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Detekce a kvantifikace inhibitorů proteáz v klíštěti \kur{Ixodes ricinus} pomocí monoklonálních protilátek
VANÍČKOVÁ, Martina
Inhibitors of proteases in tick saliva play an important role during tick feeding. Tick saliva contains a wide range of bioactive components which are able to modulate host imunity. Therefore, ticks are able to feed for a long time and transfer tick-borne diseases pathogens. The risk of transfer can be significantly reduced by deactivation of theese protease inhibitors. In this study I made monoclonal antibodies for detection and quantification of two serine protease inhibitors in tick saliva and other tick-body parts.
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Measurement of signalling pathway activation in mouse macrophage line IC-21 and primery dendritic cells after infection with tick-borne encephalitis virus.
Kožantová, Jana ; Lieskovská, Jaroslava (advisor) ; Drbal, Karel (referee)
Tick-borne encephalitis is a serious disease of the central nervous system. It is caused by tick-borne encephalitis virus, which is transmitted by ticks. The Czech Republic is one of the countries with the highest prevalence of this disease. Tick-borne encephalitis virus is able to replicate in several cell types. In this work we focused on macrophage line IC-21 and dendritic cells, because these cells are the first, which encounter the virus and support its spreading in the host at early stage of infection. So far there is not known any specific receptor for virus entry into cells or which signaling pathways activates. Therefore, we decided to investigate the activation of selected signaling pathways after infection with tick-borne encephalitis virus and influence of tick saliva on this activation. We employed methods of dual luciferase reporter assay, immunosandwich assay and western blot. The obtained results showed that in virus infected IC-21 cells are activated phosphatidyl-inositol pathway, NF-κB pathway, signaling molecule Erk1/2 and others. Testing of tick saliva effect revealed significantly decreased activity of NF-κB, AP-1 and CREB.
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Neurobehavioral consequences of experimental psychosis in laboratory rats
Svojanovská, Markéta ; Stuchlík, Aleš (advisor) ; Telenský, Petr (referee)
Schizophrenia is a serious neuropsychiatric disease with a lifetime prevalence of 1% and it disrupts almost all mental functions. It manifests with many symptoms, which can be roughly classified into three main classes - positive, negative and cognitive dysfunctions. The psychosis, which can be often seen in schizophrenia, is a very serious problem that along with all other symptoms influences the patients' clinical status as well as quality of their life. As no direct causes or causal treatments for schizophrenia are known, scientist often focus on animal models of schizophrenia as tools for investigating mechanisms that can take a part in real disease and for seeking novel antipsychotics. This thesis aims at investigating two-week subchronic treatment with dizocilpine (MK-801), a non-competitive antagonist of NMDA receptors, in Wistar and Long-Evans rats aged 30 (PND 30) and 60 days (PND 60) at the onset of the treatment. Subsequently, long-term neurobehavioral consequences of this experimental psychosis were studied by testing rats at three behavioral tasks: the Elevated-plus maze (EPM), the Morris water maze (MWM) and active place avoidance on a rotating arena (Carousel). The Western blot method was used to determine post-mortem changes in expression of the NR1, NR2A and NR2B subunits of the...
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Possibel approaches to gene therapy of cystic fibrosis
Král, Jan ; Bořek Dohalská, Lucie (advisor) ; Vaněk, Ondřej (referee)
1 Abstract Cystic fibrosis is an autosomal recesive disorder caused by a mutation in the CFTR gene, which leads to inefficiency or absence of CFTR chlorid channel. One way to induce production of CFTR protein in target cells, is to use gene therapy. The principle od gene therapy is to transfer DNA or mRNA molecules inside malfunctioning cells. The aim of this study was to optimise the detection of CFTR protein using the Western blot analysis. Then, using this method the effectiveness of CFTR-mRNA transfection was studied. To study the CFTR protein, a number of cell lines was used: a healthy human ephitelial cell line (NuLi-1), an ephitelial cell line with ΔF508 mutation (CuFi-1), and a human lung carcinoma cell line (A549). This study compared four different ways of cell lysis - lysis by sonication and lysis by three distinct lysis buffers. Lysis by RIPA buffer with protease inhibitors was determined for the detection of CFTR protein. Moreover, three different primary monoclonal antibodies were also tested. The CF3 antibody, which is specific to an extracellular epitope of CFTR protein, was found able to detect CFTR protein specifically. A couple of different glycosylated forms of CFTR protein was detected. The highest amount of CFTR protein was determined in the NuLi-1 cell line. CFTR protein was also...
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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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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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Study on the induction of intestinal conjugation enzymes by food supplements
Hrdinová, Johana ; Hodek, Petr (advisor) ; Bárta, František (referee)
Cytochrome P450s (CYP) are enzymes, that play an important role in the metabolism of foreign compounds (e.g. drugs, carcinogens, pollutants) and they also participate in the metabolism of hydrophobic endogenic substrates (e.g. sterols, fatty acids, prostaglandins). Cytochromes of subfamily CYP1A are involved in activation of precarcinogens. Whereas the consumption of dietary supplements containing chemopreventive substances (e.g. flavonoids) that can prevent the process of carcinogenesis is significantly increasing in recent years, although these substances may also have a negative effect on the organism. That because these substances can induce the cytochrome P450s and thus increase the risk of activation precarcinogens. In this study had been investigated the inductive effect of some flavonoids compounds - β-naphthoflavone, myricetin, dihydromyricetin and a drug containing an extract of red vine leafs - Antistax®.
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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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