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Monogenic susceptibility to infectious pathogens
Bloomfield, Markéta ; Šedivá, Anna (advisor) ; Koziar Vašáková, Martina (referee) ; Litzman, Jiří (referee)
(ENG) The modern approach to studies of monogenic inborn errors of immunity, driven by unprecedented advances of genetic tools, opens vast undiscovered areas of immune system components and functions. In particular, the diseases with striking clinical phenotypes with normal or near normal baseline immunophenotype, such as disorders of innate and intrinsic immunity with susceptibility to single pathogen, provide a unique window into the host-pathogen interactions. This thesis covers various novel aspects of immunopathology, genetics and clinical facets behind some such diseases, namely chronic mucocutaneous candidiasis due to hypermorphic (gain-of-function, GOF) STAT1 mutations, which hamper Th17-associated immune activities, and Mendelian susceptibility to mycobacterial diseases (MSMD) due to impairment of IL-12, IL-23/IFNγ signalling pathway. Moreover, it contributes to the mounting evidence that IL- 6 signalling is non-redundant in anti-staphylococcal immunity. Finally, it explores the novel Paediatric inflammatory multisystem syndrome temporally associated with SARS-CoV-2 (PIMS- TS) as a single pathogen-driven life-threatening immunopathology, which most likely develops due to individual, yet unknown, genetic predisposition. The findings presented in this thesis were in several cases translated...
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Regulation of transcription in mycobacteria.
Páleníková, Petra ; Krásný, Libor (advisor) ; Mrvová, Silvia (referee)
The bacterial cell has to be able to cope with environmental changes. Adaptation to these changes is achieved by changes in gene expression. Gene expression is regulated mostly at the level or transcription initiation. Transcription initiation depends on the sequence of promoters and is regulated by alternative sigma factors and many transcription factors acting either as activators or repressors. This work describes various ways of transcription regulation in the bacterial genus Mycobacterium that includes deathly pathogens such as M. tuberculosis and M. leprae. The typical characteristics of this genus are poorly conserved promoters, a high number of sigma and transcription factors, the presence of two-component systems and a lot of small RNAs that have not been characterized in detail so far.
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Interaction of nucleic acids with RNA polymerase
Janoušková, Martina ; Krásný, Libor (advisor) ; Vopálenský, Václav (referee) ; Knejzlík, Zdeněk (referee)
Regulation of gene expression by RNA polymerase (RNAP) is an essential ability of living organisms, required for their adaption to a changing environment and ultimately enabling their survival. Interaction of RNAP with ribonucleic acids (DNA or RNA) is crucial for transcription and its regulation. This Doctoral Thesis contains two projects addressing interactions of RNAP with nucleic acids: (i) Transcription of modified DNA templates and (ii) Ms1, a small RNA (sRNA) from M. smegmatis. (i) We investigated the influence of modifications in the major groove of DNA on bacterial transcription in vitro. We found out that transcription of modified DNA templates is influenced on the transcription initiation level and that the promoter sequence is important for the effect of the modifications. Furthermore, we successfully performed transcription switch ON and OFF in vitro by bioorthogonal reactions. This regulation of transcription by artificial DNA modifications has a future in biotechnologies and/or medical therapy. (ii) Regulators of transcription are also small non-coding RNAs. These molecules have an important role in gene expression regulation among prokaryotes and eukaryotes. Ms1 is an sRNA found in mycobacteria. It makes a complex with the RNAP core and it is abundant in stationary phase (in amounts...
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Regulation of expression of Ms1, a sRNA from Mycobacterium smegmatis
Páleníková, Petra ; Krásný, Libor (advisor) ; Lichá, Irena (referee)
Bacteria are exposed to various environmental conditions during their growth. They have to cope with rapid changes in temperature, lack of nutrition, etc. To survive, bacteria alter their gene expression. One type of regulation of gene expression is regulation by small RNAs (sRNAs). In bacteria, a well-studied sRNA is 6S RNA that binds to the RNA polymerase holoenzyme. However, 6S RNA has not been identified in several bacterial species. Mycobacteria are a genus that probably does not have 6S RNA. Instead, Mycobacterium smegmatis possess another sRNA - Ms1. Ms1 structurally resembles 6S RNA and indeed it was first identified as a 6S RNA structural homologue. However, Ms1 binds to RNAP devoid of any sigma factor, and, therefore, is significantly distinct from 6S RNA. This work describes regulation of expression of Ms1. DNA fragments of different length from the region upstream of the Ms1 gene were prepared. These fragments were fused to the lacZ reporter gene and their activity was tested in different growth phases and under stress. This allowed identification and characterization of the core promoter sequence and regulatory sequences that might interact with transcription factor(s). Promoter activity increased with increased length of the promoter fragment and after transition into stationary...
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Cloning, epression and purification of mycobacterial dihydrofolate reductase
Šedivá, Kateřina ; Novotná, Eva (advisor) ; Malátková, Petra (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Kateřina Šedivá Supervisor: Mgr. Eva Novotná, Ph.D. Title of diploma thesis: Cloning, expression and purification of mycobacterial dihydrofolate reductase Dihydrofolate reductase is an enzyme essential for the metabolism of folic acid - it catalyzes the reduction of dihydrofolate to tetrahydrofolate. Tetrahydrofolate is an important cofactor involved in one-cabron transfer reactions. Dihydrofolate reductase plays a key role in the synthesis of DNA, RNA and proteins. Dihydrofolate reductase was also found in M. tuberculosis. This bacterium is the most common causative agent of tuberculosis in humans. Thus dihydrofolate reductase could be a potential target for the design of new antituberculotics. The recombinant protein dihydrofolate reductase was prepared in several steps. The coding sequence of the protein was first amplified by polymerase chain reaction. A recombinant plasmid, obtained by the ligation of an amplified segment of DNA with plasmid pET-28b(+), was transformed into competent cells E. coli strain BH101 by the heat shock method. Cells E. coli strain BL21(DE3) were used for the protein expression. The expression was induced by the addition of isopropyl-β-D-...
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Regulation of transcription in mycobacteria.
Páleníková, Petra ; Krásný, Libor (advisor) ; Mrvová, Silvia (referee)
The bacterial cell has to be able to cope with environmental changes. Adaptation to these changes is achieved by changes in gene expression. Gene expression is regulated mostly at the level or transcription initiation. Transcription initiation depends on the sequence of promoters and is regulated by alternative sigma factors and many transcription factors acting either as activators or repressors. This work describes various ways of transcription regulation in the bacterial genus Mycobacterium that includes deathly pathogens such as M. tuberculosis and M. leprae. The typical characteristics of this genus are poorly conserved promoters, a high number of sigma and transcription factors, the presence of two-component systems and a lot of small RNAs that have not been characterized in detail so far.
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Mycobacterial infection of domestic and wild ruminants
ROTHOVÁ, Kristýna
The genus Mycobacterium is well represented by Mycobacterium bovis, the etiological agent of bovine tuberculosis and Mycobacterium avium subsp. paratuberculosis (MAP) infects domestic cattle, wild ruminants and zoo animals leading to chronic enteritis known as paratuberculosis. The infection is chronic, progressive and unresponsive to treatment. Most infected animals do not develop clinical disease but may excrete the bacteria. Clinically sick animals suffer emaciation and in some species diarrhoea, followed by eventual death. During the course of the disease, excretion of MAP in faeces and milk occurs. Though Paratuberculosis is not classified as a zoonosis, current opinions on the possible role of this mycobacteria in public health is discussed. Bovine tuberculosis is a disease characterised by the progressive development of characteristic granulomas, or tubercles, in the lungs, lymph nodes or other organs. This disease is a well-known zoonotic disease which affects cattle world-wide, and causes major economic losses. The public health risk has been alleviated in many countries by the introduction of pasteurisation. Bovine tuberculosis was eradicated in Czech republic in 1968, but still a risk of infection through the importation of animals mainly to the zoological gardens.
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Laboratory diagnosis of mycobacteria, with a focus on the bacteriological proof
VELKOVÁ, Martina
Laboratory methods play an important role in disease diagnostic. Although there are currently available fast molecular genetic methods and methods based on cell response, basic diagnostic method of direct evidence is still microscopy and cultivation. Microscopy (staining by Ziehl-Neelsen and fluorescence microscopy) is the primary method for most of the specimens, especially for sputum. The sensitivity of this method is not very high because for the detection of a positive finding in 1 mm3 is needed at least 105 microbes. However, the method is important because it can fastly proof extensive clinical and epidemiological serious diseases. The ?Golden standard? is today the cultivation on solid egg media. Nowadays it is filled in automatic detection system, which accelerates the time of detection. The thesis was carried out in the Laboratory of Medical Microbiology, Bacteriology department, Hospital Ceske Budejovice in the period from 1.1.2013 till 6.6.2013, and included 400 sputa, which were processed and examined. The purpose was to compare the effectiveness of decontamination methods with HCl and with NALC, and to monitor the recovery and detection for the automatic detection system and conventional cultivation. In the automatic system using the BACTECTM MGITTM 960 Non-Radiometric fluorescent technology is cultivation in liquid Middlebrook 7H9 medium supplemented with antimicrobials (polymyxin B, amphotericin B, nalidixic acid, trimethoprim, azlocilin). The classical culture used solid egg culture medium: Lowenstein-Jensen and culture medium by Ogawa. The individual results then show that the method of HCl had the overall contamination of 1 % of the samples. For each methods in 5 % BACTECTM MGITTM 960 and 4 % in the conventional cultivation. In comparison the method NALC, had the overall contamination of 21 % of the samples. For the individual methods in 26 % of BACTECTM MGITTM 960 and 50 % in the conventional cultivation. From these results it is obvious that the method with the NALC has lower efficiency, the proportion of contamination compared to the method with HCL increased by 20 %. Decontamination with the NALC method recommended by the manufacturer for BACTECTM MGITTM 960 was found to be unsatisfactory and was canceled. Comparing the recovery and the detection of the strains isolated in individual methods showed that the BACTECTM MGITTM 960 exhibits greater sensitivity than conventional cultivation, since the total of 17 strains isolated BACTECTM MGITTM 960 captured 15 against 10 strains isolated in conventional cultivation. There is also a significantly shorter time to detect positive samples. The average detection time for BACTECTM MGITTM 960 was 16.2 days, while a conventional culturing was 31 days. It was confirmed that the BACTECTM MGITTM 960 system achieves better results, but optimization is achieved by combining the two methods used. There is an apparent decrease in detection of mycobacteria from the processed statistical data of the strains isolated during the period 2010 - 2012, but a substantial reduction does not occur. Noticeable is only the decline of the isolated stains of Mycobacterium bovis BCG, which can be explained by the fact that the vaccination against tuberculosis is since 2011 no longer carried out across the board, but according to the new legislation in force only in high-risk groups. An interesting fact is that every year the highest laboratory detection is found in the age group of over 60 years.
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