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Function of Biotransformation Enzymes in Development of Nephropathies Caused by Aristolochic Acid
Bárta, František ; Stiborová, Marie (advisor) ; Koblihová, Jitka (referee) ; Eckschlager, Tomáš (referee)
- 6 - ABSTRACT Plant alkaloid aristolochic acid (AA) is a proven human carcinogen which causes two serious diseases: Aristolochic Acid Nephropathy (AAN) and Balkan Endemic Nephropathy (BEN). One of the characteristic features of both AAN and BEN is their close association with the development of upper urothelial carcinoma (UUC) in the renal tissue of patients. Although both nephropathies are mediated by the same compound (i.e. AA), their development differs slightly. The differences might be explained by a different exposure schedule of patients or interindividual differences in expression levels and activities of the enzymes metabolising AA in organisms. Detailed knowledge of these enzymes can contribute to the elucidation of the interindividual susceptibility to AA. In this thesis, enzymes participating in both oxidative detoxification of AAI, a major component of natural mixture of AA, and its reductive activation leading to the formation of AA-DNA adducts were studied. In a rat experimental model (Rattus norvegicus), NAD(P)H:quinone oxidoreductase 1 (NQO1) and its role in reductive bio-activation of AAI in vivo were examined utilising a specific inhibitor of this enzyme, dicoumarol. Oxidative detoxification of AAI resulting in formation of a demethylated derivative AAIa (8-hydroxyaristolochic...
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Use of chicken antibodies against PAIIL lectin for prevention of Pseudomonas aeruginosa infections in patients with cystic fibrosis
Kubíčková, Božena ; Hodek, Petr (advisor) ; Anzenbacherová, Eva (referee) ; Eckschlager, Tomáš (referee)
Cystic fibrosis (CF) is one of the relatively common inherited diseases caused by a mutation in the gene encoding for CFTR protein, which forms a chloride channel that significantly affects ion homeostasis and the associated fluid management of the cell. This disease mainly affects the respiratory and digestive systems, being the most life-threatening in the respiratory tract. Patients with CF suffer from frequent and recurrent respiratory infections that lead to the development of chronic inflammation and gradual destruction of lung tissue. These lung infections, which are caused mostly by the opportunistic pathogen Pseudomonas aeruginosa, are the most common cause of morbidity and mortality in these patients. At present, antibiotics are used in the treatment of Pseudomonas aeruginosa infections, but new methods of antibacterial therapy need to be found to overcome the development of resistance. In addition to active immunization of CF patients against Pseudomonas aeruginosa, their passive immunization with specific chicken antibodies directed against this pathogen offers promising possibilities. This dissertation thesis is aimed to verify the prophylactic potential of hen IgY antibodies against the virulence factor Pseudomonas aeruginosa - lectin PAIIL, and to further develop an experimental...
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Analysis of cancer predisposition and functional analysis of variants of unknown significance
Stolařová, Lenka ; Kleibl, Zdeněk (advisor) ; Eckschlager, Tomáš (referee) ; Souček, Pavel (referee)
On average, 5-10% of all cancers occur in patients with hereditary tumors, who may have mutations in tens to hundreds of tumor predisposition genes. The phenotypes in mutation carriers overlap, and parallel analyses with sequencing panels is the method of choice in diagnostics. In our laboratory, we designed a universal panel and a targeted panel for a specific cancer, which allowed us to identify genetic alterations in patients with ovarian cancer, breast cancer, melanoma, and other cancers in the Czech Republic. The results of next generation sequencing (NGS) analyses show that the most frequent genetic alteration in ovarian cancers patients in the Czech Republic are hereditary mutations in BRCA1 (in 24% of unselected patients) and in malignant melanoma patients CDKN2A (in 2% of high risk patients). The presence of hereditary alterations is a clinically significant phenomenon affecting the prognosis and treatment of the disease. However, the interpretation of NGS findings is complicated by the presence of variants of unknown significance (VUS). We participate in the interpretation of VUS in the main predisposing genes BRCA1 and BRCA2 within the international consortium ENIGMA (Evidence-based Network for the Interpretation of Germline Mutant Alleles). Our and international results of the most...
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Musculoskeletal System Dysfunction in Patients in Long-Term Remission of Wilms Nephroblastoma
Šafářová, Marcela ; Kolář, Pavel (advisor) ; Véle, František (referee) ; Eckschlager, Tomáš (referee)
Musculoskeletal System Dysfunction in Patients in Long-Term Remission of Wilms Nephroblastoma Objective: The objective of this retrospective clinical study was to describe the multidisciplinary program of childhood cancer survivors in their young adult age in an adult-based ambulatory medical setting and to report the latent sequelae of the pathology. The hypothesis is that the treatment with all three modalities (chemotherapy, radiotherapy and surgery) causes the development of the latent effects in varying degrees. The goal of this study was to investigate the typical musculoskeletal system changes of survivors of Wilms Nephroblastoma who had previously received their basic treatment more than 5 years ago. Design: 151 subjects underwent clinical assessment of body posture, postural tests and evaluation on BalanceMaster® force plate. These tests were conducted in conjunction with other clinical evaluations, namely, pulmonary and renal function tests, immunology deficiency and exercise capacity to seek its relationship to the diseases' risk factors. The correlation between postural findings and the above clinical tests were investigated. Subjects: Ambulatory patients with a primary diagnosis of nephroblastoma who have undergone treatment greater than 5 years ago. Settings: Clinics at the University...
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Importance of V-ATPase for cancer cell resistence
Suchánková, Kristýna ; Eckschlager, Tomáš (advisor) ; Indra, Radek (referee)
Chemoresistance is one of the main causes of failure of anticancer chemotherapy. Vacuolar-type ATPase (V-ATPase) is an ATP-dependent proton pump involved in the regulation of the pH in cells, cell organelles and the intracellular space. A significant acidification of the extracellular space and intracellular compartments occurs in connection with the metabolism of tumour cells (glucose metabolism, hypoxia, insufficient blood perfusion of the cancer tissue). Basic drugs are transferred into acidic organelles based on the pH gradient, where they are then protonated and accumulated. This mechanism is called lysosomal sequestration and is one of the mechanisms how tumour cells resist to applied drugs, which then do not reach their target site in cancer cell. An increased expression of V-ATPases has been described in relation to chemoresistance and the progression of tumours. This dissertation is focused on observing the membrane subunit V0d from the complex of V-ATPase and the changes in resistance to ellipticine caused by the silencing of this subunit's gene in human neuroblastoma cell lines UKF-NB-4 (sensitive) and UKF-NB-4ELLI (resistant to ellipticine). The expression of the V0d protein was first examined on mRNA level using real-time polymerase chain reaction (RT-PCR). The silencing of selected...
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lncRNA MIAT expression in cancer cells.
Jursová, Pavlína ; Eckschlager, Tomáš (advisor) ; Sztacho, Martin (referee)
LncRNAs have been shown, in many cases, to function as important regulators for gene expression and thus, they can play a critical role in various biological functions and disease processes including cancer. Myocardial infarction associated transcript (MIAT) is one of the non-coding RNAs first identified as lncRNA in 2006 and originally isolated as a candidate gene for myocardial infarction. This long non-coding RNA is also involved in other diseases such as diabetic retinopathy, paranoid schizophrenia or microvascular dysfunction. MIAT has also been identified as a carcinogenic regulator in many malignant tumors. Numerous researches have reported that MIAT silencing reduces cell viability, proliferation and invasivity and enhances cellular senescence and apoptosis of cancer cells. Therefore, it is considered a potential biomarker and therapeutic target in cancer. MIAT is involved in cellular processes through various mechanisms. It regulates alternative splicing, gene expression or functions through ceRNA mechanism and thus influences biological processes related to the tumor formation. Furthermore, in this study have been found that relative expression of MIAT was increased in Ewing sarcoma cell lines.
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Genetic variability in sporadic colorectal cancer: Searching for novel risk, prognostic and predictive biomarkers.
Jirásková, Kateřina ; Vodička, Pavel (advisor) ; Machoň, Ondřej (referee) ; Eckschlager, Tomáš (referee)
Colorectal cancer (CRC) is a major public health problem worldwide. Despite improvements in the diagnostic process and advancement in the treatment methods, the prognosis remains poor. To improve survival rates, it is important to identify people with the predisposition for CRC and to detect the potentially curable early stage of the disease. Furthermore, identifying those who would have an adverse clinical outcome associated with a particular chemotherapy would help to avoid redundant chemotherapy burden in patients and contribute to enhanced therapeutic efficacy, while minimizing treatment-related toxicity. The aim of the Thesis was to search for novel promising diagnostic, prognostic and predictive DNA-based biomarkers of sporadic form of CRC. As each patient is genetically unique, these biomarkers would aid clinicians in better diagnosis and/or in the selection of an optimal type of therapy for an individual CRC patient based on their molecular profile. In order to explore this issue, we investigated several candidate genes in healthy individuals as well as in newly diagnosed cancer patients. The major outcomes of this PhD study, which were fully reported in seven publications included in the present Thesis, are 1) The observation of several candidate single nucleotide polymorphisms in microRNA...
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V-ATPase expression in hypoxic conditions in neuroblastoma cells
Kittlerová, Kateřina ; Eckschlager, Tomáš (advisor) ; Moserová, Michaela (referee)
Tumor diseases belong to one of the most common death causes all around the world these days, therefore scientists still work on new therapeutic procedures. Tumor diseases are the second most common death causes among kids and juvenile. One of the freqeuently diagnosed tumor among kids and juvenile is neuroblastoma. Neuroblastoma is malignant embryonic tumor of the peripheral nervous system. Chemotherapy is used as tumor treatment by therapeutic procedures such as surgical removal and tumor irradiation. Cisplatin is one of the most applicated cytostatics, however it's efficiency it's lowered despite of creating resistance during the treatment. Vacuolar ATPase (V-ATPase) acidifies some of the cell organelles including lysosomes, which can lead to lysosomal sequestration of some of the substances including cytostatics and therefore the cure can't get to the therapeutic target. Key role in lysosomal activity regulation performes transcription factor EB (TFEB). This study deals with expression of d subunit of vacuolar ATPase (ATP6V0D1) and TFEB in neuroblastoma sensitive cell line UFK-NB-4 and resistant to cisplatin UKF-NB-4CDDP in normoxia and hypoxia. After exposure of neuroblastoma resistant cells to cisplatin the expression of ATP6V0D1 gene raised compared to normoxia. In case of TFEB the...
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Potentation of the effect of cytostatic by v-ATPase inhibitors
Vaňková, Kristýna ; Eckschlager, Tomáš (advisor) ; Hudeček, Jiří (referee)
Tumour diseases belong among some of the most severe problems of advanced civilization and of current medicine. Neuroblastoma is a solid embryonal children's tumour, which develops from the cells of the neural crest. The increased expression of some oncogenes and their mutation leading to dysfunction plays a major role in the pathogenesis of this disease. One of the possible treatments of the tumours is the use of cytostatics, substances with no selectivity, which kills both damaged and healthy cells by their toxic effect. An example of such a cytotoxic substance is cisplatin (CDDP) which binds with purine nucleotides and leads the cell to apoptosis. A current complication in the treatment is development of chemoresistance. In connection with this issue vacuolar-type H+ -ATPase (V-ATPase) responsible for the acidification of vacuoles is studied. This bachelor thesis concentrates on studying the expression of selected genes of the V- ATPase in the neuroblastoma stem cell line sensitive (UKF-NB-4) and resistant to cisplatin (UKF-NB-4CDDP) and after their exposure to cisplatin. That was accomplished on the genetic level by the isolation of cellular RNA, reverse transcription creating complementary DNA (cDNA) and a method of a real time quantitative polymerase chain reaction (RT-PCR). The aim of the...
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Study on metabolism of plant carcinogen aristolochic acid I
Dedíková, Alena ; Stiborová, Marie (advisor) ; Eckschlager, Tomáš (referee)
Aristolochic acids (AA) are natural compounds present in plant species of Aristolochiaceae. These plant alkaloids exhibit nephrotoxic effects and are human carcinogens. The extract of plants species Aristolochiaceae contains various kinds of substances, including aristolochic acids. Aristolochic acid I (AAI) and aristolochic acid II (AAII) are mainly present. AA compounds are associated with two diseases, Aristolochic Acid Nephropathy (ANN), previously known as Chinese Herbs Nephropathy (CHN) and Balkan Endemic Nephropathy (BEN). Both these diseases are accompanied by renal failure and the formation of tumors in urothelial tissue, which are caused by AA. However, the same clinical manifestations do not occur in both diseases. This is propably due to the variability in enzymes involved in AA biotransformation, but also depends on AA doses. In organisms, AA are metabolized by activation and/or detoxification reactions. These thesis is directed on the activating metabolism of AAI, precisely to clarify the formation of reducing metabolites of AAI. Reduction of AAI is the activation pathway responsible for the genotoxic effect of AAI. During this reaction AAI is enzymatically reduced to the cyclic acylnitrenium ion that is capable of binding to the exocyclic amino groups of adenine and guanine in DNA...
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