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Purification of patatin proteins from tubers of selected potato (\kur{Solanum tuberosum} L.) cultivars with subsequent characterization of isolated patatin proteins
ŠVEHLOVÁ, Eva
The complex of patatin proteins were extracted from potato tubers (Solanum tuberosum L.) and its purification process is described in this work. The initial material for patatin protein isolation were potato fruit juices extracted from cultivars Laura, Sibu and Westamyl. Based on the known patitin´s properties, the ion-exchange chromatography (DEAE medium) coupled with affinity chromatography was applied. We used the column containing biological ligant of Concanavalin A, which was able to bind the carbohydrates in the molecule of patatin proteins. The used purification process were successful and according to the SDS-PAGE analysis we obtained patatin proteins of a high purity from each the potato cultivars. The antioxidant activity was observed for all samples and range from 0,007 to 0,086 mg ascorbic acid concentration / g protein.
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Mechanisms of MHCII signaling in B lymphocytes
Kotlabová, Klára ; Brdička, Tomáš (advisor) ; Černý, Jan (referee)
During the initiation of an antigen-specific immune response, peptide fragments originating from the antigen are presented in complex with MHC class II glycoproteins (MHCgpII) on the surface of the antigen presenting cells (APC). Antigen recognition by T lymphocyte is accompanied by the formation of the molecular structure at the interface with APC called immunological synapse (IS). During this contact, signal transduction is initiated at both, T lymphocyte and APC, sides of the IS. For a long time it was thought that the only function of MHCgpII is presentation of antigen. However, later it was found that stimulation of MHCgpII led to triggering of signals contributing to decision about the further fate of APC. MHCgpII do not have any signaling motifs in their cytoplasmatic domains, and so associated molecules are necessary for the transduction of the signals. This work focuses on B lymphocytes in which the associated molecules are Ig alfa/beta, MPYS, CD19 and CD20. After the stimulation of MHCgpII these proteins mediate signaling events including activation of several families of protein kinases, phospholipase C, mobilization of calcium and activation of transcriptional factors NFAT and AP-1. In B lymphocytes, activities of these pathways may result in proliferation and differentiation but also in the...
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Prolyl endopeptidase of the blood fluke Schistosoma mansoni
Fajtová, Pavla ; Konvalinka, Jan (advisor) ; Ryšlavá, Helena (referee)
Prolyl endopeptidase SmPEP from the blood fluke Schistosoma mansoni is investigated here for the first time. This enzyme is potentially interesting as a drug target for the treatment of schistosomiasis. SmPEP was detected in the extract of adult worms by enzyme activity and immunoreactivity. Enzymatically active SmPEP was produced in the E. coli expression system and was chromatographically purified. The pH optimum of recombinant SmPEP was about 8. Substrate specificity analysis revealed that SmPEP cleaved peptide substrates by endopeptidase activity, however, macromolecular substrates were not fragmented. The residue preferences in the positions P3 to P1' were determined using synthetic fluorogenic peptide substrates. SmPEP was found to be highly sensitive to the inhibition by Z-Ala-Pro-CMK and Z-Arg-Pro-CHO. Primary screening of crystallization conditions for recombinant SmPEP was performed. " (In Czech)"
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Construction of various types of vaccines based on the structural proteins of mouse polyomavirus and analysis of immune response after their administration to mice
Hrušková, Veronika
|Abstract Model mouse polyomavirus like-particles carrying enhanced green fluorescence protein (EGFP-VLPs) were prepared and used for intranasal immunization of mice. EGFP-VLPs induced strong anti-VP1 but not anti- EGFP antibody production. In vitro restimulation with antigen-pulsed bone marrow-derived dendritic cells (BMDCs) induced remarkable T-cell proliferative response specific for both VP1 and EGFP antigen and IL-2 and IFN-_ production. Surprisingly, no specific cytotoxic activities against VP1 and EGFP proteins were detected. After intranasal administration of EGFP-VLPs, as well as after polyomavirus infection, a moderate reduction of CD4+CD25+Foxp3+ T cells was observed in spleens but not in lymph nodes and peripheral blood, suggesting that both MPyV virions and VLPs are able to induce changes in distribution of regulatory T cells. Treatment of EGFP-VLPs pulsed BMDCs with inhibitors of endosomal acidification proved that presentation of peptides on MHCgp class II is dependent on acidic endosomal environment. Substantial decrease of CD4-specific T-cell proliferation in the presence of proteasome inhibitor suggests that MHCgp class II might load VPL-derived peptides processed by proteasomes. Based on the model EGFP-VLPs, mouse polyomavirus-like particles carrying inside a fragment of the Bcr-Abl...
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Exctretion of urinary N7 guanine and N3 adenine DNA adducts in mice after inhalation of styrene
Mikeš, Petr ; Mráz, Jaroslav (referee)
Adenine adducts of styrene : 3-(2-hydroxy-1-phenylethyl)adenine (N3αA) and 3-(2-hydroxy-2- phenylethyl) adenine (N3βA), were found in the urine of mice exposed to styrene vapor. Vast majority of styrene is metabolized to styrene -7,8-oxide. This reactive molecule is futher bio transferred either to mandelic and phenylglyoxalic acid or bounds to nucleophilic sites in biomolecules. Adducts of styrene oxide with amino acids contained in proteins and with purine and pyrimidine bases of nucleic acid were observed during in vitro tests. These styrene-7,8-oxide derived adenine adducts, as well as previously identified guanine adducts, 7-(2-hydroxy- 1- phenylethyl)guanine (N7αG) and 7-(2-hydroxy-2-phenylethyl)guanine (N7βG) were quantified by HPLC-ESI-MS2 and the excretion profile during and after repeated exposure to 600 mg.m-3 or 1200 mg m-3 of styrene for 10 consecutive days (6 h/day) was determined. The excretion was dose dependent. Total excreted amount of N3 adenine adducts (N3αA+N3βA) represents 0.8 ×10−5 % of the absorbed dose, while overall sum of N7 guanine adducts (N7αG+N7βG) yields about 1.4 ×10−5 % of the dose. No accumulation of the adducts was observed. Due to rapid depurination of the DNA, the excretion of both N3 adenine and N7 guanine adducts ceased shortly after finishing the exposure. Both N3...
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Molecular-biological characterization of Potato virus M and Potato leafroll virus
Vaculík, Petr ; Čeřovská, Noemi (advisor) ; Fousek, Jan (referee)
The main aims of diploma thesis were: 1) The sequence analysis of the Czech isolate of Potato virus M (PVM) VIRUBRA 4/009 and phylogenetic analysis of PVM coat proteins sequences 2) The bacterial expression of recombinant triple gene block protein 1 (TGB1) of PVM derived from the Czech isolate VIRUBRA 4/007 3) The construction of expression cassette of Potato leafroll virus (PLRV) coat protein and its transformation into A. tumefaciens for transgenic PLRV resistant plant formation In theoretical part of the thesis the taxonomic classification, morphology, genomic structure and virus transmission are discussed. Furthermore, the main rules concerning the bacterial expression of recombinant proteins and construction of transgenic plants using A. tumefaciens are described. Methodical part is devoted to description of generally used molecular biological and immunochemical methods. The following results were obtained in the thesis: The complete nucleotide sequences of open reading frames coding for three movement proteins (Triple gene block -TGB), coat protein and NA-binding protein of PVM isolate VIRUBRA 4/009; phylogenetic analysis was performed; the TGB1 protein was expressed in bacterial cells and will be used for polyclonal antibodies raising. Finally, the expression cassette containing the PLRV...
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Evaluation of food and diet in disease phenylketonuria
Čermáková, Venuše ; Faměra, Oldřich (advisor) ; Renata, Renata (referee)
This diploma thesis pursues the hypothesis, that strict abiding or failures in low protein diet are deciding about the health of patients with phenylketonuria. The goal of the thesis was to evaluate the composition of the diet for selected set of patients from the dietological and nutritious point of view in dependence on individual health condition, to compare the values with the diet of healthy people and to evaluate the availability of special healthcare and nutrition for phenylketonuria patients.
The summary of literature gives informations about phenylketonuria, causes of its origin, metabolism of phenylalanin, diagnostics, possibilities of treatment, risks
of repercussion of not treating, impact of nutrition composition of patients' health and dietological recommendations.
For assessment of the bilance of nutrients and energy in the diet of phenylketonuria patients a group of 10 people was selected with different age, sex and dietary level, 5 people with phenylketonuria a 5 healthy persons. For each person a detailed weekly diet was setup and nutritious intake was evaluated, observed was daily intake of Phe, protein, fat, carbohydrate and energy. For persons with phenylketonuria the nutritious was evaluated
in two steps, with and without usage of medical agent. The values of the patients were compared with healthy persons.
From the results a necessity of using medical amino acid agents was derived, in other case there is a risk of serious deficit of protein intake, for some also of fat and energy.
A complicated situation arises for women with phenylketonuria, who plan pregnancy.
The research implies necessity of strict adherence to low protein diet before conception and frequent monitoring of Phe blood values during pregnancy.
The diet must be compiled cautiosly with considering the individual state of patiens,
a thorough education from childhood to adulthood is recommended. The situation would be simplified with a better approach of the state in the area of compensation the costs for foods, but also additional support for phenylketonuria patiens, to improve especially the availability of special foods for low protein diet and treatment possibility.
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Changes of mares milk contents during lactacion
VRÁNOVÁ, Vendula
The objective is to determine the composition of mare´s milk at different stages of lactation and changes in its content that occurs during lactation. Variability of constituents in the milk contents between mares and possible link in the content of individual components of milk was also examined. Average values of contents were found during the five months of lactation: Fat 1.5%, protein 1.62%, lactose 6.55% and dry matter 9.78%. It was found that during lactation fat content decreases from 1.7% to 1.3% and protein content decreases from 1.75% to 1.48%. Lactose and dry matter content is unchanged. A higher coefficient of variation was found in fat and protein (between 16 -17%), lower in lactose and dry matter content (5-6%). Relation was found between protein and fat. On the contrary, no evidence of correlation between protein and lactose content, nor between the fat and lactose content was found.
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Assessment of expression markers of HLA class II genes.
Zajacová, Marta ; Černá, Marie (advisor) ; Černý, Jan (referee)
BACKGROUND: HLA molecules play a central role in the immune response. HLA class II are involved in the selection of the T-cell repertoire in the thymus, and in presentation of antigenic peptides to antigen reactive CD4-positive T cells. The HLA class II act as restriction determinants in the presentation of antigens to T lymphocytes and their expression on the cell surface is necessary for triggering the immune responses. Regulated transcription of HLA class II genes is a complex system involving cis-acting sequence elements and trans-acting protein factors. It has been reported that allelic polymorphism exists in the regulatory regions of HLA class II DQA1, DQB1 and DRB1 genes. Most of the polymorphisms appear to be conserved within a haplotype. The hierarchy of sequence homology which exists among the structural genes is not paralleled among their promoter sequences. It is of interest that the most striking discrepancy was found for the DR4 linked alleles, DQB1*0301 and DQB1*0302, which were described as frequent risk factors for a variety of autoimmune diseases. The structural genes of these two alleles are the most closely related, but their regulatory sequences are the most heterogeneous among DQB1 variants. These sequence differences correspond to functional variation: The promoter strength...
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Determination of total protein, carbohydrates and polyphenols in beer
Dostálová, Blanka ; Gregor,, Tomáš (referee) ; Diviš, Pavel (advisor)
This master‘s thesis deals with the analysis of 65 different beers, with a focus on finding differences between Czech tap ales, lagers Czech, Czech special beers and foreign beers. Czech beers were compared by the results of analysis among beers bearing the indication "Czech beer" and the group without this indication. The total amount of proteins, polyphenols and carbohydrates was determined using UV-VIS spectrometry. The history of brewing, the nature and types of Czech beers and protected geographical indication "Czech beer" were described. Raw materials for beer production and brewing technology have been listed and described. In the last part of the research, components of beer and the principle of the UV-VIS spectrometry have been specified. Th determination of total protein was performed using Hartree-Lowry method, which is based on two-component reagent. The first component is the biuret agent, the second component is the Folin-Ciocalteau reagent for phenols. Determination of the polyphenols has been carried out by the Folin-Ciocalteau reagent and total carbohydrates were determined spectrophotometrically according to the Analytica EBC using anthron agent. The results were statistically processed and evaluated in the final part.
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