The introduction of calcineurin inhibitors (CNI) into immunosuppressive regimens significantly improved patients prognosis after heart transplantation. Some of the most significant complications have been recognized, such as the development of arterial hypertension and renal impairment due to calcineurin inhibitor toxicity. The aim of the study was to compare the effect of the dual blockade of the renin-angiotensin system (dual RAS combination) with standard antihypertensive medication on blood pressure control. The second aim was to evaluate whether effective antihypertensive combination therapy (dual RAS or a standard antihypertensive drugs combination) would reduce the progression of chronic kidney disease in patients with chronic immunosuppressive prophylaxis. Treatment of arterial hypertension involving the combination of angiotensin-converting enzyme inhibitor (ACEi) and angiotensin II receptor blocker (ARB) was similarly effective compared to the standard combination of antihypertensives. Blood pressure treatment targets were achieved in both studies. Administration of antihypertensive combination therapy including dual blockade of RAS alleviated the progression of chronic renal disease in the experimental and clinical part, where the nephroprotective effect of dual RAS blockade...

Possible connection between latent toxoplasmosis and schizophrenia is a very interesting and medically important topic. In this thesis I tried to map current state of knowledge in the interdisciplinary research of schizophrenia and Toxoplasma gondii and their possible connections as well as to show differences in responses between Toxoplasma-positive and Toxoplasma-negative subjects using simple computer-administered tests of prepulse inhibition of startle reaction (PPI). Such differences would suggest another similarity between schizophrenia patients and subjects with latent toxoplasmosis as the sensorimotor gating responsible for PPI was found to be disrupted in schizophrenia patients. Side goal of the study was to test newly developed PC software for testing PPI and to determine its applicability in further research. Subjects for the tests were recruited among adepts of professional military service; 409 subjects completed the test of acoustic PPI and 276 subjects completed the test of visual PPI. All the subjects were tested on presence of specific anti-Toxoplasma IgG in their blood serum. Both tests revealed significant (p<0.001) differences between responses on prepulse-preceded stimuli and plain stimuli without prepulse, no significant results were, however, gained for the effects of latent...

Equistatin from the sea anemone Actinia equina contains a protein domain Eqd2 which inhibits aspartic peptidases and has not been characterized in detail. Recombinant Eqd2 was produced in the yeast expression system, and a protocol for its chromatographic purification was designed. The inhibitory specificity of Eqd2 was determined using a fluorescence inhibition assay, showing that Eqd2 is a highly selective inhibitor of cathepsin D-like and pepsin-like aspartic peptidases of family A1. Furthermore, size exclusion chromatography was used to analyze the Eqd2-peptidase complex and Eqd2 oligomerization in solution. Initial screening of crystallization conditions for Eqd2 was performed towards its structural analysis. This work provides important new information about Eqd2 as a unique type of natural inhibitors of aspartic peptidases. Its interaction mechanism can be exploited in the development of synthetic mimetics for regulation of medically important peptidases. (In Czech) Key words: peptidase inhibitors, proteolytic enzymes, activity and inhibition of enzymes, recombinant expression, protein purification, protein crystallization, equistatin

Experimental models of a transfer of stem cells for therapeutic purposes Abstract Stem cell therapy currently represents a standard procedure of treating a wide variety of hereditary diseases and serious injuries. Development of the most suitable way of transfer of stem cells into the patient body remains very important question concerning this type of therapy. In our experiments we used nanofiber scaffolds for stem cell cultivation and their subsequent transfer. These nanofibers were prepared by the original needleless electrospun NanospiderTM technology. Allogeneic cornea or skin graft were transplanted from B6 mice to BALB/c mice. The grafts were covered by a nanofibrous scaffold with cultivated stem cells. Stem cells were stained by an imunofluorescent dye to enable us to monitore their migration from nanofibers into tissues and consequent distribution in the body and characterize changes of this distribution in the time. The methods of ELISA and PCR were used to confirm that mesenchymal stem cells support the production of antiinflammatory cytokines IL-4 and IL-10 and contribute to inhibition of production of proinflammatory cytokines IL-1, IFNγ and inducible nitric oxide synthase. We confirmed an important beneficial role of nanofiber scaffolds in transplantation of mesenchymal stem cells. Nanofiber...

Effects of Toxoplasmosis on Reaction Times and Prepulse Inhibition of Startle Reaction in Humans vi Abstract Toxoplasma gondii, a single-cell coccidia from almost exclusively parasitic phylum Apicomplexa, does not typically cause acute health issues in humans with most exceptions among immunodeficient individuals and pregnant mothers or, more precisely, their offspring. In the latent phase, the bradyzoites in tissue cysts placed most often in neural and muscle tissues can evolve pressure on the host's body both as a collateral effect of the presence of the parasitic organism in host's tissues and as a consequence of adaptive evolution leading to increase in probability of trophic transmission to the final host, a felid. In humans, this can result in slight changes in personality profiles, deterioration of psychomotor and cognitive functions, and development of serious mental disorders. The thesis focuses predominantly on one of the aspects of the changes, namely the effect of latent toxoplasmosis on the processing of startle signals themselves and when modified by a preceding low-intensity signal; this processing may be connected with the development of schizophrenia in predisposed individuals. Studies conducted within the project framework found changes int the speed of signal processing in...

83 Abstract The work focuses on the physiology of wood-rotting fungi and the possibilities of their inhibition by physical methods (microwave and gamma irradiations, dry heat) or newly developed chemical preparatives contaning metallic nanoparticles. The effect of abovementioned treatments on mycelial growth, activities of extracellular enzymes responsible for wood degradation and on spore germination or viability was studied in selected strains of wood-rotting fungi obtained from culture collection (Coniophora puteana, Fibroporia vaillantii, Gloeophyllum sepiarium) or isolated from infected buildings (Serpula lacrymans).

PDI ("potato cathepsin D inhibitor ") and NID ("novel inhibitor of cathepsin D ") from potato (Solanum tuberosum) belong to the protein family of Kunitz inhibitors (I3 family, Merops database). These 20 kDa isoinhibitors with the typical β-trefoil architecture inhibit aspartic and serine peptidases. In this thesis, the constructs for recombinant expression of PDI and NID in the yeast Pichia pastoris system were prepared and high-producing colonies were selected. Both proteins were identified in the cultivation media by mass spectrometry and N-terminal sequencing. A purification protocol for PDI with three chromatographic steps was designed. Analogous functional properties were demonstrated for the purified recombinant PDI and the native PDI isolated from a natural source. Analysis of the inhibitory specificity showed that PDI is a potent inhibitor of selected aspartic peptidases from the A1 family and serine peptidases from the S1 family, including a relevant enzyme of insect origin. This finding supports the hypothesis that Kunitz inhibitors are involved in plant defense against herbivorous insects. The inhibitors prepared within the project will be used for analysis of the reactive centers against target peptidases by protein crystallography. (In Czech) Key words: proteolytic enzymes, activity...

Title: Effect of robot-assisted therapy on upper limb function in activities of daily living in patients after acquired brain injury Aims: The main aim of the thesis was to evaluate and compare the effectiveness of two therapeutic approaches using robot-assisted glove Gloreha Sinfonia to perform Activity of daily living in patients after acquired brain injury. Methods: The study is an empirical quantitative research, a monocentric randomized controlled, simply blinded study. The study compares two groups, group A (n = 20) used a robot-assisted Gloreha glove with a bimanual approach, group B (n = 20) had therapy with a robot-assisted Gloreha glove with an unimanual approach. All patients were evaluated for eligibility and underwent initial testing (T1). Patients in both groups had therapy (unimanual / bimanual) for three weeks (15x), retest (T2) was performed after completion of the intervention, and a follow-up evaluation was performed after 1 month (T3). Patients were always evaluated by the Upper Extremity Motor Activity Log (UE MAL), Motor Assessment Scale (MAS), Action Research Arm Test (ARAT), Box and Block test (BBT) and Motricity Index (MI). Results: According to the results of the MAS test in group A, there was a statistically significant improvement in upper limb function in category 8 at...

The work was comprehensively focused on the development of adhesive forms of probiotics in the form of a biofilm on combined carriers with a prebiotic component. The second part dealed with the influence of food on the multiplication and survival of selected types of probiotic bacteria. Subsequently, the effect of individualized probiotic supplements on changes in the human intestinal microbiome was monitored. Suitable adherent probiotic strains for biofilm formation were selected and tested. Methods have been introduced and different variants of carriers for culturing and binding bacteria have been tested. In vitro experiments verified the stability of biofilm stucture and its resistance to low pH, bile and antibiotics in comparison with the planktonic cell form. The antimicrobial effect of probiotic strains in the form of a biofilm was studied. The cultivation of the multispecies biofilm on the combined carrier was optimized and the stability of the biofilm and the final viability of probiotic bacteria were confirmed. Furthermore, the influence of various foods and beverages on the viability of probiotic bacteria was evaluated with emphasis on the simulation of passage through the gastrointestinal tract. Both models, solutions with standardised concentrations of alcohol, sugar, salts, proteins or different pH and different types of real foods and beverages were tested. The effect of food and beverages was tested on monocultures of Lactobacillus acidophilus, Bifidobacterium breve and on probiotic capsules containing a mixed culture of probiotic microorganisms. The survival of probiotics in various food matrices in the simulated gastrointestinal tract was quantitatively different. We managed to define foods suitable for supporting the multiplication of probiotic bacteria. A separate part of the work was focused on the targeted modulation of the intestinal microbiome by individualized probiotics that were prepared on the basis of molecular biological analyzes of the intestinal microbiome aimed at detecting the percentage of lactobacilli, bifidobacteria and phylum Firmicutes and Bacteroidetes. Personalized probiotic supplementation confirmed the positive effect of this approach on microbiome changes, especially on the increase of the content of lactobacilli, bifidobacteria and the overall diversity of the microbiome.