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Interaction of the adenylate cyclase toxin with complement receptor 3 - Relation of structure and function
Morová, Jana
Adenylate cyclase toxin (CyaA) is a key virulence factor of Bordetella pertussiss, the agent of whooping cough (pertussis). CyaA is a secreted bi-functional toxin belonging to the RTX (Repeat in ToXin) family of bacterial cyolysins capable to permeabilize cellular membranes by forming small cation-selective pores. The major activity of CyaA, however, consists in delivery of an adenylate cyclase domain into target cell cytosol, where upon activation by calmodulin it catalyzes uncontrolled conversion of cellular ATP to cAMP, a key signallilng molecule subverting cell functions. Recently, it has been demonstrated that CyaA utilizes the CD11b/CD18 integrin as a specific cellular receptor. The CD11b/CD18 heterodimer is rather promiscuous cell surface molecule, playing an important role in several biological functions of myeloid phagocytic cell, among which are bactericidal functions, such as chemotaxis, phagocytosis, degranulation and superoxide generation. Inhibition of those functions by CyaA action then appears to play an important role in Bordetella virulence. Study of the penetration of CyaA into the cells is important in two reasons. The CyaA toxin is endowed with a unique mechanism of entry into eukaryotic cells that consists in a direct translocation of the catalytic domain across the plasma...
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Assessing biochemical properties of PDE8A1: Design of experimental system in living cells"
Galica, Tomáš ; Černý, Jan (advisor) ; Mašek, Tomáš (referee)
4 Abstract Phosphodiesterases (PDEs), enzymes that hydrolyze cyclic nucleotides, are important components of signal transduction pathways in eukaryotic cells. Second messenger 3'-5'- cyclic adenosine monophosphate (cAMP) is hydrolyzed by specific PDEs. By controlling concentration levels of cAMP in cell, PDEs preserve favorable environment for successful transmission of the cAMP signal. Moreover, PDEs are activated by protein kinase A (PKA) in response to elevated cAMP concentration, which is a feature crucial for signal termination. PDE8A1 is a high-affinity cAMP-specific IBMX insensitive phosphodiesterase, an enzyme important for cAMP signaling. However, mostly due to a lack of specific inhibitor, its role has not been assessed in detail. This thesis reports cloning of PDE8A1, identification of its posttranslational modifications and subcellular localization, as well as an alternative approach to address PDE biology by the use of cyclase toxin from Bordetella pertussis. Keywords: phosphodiesterase, cAMP, posttranslational modification, myristoylation, palmitoylation, adenylate cyclase toxin
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Posttranlational protein modifications in response to DNA damage
Kroupa, Michal ; Hodný, Zdeněk (advisor) ; Novotný, Marian (referee)
- 5 - Abstract Thousands of DNA lessions occur in each cell every day of which the most toxic are double-strand breaks (DSBs). Signaling of their presence and subsequent repair are mediated by so-called DNA-damage response mechanism (DDR), which involves accumulation of many effector proteins into DSBs sites. These molecular accumulation at DSBs are termed DNA damage foci. Depending on presence of sister chromatid, DSBs are repaired by two major mechanisms: by homologous recombination and by non-homologous end joining. Both pathways lead to activation of checkpoint kinases (Chk1 or Chk2) which iniciate checkpoints in cell cycle and allow repair of damaged DNA. Signaling of DNA damage and activation of these pathways are regulated by posttranslational protein modifications. These enzymatic reactions involve mainly phosphorylation, ubiquitination and sumoylation. Recently it was shown that ubiquitination of damaged chromatin is a prerequisite for sumoylation of tumor supressors BRCA1 and 53BP1. Failure in DNA damage recognizing mechanisms caused by disorders such as modifications or mutations of 53BP1 and BRCA1 genes can lead to subsequent disruption of genomic integrity and then a high risk for selection of cell clones with tumorigenic potencial. Current research is focused on regulation of posttranslational...
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Production of biologically active proteins in plants
Šmídková, Markéta ; Angelis, Karel (advisor) ; Bříza, Jindřich (referee) ; Griga, Miroslav (referee)
6. SOUHRN 1) Byly zavedeny tři rostlinné expresní systém umožňující produkci biologicky aktivních proteinů. První využívá dočasné exprese z vektoru dopraveného do buňky Agrobacteriem po infiltraci listů tabáku bakteriální suspenzí. Pomocí tohoto systému jsme schopni vyprodukovat množství rekombinantního proteinu dosahujícího 5% CRP během 3 dnů. Druhý expresní systém využívá suspenzní kultury stabilně transformovaného mechu Physcomitrella patens. Za kontrolovaných podmínek pěstování v bioreaktoru je výtěžek rekombinantního proteinu srovnatelný s výtěžkem z agroinfiltrace. Expresní systém využívající kombinace virové expresní kazety, založené na TRV, a jejího přenosu do rostlinné buňky pomocí Agrobacteria je pro produkční účely vzhledem k nízkým výtěžkům méně vhodný, nežli předchozí dva systémy. 2) Ve všech třech systémech lze produkovat heterologní proteiny, které se svými vlastnostmi neliší od proteinů produkovaných v původním organismu. Pro dosažení požadované aktivity heterologního proteinu produkovaného rostlinou, stejně jako pro dosažení vysokého výtěžku, je nutné provést řadu optimalizací. Významné je zjištění, že v případě virových antigenů (HPV16L1 protein) i jednořetězcových fragmentů protilátek (scFv MEM97) je pro rostlinu výhodnější exprimovat sekvence genů rekombinantních proteinů s vyšším...
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Laboratory diagnosis of tuberculosis based on Interferon-Gamma Release Assay (IGRA)
BEDNÁŘOVÁ, Marcela
Interferon-gamma release assay (IGRA) methods have become a common part of the routine tuberculosis (TB) diagnosis. These are indirect diagnostic methods, which detect the cell-mediated immune reactivity to the presence of Mycobacterium tuberculosis antigens inside the human body. The methods are based on the principle of detection of interferon-gama(IFN-gama) produced by the T-lymphocytes sensitized by TB antigens. Currently, there are two tests available (QuantiFERON -TB Gold In -Tube and T - SPOT.TB). This thesis is dealing with the principle, the practical use and the place of IGRA methods in the comprehensive diagnosis of TB. The Collection of 1554 blood samples, examined by QuantiFERON-TB Gold In-Tube in the period from 1st June 2011 to 30th June 2013 in the Mycobacteriology Laboratory of the University Hospital in Pilsen was evaluated in the practical part. The percentage of various types of health care settings indicating this examination, as well as the percentage of positive, negative and undetermined results in some selected groups of patients (screening before the initiation of biological treatment, in patients with primary lung tumors, in person with a high-risk of TB and person in contact with active form of the disease) was monitored. The results of patients with active TB verified by culture were also assessed. The results of this study indicated, that IGRA methods are of a great contribution in the TB diagnosis, in particular in the latent form, under certain conditions also in the active form. At the same time it should bementioned, that the diagnosis of tuberculosis must always be complex. This method should not be used for the TB diagnosis solely.
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Influence of growth stimulators on physiological characteristics by hops plants (""Humulus lupulus"" L.)
Bazika, Václav ; Hnilička, František (advisor) ; Josef, Josef (referee)
At growing Saaz variety is the target to reach the highest yield of hop´s cones and high content of alpha acids. The average content of this acids in Saaz variety is in the range from 2,5 to 4,5 %. The growers effort is to reach the higher number of this range because the final selling price is given by content of alpha acids. During the active growth of hops, grower can targeted stimulate the hops to positively affect the physiological processes. Then he can reach better yield of valuable alpha acids and that´s why the target of this diploma thesis was to fix the influence of growth stimulators on physiological processes on the yield of hop´s cones and the yield of alpha acids. And based on the results to identify the suitability of using growth stimulators during the intense hops growing. Following scientific hypothesis were designed: determine the plants reaction diversity on growth stimulator. For reaching the targets and confirm or reject the hypothesis was observed in natural conditions nearby the Chrastany region the plants of Saaz (clone 72) in interaction with the usage of growth stimulators. These stimulators were used: Humastar, Fertileader Vital, Lignoaktivátor. There were four variants: controled with no aplication and three other variants with usage of growth stimulators (for each variant were apllied only one stimulator for three times).
The influence of growth stimulators on hops plants (phase BBCH from 37 to 81) was observed on the basis of the rate of gas exchanges, chlorophyl fluorescence and the leaves chlorophyl content, which were measured by infrared gas analyzer LCpro+ (ADC BioScientific Ltd. Hoddesdon, Great Britain), OS 1-FL (OPTI SCIENCES), Chlorofyl CCM 200 ADC BioScientific Ltd. Hoddesdon, Great Britain) After the crop was evaluated the yield of hop´s cones and the content of alpha acids, beta acids and essentials oils.
The results of measurment indicates that the highest average transpiration rate was observed after Humastar application (+18,3 % against control variant). In the control variant was found the lowest average transpiration rate from all variants. The highest photosynthesis rate was found after Fertileader Vital application (+7 % against control variant). Decrease of photosynthesis rate about 17 % against the control variant was observed after Humastar application. The differences was also observed in chlorophyll fluorescence. The highest Fv/Fm value reached the hop´s plants after Fertileader Vital application, concretely 0,792. On the other hand the lowest Fv/Fm value was observed after Lignoaktivátor application (0,780). After the application of all tested growth stimulators were reached increase of a pigment´s content in hop´s leaves against the control variant. The highest content of pigments were observed after Fertileader Vital application (+19,2 % against control variant). Control variant reached the average content of pigments in hop´s leaves 25,5 chlorophyll units. Differences after the growth stimulation was also observed in yield and quality of dry hop´s cones. The highest yield reached the Lignoaktivátor variant (0,73 t/ha). This is an increase of 17,7 % against the control variant (0,62 t/ha). The yield of hop´s cones was decreased of 8,1 % against the control variant after Humastar application. The similiar trends were also observed in hop´s cones quality. The highest content of alpha acids (3,06 %) was reached after Lignoaktivátor application and at the same time Lignoaktivátor variant also achieved the highest content of essentials oils per 100 g of a sample (0,22 ml). Humastar variant had the lowest content of alpha acids (2,18 %) and also the lowest content of essential oils (0,14 ml).
Based on the results it can be stated that the used growth stimulators had the influence on the physiological characteristics of hop´s plants and also affected the different yield and quality of hop´s cones. Proposed hypothesis were confirmed. Based on the one-year results, application of Fertileader Vital or Lignoaktivátor seems to bee more profitable than Humastar application.
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Cloning, expression and characterization of human serine racemase mutants
Nováková, Ilona ; Brynda, Jiří (referee) ; Konvalinka, Jan (advisor)
AAbbssttrraacctt Human serine racemase (hSR) is a cytosolic pyridoxal-5'-phosphate dependent enzyme localized in the central nervous system. It synthesizes D-serine, which is an endogenous coagonist for the N-methyl-D-aspartate (NMDA) receptors and plays a key role in excitatory neurotransmission in the brain. Thus, human serine racemase is a promising target for the treatment of neurodegenerative diseases connected with NMDA receptors. However, few specific inhibitors have been identified to date and the crystal structure of hSR has become available only very recently. We decided to perform a random mutagenesis to determine the amino acid residues critical for the enzyme activity. Ser 84 was reported as a catalytic residue along with Lys 56. After analysis of a double mutant S84G/P111L which retained its capability to convert L-serine to pyruvate, we prepared and characterized the single mutant S84G in order to exclude potential effect of the P111L mutation.on the activity of the analyzed enzyme. KKeeyy wwoorrddss:: D-serine; Serine racemase; PLP-dependent enzymes; Random mutagenesis; Racemases
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Autoimmune thyroiditis in children and the role of simultaneous Helicobacter pylori infection
Pomahačová, Renata ; Sýkora, Josef (advisor) ; Hníková, Olga (referee) ; Kalvachová, Božena (referee)
1. Summary The first aim of our study was to find out the present occurence of H. pylori infection in a group of children with juvenile lymphocytic thyroiditis (JLT) and to compare with the prevalence of this infection in a group of healthy children of the same age. The group of patients with JLT included 199 children (range 0-18 years). H. pylori infection was confirmed in 15 patients (7,5%) by a non-invasive diagnostic test of H. pylori antigen positivity in stools by the ELISA test using monoclonal antibodies (Amplified IDEIQA HpStAR ELISA, DakoCytomation, Glostrup, Denmark). This H.pylori positive group included 14 girls and 1 boy aged 11,2 ±2,3. 6 children had hypofunction of the thyroid gland, in 3 children was established the atrophic form of the thyroiditis with severe hypothyroidism with presence of myxoedema, the other children suffered from the hypertrophic form of the thyroiditis. 7 subjects underwent gastroscopy which confirmed chronic active gastritis. Only 3 children had gastrointestinal symptoms caused by H. pylori infection, the other children were asymptomatic. This finding confirms common information about asymptomatic development of H. pylori infection in children in 90% of cases. The control group included 1545 asymptomatic healthy children in range of 0-15 years of the same geographic...
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Glycocalyx shedding by cercariae of bird schistosomes
Chaloupecká, Jana ; Mikeš, Libor (advisor) ; Štěrba, Ján (referee)
Trichobilharzia spp. are avian schistosomes related to medically important human parasites of the genus Schistosoma. Penetrating cercariae are well known as causative agent of cercarial dermatitis in humans. Cercariae actively penetrate the skin of definitive hosts and transform into schistosomula. This process is preceded by cercarial tail detachment and includes emptying of penetration glands and extensive surface changes. One of these changes is the loss of highly immunogenic glycocalyx which represents a protective coat in the aquatic environment. The glycocalyx has specific composition of saccharide molecules which are bound to lipids or proteins on the membrane of cercarial tegument. There is only limited information about the mechanism of shedding. Hypotheses based on indirect evidences suggest that peptidases or (phospho)lipases from penetration glands could be involved. This work describes the changes in surface glycosylation during transformation of cercariae into schistosomula by fluorescently labelled lectins and monoclonal antibodies against Lewis X antigen. Lectins UEA-I, LTA and PNA have been chosen as markers of transformation of T. regenti. Further, our experiments have been focused on shedding of cercarial glycocalyx. During in vitro induction of penetration gland emptying and...
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