|
|
Generation and Characterization of Glutamate Carboxypeptidase II (GCPII)-Deficient Mice
Vorlová, Barbora ; Šácha, Pavel (advisor) ; Eckschlager, Tomáš (referee) ; Bařinka, Cyril (referee)
Glutamate carboxypeptidase II (GCPII) is a transmembrane glycoprotein, which consists of short intracellular and transmembrane domains, and a large extracellular domain possessing carboxypeptidase activity. In the human body, GCPII fulfils a neuromodulatory function in the brain and facilitates folate absorption in the small intestine. In addition to the brain and small intestine, high level of GCPII is also present in the prostate and kidney. However, GCPII function in these tissues has not been determined yet. To study the role of GCPII in detail, several research groups attempted to inactivate GCPII encoding gene Folh1 in mice. Surprisingly, the experiments led to rather conflicting results ranging from embryonic lethality to generation of viable GCPII-deficient mice without any obvious phenotype. This dissertation project aimed to dissect the discrepancy using alternative strategy for gene modification. For this purpose, we designed TALENs that specifically targeted exon 11 of Folh1 gene and manipulated mouse zygotes of C57BL/6NCrl genetic background. We analysed all genetically modified mice of F0 generation for presence of TALEN-mediated mutations and established 5 different GCPII-mutant mouse colonies from founder mice that altogether carried 2 frame-shift mutations and 3 small in-frame...
|
|
|
Epigenetic and Cytotoxic Effects of Histone Deacetylase Inhibitors in Combination with Cytostatics on Neuroblasma
Abdel Rahman, Mohamed Ashraf Khalil ; Eckschlager, Tomáš (advisor) ; Mandys, Václav (referee) ; Krsková, Lenka (referee)
The enhanced expression of histone deacetylases (HDACs) in a variety of malignancies drew attention to investigate a new category of anti-cancer drugs that are based on the inhibition of those enzymes. Valproic acid (VPA) is a well-known antiepileptic drug that exhibits antitumor activities through inhibition of HDACs class I and IIa. Cancer stem cells (CSCs) have been recognized to drive the tumor growth and progression hence; attention has been given to target this small subpopulation of CSCs rather than the whole bulk tumor cells. CD133 is considered to be a CSC marker in several tumors and its transcription is strongly influenced by epigenetic changes that will be altered upon administration of histone deacetylase inhibitors (HDACi) in cancer treatment. Therefore, we evaluated the epigenetic and cytotoxic effects of treatment with 1 mM VPA in combination with other chemotherapeutics and its influence on the expression of CD133 in human neuroblastoma (NB) cell lines. Our results revealed that addition of VPA to DNA-damaging chemotherapeutics induced a synergistic anti-tumor effect that was associated with caspase-3 dependent induction of apoptosis in UKF-NB-4 cells. This synergism was related to the increase of the acetylation status of histones H3 and H4 and was only produced either by...
|
|
| |
|
|
Epigenetically based chemoresistance of cancer cells
Feriančiková, Barbara ; Eckschlager, Tomáš (advisor) ; Šácha, Pavel (referee)
Cancer, despite significant advances in diagnosis and treatment, is the second most common cause of death in economically advanced countries. The main reason for the failure of anticancer therapy is the development of chemoresistance, which can be either internal or acquired, and is primarily mediated by the activation of various key regulators (eg MDR, PI3K/Akt, etc.). Genetic and epigenetic mechanisms are involved in activating these pathwa- ys. Significant epigenetic mechanisms that can participate in chemoresistance include regula- tion of gene expression by microRNA (miRNA) and long noncoding RNA (lncRNA). Dere- gulated expression of these non-coding RNAs has been observed in many diseases and their involvement in the initiation and progression of malignant tumors has been demonstrated. In this study, we investigated the expression of long non-coding RNA MIAT in hypoxia (1% O2) in chemosensitive and chemoresistant neuroblastoma cell lines (NBL), as hypoxia is a significant negative prognostic factor of many tumors and is involved in chemoresistance. Relative expression of MIAT was influenced by the number of cultured cells, where expression was increased by culturing more cells. MIAT expression was also significantly increased after 6 hours of NBL culture UKF-NB-4 in hypoxic conditions, and...
|
|
|
Development of therapeutic vaccine against HPV-16 induced tumor- influence of E7 antigen modification on cell madiated immune response
Macková, Jana ; Němečková, Šárka (advisor) ; Eckschlager, Tomáš (referee) ; Reiniš, Milan (referee)
Conclusions We constructed the vaccines carrying IIPV-16 E7 antigen based on .B. pertussisCyaA toxin We introduced ELISPOT and MHC-I totaÍner assays for testing of cell-mediarcdimmrmeresponsein themousemodel We tested cell-mediated immune response following immunization with differentkinds of thevaccinescarryingthemodifiedHPV-16 E7 antigen: o Recombinantadenylďe cyclase toxoid CyaA336IE7 is able to induce E7 specific CDE- cellular immuneresponsein mice and proteď them againstTC-l firmorgrowth o Some DNA vaccines carying the E7 genefusď to anothergeneare able to induoe betteranti-tumorimmuneresponsethan non-modified E7 genein mice @NA vaccinesevďuated accordingto themagpitude of CTL responseinduced: ETCTGG.GUS > ETGGGHSP, ETHSP >> cP-E7 >E7) o Fusion of E7 with W hemagglutininleadsto cell-surfaceexpressionof E7 and following vaccinďion with W.E7.HA it induces Th-2 polarized immuner€spons€ type ďong with the absenceof anti.tumor Th-l response o Co-expression of IL-12 from double recombinant vaccinia virus (W.IL-l2-sig/E7|LAI\D) reducesCD8- cellular immuniý inducedby Sig/E7lLAMP o Immunization with dendritic cells transducedby rW improves the efficacy of rW vaccination o Combined immunization increases vaccination effectiveness (CyaA336/E7+MVA- Sig/E7lLAMP, DNA-SigETGGG/LAMP+cellular vaccine) I7
|
|
| |
|
|
Influence of V-ATPase inhibitors on chemoresistant neuroblastoma lines in vitro
Honzejková, Karolína ; Eckschlager, Tomáš (advisor) ; Martínková, Markéta (referee)
Tumor diseases are one of the most common causes of death worldwide. Despite the great advances in therapy in the last fifty years, this is still a serious health problem. Therefore, great efforts are still concentrated on development of new anti-cancer drugs and therapeutic approaches. Neuroblastoma (NBL) is the most common tumor in infants and the fourth most common in children. Successful treatment is greatly complicated by its heterogeneity. Chemoresistance is an undesirable phenomenon of chemotherapy. One of the chemoresistance mechanisms is the accumulation of weakly basic anticancer drugs in lysosomes. This work deals with the measurement of lysosomal uptake of these compounds in neuroblastoma cell lines UKF-NB-4 and derived, ellipticine-resistant, line (UKF-NB- 4ELLI ) under different conditions. A method for determining the cell lysosomal capacity (volume) by measuring fluorescence intensity of lysosome-specific LTR dye was introduced and the ability of bafilomycin A, a V-ATPase inhibitor, to potentiate the effects of an anticancer agent ellipticine by inhibiting its lysosomal accumulation was investigated. Keywords: neuroblastoma, lysosome, vacuolar ATPase, multidrug resistance
|
|
|
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...
|
|
|
H19 methylation determination and KCNQ1OT1 at nephroblasts , pheochromocytomas and paragangliomas using MLPA technique
Jenčová, Pavla ; Vícha, Aleš (advisor) ; Eckschlager, Tomáš (referee)
DNA methylation is an epigenetic mechanism that affects the level of gene expression. Methylation is a physiological for imprinted genes, when is required to express a gene derived only from a particular parent. If a fault occurs in the DNA methylation of these genes, various diseases may develop. The object of this work was to determine the level of methylation of H19 and KCNQ1OT1 genes, located on the short arm of chromosome 11 in 15.5 region. The aim of this study was to investigate the changes in methylation of these genes in nephroblastomas, pheochromocytomas and paragangliomas, determine what changes of target genes are present in the samples, examined and divided samples into groups according to the detected changes. File of nephroblastomas is divided into 3 groups according to the methylation changes in genes and compared with a similar study by Scott et al. 2012. File of pheochromocytomas and paragangliomas are found according to changes in the methylation of genes divided into 4 groups entirety. Found changes are aligned with the research Margetts et al. 2005. Key words DNA methylation, H19, KCNQ1OT1, nephroblastoma, pheochromocytoma, paraganglioma
|
|
|
The comparison of properties of cell lines resistant to ellipticine, doxorubicin, and cisplatin
Černá, Tereza ; Poljaková, Jitka (advisor) ; Eckschlager, Tomáš (referee)
7 Abstract Neuroblastoma is the most common extracranial solid tumor of childhood. Despite advances in cancer diagnosis and therapy, the treatment of some forms of neuroblastoma is still complicated. One of the major complications of the chemotherapy is a developed drug resistance. This master thesis deals with the effect of cytostatics on protein and gene expression of selected proteins, which may contribute to chemoresistance of the human neuroblastoma cell line UKF-NB-4. The sensitive line UKF-NB-4 and the resistant line UKF-NB-4CDDP , UKF-NB-4DOXO and UKF-NB-4ELLI were exposed to cisplatin, doxorubicin, ellipticine for 24, 48 and 72 hours. The Western blot analysis showed that cytostatic agents cisplatin, doxorubicin or ellipticine added to the sensitive neuroblastoma cell line UKF-NB-4 in amounts which are added to resistant neuroblastoma cell lines in order to maintain resistance induced expression of p53 and reduced expression of retinoblastoma protein pRb after 72 hours of cultivation. Differences in the expression of RAS protein, cytochrome P450 1A1, 3A4 and cytochrome b5 has not been shown. Changes in the expression of the studied proteins in resistant lines UKF-NB-4CDDP , UKF-NB-4DOXO and UKF-NB-4ELLI cultured with and without cytostatic agents were not detected by the Western blot analysis....
|
guest ::
