RNA polymerase (RNAP) is the enzyme that catalyzes synthesis of RNA. Mycobacterial RNAP significantly differs from RNAPs from other bacterial species. It requires special transcription factors such as RbpA or CarD. Another difference is the presence of a small RNA (sRNA), Ms1, that binds to mycobacterial RNAP. Ms1 regulates the amount of RNAP in the cell. In our laboratory we recently discovered MoaB2, a new binding partner of mycobacterial A (encoded by sigA), an RNAP subunit, which is essential for recognition of the initial promoter sequence and initiation of transcription. The function of MoaB2 in the regulation of transcription and gene expression is still unknown. The first aim of this Thesis is contribute to elucidation of the mechanism by which Ms1 affects the amount of RNAP. The experiments revealed that this regulation occurs at the level of transcription; Ms1 affects the activity of promoter(s) that drive the transcription of rpoB- rpoC that encode the two catalytic subunits of RNAP. The second aim of this Thesis is to characterize the interactions of MoaB2 with protein of the transcription apparatus. The results confirmed the interaction of MoaB2 with A and showed that neither RNAP nor transcription factors RbpA and CarD are required for this interaction. Finally, a role of the...

Charles University, Faculty of Pharmacy, Hradec Králové Field of study: Specialist in laboratory methods Author: Bc. Tomáš Hampl Supervisor: PharmDr. Ondřej Janďourek, Ph.D. Title of diploma thesis: Mycobacterium tuberculosis as causative agent of grave illness The aim of this diploma thesis is to deal with tuberculosis, describe the pathogen, taxonomic classification, describe the origin, course and clinical manifestations of the disease, diagnosis, including laboratory diagnostics, epidemiology of tuberculosis, treatment and prevention. Tuberculosis (TBC) is one of the deadliest infectious diseases in the world caused by bacterium M. tuberculosis in humans. TBC is sometimes referred as a disease of the past, but the recent rise in cases of the disease, and especially the spread of forms of drug-resistant TBC, makes the disease very dangerous. Currently, a major problem is the alarming rise in resistant and multi-resistant tuberculosis, which is not counteracted by commonly used first-line drugs. Therefore, there is a constant emphasis around the world on discovering and testing new potential antituberculotics that could help patients with resistant and multidrug-resistant TBC. In the experimental part, the thesis deals with screening of antimycobacterial activity of potential antituberculotics...

Charles University Faculty of Pharmacy in Hradec Kralove Department of Biological and Medical Sciences Study program: Specialist on Laboratory Methods Author: Bc. Tereza Pchálková Supervisor: PharmDr. Ondřej Janďourek, Ph.D. Title of diploma thesis: In vitro screening of potential antimycobacterial compounds active against fast growing strains of Mycobacterium genus II Key words: Mycobacteria, Tuberculosis, Antituberculotics, Microdilution broth method, Minimum inhibition concentration Background: The aim of this diploma thesis has been screening of in vitro antimycobacterial activity of novel compounds against fast growing strains of the genus Mycobacterium (Mycobacterium smegmatis and Mycobacterium aurum). Another aim has been predicting the structure-activity relationships for tested compounds. Methods: The technique used for activity determination was microdilution broth method. The value of minimum inhibition concentration for each compound was determined. The evaluation was performed visually and spectrophotometrically using the Alamar blue indicator. Results: A total of 79 compounds were tested. 22 tested compounds showed significant activity against mycobacteria. The substances were sorted according to the similarity in chemical structure into 10 groups. From a chemical point of view, these...

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...

Biofilms represent universal strategy for bacterial survival. Living in form of biofilms, bacteria acquire wide range of advantages over planktonically growing cultures. It can be assumed that nearly 99% of world bacterial population is living in form of biofilms. There are benefits and drawbacks associated with bacterial biofilms for mankind. Life in biofilms makes pathogens more effective and persistent through higher antibiotic resistance and helps them to hide before immune system of the host. Mycobacteria, which are capable of forming biofilms on variety of surfaces, differ from most of other bacteria by unique composition of their cell wall. It provides them with high resistance against physical or chemical damage. This is one of the reasons for considering Mycobacterium tuberculosis as a highly potent pathogen. The studies of mycobacterial biofilms are motivated by effort to improve or find new therapeutic methods. This work is aimed at morphological and proteomic comparative analyses of biofilms obtained from Mycobacterium smegmatis grown on surface of glass and silica/zirconium beads, on liquid medium surface or grown submerged in shaken planktonic culture. We have developed technique for preparation of "floating" biofilm sample to be observed in SEM. We have shown that the growth of...

Charles University Faculty of Pharmacy in Hradec Kralove Department of Biological and Medical Sciences Study program: Specialist on Laboratory Methods Author: Bc. Lenka Králíková Supervisor: PharmDr. Ondřej Janďourek, Ph.D. Title of diploma: Atypical mycobacteria as causative agents of human diseases The aim of this diploma thesis is to summarize the history of mycobacteria, their classification, epidemiology of nontuberculous infections, clinical significance, diagnosis, resistance, and treatment options. Within the epidemiology, the way of managing tuberculosis and non-tuberculous mycobacterioses is compared. Mycobacterioses are rare diseases, which incidence is gradually increasing with the increasing number of immunocompromised patients. Based on the spot of infection, we divide them into lymphadenitis, skin lesions, lung disease and disseminated infections. The most frequently isolated pathogens are Mycobacterium avium complex, Mycobacterium ulcerans, Mycobacterium abscessus, Mycobacterium marinum and Mycobacterium kansasii. The experimental part is devoted to the antimycobacterial activity of newly synthesized compounds, which could find application in the future in the treatment of tuberculosis and other mycobacterioses. Substance testing was performed on strains of the genus Mycobacterium...

Microorganisms grow in planktonic form, but more often they adhere to a number of surfaces and create three-dimensional structures called biofilms. Floating biofilms, which are formed at the water-air interface, are one of the life strategies, which the bacteria can take. Non-pathogenic Mycobacterium smegmatis was used as a laboratory model for the study of this kind of biofilm. The understanding of mechanisms of their formation of this species may be applicable to the pathogenic species of the genus Mycobacterium, study of which in the laboratory brings a number of disadvantages. This diploma thesis focuses on the morphological and proteome analysis of the M. smegmatis floating biofilm. Using a stereo microscope and scanning electron microscopy was observed that bacteria clump and create the "nucleation centres" at the beginning of the biofilm development. This centers grow to the surroundings and connect afterwards. In the later stages of the development the centers fuse in compact layer, which then grows into the compact and multilayer biofilm. The key method in this study was two-dimensional electrophoresis of proteins. The proteome analysis of floating biofilm was performed with this method. The preparation of protein samples and the method for protein concentration measurement was optimized....