Acute lymphoblastic leukemia (ALL) is the most common malignant dise- ase in children. Despite great advancements in treatment of this disease, around 15-20 % of patients suffer a relapse. One of the possible reasons for relapse is developed resistance to cytostatic drugs. L-asparaginase is an im- portant chemotherapy component for childhood ALL and resistance to this drug often complicates treatment. To date, causes of developing resistance have not been sufficiently described. This thesis is a part of a greater research project focusing on mechanisms of L-asparaginase's activity and reasons for developing resistance to this chemotherapeutic agent. Differential metabolic requirements of cancerous cells have been described as early as 1924 by O. H. Warburg and they have been subject to scientific inquiry since. This study aimed to describe the relationship between basal metabolic determinants of leukemia cells and their sensitivity to L-asparaginase. For this reason, two metabolic pathways, glycolysis and oxidative phosphorylati- on, were studied in detail using a Seahorse Bioanalyzer. Further, expression of specific genes involved in glycolysis was detected. Content of mitochon- drial reticulum in cells, expression of the asparagine synthetase gene, and cell size were also studied. Experiments were...

Gag polyprotein is the precursor of HIV-1 structural proteins, required for correct assembly, budding and maturation of viral particle within HIV-1 life cycle. The process of maturation into an infectious virion is dependent on Gag and GagPol cleavage at nine predefined sites by HIV-1 proteinase. Its disruption is one of the main targets of HIV treatment. HIV-1, however, develops resistance to the proteinase inhibitors by creating mutations in both the proteinase and the substrate. The Gag processing by HIV-1 proteinase is a highly sequential process, that happens in specific order and rate. Previous biochemical studies determined the kinetic data of these processes using oligopeptides representing naturally occuring cleavage sites. This thesis describes the cleavage of the Gag polyprotein itself, which is the natural substrate of HIV-1 proteinase. For this purpose, the full-length Gag polyprotein was recombinantly prepared in bacterial expression system. The cleavage was carried out and its products were analyzed via SDS-PAGE and Western blotting. The substrate specificity of the wild-type and mutant HIV-1 proteinase with respect to the full-length wild-type Gag polyprotein was compared. Substantial differences were observed between the rates of individual steps of cleavage by the wild-type and mutant...

Charles University in Prague, Faculty of Science Department of Genetics and Microbiology Ph.D. study program: Molecular and Cellular Biology, Genetics and Virology Genetic basis of multidrug resistance in Acinetobacter baumannii Lenka Křížová Supervisor: Doc. RNDr. Alexandr Nemec, Ph.D. Supervisor-consultant: RNDr. Lubomír Janda, Ph.D. Prague 2014 SUMMARY Acinetobacter baumannii has emerged as a significant bacterial pathogen pre-eminently associated with hospital-acquired infections. Strains of this species may currently exhibit resistance to nearly all or even all clinically relevant drugs. The vast majority of epidemic and multidrug-resistant A. baumannii strains belong to a few globally spread lineages, in particular to the so-called European (EU) clones I, II, and III. Complex resistance patterns displayed by these strains result from their marked capacity to develop, acquire, and combine secondary resistance mechanisms against originally effective agents. The aim of this thesis was to broaden our knowledge on the genetic basis and epidemiology of multidrug resistance in A. baumannii. The obtained results have been published in the form of six studies which are part of this thesis. In the first study, we analysed the epidemiology of carbapenem resistance among hospital strains of Acinetobacter in the...

Helicobacter pylori (hereinafter referred to as H. pylori) is a gram-negative bacteria which colonises the human stomach mucosa. Its role in the aethiopathogenesis of chronic gastritis, ulcer disorders of the gastroduodenum and MALT-lymphoma has been clearly demonstrated, and in connection with the occurrence of stomach cancer it has been indicated by the World Health Organisation (WHO) as a class I carcinogen. H. pylori infection can be detected from samples of stomach mucosa taken in an endoscopic examination (rapid urease test, microscopic examination, culture), or the non-invasive method can be used (13 C-Urea Breath Test or H. Pylori stool antigen test - HpSA). Effective therapy of H. pylori infection resides in the administration of a combination of antibiotics and a proton pump inhibitor. In recent years the resistance of bacterial strains to used antibiotics has been increasing on a worldwide scale, and we can also observe this trend in the case of H. pylori. If the level of resistance exceeds 20 % for clarithromycin and 40 % for metronidazole, these antibiotics are not recommended for the treatment of an infection caused by this bacteria. In a group of 61 patients at the Department of Internal Medicine at the University Hospital Motol who had undergone an endoscopic examination of the...

My thesis is devoted to the resistance of pollen beetle (Meligethes aeneus, son. Brassicogethes aeneus Fabricius, 1775) to pesticides in selected localities in southern Moravia. Collecting adults was performed in the spring of 2013 by tapped into polyethylene bags. Adults are collected from the stands of oilseed rape, mustard and poppy. Tests were performed on the basis of the methodology of Insecticide Resistance Action Committee number 011, 021, 025. In total, we tested the pollen population from 18 locations. For testing, we used the following chemical substances: Biscaya 240 OD, cypermethrin, lambda-cyhalothrin, tau-fluvalinate, etofenprox, chlorpyrifos(ethyl). We evaluated 100% of the registered dose of pesticides after 24 hours. From the results of the individual populations, we found large differences in resistance to chemicals.

The aim of this thesis was to determine presence of resistant populations pollen beetle (Brassicogethes aeneus). Determination their resistance level and proposing an anti-resistant strategy was solved too. The adults of pollen beetle were collected in year 2012 and 2013 from 16 locations in South Moravia. Collections were done on rape, but also on poppy and mustard. Pyrethroids - cypermethrin, lambda-cyhalotrin, tau-fluvalinate; neonicotinoids -- commercially used preparation Biscaya 240 OD and organophosphorous insecticide active substance chlorpyrifos ethyl were tested. The tests were conducted according to the methodology of Insecticide Resistance Action Committee (IRAC) number 011, 021, 025. Each active substance was tested in three repetitions. The adults were exposed to 100 % registered dose after 24 hour. Substance efficiency in 2012 and 2013 was different. There was an efficiency drop in Biscaya 240 OD preparation. On the contrary, lambda-cyhalotrine substance was more effective in 2013.

In 2011 was conducted experimental plantings on land ZF Mendelu in the Lednice for determining the relative resistance of selected varieties of apricots, and to determine the potential substances to enhance resistance to P. syringae pv. syringae. The varieties were inoculated with strains FN3, Lmg1247 and CCM4073. In 2013 and 2014 were observed selected symptoms on plants, which was determined the level of relative resistance of each variety. As resistant varieties were determined 'Kuresia', 'Orangered', 'Bergeval' and 'Bergarouge'. As moderately susceptible varieties were determined 'Tardicot', 'Leskora', 'Aurora', 'Harogem', 'Pinkcot' and 'Sylvercot' on rootstock 'Waxva'. As susceptible varieties were determined 'Goldrich', 'Kioto', 'Bergeron' and 'Sylvercot' on rootstock 'Rubira'.

The present study focused on clarification of the roles of environmental osmolality and ion composition in the initiation of spermatozoa motility in different freshwater fish species. Additionally, the role of osmotic and ionic agents in sturgeon spermatozoa maturation was investigated. A first aim of the present study focused on studies of osmotic regulation of spermatozoa volume alterations in different fish species. This led to the discovery that spermatozoa swelling are species-specific: fish sperm with ionic mode of motility activation (sturgeon, trout) has no capacity for swelling, In contrast to carp sperm (osmotic mode of activation) for which volume correlates with environmental osmolality. A second aim was to apply the capacity for swelling in carp spermatozoa to improve the cryopreservation procedure relative to post-thaw sperm quality. Post-thaw motility parameters and ability to fertilize eggs were measured and compared with non-treated sperm. As a result, higher post-thaw motility rate, spermatozoa velocity, and fertilization rate were observed in sperm pretreated with hypotonic solutions compared to non-treated sperm. Thus, our results demonstrate for the first time, that spermatozoa ability for hypotonic changes could be used for improvement of cryopreservation procedure. A third aim of this study was the investigation of the role of environmental osmolality and ion composition in fish sperm motility. Sturgeons and salmonids sperm was activated in media of differing ionic and osmotic composition. Even though environmental Ca2+ concentration is not crucial in conditions of Na+ presence, a minimal intracellular free Ca2+ concentration should be present in sturgeon spermatozoa for initiation of motility. Alteration of osmolality does not seem to play a major regulatory role in trout spermatozoa activation. Alteration of sperm sensitivity to Ca2+ was detected during spawning, according to seasonality. Low motility rate was detected in brook trout spermatozoa at the end of the spawning period. However, a Ca2+ concentration increase up to 10 mM in activating media led to activate motility in at least 85% of the spermatozoa, which suggests a decrease of sperm sensitivity to Ca2+ at the end of the spawning season. The fourth aim was the study of the role of osmotic and ionic agents in sterlet spermatozoa maturation. Our results demonstrate that sturgeon testicular spermatozoa lack capability for motility activation and fertilization. During spawning, sperm is released from testis, pass through the kidney where it is mixed with urine, into the Wolffian duct. We demonstrated for the first time, that testicular sperm incubation with urine controls in vivo maturation, but can be performed in vitro as well (Chapter 6). We detected that sperm maturation does not occur under conditions of Ca2+ deficiency, suggesting that Ca2+ plays an essential role in this process (Chapter 7). During this maturation process, we observed a gradual decrease of spermatozoa sensitivity to environmental Ca2+. We found that testicular spermatozoa motility could be activated only when the Ca2+ concentration is high. Thus, we hypothesize that maturation process might be controlled by Ca2+ influx into the cell with subsequent loading of some Ca2+ stores. To our knowledge, these results provide, for the first time, evidence for the presence of Ca2+ stores in sturgeon spermatozoa. As a conclusion, the results of this study shed light on osmotic and ionic regulation of sperm motility in different fish species. The involvement of specific ions together with osmotic shock in fish spermatozoa maturation and aging were clarified. These findings broaden the possibilities of in vitro sperm manipulation in fisheries practice such as the use of sturgeon testicular spermatozoa, improvement of poor quality sperm in salmonids at the end of spawning season, and increase of carp sperm cryoresistance.

This thesis deals with viral pathogens of pea and its seed transmission. It describes characteristic of pea seed-borne mosaic virus (PSbMV) and Pea enation mosaic virus (PEMV), mechanism of seed transmission, methods for detection viruses in seeds and pea resistence to both of viruses. In the experimental part was tested different stages of development of three breeding on new varietes (SM 247, 1019/12 and 1011/12) for presence PSbMV by DAS-ELISA. The aim of the experiment was to determine the developmental stage of pea relevant to determine the transmission PSbMV on pea plants. After the evaluation, it was concluded that this developmental stage is the embryo or one week plant. Furthermore, the selected lines of pea were tested for resistance to PEMV. On the basis of this experiment were obtained pea populations resistant to this virus.

This thesis is focused on polymorphism of MDR1 gene and related drug resistance. Resistance is caused by deletion of four nucleotids, that resulting in a frame shift and synthesis of nonfunctional transport of P-glycoprotein. The text describes a polymorphism of MDR1 (ABCB1) gene, which results in reduced resistance to drugs belonging to the group of macrocyclic lactones. It also describes inheritance of this phenomenon and it deals with the detection of mutation using PCR (polymerase chain reaction) and by fragmentation analyses. A review of literature study is a form of research solely from scientific publications. 128 dogs were included into the own analysis. The results confirmed that Collies had the highest presence of deletions (29,73 %) with a high number of carriers in the study population of dogs (54,05 %). The percentage of affected individuals in the breed of Australian Shepherd and Sheltie was significantly lower (7,32 % and 6 %), but the percentage of carriers were also high in both Australian Shepherds (34,14 %) and the breed Sheltie (48 %).