Streptomyces, filamentous Gram-positive bacteria are producers of more than 70% of antibiotics used in human therapy and agriculture. They are remarkable because of their complex life cycle (morphological differentiation) which leads to a formation of dormant spores able to survive unfavorable living conditions and allowing long-term survival of the organism. Soil represents their mostly natural living environment. In laboratory conditions they are cultivated in liquid media or on agar. We have developed in our laboratory two phase cultivation system which allows quantitative and reproducible preparation of samples for proteomic, transcriptomic and metabolomic analyses of Streptomycetes differentiation. The system is composed of inert micro- beads submerged in liquid medium. We used two types of micro-beads in our studies, glass and zirconia/silica beads. We followed the surface growth and differentiation of Streptomycetes on both types of beads using optical and electron microscopy (SEM) techniques. We observed major growth and higher antibiotic production on glass beads. Another difference we observed was in size and shape of colonies. In further research, using comparative proteomics, we attempted to identify proteins which might be responsible for recognition and adhesion of Streptomycetes to...

Acute and chronic morphine administration can significantly reduce ischemia- reperfusion injury of the rat heart. However, the molecular mechanisms mediating the protective effect of morphine are not yet fully elucidated. Concurrently, there is a lack of information about the effects of the long-term action of morphine on heart tissue. Therefore, in the first part of the project, we studied the effect of long-term administration of high doses of morphine (10 mg/kg/day, 10 days) on rat heart tissue. In the second part of the project, we investigated the effect of 1 mM morphine on viability and redox state of rat cardiomyoblast cell line H9c2 that was influenced by oxidative stress elicited by exposure to 300 μM tert-butyl hydroperoxide (t-BHP). Our experiments have shown that long-term morphine administration affected neither the amount nor the affinity of myocardial β-adrenergic receptors (β-AR), but almost doubled the number of the dominant isoforms of myocardial adenylyl cyclase (AC) V/VI and led to supersensitization of AC. At the same time, proteomic analyses revealed that long-term morphine administration was associated with significant changes in the left ventricular proteome. In particular, there was an increase in the expression of heat shock proteins (HSP). Increased expression of HSP27...

Proteomics as a modern comprehensive approach to the analysis of proteomes was applied in three projects aimed at diagnosis and therapy of cancer. The aim of the first the project was to find a new diagnostic biomarker for ovarian cancer. Two different comparative proteomic approaches were used for comparative analysis of sera from patients diagnosed with ovarian cancer and from healthy age-matched women. We identified -1-antitrypsin with increased concentration in patien sera, and apolipoprotein A4 and retinol-binding protein 4 (RBP4) with significantly decreased concentration in patients. The significantly decerased concentration of RBP4 in patients is a new observation. We propose that RBP4 is either decreased in ovarian cancer patients as a result of its reduced production by ovary or it may reflect less specific systemic changes, for instance early onset of cancer cachexia. The second project was focused on gaining insight into the molecular mechanism of cytarabine resistance in mantle cell lymphoma (MCL). Proteomic and transcriptomic analyses of cytarabine-resistant cells revealed marked downregulation of deoxycytidine kinase (DCK) - a protein essential to intracellular activation of purine and pyrimidine nucleosides and their analogues including cytarabine. The cytarabine-resistant MCL...

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....

Proteomics is a set of analytical methods which enable qualitative and quantitative characterization of the proteome. Expression proteomics quantitatively compares proteomes of cells, tissues, body fluids or other biological materials to find differencies in protein expression and, based on these differencies, to describe the biological processes occuring in investigated organisms. An initial material for expression proteomic studies are complex mixtures containing thousands of proteins, which are analyzed using separation (electrophoretic and chromatographic) methods, and identified, possibly quantified using mass spectrometry. The aim of this Thesis is to demonstrate the application of the tools of expression proteomics in solving diverse challenges in biomedicine. We employed various proteomic approaches and tools for studying molecular mechanisms of human diseases using pacient biological samples, or a model organism and a cell culture. We were conducting three different research projects, namely: A quest for potencial molecular targets for selective elimination of TRAIL-resistant mantle cell lymphoma cells; Investigation of molecular mechanisms of heart failure using a rat model of the disease induced by volume overload; and Searching for diagnostically usable serum biomarkers of ovarian...

Proteomics is a set of analytical methods which enable qualitative and quantitative characterization of the proteome. Expression proteomics quantitatively compares proteomes of cells, tissues, body fluids or other biological materials to find differencies in protein expression and, based on these differencies, to describe the biological processes occuring in investigated organisms. An initial material for expression proteomic studies are complex mixtures containing thousands of proteins, which are analyzed using separation (electrophoretic and chromatographic) methods, and identified, possibly quantified using mass spectrometry. The aim of this Thesis is to demonstrate the application of the tools of expression proteomics in solving diverse challenges in biomedicine. We employed various proteomic approaches and tools for studying molecular mechanisms of human diseases using pacient biological samples, or a model organism and a cell culture. We were conducting three different research projects, namely: A quest for potencial molecular targets for selective elimination of TRAIL-resistant mantle cell lymphoma cells; Investigation of molecular mechanisms of heart failure using a rat model of the disease induced by volume overload; and Searching for diagnostically usable serum biomarkers of ovarian...

The Gram-negative aerobic coccobacillus Bordetella pertussis is one of the few exclusively human pathogens and the main causative agent of the respiratory infectious disease called pertussis, or whooping cough. Despite global vaccination programs, pertussis remains an important public-health burden and still accounts for over 100,000 infant deaths and over a dozen of millions of whooping cough cases every year. Substantial effort is devoted to studies on the mechanisms of action of virulence factors of B. pertussis, but the biology of interactions of B. pertussis with its human host remains largely underexplored. Evolution, genetics and adaptation of B. pertussis to the complex environment of human nasopharynx and the mechanisms enabling B. pertussis to overcome host innate and adaptive mucosal immune defenses, remain poorly understood. In such situations, unbiased exploratory omics approaches represent valuable tools for uncovering of unknown aspects of host-pathogen interactions and open the path to detailed analysis of virulence-underlying processes by mechanistic studies. In this thesis, I am presenting the results of three omics projects on B. pertussis biology that involved high-throughput proteomics. In the inital phosphoprotemics project, we analyzed the kinase signaling pathways hijacked...

Biofilm formation is one of the most common bacterial survival strategies. Majority of bacterial species are able to form these three-dimensional structures, including pathogens like Mycobacterium tuberculosis. Representatives of Mycobacterium genus widely occur in the nature, although they can cause serious problems when they appear in medical equipment and artificial replacements of the human body. Non-pathogenic Mycobacterium smegmatis mc2 155 was used as a model organism in our experiments. We investigated morphology of the three- and six-day-old colonies (in fact biofilms) on agar and agar covered with cellophane using Stereo microscope and Scanning Electron Microscope. We found that a type of surface as well as a carbon source has a great influence on the morphology of the M. smegmatis colonies. We isolated proteomes from the agar and cellophane cultures and from planktonic culture. Two-dimensional electrophoresis was used as the main proteomic method. Proteomic data were analyzed using PDQuest software. Then the sets of proteins detected by qualitative and quantitative analyses were compared using Venn diagrams. As a result, we recognized 7 unique proteins that might be specific for recognition and adhesion of bacteria to the cellophane, no unique protein in agar proteome and 46 unique...

The theoretical part of work is focused on the issue of biomass which can be used for energy purposes, inparticular agricultural waste, as well as can serve as a substrate for biogas station. It also deals with proteomics, its goals and approaches, separation methods. The aim of this work was to measure each sample of enzyme activity of biomass, which are used as a raw materials for biogas plants and their proteomic identification.

Despite significant progress in the development of anti-cancer drugs, there is still a need for novel therapeutic strategies that would improve the outcome of cancer patients. Using proteomic technologies and cell lines with different phenotype of p53 tumour suppressor, we monitored cancer cell response to anti-cancer treatment with focus on the development of drug resistance. The different levels of metabolic proteins were identified in our study which may help to explain different anti-cancer activity of drugs with only a subtle difference in structure. More importantly, proteins associated with the development of drug resistance were identified and such expression changes have become a focus of interest. Our findings demonstrate a higher protein level of serine hydroxymethyltransferase, serpin B5 and calretinin in cancer cells resistant to Aurora kinase inhibitors. Such proteins promote the tumour growth with no apparent impact of p53 phenotype whilst voltage-dependent anion-selective channel protein 2 contributes to the development of resistance only in cells with functional p53 which is accompanied by the decreased level of elongation factor 2. On the other hand, cancer cells with loss of p53 appear to amplify alternative mechanisms such as protection against oxidative stress. The results...