oxidoreductase. Combination of this features lead to serious illnesses, which are almost fatal and we still haven't any useful therapy. Aim of this study is to summarize present knowledge about...
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Fumarate hydratase as tumor suppressor
Kedrová, Kateřina ; Hansíková, Hana (advisor) ; Befekadu, Asfaw (referee)
1 Abstract Fumarate hydratase (fumarase, EC 4.2.1.2) catalyzes the reverse hydration of fumarate to S malate. In mammalian cells, it changes fumarate in the mitochondrial matrix as a part of the citric acid cycle and in the cytosol, where functions to metabolize fumarate the product of the degradation of some amino acids, of ammonia transformation to urea acid or of the purine nucleotide synthesis. . In human cells, fumarase is encoded by FH gene localized on chromosome 1 (1q42.1). The FH gene consists of 10 exons and encodes for a 510 amino acids-long protein including the N-terminal mitochondrial signal sequence. Germline heterozygous FH mutations were found in two autosomal dominant syndromes. These are multiple cutaneous and uterine leiomyomatosis (MCUL1 or MCL) and hereditary leiomyomatosis and renal cell cancer (HLRCC). In the most of tumors from these patients, loss of FH gene heterozygosity was also found. It has been suggested that fumarase acts as a tumor suppressor according to Knudson's two-hit hypothesis. The aim of the bachelor thesis was to study the activity and amounts of fumarase in a series of 22 samples of uterine leiomyomas from 22 young women patients (21-31 years) with sporadic uterine leiomyomas. As a control sample, uterine leiomyoma from a 38-year-old patient was used. Activity of...
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Study of mitochondrial ultrastructure and functions in selected mitochondrial and lysosomal storage disorders
Kostková, Olga ; Hansíková, Hana (advisor) ; Šmíd, František (referee) ; Hyánek, Josef (referee)
This thesis has been worked out in The laboratory for study of mitochondrial disorders (Departement of Pediatrics, 1st Faculty of Medcine, Chales university in Prague) and in cooperation with The Institute of Inherited Metabolic Disorders. Mitochondrial disorders represent a heterogeneous group of diseases with the onset at any age from neonatal period till adulthood, mostly presented with very severe clinical courses of disease. The mammalian organism is fully dependent on mitochondrial oxidative phosphorylation system as on the major energy producer of the cell. Therefore the mitochondrial disorders affect mainly high energy demanded tissues such as brain, heart or muscle. Simillar phenotype is observed in many lysosomal storage disorders. Despite of expanding knowledge of molecular basis of mitochondrial and lysosomal disorders, it may be still difficult to explain the exact pathogenesis of disease as well as the prognosis for patients and their families. Mitochondrial functions affect more than just energy production; they contribute in initiation of apoptosis, in cellular calcium homeostasis, and in production of reactive oxygene species. Disturbed mitochondria become a goal of autophagy mediated by the lysosomal compartement. The results of our study enable: 1. better understanding of the tissue...
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Flow cytometry in the diagnostics and characterization of congenital disorders of glycosylation
Veselá, Šárka ; Hansíková, Hana (advisor) ; Hodek, Petr (referee)
Congenital disorders of glycosylation (CDG) are rare multisystem metabolic diseases and their number has rapidly grown in recent years. The clinical manifestation includes very broad spectrum of symptoms. In most of all cases CDG are caused by mutations in genes encoding the enzymes of glycosylation pathway. Based on the type of defect, CDG are divided into the following groups: disorders of N-glycosylation or O-glycosylation of proteins, defects in modification of proteins by GPI anchor, disorders of lipid glycosylation and defects that impact multiple glycosylation pathways. The aim of the thesis was to find new biochemical analyses suitable for diagnostics and characterization of CDG patients. The experimental conditions were optimized for selected markers (Sambucus Nigra (SNA) lectin, proaerolysin (FLAER), antibodies to proteins CD55 and CD59) and the staining was applied to cultivated skin fibroblasts from controls and patients diagnosed with CDG by whole-exome sequencing (ATP6AP1-CDG, PIGN-CDG, SLC10A7-CDG, PISD deficiency). The experiments were performed using flow cytometry (FACS) and fluorescent microscopy (FM). The detection of sialylation by SNA lectin and analysis of the mitochondrial membrane potential changes by a fluorescent labelled probe JC-1 with FCCP simulation of mitochondrial...
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Biochemical and molecular studies of the congenital disorders of glycosylation
Ondrušková, Nina ; Hansíková, Hana (advisor) ; Stiborová, Marie (referee) ; Hřebíček, Martin (referee)
Congenital disorders of glycosylation (CDG) represent a rapidly growing group of rare inherited metabolic diseases with estimated prevalence as high as 1:20 000, which are caused by genetic defects that impair the process of glycosylation, i.e. the enzymatic addition of a specific saccharide structure onto a protein or lipid backbone. Due to non-specificity and variability of clinical symptoms in the patients, the medical diagnosis of CDG remains extremely challenging and significantly relies on accurate biochemical and genetic analyses. The overall goal of the present dissertation thesis was to study CDG at the biochemical and molecular genetic level in the context of the Czech and Slovak Republic, which involved three specific aims: A.) to introduce and optimize laboratory screening methods for CDG detection in a group of clinically suspected patients, B.) to determine the corresponding genetic defect in the positive patients selected via CDG screening and to study the pathobiochemical aspects of specific CDG types at the cellular level, and C.) to analyze glycosylation disturbances of non- CDG etiology. Contributions of this work include optimization of isoelectric focusing of apolipoprotein C-III (ApoC-III) as a screening method for O-glycosylation abnormalities, as well as the description of...
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Mitophagy in Huntington's Disease
Šonský, Ivan ; Hansíková, Hana (advisor) ; Macůrková, Marie (referee)
Mitochondrial dysfunctions contribute to the progression of many neurodegenerative diseases, including Huntington's disease (HD). In HD, mutation in the huntingtin gene (HTT) results in the expansion of CAG repeats, causing the growth of the polyglutamine tract. This growth is responsible for the gain of toxicity function of the protein. The turnover of dysfunctional and damaged mitochondria is mediated via mitophagy - a selective form of autophagy. Additionally, mitophagy impairments have recently been described to play a key role not only in neurodegenerative diseases. The protrusion of mitophagy results in the clustering of defective mitochondria, organelles which are responsible for fulfilling the energetic demands of neural cells. The most distinctive impact of the impairment is on the striatal medium spiny neurons and results in the development of motor and cognitive dysfunctions. This thesis describes how HD affects mitophagy and reveals the biggest obstacle of mitophagy - disruption of mitochondria targeting into emerging autophagosomes caused by the abnormal interaction of mHTT and p62. Induction of mitophagy at this stage could be crucial for the future therapeutic research of HD. Generally, initiation of mitophagy could become a relevant therapeutic target for many other...
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