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Retroviral infection of chicken testicular cells in a process of construction of transgenic poultry
Kalina, Jiří ; Škvor, Jiří (advisor) ; Petr, Jaroslav (referee) ; Jílek, František (referee)
Biotechnological research in chicken transgenesis is still lagging beyond the mammals mainly due to the specificities of avian reproductive system. This thesis is trying to offer functionally complex approach to the chciken transgenesis through one of the techniques. Common transfection techniques were applied on chicken blastodermal and testicular cells to affirm this approach. Our original techniqe of sterilization of chicken testes was improved and applied. Stained testicular cells of donor male were transplanted into sterilized acceptors testes and subseqent tubuli recolonization and sperm production were described. The retroviral- based vector was developed and transplanted testicular cells were successfuly infected. Carried marker transgene (Green fluorescence protein, GFP) was detected in DNA of produced sperms and no significant CpG methylation was detected when sreening infected cells. Through the flow cytometry, testicular cells used for transplantation were analyzed. All major spermatogonia-derived populations were described including the side population (SP), possible group of spermatogonial stem cells.
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Genetic and Proteomic Screening in Patients with Cardiovascular Disease.
Šímová, Jana ; Škvor, Jiří (advisor) ; Gregor, Pavel (referee) ; Čurila, Karol (referee)
The aim of this study is to analyse a genetic and proteomic aspects that could play an important role in development of chosen cardiovascular disease. Matrix metalloproteinases are enzymes that contribute strongly to the degradation of extracellular matrix components. In this study the serological levels of MMP-2 and MMP-9 were investigated using immunological testing in patients with aortic valve disease and in patients with myocardial infarction. Significantly higher levels of MMP-2 and MMP-9 were determined in both above mentioned groups of patients. Association of serum levels of MMP-2 and MMP-9 and development of concomitant aortic dilatation was not confirmed in patients with aortic valve disease. Changes in serum levels within 24 hours and after 6 months post myocardial infarction were characterized. About 10 % of patients operated for aortic valve disease suffer simultaneously from ascending aortic dilatation. The current study did not reveal any significant genetic variation in TGFBR2 gene and in chosen exons of FBN1 gene in these patients. Further genetic research is needed to identify the cause of the pathology in aortic wall. Gene expression of selected genes was measured by microarray screening in patients with myocardial infarction. These genes were related to MMPs and did not show...
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Establishment and characterization of novel IL-6-dependent myeloma cell line ÚHKT-893
Vančurová, Irena ; Stöckbauer, Petr (advisor) ; Škvor, Jiří (referee)
Multiple myeloma is an incurable fatal neoplasm of plasma cells affecting mainly elderly people. There are many research laboratories in the world where multiple myeloma is studied. Permanent cell lines are indispensable tools for both basic and applied research. However, the establishment of new cell lines is difficult with poor success. We established 96 primary cultures of bone marrow samples from myeloma patients. Only one culture succeeded in permanet myeloma cell line. The novel plasmacytic cell line ÚHKT-893 was established from bone marrow sample of 57 years old female relapsed with multiple myeloma of IgGκ isotype. The cell line is however dependent on the continuous presence of interleukin-6 in culture media. ÚHKT-893 cells grow continuously already more than 1 year. The growth of cells was regularly monitored according to growth curves and by measurement of cell viability. Surface antigenic profile was repeatedly determined by flow cytometry. The simultaneous expression of both CD138 and CD38 surface molecules confirmed the plasma cell origin of cells. The establishment of the ÚHKT-893 myeloma cell line will extend the panel of existing myeloma cell lines as a new ex vivo model for the study of the etiology and pathogenesis of multiple myeloma. It will also provide the source of new...
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Application of molecular and cellular biology methods in research of protozoa Eimeria
Vrba, Vladimír ; Škvor, Jiří (advisor) ; Čepička, Ivan (referee) ; Hampl, Vladimír (referee)
Eimeria is an apicomplexan parasite causing disease coccidiosis that is most prominent in poultry farming industry. This thesis is aimed to develop new molecular tools and resolve issues that would be a valuable contribution in the field from both research and industry perspective. Because immunity to Eimeria is strictly species- specific, it is important to know and recognize correctly all species that parasitize the host. Traditional diagnostic approaches rely on classical methods such as oocyst morphology determination under the microscope, measurement of prepatent period or in-vivo assessment of lesions caused by this parasite. However, diagnostics of individual species using these methods is very time-consuming and it is often unreliable, especially when mixture of multiple species whose parameters overlap is analyzed. Methods utilizing conventional PCR to distinguish species already exist, however, they lack advantages offered by quantitative real-time PCR (qPCR). The first aim of this thesis was to develop qPCR assays for detection and quantification of seven Eimeria species which infect chicken utilizing single-copy non-polymorphic targets in order to ensure maximal specifity and coverage of all strains of each species. Usefulness of this method was demonstrated by analysis of field...
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Immunologic and epigenic modulation of gene expression of human leukemia cell lines
Elknerová, Klára ; Stöckbauer, Petr (advisor) ; Špíšek, Radek (referee) ; Škvor, Jiří (referee)
Therapy by monoclonal antibody to CD34 molecule Growth-inhibitory and proapoptotic effect of the monoclonal antibody to CD34 molecule, clone 4H11, were tested on CD34+ leukemic cell lines (MOLM-9, JURL-MK1) and CD34- cell line (PS-1). We have found that the monoclonal antibody to CD34 inhibited the proliferation and induced apoptosis of all CD34+ cell lines tested, however it has similar effect on CD34- leukemic cell line (PS-1) in concentration higher than 32 µg/ml. We did not observe induction of differentiation by anti-CD34 antibody, but a growth arrest of cells in the G0/G1 phase of the cell cycle was detected in CD34+ cell lines. Combinations of anti-CD34 antibody with both type I (α, β) or type II ( ) interferons did not enhance the effects on the cell growth or inhibition of cellular proliferation of the antibody alone. Combinations of anti-CD34 antibody with hematopoietic cytokines or INF-γ did not induced diferentiation. Our data suggest that the monoclonal antibody to CD34 molecule prepared from clone 4H11, after sufficient experimental and preclinical testing on laboratory animals, may provide a new basis for possible targeted antibody therapy of acute or chronic myeloid leukemia. Therapy by histone deacetylase inhibitors Histone deacetylase inhibitors (HDACi) are emerging new class of...
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