|
|
Apoptosis of tumor cells : role of TRAIL and caspase 10
Truxová, Iva ; Živný, Jan (advisor) ; Anděra, Ladislav (referee)
One of the key features of cancer cells is the ability to escape programmed cell death (apoptosis). As a mechanism of apoptosis inactivation in cancer cells, somatic mutations of pro-apoptotic genes have been reported in many cancers. Caspase 10 is an initiator caspase whose physiological function remains poorly understood. Also the ability of caspase 10 to substitute for caspase 8 in the death receptors apoptotic pathway is still controversial. However, the fact that some of the mutations found in CASP10 gene was associated with apoptosis defects (79, 81) suggest that caspase 10 could be also important in apoptosis initiation. In our lab, there was found a heterozygous mutation in CASP10 gene of Jurkat (human T-acute lymphoblastic leukemia) clone resistant to TRAIL (J-TR1). This mutation influence the amino acid composition close to the active site of the enzyme. The aim of this thesis was to confirm the mutation by ARMS-PCR and to determine if an overexpression of normal (unmutated) or mutated caspase 10 D in TRAIL sensitive and/or TRAIL resistant Jurkat cells (J-WT and/or J-TR1) will influence TRAIL induced apoptosis. Mutation was confirmed. We created J-WT and J-TR1 stable clones transfected by vector with unmutated or mutated CASP10 D (CASP10 D WT or CASP10 D MUT). CASP10 D MUT overexpression in J-WT...
|
|
|
Th1/Th2 cytokine gene polymorphisms in patients with urine fibroid
Sosna, Ondřej ; Kužel, David (advisor) ; Živný, Jan (referee) ; Novotný, Zdeněk (referee)
Background: Uterine fibriod (UF) or leiomyoma is the most frequent benign tumour upon lower genital tract and represents the most frequent indication for hysterectomy. The aetiology remains still unknown. The genetic factors contributing for the development of UF are being intensively investigated. The aim of our study was to look for possible genetic markers which could be used as prognostic tools for evaluation of an increased risk for development of UF. Methods: The study group enrolled 102 patients diagnosed with UF and 145 healthy controls. Ultrasonographic examination of the pelvis was performed and a single blood sample was taken in all women. Histological verification followed the surgery in the patient group. The principal of the cytokine gene polymorphisms detection is based on PCR reaction with sequence-specific primers. Results: A large spectrum of Th1/Th2 cytokine gene polymorphisms in patients with uterine fibroid was compared with control group. The frequencies of the majority of tested cytokine gene SNP in the patient cohort were not statistically different from the cytokine SNP in the control group. However, an intriguing association between polymorphisms of the IL-4 gene promotor at positions -590 C/T and -33 C/T, and the risk of leiomyoma was observed. The CC genotype of IL-4 at position...
|
|
|
The relationship between selected inflammation markers and markers of the endothelial dysfunction to preterm labor and fetal inflammatory response
Koucký, Michal ; Hájek, Zdeněk (advisor) ; Krofta, Ladislav (referee) ; Živný, Jan (referee)
The doctoral dissertacion is focused on the role of inflammation in the pathogenesis of preterm labor. In the first part, we describe the current view on pathophysiology of preterm labor. In the second part, we evaluated the relationship of specific markers of inflammation and endothelial dysfunction to preterm birth and fetal inflammatory response. The most important findings of our study was that we found decreased levels of MMP-2 and decreased levels of sRAGE in women with preterm labor in comparison with the control group of pregnant women. Similarly, we found decreased levels of MMP-2 in women with subsequent diagnosed fetal inflammatory response. sRAGE is currently ranked among patttern recognition receptors. In the case of sRAGE we followed the results of our pilot project, it can be assumed that the its low level are connected with tissue damage. We confirmed that it can play an important role in the pathogenesis of preterm labor. We assume abnormal regulatory mechanisms of the production of MMP-2. In both cases, however, further studies are required to elucidate the functional significance of our results.
|
|
|
The Role of Cellular Prion Protein in Erythroid Differentiation
Panigaj, Martin ; Holada, Karel (advisor) ; Živný, Jan (referee) ; Rusina, Robert (referee)
4 Abstract The cellular prion protein (PrPC ) is evolutionary conserved protein expressed in cells of various origins. Although PrPC plays a basic role in the pathogenesis of the fatal neurodegenerative prion disorders, its physiological role remains enigmatic. Prion diseases are characteristic by long latency period during which they are not identifiable by any conventional methods. Although the blood is an ideal material for developing of screening tests, little is known about traits of PrPC and its role in blood cells. We showed that human erythrocytes express low amounts of PrPC per cell, but due to the high numbers of erythrocytes, they are major contributors to the pool of blood cell-associated PrPC . Based on our biochemical characterization we propose that PrPC on human erythrocytes is uniquely modified. Such a modification in abnormal prion protein may complicate screening tests for prion diseases in blood. It was reported that prion diseases deregulate the transcription of erythroid genes, and PrP-/- mice demonstrate a defective response to experimental anemia. To investigate the role of the PrPC in erythropoiesis, we studied the protein's expression on mouse erythroid precursors in vivo and in vitro. We showed that surface expression of PrPC on erythroid precursors in bone marrow and spleen...
|
|
|
Molecular and functional characterization of the death receptor 6
Klíma, Martin ; Anděra, Ladislav (advisor) ; Živný, Jan (referee) ; Kovář, Marek (referee)
Death receptor-6 (DR6/TNFRsf21/CD358) is a receptor from the TNFR superfamily that likely participates in the regulation of proliferation and differentiation of T- and B-lymphocytes and neural cells. The 655-amino acid human DR6 is a type I transmembrane protein containing four cysteine-rich domains in its extracelular part and a death domain followed by the CARD-like region in its cytoplasmic part. Overexpression of DR6 in some cell lines leads to apoptosis, and/or to activation of nuclear factor NF-κB and stress kinases of the JNK family. In the first part of our work we focused on molecular characterization of DR6, including the analysis of its posttranslational modifications. We found that DR6 is an extensively posttranslationally modified protein including S-palmitoylation and both N- and O-glycosylation. Six N-glycosylation and one S-palmitoylation sites were precisely mapped to appropriate asparagines and cysteine respectively. The juxtaposed linker region (between cystein-rich domains and the transmembrane part), which also contains Ser/Thr/Pro-rich region with clustered putative O-glycosylation sites, is required for the plasma membrane localization of DR6. N-glycosylation, but interestingly not S-palmitoylation, may play a role in targeting of DR6 into detergent-resistant...
|
|
|
Cloning, expression and characterization of recombinant growth factors
Svoboda, Ondřej ; Živný, Jan (referee) ; Bartůněk, Petr (advisor)
Cloning, expression and characterization of recombinant growth factors The role of growth factors and cytokines to regulate a variety of biological processes is ever increasing. Many studies have illustrated that this diverse group of proteins are capable of stimulating cellular growth, proliferation, survival, and cellular differentiation. This thesis mainly focuses on the cytokine interleukin-6 and its specific receptor (IL-6R). The IL-6R protein has been shown to exist as both a membrane-bound and soluble form. The soluble form of IL-6R (sIL-6R) signals via a mechanism called "transsignaling" that involves the receptor associating extracellularly with its ligand IL-6 to generate an IL-6/sIL- 6R agonistic complex. This complex subsequently binds directly to another IL-6 receptor, gp130 that in turn activates it to augment a specific response. It is known that the formation of the IL-6/sIL-6R complex can be greatly enhanced by converting it into a single molecule, called Hyper-IL-6. This fusion protein consists of a sIL- 6R located at the N-terminus and IL-6 at the C-terminus separated by a flexible linker sequence. It was more than ten years since human Hyper-IL-6 was first described and since then, this molecule has proven to be an ...
|
|
|
Origins of vertebrate hematiopoiesis
Svoboda, Ondřej ; Bartůněk, Petr (advisor) ; Divoký, Vladimír (referee) ; Živný, Jan (referee)
(ENGLISH) Hematopoiesis is dependent on the actions of hematopoietic stem cells (HSCs). This process is tightly controlled through a complex array of extrinsic and intrinsic factors. Even though the hematopoiesis seems to be well conserved across the disparate vertebrate animals, erythroid and thrombocytic differentiation have changed during the evolution of mammals. Specifically, adult mammalian red blood cells have the unique feature of being enucleated, and mammalian thrombocytes are not individual cells, but fragments of megakaryocytes, instead. It is likely that these enhancements provided a survival advantage to early mammalian species; however, they also bring up the question of evolutionary origin of these cells that studied using zebrafish (Danio rerio) model. First, it was necessary to generate a toolbox of a recombinant cytokines and optimized culture media that allowed us to manipulate zebrafish hematopoietic cells ex vivo in liquid and clonal cultures. Interestingly, teleost species underwent an extra duplication event during their evolution and as a result, two copies (paralogs) of some of the genes are present in zebrafish. This was also the case for majority of the cytokines from our toolbox and here, we provide functional characterization of these paralogs. Strikingly, our results...
|
|
| |
|
| |
|
| |
guest ::
RSS feed.