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Evaluation of gene expression of selected ABC and SLC transporters in the HTR-8/SVneo cell line during stimulation with pro-inflammatory cytokines
Pokorná, Petra ; Čečková, Martina (advisor) ; Mladěnka, Přemysl (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Petra Pokorná Supervisor: Assoc. Prof. PharmDr. Martina Čečková, Ph.D. Consultant: Mgr. Simona Dudičová Title of diploma thesis: Evaluation of gene expression of selected ABC and OATP transporters in the HTR-8/SVneo cell line during stimulation with pro-inflammatory cytokines Placenta is the first and the largest fetal organ that gradually develops during pregnancy and plays an essential role in the development of the fetus. It fulfills the entire spectrum of functions, ensures the transport of nutrients to the fetus and the removal of waste substances back into the maternal circulation, protects the fetus from toxins, and at the same time fulfills a certain mechanical and especially immunological barrier between mother and fetus. One of the main functions of the placenta is the transport function which is made possible by membrane transporters present mainly in the syncytiotrophoblast layer of the placenta. Transporters in the human placenta can be divided into two families, SLC and ABC which are further divided into several subfamilies. The expression of transporters changes physiologically during pregnancy, but pathological conditions such as inflammation can also influence the expression....
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Regeneration initiation of Xenopus laevis tail-functional study
Netušil, Jiří ; Šindelka, Radek (advisor) ; Petr, Jaroslav (referee)
The recent introduction of high-throughput sequencing techniques rapidly changes our perception of vertebrate regeneration. One of the model organisms intensely studied for its regenerative potential is the embryonic tadpole stage of African clawed frog (Xenopus laevis). This thesis aims to review and functionally validate some of the latest findings gained by RNA sequencing of regenerating Xenopus laevis tail. We specifically focus on gene expression changes during the early phases of regeneration and how their absence affects the progression and phenotypic outcome of this process. Our analysis confirms that Regeneration initiating cells (RICs) identified by single-cell and spatial RNA sequencing represent a vital element in successful tail regeneration. The RIC marker genes mmp9 and pmepa1 affect the processes linked to extracellular matrix remodelling and the migration pattern of previously described Regeneration organizing cells (ROCs) while not interfering with the myeloid cell lineage. We further demonstrate a novel use of Vivo Morpholino oligonucleotides in a transient knockdown assay and offer an assessment of its benefits and limitations. This thesis highlights the importance of spatio-temporal regulation happening on both cellular and molecular levels during the initial 24 hours of...
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A tool for prediction of small RNA in RNA-Seq data
Pomykalová, Barbora ; Čejková, Darina (referee) ; Jurečková, Kateřina (advisor)
This diploma thesis focuses on the detection of small RNA (sRNA) in the bacterial genome. sRNAs are short non-coding transcripts that play a key role in gene expression. To date, there are several algorithms focusing on the detection of sRNAs from RNA-Sequencing (RNA-Seq) data that can be obtained by some of the sequencing platforms. The most frequently used platforms are Illumina and Ion Torrent belonging to the next generation sequencing and PacBio with Oxford Nanopore belonging to the third generation of sequencing. In this work, the workflow of sRNA detection using freely available tools was described and then an own unique tool for sRNA detection – the SEARCHsRNA tool – was designed. Two open-source software tools – Rockhopper and DETR'PROK, together with newly created tool, were tested on RNA-Seq data for bacteria Vibrio atlanticus LGP32.
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Study of the expression and toxicity of catechol derivates in breast cancer cell lines
Vyoralová, Tereza ; Carazo Fernández, Alejandro (advisor) ; Jirkovský, Eduard (referee)
Studium exprese a toxicity derivátů katecholu v prsních nádorových buněčných liniích, Tereza Vyoralová ABSTRACT Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Tereza Vyoralová Supervisor: PharmDr. Alejandro Carazo Ph.D. Title of the diploma thesis: Study of the expression and toxicity of catechol derivates in breast cancer cell lines Estrogens have several important functions in the human organism and are also involved in the formation and development of breast cancer. These effects are mediated by binding to estrogen receptors (ER) and show complex spectrum of effects such as regulation of reproduction, menstrual cycle, brain function, bone density or mobilization of cholesterol. In addition the presence and quantification of ER in breast cancer is currently used as one of the most important predictive biomarkers in patients suffering from this disease. The occurence and activity of ER correlates with the response of breast cancer to hormonal treatment and with the patient's prognosis. Despite intensive research, breast cancer is still ranked among the cancers with the highest lethality. Catechols are organic compounds that are naturally present in food as pollutants, but also can be found in the human body, where they play an important role as...
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Low-temperature injury in insect tissues and mechanisms of its repair
ŠTĚTINA, Tomáš
While physiological adjustments accompanying insect cold acclimation prior to cold stress have been relatively well explored, processes of recovery after cold stress received much less attention. Using a broad array of physiological, molecular biological and microscopic techniques, I aimed to capture the complexity of processes occurring after the exposure to cold stress in the larvae of two drosophilid species - freeze tolerant Chymomyza costata and freeze sensitive Drosophila melanogaster. Several lines of evidence pointed toward impaired mitochondrial functions in lethally frozen larvae. The freeze-tolerant phenotypes of C. costata retain integrity of mitochondria even after deep freezing.
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The role of Nuclear Phosphatidylinositol 4,5 bisphosphate in RNA Polymerase II Transcription
Balaban, Can ; Hozák, Pavel (advisor) ; Šebesta, Marek (referee) ; Castano, Enrique (referee)
(in English) The phosphatidylinositols are a subclass of glycerophospholipids with their inositol head group linked to the diacylglycerol backbone. The differential phosphorylation of the inositol head group yields seven different phosphoinositide phosphates (PIPs) which can be mono-, bis,- or tris-phosphorylated. The roles of the cytoplasmic PIPs have been extensively studied in vesicular trafficking, ion channels, generating second messengers and, membrane and cytoskeletal dynamics. While their cytoplasmic functions are very well described, the molecular mechanism of their nuclear functions are still poorly understood. From the nuclear PIPs, the Phosphatidylinositol 4,5-bisphosphate (PIP2) is the most abundant phosphoinositide in the cell nucleus and it participates to the nuclear architecture by regulating processes such as chromatin remodeling, DNA-damage response and gene expression. In the cell nucleus, it localizes mostly to nuclear speckles where it interacts with the splicing machinery. In nucleolus, PIP2 is involved in the RNA Polymerase I machinery to regulate rDNA transcription. Recently, we have defined a nucleoplasmic pool of PIP2 which is observed in 40 to 100nm foci. The nascent transcripts of RNA Polymerase II (RNAPII) were visualized at their periphery and RNA was shown to be...
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CRISPR/Cas9 editing of leukemic B-cells: searching for microRNA-155 targets involved in the process of leukemogenesis
Sypecká, Markéta ; Savvulidi Vargová, Karina (advisor) ; Zadražil, Zdeněk (referee)
CRISPR/Cas9 editing of leukemic B-cells: searching for microRNA-155 targets involved in the process of leukemogenesis Introduction: Chronic lymphocytic leukemia (chronic lymphoid leukemia, CLL) is a monoclonal disorder characterized by a progressive accumulation of functionally incompetent B-lymphocytes. CLL is the most common form of leukemia found in adults in Western countries. Course of the disease can differ: some patients die rapidly, within 2-3 years of diagnosis, mainly due to complications from CLL, but most patients live 5-10 years. However, with disease progression significantly increases level of miR-155, which is known as oncomiR. MicroRNAs (miRNAs) represent negative regulators of gene expression. MiR-155 affects genes, which are involved in leukemogenesis and cell cycle. And it is known, that miR-155 suppresses its targets (similarly as other miRNAs). We hypothesized that by gene editing of CLL cells we unblock miR-155 targets and find out correlation between these targets (known and unknown) with CLL leukemogenesis. Methods: We used CRISPR/Cas9 method for gene editing, which enables the deletion of mature miR-155 sequence in the genome of leukemic B-cells. CRISPR/Cas9 plasmid was transferred to the leukemic B-cell cell line HG-3 via nucleofection. Clones with successful transfer of...
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Identification of expression profile changes of resistance-associated genes in experimental in vivo model of ovarian carcinoma
Bryndová, Barbora ; Václavíková, Radka (advisor) ; Dračínská, Helena (referee)
Ovarian carcinoma is one of the most serious gynecological malignancies in the world. High mortality of this cancer is mainly due to the development of multiple drug resistance to conventional treatments involving taxanes. Therefore, current research is focused on the study of new taxane-based derivatives capable to overcome known mechanisms of drug resistance. At present, there are several promising Stony Brook taxane derivatives (SB-T-taxanes), investigated in frame of efficacy, toxicity and mechanism of action. The aim of this bachelor thesis was to study changes in the expression of genes involved in the development of resistance in tissues and tumor obtained from in vivo mo- dels after the administration of derivatives SB-T-121605 compared to conventional taxane paclitaxel and non-treated models. Expression of selected ABC transporter genes (ABCB1, ABCC1 and ABCG2), taxane metabolizing enzyme genes (CYP3A11, CYP3A13 and CYP2C29) and their mouse orthologs was estimated in liver, intestinal and tumor tissue samples taken from in vivo mouse xenograft models. Tumor tissue was derivated from the highly resistant NCI/ADR-RES ovarian cancer cell line. Gene expression was determined by real-time quantitative polymerase chain reaction. Administration of the SB-T-121605 derivative led to several...
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Characterization of pancreatic beta cells after their in vitro proliferation induced by synthetic modified mRNA
Veľasová, Adriana ; Koblas, Tomáš (advisor) ; Černá, Věra (referee)
The origin and development of type I. and II. diabetes mellitus is directly related to homeostasis of proliferation and apoptosis of pancreatic β-cells. Any imbalance that leads to a decrease in the number of β-cells consequently increases the pro- bability of developing this disease. Patients suffering from diabetes mellitus are de- pendent on partial or complete exogenous insulin replacement, as their pancreas is unable to meet the body's insulin needs. Therefore a need for restoration of normal β-cell mass in diabetic patients leads to the attempts to develop new therapeutic approaches that could expand remaining β-cells of the organism and restore phys- iological insulin production. A major obstacle in this regard is a low sensitivity of terminally differentiated β-cells to mitogenic stimuli that could induce the entry of β-cells into the cell cycle. Activation of β-cell proliferation is associated with the G0/G1/S cell cycle transi- tion, which is under the control of retinoblastoma protein (RB). In order to activate cell cycle entry RB must be phosphorylated. RB phosphorylation is provided by specific cell cycle regulators, particularly cyclin-dependent kinases 4 and 6, which associate with family D cyclins. In accordance with the aim of this Diploma thesis, the effect of these cell cycle...
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Interaction of the human pathogen Bordetella pertussis with blood serum
Štipl, Daniel ; Večerek, Branislav (advisor) ; Kamanová, Jana (referee)
Bordetella pertussis is a Gram-negative strictly human pathogen and the major causative agent of whooping cough or pertussis. The incidence of this highly contagious respiratory disease in developed countries has increased in the last decades. One of the less characterized virulence factors of B. pertussis is the type three secretion system (TTSS) which is responsible for the secretion of the effector proteins into host eukaryotic cells. This diploma thesis sheds light onto factors influencing TTSS in vitro activity. Although TTSS of laboratory strain Tohama I was induced by biologically active compounds present in blood (e. g. complement proteins), TTSS of recent clinical isolate B1917 seems to be induced permanently. Furthermore, BB0302 encoding a GntR family transcription regulator in B. bronchiseptica RB50 (homologous to BP0209 of Tohama I) was studied, however, the deletion of this gene did not affect the TTSS functionality. Serum resistance is a factor that plays a key role in the pathogenesis of B pertussis. We show that Czech recent isolates (2008-2015) are significantly more resistant to serum killing in vitro than the original vaccine strains (1954-1965). This phenomenon seems to result from the adaptation of global B. pertussis population to its human host. In addition, this diploma...
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