The presence of infections caused by Staphylococcus aureus (S. aureus) and its resistant form - methicillin-resistant S. aureus (MRSA) - are more frequent and treatment with increasing resistance becomes increasingly complicated. Early identification of microbiome in the infectious wounds leads to implementation of the correct treatment and thereby saving the patient from the formation of abscesses, amputation of limbs or even death. Presented thesis entitled Progressive tools for the identification of Staphylococcus aureus is focused on the study of the occurrence of these bacteria in clinical samples, design and construction of new detection methods based on magnetic separation and evaluation of the effects of antibiotics and metal nanoparticles on growth and biofilm formation, gene expression and protein composition in S. aureus and MRSA. The most abundant microorganism in microbiome of infections is S. aureus and in our studies was used as model bacteria for the design of fast, accurate and sensitive detection techniques utilizing indirect detection such as: i) detection of product of biochemical reaction between bacterial enzymes and matrix, ii) detection of the amplified gene using gold nanoparticles as an indicator probe, iii) determination of the detection oligonucleotides after sandwich capture of bacteria between micro and nanoparticles, and iv) a multiplex detection of quantum dots using sandwich capture of specific gene from selected bacteria. Subsequently, an influence effect of beta-lactam antibiotics and their complexes with selenium nanoparticles to non-resistant and resistant form of S. aureus was investigated. The significant inhibition of bacterial growth and biofilm formation as well as changes in gene expression and protein composition were achieved in MRSA by using the complexes of antibiotics and selenium nanoparticles.

This thesis focuses on the possibility of environmental protection of PIWI vine varieties against fungal diseases. The theoretical part of the thesis summarizes information about organic viticulture, vine resistance against fungal diseases, explains the importance of PIWI varieties and describes the most important fungal diseases of vine. In the experimental part, an experiment using ecological preparations against fungal diseases is described. The effectiveness of these formulations and the content of grapevine elements is evaluated here. Furthermore, qualitative parameters of grapes and aromatic substances in wine are analyzed and statistically processed.

Cytoplasmic membrane adaptation to surfactin in Bacillus subtilis 168 non-producing strain Surfactin, the most potent surface active compound and antibiotic is produced by bacteria of the genus Bacillus. Surfactin interacts with membrane bilayers, that results in destabilization and permeabilization of this structure. However mechanism of surfactin self-resistance in the producer's membrane is not understood. The aim of this study was to characterize the adaptive processes occurring at the level of cytoplasmic membrane of surfactin non-producing strain B. subtilis 168, which was exposed to exogenously added surfactin during the exponential phase of growth. The cultivation protocol of B. subtilis growth on agar media plates supplemented with surfactin was developed. Two surfactin concentrations that inhibit (400 g/ml) and even stimulate (300 g/ml) the growth of B. subtilis 168 strain were assessed. Surfactin brought about the growth arrest for 3 hours and the restored growth rate decreased in the case of inhibitory concentration, whereas the stimulatory concentration increased the growth rate and resulted in higher final density of the population. TLC was performed to analyze the polar head groups of membrane phospholipids. The portion of phosphatidylserine was found to increase at both surfactin...

Charles University Faculty of Pharmacy in Hradci Králové Departement of Pharmacology & Toxicology Student: Magdalena Moriová Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Study on the role of selected cytochrome P450 isoforms in cytostatic resistance at apoptosis level Cytostatic resistance is one of the most problematic obstacles in oncological treatment. Beside pharmacodynamic mechanisms, pharmacokinetic factors play an important role in drug resistance as well. Enzymatic transformation of active substance to inactive metabolite in tumor cells probably belongs to these mechanisms, however, evidences concerning the relevance of this phenomenon are predominantly either indirect and/or affected by interference elements. Using comparative experiments with HepG2 cell lines with/without CYP3A4 overexpression, we focused on the evaluation of the role of this clinically important enzyme in the resistance against docetaxel. Methodologically, it was the assessment of apoptosis induction (activation of caspases 3/7, 8 and 9) using commercial luminescent kits. Our results suggest significant participation of CYP3A4 enzyme on the reduction of docetaxel anticancer efficacy after 48 h from treatment, whereas this effect was not recorded in earlier intervals. These findings perfectly correlate...

This thesis is focused on polymorphism of MDR1 gene c.-6-180T>G, which causes phenobarbital resistance. Phenobarbital is one of the most commonly used drug against epilepsy, which is one of the most common neurological disease in dogs with frequency between 0,5-5 % according to breed. This mutation has so far been detected only in the border collie breed, which manifests up to 30 percent resistance to the treatment of epilepsy. In the text is subscribed border collie breed, dog epilepsy and gene MDR1. Methodical part is focused on detection of the polymorphism in model population of 82 dogs. The results agree with previous studies and confirm frequency of mutant alleles 32 % within the population.

This master thesis is focused on the role of horizontal gene transfer in disseminating tetracycline resistance among bacteria in farm soils. In the experimental part, plasmids carrying antibiotic resistance, were exogenously isolated in biparental matings with cattle manure and Escherichia coli K-12 CV601 gfp recipients.

Glycopeptides are the so-called last-resort antibiotics in clinical practice used to treat heavier, predominantly nosocomial infections caused by multi-resistant coagulase-negative staphylococci. The origin and genetic basis of resistance to glycopeptide antibiotics has not yet been elucidated within coagulase-negative staphylococci. Research on Staphylococcus aureus has shown, that intermediate resistance to glycopeptide antibiotics is associated with the presence of one or more mutations, rather than being conditioned by the support of a particular genetic element, such as in enterococci. By using various types of in vitro resistant mutant selection, we were able to obtain isogenic pairs of glycopeptide sensitive and resistant strains of Staphylococcus epidermidis and Staphylococcus haemolyticus. By sequencing the genomes of these pairs, one nucleotide polymorphisms were identified and predominantly found in metabolic and cell wall control systems. Phenotypic analysis did not reveal a direct association of glycopeptide resistance with increased biofilm formation. In clinical practice, the cross-resistance of glycopeptides and other antibiotics is problematic. For the non-glycopeptide antibiotics imipenem and rifampicin, the incidence of cross-resistance with glycopeptide antibiotics in S. aureus...

Therapeutic resistance of tumours represents an important clinical issue. We can classify the therapeutic tumour resistance in two ways. According to the clinical course, tumours can behave either as primary resistant, i.e. from the very beginning not responsive, or they can display a secondary (also called acquired) resistance, whereby an initial clinical response is lost and the tumour develops into chemo-, radio- or immunoresistant disease. An alternative classification distinguishes cell autonomous resistance mechanisms from resistance that relies on complex interactions within the context of tumour microenvironment. From the research perspective, modelling therapeutic resistance frequently involves experimental treatment of sensitive cancer cells and selection of daughter resistant cell lines. The Ph.D. thesis includes derivation of two unique models of urothelial bladder carcinoma therapeutic resistance. The first model involves newly established urothelial carcinoma cell lines BC44 and BC44DoxoR, which resulted from a prolonged doxorubicin exposure of the mother cell line. The daughter chemoresistant cell line exhibits multidrug resistant phenotype, which extends beyond the selecting drug - doxorubicin - to four additional chemotherapeutic drugs (cisplatin, methotrexate, vinblastine, and...

Hormonal therapy is a common part of breast carcinoma treatment in patients whose tumors express estrogen and progesterone receptors. The aim of hormonal therapy is to prevent proliferative effect of hormones througt their receptor proteins in order to inhibit tumor growth. However, certain number of tumors is resistant to hormonal therapy despite expression of hormonal receptors. Presently, the reasons of this resistance are not fully understood. Oxysterols are hydroxylated cholesterol derivates, which may play some role in development of the resistance. They may interfere with hormonal therapy effect and influence some signal pathways leading to cancer progression. This study comes with results of gene expression of proteins influenced by oxysterol action, metabolic and transport proteins, transcription factors and members of signaling pathways that may be related to oxysterol effect. This thesis identifies some candidate genes for future analysis on the basis of comparison of gene expression between estrogen receptor positive and negative tumors and correlation with clinopathological data. The final goal should lead to discovery of new diagnostic markers for breast cancer therapy. Powered by TCPDF (www.tcpdf.org)

Estrogen signalling pathway plays crucial role in carcinogenesis of breast cancer. Estrogen receptor (ER) is a prototypical hormone receptor that upon binding its ligand, estradiol, translocates into the nucleus and turns on target genes related to cellular proliferation and survival. Although estrogen signalling physiologically supports normal breast tissue development, deregulations of this pathway contribute to development of breast tumours that are estrogen receptor dependent. One of the main obstacles in breast cancer treatment is acquired resistance to common anticancer drugs also known as multidrug resistance (MDR). The switch between chemotherapy responsive to chemotherapy resistant cell phenotype is usually accompanied by increased expression of ABC transporters, special membrane proteins responsible for export of various kinds of commonly used anticancer drugs from the intracellular to extracellular space and is also linked to the existence of cancer stem cells (CSCs). ABC transporters can not only export chemotherapeutic drugs but may modulate tumour microenvironment through the transport of endogenous intracellular substrates such as leukotrienes (LTs), sphingolipids and prostaglandins PGs). This function may also play important role in carcinogenesis. The aim of the thesis was to...