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Comparison confirmation ELISA to recomLine test for the determination of hepatitis C
BAČKOVÁ, Marcela
My bachelor thesis deals with hepatitis C disease and its laboratory diagnosis, especially confirmatory tests. The theoretical part focuses on the disease in general, describes the hepatitis C virus that causes the disease. It also discusses the acute and chronic phases, virus transmission and treatment options. In the practical part I describe the examination of anti-HCV screening antibodies and how to proceed in case of a positive finding. The main task of my practical part is to compare two confirmatory tests - ELISA and immunoblot recomLine. At the end of the work I evaluate both tests and compare the final results.
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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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Glycosylation and antigenic properties of Phlebotomus perniciosus and P. orientalis salivary proteins
Sumová, Petra ; Volf, Petr (advisor) ; Grubhoffer, Libor (referee)
The goal of this study was to map the glycosylation pattern and antigenic properties of the salivary proteins of two closely related sand fly species, Phlebotomus perniciosus and P. orientalis. Affinity blotting with commercially available lectins revealed that many salivary proteins of these species are N-glycosylated, while the presence of O-glycosylation could not be confirmed. The level of N-glycosylation of most of these proteins is quite low, a larger number of potential N-glycosylation sites were found only in the amino acid sequences of P. orientalis hyaluronidase and endonucleases of both species tested. Four antigens from P. perniciosus salivary glands were selected for expression in a bacterial expression system; two of these proteins (PpeSP01 and PpeSP01B) were not glycosylated and the glycosylation level of the remaining two (PpeSP03B and PpeSP07) was low. The antigenic properties of the four chosen recombinant proteins were subsequently tested using immunoblot and ELISA. During the initial experiments with the sera of dogs experimentally bitten by P. perniciosus, two proteins (rSP07 and rSP01B) were proven unsuitable and they were excluded from further experiments. Recombinant proteins rSP03B and rSP01 were recognized by the same IgG antibodies as the native forms of these proteins...
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Glycosylation and antigenic properties of Phlebotomus perniciosus and P. orientalis salivary proteins
Sumová, Petra ; Volf, Petr (advisor) ; Grubhoffer, Libor (referee)
The goal of this study was to map the glycosylation pattern and antigenic properties of the salivary proteins of two closely related sand fly species, Phlebotomus perniciosus and P. orientalis. Affinity blotting with commercially available lectins revealed that many salivary proteins of these species are N-glycosylated, while the presence of O-glycosylation could not be confirmed. The level of N-glycosylation of most of these proteins is quite low, a larger number of potential N-glycosylation sites were found only in the amino acid sequences of P. orientalis hyaluronidase and endonucleases of both species tested. Four antigens from P. perniciosus salivary glands were selected for expression in a bacterial expression system; two of these proteins (PpeSP01 and PpeSP01B) were not glycosylated and the glycosylation level of the remaining two (PpeSP03B and PpeSP07) was low. The antigenic properties of the four chosen recombinant proteins were subsequently tested using immunoblot and ELISA. During the initial experiments with the sera of dogs experimentally bitten by P. perniciosus, two proteins (rSP07 and rSP01B) were proven unsuitable and they were excluded from further experiments. Recombinant proteins rSP03B and rSP01 were recognized by the same IgG antibodies as the native forms of these proteins...
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Characterization of excretory-secretory proteins of liver fluke Fascioloides magna.
Beránková, Kateřina ; Kašný, Martin (advisor) ; Horn, Martin (referee)
Fascioloides magna (the giant liver fluke) originated from North America, is known in the Czech Republic since 1930s. This pathogenic fluke invades mostly cervids, but livestock too. Excretory-secretory products (ES products) contain number of esential biomolecules which are produced by excretory and secretory system of the fluke. These molecules play key role in many biological process during the life cycle not only of fascioloid flukes (e.g. migration in the host tissues, immune evasion and digestion). Due to their antigenic properties they could be also used in immunodiagnostics. Excretory-secretory proteins from adult Fascioloides magna and comparative related species Fasciola hepatica were purified and separated by the basic biochemical methods (1D, 2D electrophoresis, ion-exchange chromatography) and their activity was confirmed by specific (fluorogenic peptide) and nonspecific (gelatine) substrates. By using the mass spectrometry methods (MALDI TOF/TOF), the most abundant peptidolytically active proteins from ES products of F. magna were identified as cathepsin L (FmCL). Recombinant analog of FmCL was expressed in Pichia pastoris expression system. The peptidolytic activity was again confirmed using the synthetic fluorogenic substrates; the specifity of recombinant FmCL active site was...
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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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