Neste trabalho foram investigadas a atividade antialérgica de extratos, ou substâncias isoladas, obtidos de fontes naturais. Para isso foi utilizado o sistema biossensor baseado em mastócitos os quais liberam a enzima beta-hexosaminidase usada como marcador da degranulação. Para algumas substâncias naturais da classe dos flavonóides (quercetina-Qc e rutina-Rt) e ácidos polifenólicos (ácido dimetoxicinâmico-Dm e ácido cafeico-Cf), os ensaios biológicos foram conduzidos na presença de beta-ciclodextrina (beta-CD) a fim de estudar a eficiência do ensaio biológico e o efeito de complexação na atividade antialérgica. Inicialmente, foram investigadas, as propriedades espectroscópicas destes flavonóides e ácidos polifenólicos, na ausência, e presença de beta-CD. As mudanças nos espectros de absorção e fluorescência, em presença de beta-CD, mostraram que ocorre a associação dos fármacos com a beta-CD. Assim, as constantes de incorporação (Kc) foram determinadas pelo método de Higuchi e Connors e os resultados mostraram maior incorporação da Qc (Kc = 172 /M) na cavidade da beta-CD quando comparada a Rt (Kc = 139 /M). No caso dos polifenóis, Dm mostrou incorporação maior em relação ao Cf, com valores de Kc iguais a 718 e 278 /M, respectivamente. Os valores de Kc foram considerados apropriados para a aplicação de compostos de inclusão como agentes terapêuticos. Assim, os complexos de inclusão sólidos, foram preparados por uma adaptação do método da co-evaporação e caracterizados por Análise Termogravimétrica (TGA), Análise Térmica Diferencial (DTA), Calorimetria Diferencial de Varredura (DSC), espectroscopia na região do Infravermelho (FTIR) e Ressonância Magnética Nuclear de Prótons (1H-RMN). O parâmetro físico-químico para interação hidrofóbica (log P) foi determinado para os flavonóides e acidos polifenólicos e os resultados indicaram que a hidrofobicidade seguiu a seguinte ordem: Dm > Cf > Qc > Rt. Os complexos de inclusão foram mais eficazes para inibir a liberação da beta-hexosaminidase do que os fármacos na forma livre. A atividade anti-alérgica da Qc livre (IC50= 5,1 µM) mostrou um aumento de oito vêzes quando complexada com a beta-CD (IC50= 0,62 µM). Um aumento da atividade foi observado, também, para os complexos Rt/CD, Cf/CD e Dm/CD. Este efeito foi mais forte para os compostos com maior hidrofobicidade. A atividade antialérgica das substâncais naturais livres provenientes de várias classes de plantas tais como flavonóides, ácidos polifenólicos, terpenos, alcalóides e iridóides foi, também, investigada. Os flavonóides tais como quercetina (IC50= 5,1 µM), 7-metil quercetina (IC50= 6,2 µM), caempferol-3-glicosideo (IC50= 6,7 µM) and 4-O-(6-trans-p-coumaroil)--D-glicopyranosil okanina (IC50= 5,8 µM) mostraram a maior atividade antialérgica comparados ao fumarato de cetotifeno (IC50= 15,1 µM). Os extratos provenientes de diversas espécies de plantas tais como Bidens sulphurea, Bidens gardneri, Bidens graveolens, Mikania parodii Cabrera e Mikania pilosa Baker foram, também, investigados. Os resultados mostraram maior atividade para o extrato de Bidens obtido de acetato de etila. Este extrato é rico em derivados metilados de quercetina os quais exibiram forte atividade antialérgica quando utilizados no ensaio biológico como substância isolada. / Anti-allergic activity of extracts and isolated compounds obtained from natural sources was investigated using the mast-cell based biosensor system. Mast cells release beta-hexosaminidase enzyme which is used as a marker of degranulation. Flavonoids (quercetin-Qc and rutin-Rt) and polyphenolic acids (caffeic acid-Cf and dimethoxy cinnamic acid-Dm) were used as inclusion compounds with beta-cyclodextrin (beta-CD) in order to compare the efficiency of the biological assay and the anti-allergic activity of the drugs free or associated with beta-CD. Spectroscopic properties of the flavonoids and polyphenolic acids were monitored in the absence or presence of beta-CD. The absorbance and fluorescence spectra showed drug association with beta-CD; subsequently the stability constants (kc) of the drugs were obtained in accordance with the method of the Higuchi-Connors. The results showed higher association of Qc with beta-CD (Kc= 172 /M) compared to Rt (Kc= 139 /M). For the polyphenolic acids, Dm exhibited the higher association with beta-CD compared to Cf (718 and 278 /M respectively). The Kc values felt within the range considered adequate for the formation of inclusion complex, and they can be used to improve the bioavailability of the flavonoids and polyphenolic acids. The solid inclusion compounds were obtained by an adaptation of the co-evaporation method and characterized by Thermo Gravimetric Analysis (TG), Differential Thermal Analysis (DTA), Differential Scanning Calorimetry (DSC), Fourier Transform Infrared Spectroscopy (FTIR) and Proton Nuclear Magnetic Resonance Spectroscopy (1H NMR). Physico-chemical parameter for hydrophobic interaction (log P) was determined for the flavonoids and polyphenolic acids and the results indicated that the hydrophobicity followed the order: Dm > Cf > Qc > Rt. The inclusion complexes were more effective at inhibiting beta-hexosaminidase release than plain drugs. The anti-allergic activity of plain Qc (IC50= 5.05 M) showed eightfold improvement when included inside the beta-CD cavity (IC50= 0.62 M). Higher biological activity on the part of the complex was also observed for the complexes Rt/CD, Cf/CD and Dm/CD. This effect was stronger for the compounds with higher hydrophobicity. The anti-allergic activity of plain natural compounds from several classes of plants such as flavonoids, polyphenolic acids, terpenes, alkaloids and iridoids was investigated. Flavonoids such as quercetin (IC50= 5.1 µM), 7-methyl quercetin (IC50= 6.2 µM), kaempferol-3-glycoside (IC50= 6.7 µM) and 4-O-(6-trans-p-coumaroil)--D-glucopyranosyl okanin (IC50= 5.8 µM) showed the stronger anti-allergic activity compared with ketotifen fumarate, a reference drug (IC50= 15.1 µM). Extracts proceeding from different species of plants such as Bidens sulphurea, Bidens gardneri, Bidens graveolens, Mikania parodii Cabrera and Mikania pilosa Baker were also investigated. The results showed stronger anti-allergic activity for ethyl acetate extracts obtained from Bidens specie. This extracts are rich in methylated quercetin derivatives which showed strong anti-allergic activity when assayed as isolated substances

Neste trabalho foram investigadas a atividade antialérgica de extratos, ou substâncias isoladas, obtidos de fontes naturais. Para isso foi utilizado o sistema biossensor baseado em mastócitos os quais liberam a enzima beta-hexosaminidase usada como marcador da degranulação. Para algumas substâncias naturais da classe dos flavonóides (quercetina-Qc e rutina-Rt) e ácidos polifenólicos (ácido dimetoxicinâmico-Dm e ácido cafeico-Cf), os ensaios biológicos foram conduzidos na presença de beta-ciclodextrina (beta-CD) a fim de estudar a eficiência do ensaio biológico e o efeito de complexação na atividade antialérgica. Inicialmente, foram investigadas, as propriedades espectroscópicas destes flavonóides e ácidos polifenólicos, na ausência, e presença de beta-CD. As mudanças nos espectros de absorção e fluorescência, em presença de beta-CD, mostraram que ocorre a associação dos fármacos com a beta-CD. Assim, as constantes de incorporação (Kc) foram determinadas pelo método de Higuchi e Connors e os resultados mostraram maior incorporação da Qc (Kc = 172 /M) na cavidade da beta-CD quando comparada a Rt (Kc = 139 /M). No caso dos polifenóis, Dm mostrou incorporação maior em relação ao Cf, com valores de Kc iguais a 718 e 278 /M, respectivamente. Os valores de Kc foram considerados apropriados para a aplicação de compostos de inclusão como agentes terapêuticos. Assim, os complexos de inclusão sólidos, foram preparados por uma adaptação do método da co-evaporação e caracterizados por Análise Termogravimétrica (TGA), Análise Térmica Diferencial (DTA), Calorimetria Diferencial de Varredura (DSC), espectroscopia na região do Infravermelho (FTIR) e Ressonância Magnética Nuclear de Prótons (1H-RMN). O parâmetro físico-químico para interação hidrofóbica (log P) foi determinado para os flavonóides e acidos polifenólicos e os resultados indicaram que a hidrofobicidade seguiu a seguinte ordem: Dm > Cf > Qc > Rt. Os complexos de inclusão foram mais eficazes para inibir a liberação da beta-hexosaminidase do que os fármacos na forma livre. A atividade anti-alérgica da Qc livre (IC50= 5,1 µM) mostrou um aumento de oito vêzes quando complexada com a beta-CD (IC50= 0,62 µM). Um aumento da atividade foi observado, também, para os complexos Rt/CD, Cf/CD e Dm/CD. Este efeito foi mais forte para os compostos com maior hidrofobicidade. A atividade antialérgica das substâncais naturais livres provenientes de várias classes de plantas tais como flavonóides, ácidos polifenólicos, terpenos, alcalóides e iridóides foi, também, investigada. Os flavonóides tais como quercetina (IC50= 5,1 µM), 7-metil quercetina (IC50= 6,2 µM), caempferol-3-glicosideo (IC50= 6,7 µM) and 4-O-(6-trans-p-coumaroil)--D-glicopyranosil okanina (IC50= 5,8 µM) mostraram a maior atividade antialérgica comparados ao fumarato de cetotifeno (IC50= 15,1 µM). Os extratos provenientes de diversas espécies de plantas tais como Bidens sulphurea, Bidens gardneri, Bidens graveolens, Mikania parodii Cabrera e Mikania pilosa Baker foram, também, investigados. Os resultados mostraram maior atividade para o extrato de Bidens obtido de acetato de etila. Este extrato é rico em derivados metilados de quercetina os quais exibiram forte atividade antialérgica quando utilizados no ensaio biológico como substância isolada. / Anti-allergic activity of extracts and isolated compounds obtained from natural sources was investigated using the mast-cell based biosensor system. Mast cells release beta-hexosaminidase enzyme which is used as a marker of degranulation. Flavonoids (quercetin-Qc and rutin-Rt) and polyphenolic acids (caffeic acid-Cf and dimethoxy cinnamic acid-Dm) were used as inclusion compounds with beta-cyclodextrin (beta-CD) in order to compare the efficiency of the biological assay and the anti-allergic activity of the drugs free or associated with beta-CD. Spectroscopic properties of the flavonoids and polyphenolic acids were monitored in the absence or presence of beta-CD. The absorbance and fluorescence spectra showed drug association with beta-CD; subsequently the stability constants (kc) of the drugs were obtained in accordance with the method of the Higuchi-Connors. The results showed higher association of Qc with beta-CD (Kc= 172 /M) compared to Rt (Kc= 139 /M). For the polyphenolic acids, Dm exhibited the higher association with beta-CD compared to Cf (718 and 278 /M respectively). The Kc values felt within the range considered adequate for the formation of inclusion complex, and they can be used to improve the bioavailability of the flavonoids and polyphenolic acids. The solid inclusion compounds were obtained by an adaptation of the co-evaporation method and characterized by Thermo Gravimetric Analysis (TG), Differential Thermal Analysis (DTA), Differential Scanning Calorimetry (DSC), Fourier Transform Infrared Spectroscopy (FTIR) and Proton Nuclear Magnetic Resonance Spectroscopy (1H NMR). Physico-chemical parameter for hydrophobic interaction (log P) was determined for the flavonoids and polyphenolic acids and the results indicated that the hydrophobicity followed the order: Dm > Cf > Qc > Rt. The inclusion complexes were more effective at inhibiting beta-hexosaminidase release than plain drugs. The anti-allergic activity of plain Qc (IC50= 5.05 M) showed eightfold improvement when included inside the beta-CD cavity (IC50= 0.62 M). Higher biological activity on the part of the complex was also observed for the complexes Rt/CD, Cf/CD and Dm/CD. This effect was stronger for the compounds with higher hydrophobicity. The anti-allergic activity of plain natural compounds from several classes of plants such as flavonoids, polyphenolic acids, terpenes, alkaloids and iridoids was investigated. Flavonoids such as quercetin (IC50= 5.1 µM), 7-methyl quercetin (IC50= 6.2 µM), kaempferol-3-glycoside (IC50= 6.7 µM) and 4-O-(6-trans-p-coumaroil)--D-glucopyranosyl okanin (IC50= 5.8 µM) showed the stronger anti-allergic activity compared with ketotifen fumarate, a reference drug (IC50= 15.1 µM). Extracts proceeding from different species of plants such as Bidens sulphurea, Bidens gardneri, Bidens graveolens, Mikania parodii Cabrera and Mikania pilosa Baker were also investigated. The results showed stronger anti-allergic activity for ethyl acetate extracts obtained from Bidens specie. This extracts are rich in methylated quercetin derivatives which showed strong anti-allergic activity when assayed as isolated substances

Iminosugars are known to behave as carbohydrate mimics in biological systems by virtue of their similar structures. However as the ring nitrogen prevents metabolism it means that iminosugars have the potential to become inhibitors of these systems. It is known, for example, that iminosugars can behave as mimics in the hydrolysis mechanism. This leads to possible medicinal applications of iminosugars. One such case is lysosomal storage disorders which arise as a result of a genetic defect which causes missense mutations coding for the N-acetylhexosaminidase enzymatic protein. N-Acetylhexosaminidases are a sub-member of the class of glycosidase enzymes. They are responsible for the cleavage of N-acetylhexosamine residues from glycoconjugates in the lysosome. Mutations in the gene coding for this protein lead to a deficiency in the enzymatic activity resulting in accumulation of unhydrolysed substrate in the lysosome. Lysosomal storage disorders have a phenotype of poor motor development and neurological problems. The infantile form usually leads to death before the age of five. An iminosugar mimic could give rise to a possible treatment for lysosomal storage disorders by acting as a molecular chaperone during protein folding, promoting correct folding by its intrinsic affinity for the native fold of the enzyme. Likewise in the treatment of cancer, the inhibitory ability of iminosugars has potential applications. In cancer, extracellular hydrolysis occurs which favours cancer cell survival. Macrophages, which attack and eliminate cancer cells, can be activated by macrophage activating factor (MAF) which displays an α-N-acetylgalactosamine residue that appears essential for the activation cascade. Cancer cells secrete an α-N-acetylgalactosaminidase enzyme that acts to decrease the potency of MAF, thus promoting cancer cell survival. Inhibition of cancer cell α-N-acetylgalactosaminidase may restore macrophage activation and generate potential therapeutics. Chapter 1 of this thesis contains extended discussion of the aforementioned, and related, diseases and the therapeutic applications of iminosugars. Some historically and biologically important iminosugars are described along with some current iminosugar drugs. Chapter 2 describes the synthetic strategies explored in an attempt to synthesise all the members of the 2-acetamido pyrrolidine iminosugars. An overview of the compounds synthesised towards this end by a past group member is given along with the work performed as part of this thesis to complete this goal. Both enantiomers with arabino- and ribo- stereochemistry and D-lyxo- were previously synthesised. The syntheses of both enantiomers with xylo- stereochemistry along with the L-lyxo- compound were completed as part of this thesis, from either D- or L-glucuronolactone and D-ribose, respectively. Chapter 3 details the synthetic strategy adopted to synthesise the enantiomer of D-DNJNAc, the first potent α-N-acetylgalactosaminidase inhibitor to be found. The synthesis towards another piperidine iminosugar, 6-deoxy DGJNAc, is presented in the second half of this chapter, along with two related compounds.

Iminosugars are polyhydroxylated alkaloids, and can be viewed as sugar analogues in which the endocyclic oxygen atom has been replaced with nitrogen. These compounds are highly medically relevant and their biological activity is largely due to their inhibition of glycosidases. Several examples of the iminosugar class are currently marketed as drugs, and many more are in earlier stages of development for a variety of diseases and disorders. The most fruitful approaches to the chemical synthesis of iminosugars have utilised carbohydrate starting materials as optically pure chiral building blocks, or chirons. Most of the monosaccharides are not readily available, but the relatively few naturally abundant cheap sugars have been exploited as chirons for over a century. The availability of the rare sugars is growing with the development of a new biotechnological approach to their synthesis, known as Izumoring. This thesis is primarily concerned with the chemical synthesis of iminosugars from carbohydrate starting materials. The synthesis of unnaturally functionalised sugar polyols and their suitability as substrates for the Izumoring process is also discussed. Chapter 1 provides a brief general overview of the history, natural occurrence and therapeutic application of iminosugars. General strategies for their synthesis from carbohydrate chirons are discussed. Chapter 2 concerns divergent syntheses of several iminosugar targets from both enantiomers of glucuronolactone and their biological evaluation. A new scaleable synthesis of the natural product 1-deoxynojirimycin is presented that has since been adopted for commercial purposes, as well as an efficient strategy for the synthesis of both enantiomers of 2,5-dideoxy-2,5-imino- mannitol and their novel amino acid analogues. Access to hexosaminidase inhibiting acetamido- substituted piperidines is presented, including 2-acetamido-1,5-imino-1,2,5-trideoxy-D- galactitol, which has been found to be one of the few known potent and specific inhibitors of α- N-acetyl-galactosaminidase. This inhibitory profile may allow the compound’s use for further investigation of a strategy for cancer treatment. Chapter 3 concerns the synthesis of carbon branched pyrrolidines and their biological evaluation. A novel and highly potent α-glycosidase inhibitor has been discovered, synthesised by a strategy that utilises the benzhydryl ether as key protecting group. A mild method for the introduction of this protecting group has been shown to be general to a range of sterically congested and/or acid/base sensitive carbohydrate lactones. Chapter 4 concerns the synthesis of deoxygenated and fluorinated sugar alcohols and their successful biotechnological transformation into ketoses by the Izumoring process. Publications arising from this work are included in the Appendix.

Computational tools play an important role in the description of biological systems. Scientists describe and study structure, conformational changes and interactions between molecules in silico, often as a cheaper and faster alternative for biosynthesis. The simulated dynamic behavior in time of a molecular system is a straight forward source of information about substrate-enzyme interactions at the atomic level, and a powerful tool for the identification of molecular properties important in enzymatic reactions. Our study is focused on the computational investigation of structure and substrate specificity of hydrolases important in biotransformation. The computational work was performed in close collaboration with biochemists-experimentalists from Charles University and the Microbiological Institute of the Academy of Sciences of the Czech Republic. Hydrolases have great a potential in the chemoenzymatic synthesis of modified carbohydrates with regulated properties. Carbohydrates, as substrates of hydrolases, are important in normal functionality of many organisms. They have a dual role in immune response regulation: some carbohydrates (like GlcNAc and ManNAc) participate in activation and some (like GalNAc) in suppressing immunity; glycosidase deficiency is associated with a number of lysosomal disorders. We used homology modeling, computational docking and molecular dynamics simulation (MD) methods for the complex study of fungal hydrolases: alpha-galactosidase/alpha-N-acetylgalactosaminidase from Aspergillus niger; beta-N-acetylhexosaminidases (HEX) (from Aspergillus oryzae and Penicillium oxalicum); nitrilase from Aspergillus niger. Our structural study unambigously demonstrates that the enzyme encoded by genes variant A (aglA) from A. niger is able to accept alpha-N-acetylgalactosamine as its substrate and explains structural features responsible for its specificity. Homology models of HEXs from P. oxalicum and A. oryzae were built and compared. Homology models were used to study the role of protein glycosylation, disulfide bonds, dimer formation and interaction with natural and modified substrates. Model of nitrilase from Aspergillus niger helped to analyze multimer formation.

in English β-N-Acetylhexosaminidase (EC 3.2.1.52) is exoglycosidase, which exhibits the unique properties in the filamentous fungi. Enzyme from these organisms are dimeric, inducible and secreted extracelluary. It is expresed as preproprotein, consists of a signal sequence, a large propeptid and a catalytic subunit. Although the enzyme is widely distributed, its structure differs in varies organisms. Bacteria have only monomeric hexosaminidase. Human enzymes are dimeric as well as fungal, but only hexosaminidase from filamentous fungi have the catalytic subunit noncovalently associated with the propeptide. Propeptide is a essential for the enzyme activity. It exists a homologues model of the catalytic subunit of β-N-acetylhexosaminidase from Penicillium oxalicum, but the structure of the propeptide has not yet been solved. The first part of this diploma thesis deals with the optimization of production and purification conditions. The second part deals with structural studies of β-N-acetylhexosaminidases from the filamentous fungi Penicillium oxalicum CCF 3438. These studies were carried out using chemical cross-linking and high resolution mass spectrometry. The combination of these methods revealed region of the noncovalent interaction of the catalytic subunit with the propeptide.

Tomáš Křížek: Applications of Capillary Electrophoresis in Life Sciences (Dissertation thesis) ABSTRACT This thesis is focused on the applications of capillary electrophoresis in two important areas of life sciences, proteomics and enzyme assays. In the first part, Pluronic F-127 copolymer was studied as a sieving matrix for proteomic applications of capillary gel electrophoresis. The effect of thermoassociation of Pluronic F-127 on the separation selectivity was investigated and no difference in selectivity of the separation below, inside and above the thermoassociation temperature region was observed. The performance of Pluronic F-127 in capillary gel electrophoresis was compared with dextran as a commonly used sieving matrix. The results showed, that Pluronic F-127 offers superior performance for low-molecular-mass proteins because it provides higher separation power than dextran with significantly lower viscosity of the background electrolyte. The lower viscosity makes the polymer easier to replace after each analysis, which leads to remarkably higher repeatability of the experiments. On the other hand, dextran, due to its higher viscosity, was shown to be more convenient for separations of protein digests, where extremely high separation efficiency is required. The second part focuses on...

in English β-N-acetylhexosaminidase (EC 3.2.1.52) belongs to exoglycosidase, and is one of the most abundant enzymes found in organisms from bacteria to human. The fungal β-N-acetylhexosaminidase from Aspergillus oryzae is composed of propeptide and catalytic domain. The propeptide is noncovalently associated with the catalytic domain of the enzyme. Propeptide is essential for the enzyme activity. While the structure of the catalytic domain was desidned by homology modeling, the structure of the propeptide has not been resolved yet. In this study, the position where the propeptide is associated with the catalytic domain, was uncovered. Presented work consists of two parts. First part deals with optimization of production conditions, purification and crystallization of β-N-acetylhexosaminidase from the filamentous fungi Aspergillus oryzae. Second part is devoted to the study of interaction between propeptide and catalytic domain, which was characterized by chemical cross-linking and high-resolution mass spectrometry. It was found that the structural changes of the catalytic domain depend on the presence of the propeptide molecule. Moreover the region of propeptide-catalytic domain interaction was revealed.

The aim of the work described in this thesis was to explore the synthesis of glycosidase inhibitors and to evaluate their potential as anti-cancer agents. Glycosidases catalyze the fission of glycosidic bonds and are involved in vital biological functions. With regard to their potential for anti-cancer therapy, two glycosidases were identified: α-N-acetyl-galactosaminidase and β-N-acetyl-hexosaminidase. The former has been implicated in causing immunosuppression in advanced cancer patients by negating the effect of the macrophage activating factor (MAF), while the latter is secreted by invading cancer cells and hence associated with metastasis formation. The synthetic focus was on generating piperidine and azetidine iminosugars, carbohydrate mimetics with their endocylic oxygen replaced by nitrogen. Their structural similarity to carbohydrates make iminosugars excellent inhibitors of glycosidases. Following synthesis of a pipecolic amide, its previously reported potent β-N-acetyl-hexosaminidase inhibition was confirmed. This data, along with inhibition profiles of several pyrrolidines, allowed the generation of a molecular model for predicting activity of β-N-acetyl-hexosaminidase inhibitors. The model was used to select azetidines in the D/L-ribo and D-lyxo configuration as suitable candidates to be explored in novel chemical space, leading to the first synthesis of a fully unprotected 3-hydroxy-2-carboxy-azetidine. The potent α-N-acetyl-galactosamindase inhibitor 2-acetamido-1,2-dideoxy-D-galacto-nojirimycin (DGJNAc) was successfully derivatised via N-alkylation. Important structural discoveries with regard to glycosylation of vitamin D₃-binding protein, the precursor of MAF, were made using MALDI mass-spectrometry. By comparing the enzymatic and cellular inhibition of N-alkylated derivatives of DGJNAc and a pyrrolidine the following generalization on iminosugar biodistribution was found: N-butylation promotes uptake into the cell/organelles, while hydrophilic side-chains restrict cellular access. An in vitro assay evaluating cancer cell invasion was devised and β-N-acetyl-hexoamindase inhibitors were shown to retard cell migration, including with the highly metastatic breast cancer cell line MDA-MB-231. Additive effects where found when the iminosugar was combined with a protease inhibitor, suggesting potential for future combination therapy.

Plants have developed a number of ways how to minimise negative influence of the environment. As a consequence of stress action, plants carbohydrate metabolism is quite often influenced, esp. on the level of expression and activities of different enzymes and also several metabolites concentration. One of key enzymes of carbohydrate metabolism is invertase. The aim of this work was to find out, whether the activity of its isoforms (cytoplasmic, vacuolar and extracellular) in tobacco plants is influenced by Potato virus Y (PVY). It was shown, that activity of cytoplasmic invertase was not affected, but the activity of vacuolar and extracellular isoform was enhanced during potyviral infection. Hence, it is likely, that vacuolar and extracellular invertases are related to plant antiviral defence. The effect of PVY on other enzymes of carbohydrate metabolism and several metabolites content was studied. Activity of α-amylase and phosphorylase, starch-degrading enzymes, was strongly enhanced during potyviral infection. That is probably how plant cells get glucose, which is a key source of energy and metabolites for biosynthesis of different compounds. It may also serve as a signal molecule. Activity of other hydrolytic enzymes, β-glucosidase and β-hexosaminidase, was also slightly increased. There was no...