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Methodology for assessing the effect of sublethal doses of pesticides on solitary bees employing the OMICs approach
Erban, Tomáš ; Shcherbachenko, Elena ; Šlachta, Martin ; Cudlín, Pavel ; Chalupníková, Julie ; Halešová, Taťána ; Tomešová, Daniela ; Václavíková, Marta ; Votavová, Alena
Pollinators are potentially exposed to a number of pesticides in the environment. The most important group of hazardous substances comes from the category of plant protection products (PPPs), which are applied not only by farmers but also by small gardeners. Each PPP including the active substance and formulation undergoes a very detailed and thorough evaluation, which is in the European Union (EU) based on Regulation (EC) No 1107/2009 of the European Parliament and of the Council. Although the negative effects of PPPs on the environment and non-target organisms are minimized with proper use, we cannot rule out possible hidden effects, especially those of a sublethal nature. Modern analytical approaches, such as the application of high-throughput methods, help to identify hidden side effects. This methodology is focused on the evaluation of the effect of sublethal doses of pesticides on solitary bees employing the OMICs approach. In the risk assessment of pesticides, the honey bee has long been preferred as a model species of pollinator, while solitary bees stay in the background. The methodology contains innovative aspects for experiments, such as a modified isolator, preparation of plants for exposure, the timing of the experiment, monitoring of active substance distribution or proteomic analysis of solitary bees to identify hidden effects of the test substance or its formulation. In this methodology, the model of the solitary bee, red mason bee (Osmia bicornis), is considered. However, the methodology is also applicable to other, especially related species of solitary bees. The methodology can be used in areas of state administration, private laboratories and research activities in the assessment of environmental risks of pesticides on solitary bees. The methodological procedure can confirm or even eliminate environmental risks when registering new products or reevaluating existing products. This methodology therefore has the potential for use in testing new substances intended for plant protection before their registration. In an exemplary embodiment, the effect of acetamiprid in a Careo sticks formulation was tested. The distribution of acetamiprid in the soil substrate and the plant was evaluated. The hazardous metabolite acetamiprid IM-2-1 was identified in plant tissues. However, proteomic analysis indicated a very low risk of acetamiprid for O. bicornis. This result is in agreement with the fact that acetamiprid has been assessed by EFSA to be a low risk for bees and is registered in the EU until 28 February 2033.
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A method for identification of foreign amylases in honey
Erban, Tomáš ; Shcherbachenko, Elena ; Talacko, Pavel ; Harant, Karel
Honey is a unique natural product. Honey has been used as a sweet and delicious foodstuff since ancient times. However, it is also valued for its multifaceted currative properties. Unfortunately, honey is one of the most adulterated foods. Nothing may be added to or modified from the honey. Honey also needs to be handled with care. Honey quality can negatively be affected by the way of processing such as heating and storage. Despite great progress in analytical methods, it is not possible to prove all adulterated honeys. Some methods of adulteration are quite sophisticated. Therefore, it is necessary find new approaches and methods for identification of honey adulteration. To be sold, honey must comply with internationally valid standards, which are also valid at national levels with possible minor modifications. One of the important parameters for honey is the level of diastase or amylase enzyme activity, which is a recognized indicator of the freshness and quality of honey. Lower diastase activity below the established level may indicate old honey, but it may also be the result of careless handling of honey. Last but not least, diastase activity may be reduced due to adulteration of honey such as its dilution with sugar substitutes. It is possible that amylase activity can be artificially adjusted by the addition of enzymes. Honey adulterated by the artificially added amylase meets the legislative requirements for placing honey on the market, but this violates the rules laid down by law. The methods used so far have not made it possible to prove this way of honey adulteration. Therefore, this methodology focuses on the identification of foreign amylases that may occur in honey. The methodology enables to identify practically any foreign amylase in honey by bottom-up shotgun proteomic approach. Based on the obtained results with specific peptides can be further used for the development of a targeted method for the identification of foreign amylases.
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Reaction intermediates in homogeneous gold catalysis
Shcherbachenko, Elena ; Roithová, Jana (advisor) ; Matoušová, Eliška (referee)
The presented master thesis is devoted to the investigation of reaction intermediates in homogeneous gold catalysis. Electrospray ionization mass spectrometry (ESI-MS) was used as the primary research technique in this study. Delayed reactant labeling was used as the main method. I have focused mainly on the hydration of 1-phenyl-1-propyne catalyzed by the gold complex [Au(IPr)(MeCN)]BF4 (IPr = 1,3-bis(2,6-di-iso-propylphenyl)imidazol-2- ylidene). I have detected two main intermediates containing one or two gold atoms, respectively (monoaurated and diaurated intermediate). I have obtained rate constants for the degradation of the reaction intermediates and their half-lives. I have derived kinetic isotope effects for the formation and the decomposition of the detected intermediates. I have shown that the kinetics of the degradation of both intermediates is identical, therefore I conclude that hydration of alkynes catalyzed by gold complex [Au(IPr)(MeCN)]BF4 proceeds most probably via neutral monoaurated intermediates. These neutral intermediates are detected by ESI-MS as protonated (monoaurated intermediate) or tagged by a second gold cation (diaurated intermediate). Key words: gold catalysis, reaction intermediates, electrospray ionization, mass spectrometry.
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Characterization of organogold compounds
Shcherbachenko, Elena ; Roithová, Jana (advisor) ; Míšek, Jiří (referee)
Geminally diaurated complexes are important intermediates involved in some of the gold catalyzed reactions and therefore they have become a subject of the current research. The aim of this thesis was to assess the influence of electronic structure of an aryl ligand on the stability of gold complexes with three-center-two-electron interaction Au2C. A series of geminally diaurated complexes was obtained by a reaction of gold complex [(IPr)Au(NCMe)]BF4 (IPr = 1,3-bis(2,6-di-iso-propylphenyl)imidazol-2-ylidene) with arylboronic acids ArB(OH)2 bearing different substituents at the aryl group (Ar = para- O2NC6H4, para-NCC6H4, para-ClC6H4, C6H5, para-H3CC6H4, para-H3COC6H4, meta- O2NC6H4, meta-NCC6H4, meta-H3CC6H4, ortho-O2NC6H4, ortho-NCC6H4, ortho-H3CC6H4). Mass spectrometry (MS) was used as the main experimental method in this study. Appearance energies (AE) for the fragmentation of geminally diaurated complexes were estimated by the methods MS/MS. The obtained energies were plotted against the corresponding Hammett σ-constants to assess the correlation between the electronic structure of a given aryl ligand and the stability of the Au2C bond. It was found that structures of gold complexes derived from the cyanophenylboronic acids do not correspond to the geminally diaurated complexes but to the...
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Reaction intermediates in homogeneous gold catalysis
Shcherbachenko, Elena ; Roithová, Jana (advisor) ; Matoušová, Eliška (referee)
The presented master thesis is devoted to the investigation of reaction intermediates in homogeneous gold catalysis. Electrospray ionization mass spectrometry (ESI-MS) was used as the primary research technique in this study. Delayed reactant labeling was used as the main method. I have focused mainly on the hydration of 1-phenyl-1-propyne catalyzed by the gold complex [Au(IPr)(MeCN)]BF4 (IPr = 1,3-bis(2,6-di-iso-propylphenyl)imidazol-2- ylidene). I have detected two main intermediates containing one or two gold atoms, respectively (monoaurated and diaurated intermediate). I have obtained rate constants for the degradation of the reaction intermediates and their half-lives. I have derived kinetic isotope effects for the formation and the decomposition of the detected intermediates. I have shown that the kinetics of the degradation of both intermediates is identical, therefore I conclude that hydration of alkynes catalyzed by gold complex [Au(IPr)(MeCN)]BF4 proceeds most probably via neutral monoaurated intermediates. These neutral intermediates are detected by ESI-MS as protonated (monoaurated intermediate) or tagged by a second gold cation (diaurated intermediate). Key words: gold catalysis, reaction intermediates, electrospray ionization, mass spectrometry.
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Characterization of organogold compounds
Shcherbachenko, Elena ; Roithová, Jana (advisor) ; Míšek, Jiří (referee)
Geminally diaurated complexes are important intermediates involved in some of the gold catalyzed reactions and therefore they have become a subject of the current research. The aim of this thesis was to assess the influence of electronic structure of an aryl ligand on the stability of gold complexes with three-center-two-electron interaction Au2C. A series of geminally diaurated complexes was obtained by a reaction of gold complex [(IPr)Au(NCMe)]BF4 (IPr = 1,3-bis(2,6-di-iso-propylphenyl)imidazol-2-ylidene) with arylboronic acids ArB(OH)2 bearing different substituents at the aryl group (Ar = para- O2NC6H4, para-NCC6H4, para-ClC6H4, C6H5, para-H3CC6H4, para-H3COC6H4, meta- O2NC6H4, meta-NCC6H4, meta-H3CC6H4, ortho-O2NC6H4, ortho-NCC6H4, ortho-H3CC6H4). Mass spectrometry (MS) was used as the main experimental method in this study. Appearance energies (AE) for the fragmentation of geminally diaurated complexes were estimated by the methods MS/MS. The obtained energies were plotted against the corresponding Hammett σ-constants to assess the correlation between the electronic structure of a given aryl ligand and the stability of the Au2C bond. It was found that structures of gold complexes derived from the cyanophenylboronic acids do not correspond to the geminally diaurated complexes but to the...
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