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Transcriptional regulation of miR-17-92 microRNA cluster during macrophage differentiation.
Rybářová, Jana ; Stopka, Tomáš (advisor) ; Pospíšek, Martin (referee)
miR-17-92 cluster (Oncomir1) encodes seven microRNAs (miRNA, miR) regulating many biological processes including proliferation, differentiation or apoptosis. Overexpression of microRNAs encoded by miR-17-92 cluster is found in a number of tumors including acute and chronic myeloid leukemias (Dixon-McIver et al., 2008; Li et al., 2008; Venturini et al., 2007). Myeloid progenitors express miR-17-92 cluster at a high level, while macrophage differentiation associates with its downregulation. Our laboratory found, that miR-17-92 cluster is repressed by transcription factor Early growth response 2 (Egr2) upon differentiation of primary myeloid PUER progenitors, induced with transcription factor PU.1. Aim of this thesis is to further test the abovementioned data by preparing a reporter vectors set, carrying various fragments of miR-17-92 putative promoter, which enables us to study regulation of transcription of miR-17-92 cluster. This task complicated by presence of increased GC content of the miR-17-92 promoter was successfully accomplished resulting in amplification of eight fragments containing the various parts of miR-17-92 promoter including region -3.3 to 0 kb relative to the start of miR-17-5p sequence, that were inserted into pGL3 reporter vector. Transfection of pGL3 reporter vector carrying...
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Francisella Tularensis infection of macorphage-like cell line J774.2 - changes of surface and intracellular molecules
Novosad, Jakub ; Krčmová, Irena (advisor) ; Raška, Milan (referee) ; Kročová, Zuzana (referee)
Summary: Background: Francisella tularensis (F.t.) is a facultative intracellular bacteria, enrolled at the list of Centre for Disease Control (CDC) as a high risk bioterrorism agent, category A. There is a long-term effort to understand to the immunopathogenesis of F.t. infection. The aim of our study was focused on phenotype analysis (CD54, CD16/32 and CD86) and nitric oxide (NO) production analysis of murine macrophage-like cell line J774.2 durig F.t. live vaccine strain (LVS) in vitro infection. J774.2 cells were either untreated or stimulated either before or after F.t. infection by interferon gamma (IFNγ), lipopolysaccharide (LPS) separately or in its combination. Method: We followed up the expression of cell surface markers and NO production 3, 6, 9, 12 and 24 hours after initiation of infection with or without stimulation using flow cytometry and Griess method respectively. The expression was followed as either absolute value of mean fluorescence index (MFI) or as relative change of MFI (∆MFI). Murine macrophage-like cells (J774.2) were incubated in cultivation flasks (2x106 cells/10ml of medium Dulbecco's MEM with Glutamax-1 with 10% BSA). The cells were activated with 10 or 50 ng of LPS / 1ml of medium or with 100 or 1 000 I.U. of IFNγ / 1ml of medium separately or in combination (10 ng/ml and 100...
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Bordetella Adenylate Cyclase: Molecular Mechanism of Action and Its Use for Antigen Delivery
Kamanová, Jana
Dissertation title: Bordetella Adenylate Cyclase: Molecular Mechanism of Action and Its Use for Antigen Delivery Author: Jana Kamanová The first part of this PhD. thesis deals with molecular mechanism of action of the adenylate cyclase toxin (CyaA), a key virulence factor of the whooping cough agent Bordetella pertussis. CyaA belongs to the family of RTX (Repeat-in-ToXin) proteins secreted by Gram-negative bacteria and primarily targets myeloid phagocytes, expressing the CD11b/CD18 integrin receptor (also known as αMβ2, CR3 or Mac-1). Upon binding, CyaA permeabilizes cell membranes by forming small cation-selective pores, and subverts cellular signaling by delivering into host cells an adenylate cyclase (AC) enzyme that converts ATP to cAMP. Elevation of the cytosolic cAMP levels by CyaA then knocks down bactericidal functions of host innate immunity. CyaA is unique among other enzymatically active toxins in its capacity to penetrate cells directly from cell surface across the cytoplasmic membrane, without the need for endocytosis. Penetrating activity of CyaA depends on plasma membrane potential and on an intact, acylated and calcium-loaded RTX cytolysin moiety. By examining a set of 18 CyaA constructs that bear overlapping deletions within AC domain and a CD8+ OVA T-cell epitope tag, we showed that the...
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ANCA-Associated Vasculitides : Complex Diagnostic Approach
Hrušková, Zdenka ; Posová, Helena (advisor) ; Stříž, Ilja (referee) ; Žák, Aleš (referee)
Objectives: The aim of this study was to assess cellular immunity parameters in patients with ANCA (Anti-Neutrophil Cytoplasmic Autoantibodies)-associated vasculitides (AAV) at different stages of the disease, with different treatment modalities, and with respect to the long-term prognosis of the patients. Methods: We examined 69 patients with AAV, 30 healthy individuals and 20 patients with chronic kidney disease. Using flow cytometry, the following markers were assessed in peripheral blood cells: surface molecules (CD4, CD8, CD3, CD19, CD80, CD86, HLA-DR, CD28, CXCR3, CCR5, CD30 and CRTH2) and intracellular cytokines (interferon gamma (IFN), tumor necrosis factor alpha (TNF), interleukin (IL)-2 and IL-4 in CD3+ T cells and IL-10 and IL-12 in monocytes). Results: Patients with AAV had decreased total number of lymphocytes, CD4+ cells, and CD4+CD45RA+ cells compared to healthy controls (p<0.001). Active patients had increased CD30 and CRTH2 expression (p<0.05). Increased CCR5 expression persisted in remission. Increased HLA-DR expression, expansion of CD28 subpopulation and increased IFN production were noted in remission but not in active disease. Patients in remission who developed a relapse during follow-up had significantly lower IL-10 production than those without relapse (p<0.01). Conclusions: Taken...
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The role of erbB-2 oncogene in the biology of cancer stem-like cells
Prokopová, Kateřina ; Neužil, Jiří (advisor) ; Anděra, Ladislav (referee)
Recent studies indicate the existence of a subpopulation of cells within tumours with stem cell-like characteristics. These "cancer stem-like cells" (CSCs) are relatively resistant to established therapies, usually targeting differentiated and fast proliferating cells. Therefore, CSCs may be a reason for the relapse of neoplastic diseases. CSCs can be characterised by a specific gene expression profile and deregulated signalling pathways. Of these, upregulation of the erbB-2 (HER2) receptor, a hallmark of ~25-30% breast cancer patients, is related to dismal prognosis, elevated proliferation potential and resistance to chemotherapy. Recent evidence has suggested that upregulation of erbB-2 leads to increase in the pool of CSCs. In our study we used mammospheres, cells grown in the absence of serum, an in vitro model of breast CSCs, which were prepared by "weaning" breast cancer MCF7 cells to a special medium. These cells were CD44high and showed increased expression of ABCG-2, Sox-2, Vimentin as well as high levels of erbB-2. Next, we prepared a stable line of MCF7 cells with low levels of erbB-2 by shRNA. ErbB-2low cells were characterised for expression of set of CSCs markers and tested for tumour forming efficacy in nude mice using ultrasound imaging. Keywords Cancer stem-like cells, erbB-2,...
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Molecular mechanisms of apoptosis regulation by fatty acids in pancreatic β-cells
Němcová, Vlasta ; Kovář, Jan (advisor) ; Anděra, Ladislav (referee) ; Mělková, Zora (referee)
The incidence of type 2 diabetes is growing rapidly and represents a big threat for the human health care and economy system as well in the 21st century. The association of type 2 diabetes with obesity is apparent and dysfunction and apoptosis of pancreatic β-cells caused by elevated levels of fatty acids in circulation are considered as an important factor contributing to the development of this disease. However, molecular mechanisms that underlie these detrimental effects of fatty acids are only partially understood. The aim of this research project was to contribute to elucidation of mechanisms by which saturated and unsaturated fatty acids regulate viability and apoptosis induction in human pancreatic β-cells in vitro. Employing human pancreatic β-cell line NES2Y, we showed that increased levels of relevant dietary saturated fatty acids (palmitic and stearic acid) induce apoptosis of pancreatic β-cells, in contrast to relevant dietary unsaturated fatty acids (e.g. palmitoleic and oleic acid). We found that stearic acid-induced apoptosis is accompanied by significant activation of caspase-2, -6, -7, -8 and -9, but not by significant activation of caspase-3. Nevertheless, it was not associated with significant cytochrome c release, alteration in PIDD, Fas receptor and Fas ligand expression and...
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The role of GH/IGF-1 axis components in the etiopathogenesis of metabolic disturbances in type 2 diabetes mellitus and acromegaly
Toušková, Věra ; Haluzík, Martin (advisor) ; Müllerová, Dana (referee) ; Saudek, František (referee)
(EN) GH/IGF-1 axis components (GH, growth hormone receptor (GH-R), IGF-1, IGF-1 receptor (IGF-1R), IGF-binding proteins (IGFBPs)) participate in the control of glucose metabolism, inflammatory processes as well as cell proliferation and differentiation, including adipocytes and monocytes. The aim of the present study was to evaluate the role of local mRNA expression of GH/IGF-1 axis components in subcutaneous adipose tissue (SCAT) and peripheral monocytes (PM) in the development of insulin resistance and differences of adipose tissue mass in following groups of patients: obese females with and without type 2 diabetes mellitus and subjects with active untreated acromegaly. A total number of 66 subjects were included in the study: obese females without type 2 diabetes mellitus (OB), obese females with type 2 diabetes mellitus (T2DM), acromegalic patients (AC) and healthy lean control subjects (C). T2DM underwent 2 weeks of very-low- calorie diet (VLCD - energy content 2500 kJ/day). According to our results we suggest that decreased mRNA expression of IGF-1, IGF-1R, IGFBP-2 and IGFBP-3 in adipose tissue of T2DM subjects may contribute to changes of fat differentiation capacity and the increased IGF-1R mRNA expression in peripheral monocytes in these patients may play a role in the regulation of...
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Molecular- genetic analysis of the Thyroid carcinomas
Sýkorová, Vlasta ; Bendlová, Běla (advisor) ; Zamrazil, Václav (referee) ; Peterka, Miroslav (referee)
Introduction: Thyroid cancer represents more than 90% of endocrine tumors and its incidence, predominantly of papillary thyroid carcinoma (PTC), is still increasing in the Czech Republic. Several genetic changes are known, but thein impact to phenotype is still controversial. Aims: To study of the genetic causes (RET/PTC, BRAF and RAS alterations) and the role of RET polymorphisms in thyroid cancer (predominantly PTC), and to correlate genotype with phenotype. Subjects and Methods: Overall 234 PTC tissues, 8 poorly differentiated carcinomas, 3 anaplastic carcinomas, 23 medullary carcinomas, 6 follicular carcinomas and one follicular adenoma were analyzed. Samples of fresh frozen thyroid tissues, fine-needle aspiration biopsies and paraffin-embedded formalin-fixed tissue sections of patients with thyroid cancer and blood samples of healthy controls were used for analysis. The expression of RET/PTC rearrangements was detected on agarose gel. Five RET polymorphisms were analyzed using specific TaqMan probes. Detection of mutations in the BRAF gene and three RAS genes was performed by direct sequencing. Presence of alteration was correlated with clinicopathological parameters. Results: We found out that some RET polymophisms are associated with development of RET/PTC rearrangements in PTC and proved,...
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Plasma-membrane alkali-metal-cation transporters involved in salt tolerance of pathogenic Candida species
Krauke, Yannick ; Sychrová, Hana (advisor) ; Malcová, Ivana (referee) ; Heidingsfeld, Olga (referee)
of Ph.D.Thesis Conclusions All the aims of the thesis were achieved.The toleranceto alkali metalcationsof four pathogenic Candida species was studied in detail and revealed differencesamong the yeasts. These differences in sall tolerance remained the same under various growth conditions.For the first time, the internalsodium and potassiumconcentrationsof several Candidaspecieswere estimatedunder highsalt-stressgrolvth.The internalK./Na* ratiowas not in relationwith the salt tolerancerevealingdifferentadaptationmechanismsto salt stress in Candida species. A first study on combinatoryuse of fluconazoleand NaCl revealed severe synergisticeffects of both compounds, leading to grovvthinhibitionand increased internalNa* concentrationsin C. albicans.The molecularbasis of this synergismremainsto be established. Ihe C. dubliniensis, C. glabrata and C. parapsilosis Cnhl NalH- antiporters were cloned and functionallycharacterizedupon heterologousexpression in S. cerevlslae to understandthe mechanismsinvolvedin the differentsalttolerancesof Candidaspecies.The three antiportersdifferedin theiractivityfor alkalimetalcations,which roughlycorrelatedwith the observed differences in salt tolerance among the species. Additionally,during the characterizationof heteroiogouslyexpressed antiporters,two antiporter chimeras...
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Development of photic entrainment of the circadian clock of the rat during early postnatal ontogenesis
Matějů, Kristýna ; Sumová, Alena (advisor) ; Höschl, Cyril (referee) ; Langmeier, Miloš (referee) ; Nevšímalová, Soňa (referee)
In most organisms, behavioral and physiological events oscillate with period ± 24 h, i.e. exhibit circadian rhythms. In mammals, circadian rhythms are generated by circadian clock within the suprachiasmatic nuclei of the hypothalamus (SCN). Light entrains circadian rhythms to the 24 h period of solar day. Information about light is conveyed from the retina to the SCN and induces expression of clock genes Period1 (Per1) and Period2 (Per2) that represent photosensitive parts of molecular circadian clockwork within SCN. Light sensitivity of Per1 and Per2 within SCN is temporally restricted to the subjective night phase. In addition, daily profiles of clock gene expression within SCN are modulated by daylenght, i. e. the photoperiod. The aim of our study was to elucidate how the mechanism of photic entrainment of the rat circadian clock develops during prenatal and early postnatal ontogenesis. Our results demonstrate that maternal circadian system provides information about external light to the fetal and early postnatal circadian clock. Circadian clock within the SCN of rat pups is light sensitive already at the first postnatal day. Mechanism gating the light sensitivity is present at postnatal day 3 and gradually maturates until postnatal day 10. The data suggest that the developing retina is responsive to...
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