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Příprava mutantního serpinu z klíštěte \kur{Ixodes ricinus}
EDEROVÁ, Monika
Point mutation altering arginin for tryptophan amino acid residue in P1 site of tick salivary serpin Iripin-1 was created using specific primers. Recombinant protein with this mutation in nucleotide sequence was then expressed in chemically competent Escherichia coli cells, extracted from them and purified by affinity and size-exclusion chromatography. To see the impact of the mutation on inhibitory function of Iripin-1, its ability to bind trypsin and form covalent complexes was evaluated.
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Problems of laboratory examination in Lyme disease
HAVLEOVÁ, Lucie
Lyme borreliosis is a multisystem infectious disease most often transmitted by ticks of the genus Ixodes. In the Czech Republic, specifically by ticks Ixodes ricinus. The initiator of this disease is a complex of spirochetal bacteria Borrelia burgdorferi sensu lato. The incidence of the disease is relatively often as several thousand cases are reported in the Czech Republic every year. However the knowledge about Lyme borreliosis is relatively extensive and simultaneously an intensive research is constantly ongoing, the opinions of experts on this issue are still unfolding accordingly. So far, it is still rather difficult to diagnose Lyme disease, especially because of the various and sometimes non-specific clinical symptoms that might often lead to failure in diagnosis. As no effective vaccine has yet been developed, the only way to reduce the risk of infection is prevention and protection against the ticks. This work investigates and summarizes current knowledge about the history, etiology, epidemiology, pathogenesis, clinical course, prevention, therapy, and diagnosis of Lyme disease. Patients suspected of having contracted Lyme disease were tested using a two-level screening, firstly by applying ELISA method in order to select either positive patients or patients with uncertain results so that a confirmation Western blot method could follow subsequently. The outcome of my work confirmed the well-known fact that Lyme disease is seasonal. The higher number of positive samples in the summer months is related to the increased activity of ticks during this period. However, the results of positive patients who are divided into groups by age or gender represent very interesting findings. Particularly the higher incidence of the positive samples can be found among women.
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Vytvoření a charakterizace transgenních linií drozofil manifestující neurodegenerativní onemocnění spinocerebelární ataxi typu 1 (SCA1)
JIČÍNSKÁ, Veronika
The aim of this thesis was to generate and characterize transgenic lines of Drosophila melanogaster manifesting spinocerebellar ataxia type 1 (SCA1), using GAL4/UAS system. Specific driver lines were used to express ataxin gene in pan neuronal, eye and motor neurons. Adult males from different crosses expressing abnormal ataxin gene with parallel controls aged 5 day and 30 day were used for all experiments. The effects of such expression on mobility, longevity and neurodegenerative changes in the brain was studied. Moreover, specific techniques such are RT-PCR, and western blotting were used to test expression of specific genes and to quantify level of specific proteins. Neurodegenerative changes in transgenic lines were documented using confocal microscopy. The expression of specific genes such as ataxin and matrix metalloproteinase 1 and 2 (dMMP1, dMMP2) was studied in all used fly lines. The results showed that gene expression of hAtXN1 was higher in lines manifesting SCA1 compared to controls. However, the trend of MMP expression was not completely clear and will be the subject of future research.
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In vivo biotinylation as a new method of galectin labeling
Balogová, Soňa ; Bojarová, Pavla (advisor) ; Kavan, Daniel (referee)
Galectins are animal lectins with affinity to terminal β-galactosides. These lectins are characterized by an evolutionarily conserved carbohydrate recognition domain (CRD). Galectins are involved in many processes in vivo, such as cell adhesion; signaling; cell proliferation and carcinogenesis. They can be used as markers in certain types of cancer and also for the targeted binding of therapeutics in the diagnosis and therapy of cancer. Galectins have been defined as a protein family composed of 15 members (11 of which are known in humans). They are classified into three subgroups, according to their structure: prototypical; chimeric; tandem-repeat type. Galectin-8 belongs to tandem-repeat galectins, which consist of two different subunits connected with a peptide linker; they contain two different CRD domains, and are therefore bivalent. Galectin-glycan interactions are based on the affinity of galectin CRDs to glycans containing a terminal β-galactoside, and this affinity is formed by the set of conserved amino acids within each CRD. These interactions can be investigated by many methods, such as X-ray crystallography; competitive ELISA; biolayer interferometry. Due to the extensive involvement of galectins in biological processes, there are many methods of labeling galectins for their detection....
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The influence of heterodimezation of splicing variants of metobotropic glutamate receptor 1a and 1b on the intracellular distribution of receptor complexes
Dvořáková, Michaela ; Konvalinka, Jan (advisor) ; Vaněk, Ondřej (referee)
L-glutamate is a major excitatory neurotransmitter in vertebrate central nervous system. L-glutamate enables synaptic transmission through ionotropic and metabotropic glutamate receptors. These receptors are indispensable in the brain. The main role of metabotropic glutamate receptors is to mediate slow excitatory and inhibitory responses by activation of intracellular messengers and to regulate cationic channels. Metabotropic glutamate receptors are involved in synaptic plasticity, different types of memory, learning, motoric coordination and neural development. On the other hand excitotoxicity of glutamate is often associated with neurodegenerative processes such as Alzheimer, Huntington and Parkinson disease. Metabotropic glutamate receptors are promising therapeutic targets for a treatment of psychiatric and neurological diseases. Targeted trafficking of metabotropic glutamate receptors to distinct parts of neurons is influenced by neuronal polarity and thus regulates sensing and transmission of extracellular signals. Newly detected heterodimeric receptors might be trafficked in a different way than homodimers and therefore our knowledge of molecular pathways of these complexes could help us with subsequent drug targeting. This work confirms heterodimerization of metabotropic glutamate receptor 1 into...
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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....
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Preparation and characterisation of antibodies against cytochrome P450 1A1
Vlasáková, Kateřina ; Hodek, Petr (advisor) ; Mácová, Iva (referee)
Cancer is the second most frequent cause of death in the Czech Republic. The malignant transformations of the tumour cells are thought to be caused by formation of DNA adducts or by reactive forms of oxygen. One of groups of enzymes, that catalyse the formation of these carcinogenic compounds, are cytochromes P450, for example the type CYP1A1 or CYP1A2. Recently, chemopreventive food supplements that should decrease the risk of cancer have become very popular. However, overusing of these supplements can induce CYP and increase the risk of cancer. Due to CYP proteins cognition could be the pharmacotherapy more efficient. Suitable tool for the study of CYP proteins is "immunoblotting", which utilizes specific bound between an antigen and an antibody. The chicken anti-peptide antibodies were used for this purpose. The goal of this experiment was to obtain chicken anti-peptide antibodies, using a peptide based on the sequence of rat CYP1A1. The chicken antibodies can be used in further research. (In Czech) Keywords: Yolk antibodies, peptide antigen, ELISA, Western blot
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Preparation and characterization of antipeptide antibodies for immunodetection of cytochromes P450
Mácová, Iva ; Hodek, Petr (advisor) ; Koblas, Tomáš (referee)
The cytochromes P450 are enzymes participating in metabolism of endogenous and exogenous compounds. Their substrates include also carcinogens which may initiate carcinogenesis after activation by CYP450. Inductors of these enzymes are also chemopreventive compounds which are very popular and recommended in current time. Thus, studying of the effect of the chemopreventive compounds on cytochromes P450 induction and cancer development is of a high clinical importance. The CYPs are most commonly found in the liver. However, there are forms that have not been detected in any human healthy tissue but their overexpression was observed in tumors. For this reason, they could serve for diagnosis and prognosis of cancer. Among these cytochromes are CYP2S1 and 2W1 which can be prognostic markers of colorectal cancer. Therefore, it would be opportune to have some tools for these enzyme detection. One option is immunodetection of cytochromes P450 by Western blot using the specific antibodies. Today mammalian antibodies (IgG) are the most widely used but antibodies isolated from egg yolk (IgY) become popular mainly due to the large number of undisputed advantages. For the preparation of the peptide immunogen, suitable peptide sequences were selected from CYP2S1 and 2W1 primary structure. The synthesized peptides...
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The expression of macrophage migration inhibitory factor in neuroblastoma cell lines
Polatová, Daniela ; Poljaková, Jitka (advisor) ; Groh, Tomáš (referee)
A macrophage migration inhibitory factor (MIF) is a cytokine which participates in immune responses induced by outer stimuli such as lipopolysaccharides of bacteria or glucocorticoids. It is produced mainly by macrophages. It activates other macrophages and T-lymphocytes and allows anti-inflammatory cytokines to be released. It contributes to angiogenesis - endogenously produced MIF is crucial for the proliferation of endothelial cells. The formation of new blood vessels stimulated by MIF was also described in tumors and it leads to tumor growth. The natural receptor for MIF is an integral membrane protein CD74, which could be presented on the cell surface. If it is presented on the surface of T-lymphocytes, it could be modified by chondroitin- sulphate and interact with a protein CD44. This modification and interaction is important for an effective activation of T-cells. MIF then binds to the complex CD74-CD44. The presence of CD74 in human neuroblastoma cell lines UKF-NB-3 and UKF-NB-4 was studied in this bachelor thesis. Using electrophoresis and Western blot CD74 was detected in the UKF-NB-4 neuroblastoma cell line but it wasn't detected in the UKF-NB-3 neuroblastoma cell line. Nevertheless, the presence of this receptor in neuroblastoma cells indicates, that even neuroblastomas could be...
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