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EPR Study of Radical Intermediates of H-transfer from Oxygeneous, Carboneous and Nitrogeneous Donors
Marešová, Renata ; Mazúr, Milan (referee) ; Stopka, Pavel (referee) ; Omelka, Ladislav (advisor)
Within the PhD. thesis the problems of H-transfer from selected types of phenols, aromatic secondary amines and compounds with acidic CH bond were investigated. This process was initiated by the action of redox agents, mostly PbO2, in nonpolar solvents. The abstraction of hydrogen atom results in the formation of radical intermediates, which were detected by EPR spectroscopy. Because in most cases, highly unstable radicals were produced, it was neccessary to apply the technique of indirect detection, so called spin trapping. This method enables to interpret the character of generated radicals on the basis of EPR parameters of radical adducts, which are formed by their reaction with suitably chosen compounds, spin traps, which are added to the reaction system. As spin traps, the aromatic nitrosocompounds were preferentially used. Due to the fact that EPR spectra of radical adducts were in most cases very complex, their interpretation was possible only using the computer simulation. Based on this approach, new information about the character of addition of phenoxyl tradicals to aromatic nitrosocompounds, stability of alkyl substituents in methyl substituted phenols and character of C-radicals, generated from substituted coumarines and beta-diketones, was obtained. Simultaneously, the small ability of nitrogen radicals, primary products of the splitting of NH bond, to enter the reaction with spin traps was proved by aromatic secondary amines.
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Radical Reactions of the Decomposition of N-H, O-H and O-O Bonds Initiated by Homogeneous and Heterogeneous Redox Agents
Majzlík, Petr ; Mazúr, Milan (referee) ; Stopka, Pavel (referee) ; Omelka, Ladislav (advisor)
The Ph.D. thesis was focused on EPR study of redox reactions of selected types of phenols, secondary amines and diperoxy coumpounds. Within the study some redox agents were employed in nonpolar, in some special cases also in polar solvents. EPR spectra of generated radical products were interpreted using spectral simulation. Study of radical reactions under participation of phenols was preferentially concentrated on the behavior of para methyl substituted phenols, where the instability of this substituent in relation to the applied redox agents was evaluated. The tendency towards the abstraction of hydrogen from methyl group, representing the paralell mechanism to the hydrogen abstaction from phenolic OH group was proved. The generated benzyl radicals were undirectly detected in the form of adducts with aromatic nitroso compounds. These adducts undergo the consecutive transformations, leading to the formation of new types of phenoxy radicals. The study of the decomposition of NH bonds was performed with substituted N,N´-paraphenylenediamines, 1-anilino-1-phenylpentane-3-ones and amino substituted 1,3-dimethyluracils. By the oxidation with 3-chloroperbenzoic acid the corresponding nitroxyl radicals were prepared. In the case of 1-anilino-1-phenylpentane-3-ones the aminyl radicals were prepared by the oxidation with PbO2, which existence was evidenced by spin trapping method with nitrosobenzene. In the framework of the investigation of the decomposition of -O-O- bonds the oxygen centred radicals, generated from peroxidic compounds of Luperox type using selected redox agents were detected by spin trapping method. The analysis of EPR spectra documented that primary alkoxyl radicals undergo the consecutive fragmentation, which leads in the presence of oxygen to the formation of secondary alkoxyl radicals. The generation of oxygen centred radicals during the decomposition was indirectly proved in the presence of model phenolic compounds, where due to the abstraction of hydrogen from phenolic OH group the phenoxyl radicals are formed.
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Proteomic architecture of sperm-egg interactions
Otčenášková, Tereza ; Stopka, Pavel (advisor) ; Petr, Jaroslav (referee) ; Vrbacký, Marek (referee)
Recent advances in proteomic methods provide new insights for biological research including the field of reproductive biology. Determination of the proteomic basis of spermatozoa is pivotal for understanding the complex process of gamete interactions during fertilization such as acrosome reaction. Great differences imposed by postcopulatory sexual selection and phylogeny can be observed regarding the size, shape, and molecular composition of sperm across animal taxa. The first objective of this doctoral thesis is to characterize the protein contents of the acrosome to ascertain its further functional significance in sperm-egg interaction. Also, we aim to investigate the potential relationships between sperm protein composition and sperm morphology diversification, risk of sperm competition, and species phylogenetic background. Wild-caught males from natural populations of species of Mus musculus musculus, Apodemus flavicollis, Microtus arvalis (order Rodentia), Acrocephalus palustris, Chloris chloris, Phylloscopus collybita, Cinclus cinclus, Hirundo rustica, and Taeniopygia guttata from a captive population (order Passeriformes) were subject to the analyses. Nano-liquid chromatography with tandem mass spectrometry was applied as the main methodological approach in this thesis. Our data implicate...
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Integrated multi-omics analysis of chemical signaling in wild rodents
Matějková, Tereza ; Stopka, Pavel (advisor) ; Macholán, Miloš (referee) ; Bryja, Josef (referee)
Symbiotic bacteria living with the host in so-called microbiomes have been one of the significant pillars of all aspects of animal evolution, chemical communication included. However, the phenotype, genotype, and microbiome of laboratory animals kept for generations in sterile conditions changed from their wild ancestors leading to profound differences in the laboratory results and the reality of wild animals. To describe the chemical communication in neglected wild rodents, this thesis focuses on the body parts involved in chemical communication (i.e. mouth, vagina, and intestines) and are also inhabited by microbiomes that produce metabolites with the capability of transmitting chemical signals. Using next-generation sequencing and state-of-the-art proteome and metabolome chromatography-mass spectrometry, this thesis covers the analysis of changes in the microbiome, proteome, and metabolome of wild mice in the context of transferring the wild individuals into the captivity, cohousing wild, and laboratory animals and hormonal changes during the estrous cycles. Moreover, this thesis describes and discusses the differences and similarities in the microbiome, proteome, and metabolome on the level of different species (Apodemus sp.), subspecies (Mus musculus domesticus vs. musculus), and environment...
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Evolution of sperm morphology in birds
Zemanová, Aneta ; Stopka, Pavel (advisor) ; Frolíková, Michaela (referee)
The sperm is one of the most variable cells that carry various morphological differences across species and is subject to selective pressures that may drive changes in sperm morphology. Different morphological adaptations that emerged may enhance successful fertilization. This thesis focuses on the analysis of sperm morphology in different species of songbirds, the representation of proteins in both sperm and seminal fluid, and their classification into different categories of gene ontology. The main aim of my thesis was to determine whether differences in proteomes follow the level of sperm competition or rather their phylogeny. Six species of songbirds were selected for this study: Marsh Warbler (Acrocephalus palustris), White-throated dipper (Cinclus cinclus), European greenfinch (Chloris chloris), Barn swallow (Hirundo rustica), Common chiffchaff (Phylloscopus collybita) and Zebra finch (Taeniopygia guttata). Sperm morphometric measurements were made in all the species, with the greatest variability recorded in the central part of the sperm flagellum. Using mass spectrometry, were analysed sperm and seminal fluid proteins separately and discriminant analysis was performed to determine the proteins that most differentiated the selected songbird species from each other. Obtained data provide...
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Chemical communication in field mice of the genus ApodemusApodemus
Obstová, Lucie ; Stopka, Pavel (advisor) ; Vohralík, Vladimír (referee)
The bachelor thesis deals with a special sebaceous organ that probably serves as a chemical communication means with four species of Apodemus field mouse - A. flavicollis, A. sylvaticus, A. agrarius and A. microps. This organ is found on the ventral side of a tail, so it is classified as a caudal organ. It consists of significantly enlarged sebaceous glands. It is particularly developed mainly with males; however, both females and infants have it as well. Its morphological structure has the same base regardless of the species, gender, and/or age; however the extent of development varies among species. The organ releases a secretion whose chemical content is unknown so far. Thanks to the gas-liquid chromatography, researchers have found out that there are both variations in the secretion contents among individual species, as well as within the same species. This kind of secretion is basically a characteristic feature of an individual, as it bears information about the species, gender, age and even the population affiliation. Functions of the organ stay unknown as well. It might serve to mark the territory, to play a role in social interactions (i.e. agonistic behaviour) and/or to be an important part of copulatory behaviour.
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Factors affecting oetrous cycles in the house mouse (Mus musculus)
Havrdová, Leona ; Stopka, Pavel (advisor) ; Němec, Pavel (referee)
Factors affecting oetrous cycles in the house mouse (Mus musculus) Abstract This bachelor thesis deals with factors which have significant influence on house mouse (Mus musculus) oestrous cycle. Main theme is oestrous cycle modulation caused by male or female chemical signals. Familiar phenomena like Bruce effect, Whitten effect and Lee-Boot effect are described in detail along with newly explored social modulation issues (ovulation trigger, male genotype role etc.). Environmental impact is also included, especially role of Bisphenol A in connection with negative impacts on human reproduction (tested on mice). Key words: oestrus, pheromone, MUP, Mus musculus, vomeronasal organ
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Analysis of gene expression during spermatogenesis in mice
Suchan, Jan ; Stopka, Pavel (advisor) ; Hortová, Kateřina (referee)
Spermatogenesis is very unique and complex process regulating and be amenable to reproductive behaviour equally. These processes are affected by outer factors such as social stimuli, represented by interaction between male and female. Mice belong to mammals with high sensitivity to chemical signals. Hence pheromonal communication takes important part in their life. Pheromones have effect on expression activity in testicular tissue throught hormonal pathways. A target of these regulations are for example proteins participating in immunity or competition actions. Phenotype plasticity might proceed on the level of DNA as well as on the level of gene expression. That's why we take interest if factors like social contacts are able to significantly change expression of genes involved in reproduction pathways. We are also interested in possibility that expression changes of target genes take part in speciation events. In this study we demonstrate the effect of short-term social contact to gene expression of biochemical pathways involved in spermatogenesis and by other pathways linked together with reproductive bahaviour. This involvement might be direct or indirect. We found genes, whose level of expression in testicular tissue is different between species Mus m. musculus and Mus m. domesticus. Here I provide...
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Identification and modeling of gene expression regulatory networks during streptomycetes germination
Straková, Eva ; Vohradský, Jiří (advisor) ; Stopka, Pavel (referee) ; Vondrášek, Jiří (referee)
Streptomycetes have been studied mostly as producers of antibiotics and for fundamentals of complex bacterial cell development. Here, transcriptomic and proteomic approaches were applied to systems study of Streptomyces coelicolor germination as a developmental transition from dormancy to the vegetative stage. The time dynamics of the gene expression levels represented by mRNA and intracellular protein accumulation and synthesis were measured throughout 5.5 h of germination at 13 time points by employing both DNA microarray and two-dimensional gel electrophoresis techniques. Using a numerical model of gene expression, genetic networks were reconstructed and functional groups of genes controlled by the sigma factors were identified. Modeling of the regulatory interactions provided a set of parameters allowing simulate kinetics of gene expression control among the sigma factors and their target genes. Particularly regulons of two sigma factors, SigR and HrdD, were identified. The analysis assigned their key role during the germination process. Analysis of global trends in the gene/protein expression revealed that the full capability of regulatory mechanisms responding to the environmental cues is reached within the first hour of germination, and identified the basic gene/protein functional groups...
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