Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Martina Sýkorová Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Study on impact of selected tyrosine kinase inhibitors on multidrug resistance mediated by ABC drug efflux transporters Tyrosine kinases are an important class of enzymes controlling cell proliferation, carcinogenesis, apoptosis and cell differentiation. Deregulation of these enzymes can transform normal cell into a cancerous one. Blocking their function by tyrosine kinase inhibitors (TKi) is considered a promising treatment for various types of cancer. ATP-binding cassette (ABC) transporters form a family of transmembrane proteins that can transport a wide variety of substrates across biological membranes via ATP-dependent drug efflux pumps. They modulate drug pharmacokinetics, but on the other hand, lead to therapy failure due to overexpression in cancer cells. In our previous study, we evaluated inhibition properties of two selected TKi (alectinib, brivanib) in MDCKII cell lines (parent one and those transduced with human ABCB1, ABCC1 and ABCG2). Alectinib significantly inhibited ABCB1, ABCG2 but not ABCC1 transporter. Brivanib showed triple inhibition of all studied transporters. In the present work, we...

Application of cytotoxic chemotherapy still remains the essential treatment strategy in advanced colorectal cancer. The intrinsic and acquired drug resistance represents one of the reasons that may even lead to failure of cancer therapy. The DNA damage response pathways have been shown to play an important role in the development of chemoresistance. There is sufficient evidence showing the high-frequency deregulated expression of many DNA repair genes across multiple cancer types. An example of such gene in colorectal cancer is MRE11, which encodes protein known as a sensor of DNA double-strand breaks. In year 2016, there was a substantial study published by our group at The Department of Molecular Biology of Cancer (IEM CAS, Prague), the study analysed the association of polymorphisms in predicted microRNA target sites of double-strand breaks (DSBs) repair genes, including MRE11, and clinical outcome and efficacy of chemotherapy in colorectal cancer. Our hypothesis, based on the mentioned study, is that specifically and exactly defined microRNAs with ability to regulate certain DNA repair proteins may not only affect the survival of colorectal cancer cells, but also the sensitivity to chemotherapy. In practical part of the submitted thesis we have identified miR-140 as a potential regulator of...

This diploma thesis focuses on the topic: "Influence of Exercise Intervention on Selected Physiological Parameters of Patients with DM Type 2." In the theoretical part, the main concepts are characterized - physical activity and inactivity, prevention, health, and civilisation diseases. From the civilization diseases, the issue of diabetes mellitus type 2 is substantially developed. In the practical part, the effect of physical activity, particularly walking 3 times a week for 60 min with low to medium loads, on selected anthropometric, biochemical, and physiological parameters is described in detail. The thesis compares an experimental group of 10 women and 10 men in the age of 45-60 years with a control group of 10 women and 10 men of the same age. The experiment was designed to be performed for three months from 09/2015 to 11/2015 and at the end the initial and final data on weight, waist circumference, blood pressure, glycaemia on an empty stomach, postprandial glycaemia, and glycated haemoglobin were compared.

The main aim of this thesis is to investigate the role of Impl2 gene in the regulation of immune response to bacterial infection in Drosophila melanogaster. The theoretical part provides a conceptual background concerning immunity in drosophila and also it summarizes what is known about the Impl2 gene. In the practical part, the function of Impl2 gene during infection is examined, starting with the location of its production during immune response, the changes of Impl2 gene amount during infection and its essentiality for a proper immune response. Furthermore, it examines the metabolic changes underlying an immune activation in connection to Impl2 gene. Last but not least, it tests how Impl2 gene is connected with the mobilization of energy during Chill Coma Recovery assay.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Simona Suchá Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Study of ABC drug efflux transporter inhibition by selected tyrosine kinase inhibitors using accumulation methods with cytostatic substrates ATP-binding cassette (ABC) drug efflux transporters are transmembrane proteins that utilize the energy from ATP hydrolysis to drive transport of endogenous and exogenous compounds out of the cell. The overexpression of ABC transporters plays a crucial role in the development of multidrug resistance (MDR), a phenomenon responsible for the failure of chemotherapy. Tyrosine kinase inhibitors (TKI) represent novel beneficial therapeutic approach in cancer treatment. TKI block tyrosine kinases which regulate important cellular processes. Deregulation of these enzymes can lead to various types of cancers. In the present work, we investigated interaction potential of selected TKI (alectinib, brivanib, osimertinib, selumetinib) in MDCKII parent cell line and those transduced with human efflux transporters ABCB1, ABCC1 and ABCG2. Using the accumulation studies, we determined the amount of accumulated model substrates (daunorubicin, mitoxantrone) and evaluated the inhibitory effect of...

The diploma thesis is devoted to issue of excessive use of antibiotics and ist consequences, the emphasis here is put especially on resistance. Due to the treatment were developed resistance stones, that did not exist earlier. Germs gained new behaviour as time went on and its sensitivity was changed to the given antibiotics. Established goals of this work were to analyse and find out which antibiotics are the most prescribed by practical doctors and what kind of antibiotics are the most used by inhabitants. The next target was to find out if people are aware of consequences of excessive use of antibiotics. The last aim was to capture the development of antibiotics consumption by inhabitants in the Czech Republic. Results show that antibiotics of the penicillin series are among the most commonly prescribed and used antibiotics, 73% of respondents have knowledge of the side effects and the consumption of antibiotics has an increasing tendency.

Proteomics as a modern comprehensive approach to the analysis of proteomes was applied in three projects aimed at diagnosis and therapy of cancer. The aim of the first the project was to find a new diagnostic biomarker for ovarian cancer. Two different comparative proteomic approaches were used for comparative analysis of sera from patients diagnosed with ovarian cancer and from healthy age-matched women. We identified -1-antitrypsin with increased concentration in patien sera, and apolipoprotein A4 and retinol-binding protein 4 (RBP4) with significantly decreased concentration in patients. The significantly decerased concentration of RBP4 in patients is a new observation. We propose that RBP4 is either decreased in ovarian cancer patients as a result of its reduced production by ovary or it may reflect less specific systemic changes, for instance early onset of cancer cachexia. The second project was focused on gaining insight into the molecular mechanism of cytarabine resistance in mantle cell lymphoma (MCL). Proteomic and transcriptomic analyses of cytarabine-resistant cells revealed marked downregulation of deoxycytidine kinase (DCK) - a protein essential to intracellular activation of purine and pyrimidine nucleosides and their analogues including cytarabine. The cytarabine-resistant MCL...

In the works presented here, we studied molecular changes associated with drug resistance in human mantle cell lymphoma (MCL) cells using proteomics. Our analyses allowed us to identify causal and/or secondary changes in protein expression associated with the development of resistance to the experimental drug TRAIL and the clinically used antimetabolites cytarabine and fludarabine. Resistance of MCL cells to the recombinant proapoptotic cytokine TRAIL was associated with downregulation of key enzymes of purine metabolism. This pathway potentially represents a molecular "weakness", which could be used as a therapeutic target for selective elimination of such resistant cells. Resistance to the pyrimidine analog drug cytarabine was associated with cross-resistance to other antinucleosides. Proteomic and transcriptomic analyses showed pronounced downregulation of deoxycytidine kinase (dCK), which activates both purine and pyrimidine antinucleosides. This change explains the cross-resistance and is the causal mechanism of resistance to cytarabine. Our observations suggest that MCL patients, who do not respond to cytarabine-based therapy, should be treated with non-nucleoside drugs. MCL cells resistant to purine-derived antinucleoside fludarabine were cross-resistant to all tested antinucleosides and...

Multiple myeloma and its aggressive variant, plasma cell leukemia, are still considered to be incurable diseases despite the progressive treatment approaches comprising novel drugs. This can be attributed to the presence of the bone marrow microenvironment which plays an important role in drug resistance of myeloma cells. Hematopoietic cell lines derived from hematologic malignancies are suitable models for the study of etiopathogenesis of these malignant diseases and for testing new potential drugs. Establishment of these cell lines is still considered to be coincidental and rare event. The first part of the thesis is focused on establishment and characterization of the cell line UHKT-944 derived from a patient with primary plasma cell leukemia, and on completion of characterization of the cell line UHKT-893 derived from a patient with multiple myeloma. Additional analysis of UHKT-893 cell line were performed including sequence analysis of IgVH gene rearrangements and cytogenetic analysis which contributed to more detailed characterization of this cell line. During cultivation of UHKT-944 cells, we monitored the cell growth and confirmed dependence on interleukin-6 (IL-6). Immunophenotype analysis revealed the presence of surface markers characteristic of malignant plasma cells. UHKT-944 cells...

Anther smuts (Microbotryum violaceum s.l.) represent a well-known system for studying pathogens of wild plants and coevolution between pathogens and hosts. Infected plants produce sterile flowers with anthers filled with fungal spores which are transmitted to other host plants by pollinators. Data from existing research come mainly from the genus Silene. However, the applicability of these findings to other host plants of anther smuts is largely unknown. Therefore, this thesis focuses on a different host species - Dianthus carthusianorum. First, the pattern of disease was surveyed in natural populations of D. carthusianorum in a small area in Střední Povltaví. Most populations were infected to various degrees and only a few populations remained completely healthy. The prevalence of disease was positively correlated with size and density of host populations. On the other hand, environmental factors and the degree of connectedness had very little effect on disease prevalence. Second, additional data were collected from a subset of populations to determine plant resistance and densities of pollinators. Differences in resistance among populations (as inferred from flower inoculations) were not significant. Populations differed in densities of pollinators, but there was no correlation between densities...