Significant morbidity and mortality continues to be associated with breast cancer and its treatments. Fisetin, a phytochemical that is present in many fruits and vegetables, has demonstrated anticancer activity. My research explores fisetin as a possible novel therapeutic modality for breast cancer. Breast cancer cell lines (MDA-MB-468, MDA- MB-231, MCF-7, T47-D, SKBR-3; mitoxantrone-resistant (MITX) and paclitaxel- resistant (Tx400) cell lines) were exposed to fisetin and cell survival was assessed by MTT, crystal violet, acid phosphatase, and colony-forming assays. Normal cells (human mammary epithelial cells, fibroblasts, human umbilical vein endothelial cells) were used as negative controls. The mechanism of action of fisetin was explored using cell cycle analysis and assays for apoptosis/necrosis, including Annexin V-propidium iodide staining and LDH-release. Apoptosis induction pathways were studied using Western blotting, as well as caspase inhibitors and cell viability assays. Flow cytometry was used to assess mitochondrial membrane stability (DiOC6 staining) and reactive oxygen species (ROS) production (dihydroethidium staining). Fisetin had a dose- and time-dependent cytotoxic effect on breast cancer cell lines (e.g., 100 ?M fisetin decreased MDA-MB-468 cell number by 70% at 72h in both crystal violet and acid phosphatase assays). In contrast, the viability of normal cells was not substantially affected by concentrations of fisetin that killed breast cancer cells. Fisetin-treated breast cancer cells showed cell cycle arrest (MDA-MB-468 cells arrested at G2/M phase; MDA-MB-231 cells arrested in S- phase) and death by apoptosis (e.g., MDA-MB-468 cells showed up to 50% apoptosis and 8% late apoptosis/necrosis by Annexin V-staining; cell cycle analysis and LDH- release assays supported these results). Fisetin-induced apoptosis was associated with mitochondrial membrane permeabilization, as well as activation of the caspase cascade since the pro-apoptotic effect of fisetin was reduced in the presence of a pan-caspase inhibitor. In addition, fisetin did not cause ROS production in MDA-MB-468 or 231 cells, ruling out a role for ROS in fisetin-mediated cytotoxicity. My findings suggest that fisetin may be useful in the treatment of breast cancer.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Nikola Kopková Supervisor: Assoc. Prof. Přemysl Mladěnka, Pharm.D., Ph.D. Title of diploma thesis: Antiplatelet effects of flavonoids The most common antioxidants in ordinary food are flavonoids. They show antioxidant and anti-aggregation effects and other positive effects on cardiovascular diseases. Flavonoids are promising candidates to be antiplatelet drugs. Although several mechanisms responsible for antiplatelet activity have been suggesteed, only a few have been documented by published studies. The inhibition of blood platelet aggregation by flavonoids is reversible, which is another important factor. Data on thrombin-induced aggregation are controversial, some claim that flavonoids have no effect, the others says they have positive effects. (In the case of quercetin and genistein, inhibition of aggregation induced by thrombin was documented). The effect on arachidonic acid in the aggregation cascade is well documented, but there are several inconsistencies resulting from the use of different materials. Other mediators of aggregation are phospholipase A2, which plays a key role in the formation of inflammatory mediators. In this case, it has been shown that mainly genistein is capable of...

Heat stress poses a serious threat to plant survival and productivity, and has a direct influence on crop yield stability. Plants response to high temperature is tightly controlled by complex genetic networks. Plants can be acclimated through gradual pre-exposure to increasing temperatures and that in turn causes higher survival in subsequent and otherwise lethal heat stress conditions. To investigate the physiological and molecular processes underlying heat acclimation and recovery, we examined changes in Arabidopsis thaliana transcriptome throughout the acclimation and the subsequent heat shock treatment. Groups of differentially expressed genes and enriched biological pathways that constitute the heat transcriptional memory were identified. The function of flavonoids in plant heat stress were further explored experimentally. In addition, we observed altered stomata density and aperture responses in heat acclimated plants, and this might be partially controlled by AGAMOUS-LIKE16 (AGL16) transcription factor and its negative regulator microRNA824 (miR824). By utilizing an automated non-invasive phenotyping facility, we have developed a protocol to record plant growth and photosynthetic performance after heat stress in wild type Arabidopsis thaliana and mutant lines at daily intervals. Through an imaging-based analysis of plants growth, we confirmed impaired thermotolerance of hsp101 compared to wild type plants by a time-series growth, morphology and chlorophyll responses. This offers a novel experimental setup for thermotolerance screenings in Arabidopsis, with defined digital markers linking the function of selected genes in heat stress responses to phenotypic traits.

Flavonoids are important and ubiquitous secondary metabolites and are known to participate in various developmental and stress response processes in plants. Common flavonoids include anthocyanins, proanthocyanidins and flavonols. This thesis aims to determine, at the molecular level, how the biosynthesis of flavonoids, in particular the proanthocyanidins, is regulated in poplar. Poplars accumulate large amount of flavonoids and the major flavonoid biosynthetic genes in poplar have been identified. Flavonoid biosynthesis is known to be regulated by MYB transcription factors. Previous work had identified MYB134 as a key regulator of proanthocyanidin synthesis in poplar. Here I describe experiments on five additional genes encoding MYB activators (MYB115 and MYB117), MYB repressors (MYB165 and MYB194), and one bHLH cofactor (bHLH131) as possible flavonoid regulators in poplar. The objective of this work is to determine the in planta functions of these new flavonoid regulators using reverse genetic methods, phytochemical and transcriptome analysis, to identify their target genes and to determine how these transcriptional regulators interact using promoter transactivation and yeast two- hybrid assays. MYB115 was identified as a second proanthocyanidin regulator. Similar to the effects of MYB134, overexpression of MYB115 in poplar led to increased proanthocyanidin content and upregulated flavonoid biosynthesis genes, but reduced the accumulation of salicinoids. Overexpression of repressor type MYBs, MYB165 or MYB194 led to reduced anthocyanin, salicinoid and hydroxycinnamic ester accumulation in leaves, while reducing proanthocyanidin content in roots. Transcriptome analysis demonstrated the downregulation of most flavonoid genes in these transgenics, as well as some shikimate pathway genes, confirming the broad repression function on the phenylpropanoid pathway. By contrast, MYB117 encodes an anthocyanin activator, and was shown to be specific to this branch of the flavonoid pathway. Overexpression of MYB117 in transgenic poplar increased accumulation of anthocyanin in all tissues, resulting in red poplar plants. One bHLH cofactor, bHLH131 was shown to interact with both MYB activators and repressors and required by MYB activators to activate flavonoid gene promoters. This indicate an important role of bHLH131 in the flavonoid biosynthesis. Proanthocyanidin MYB activators, MYB134 and MYB115 could activate each other. This indicates a positive feedback loop of proanthocyanidin MYB activators. Interestingly, repressor MYB165 suppressed expression of other flavonoid MYB repressors including MYB194 and MYB182, which shows a negative feedback loop of MYB repressors. The expression of bHLH131 was also regulated by MYB activators and repressors. These results reveal the complex interaction between these regulators. iii iv Unexpectedly, overexpression MYB134, MYB115 or MYB117 poplars upregulated flavonoid 3’5’-hydroxylase and cytochrome b5 genes, and lead to enhanced flavonoid B- ring hydroxylation and an increased proportion of delphinidin, myricetin Overexpression of flavonoid 3’5’-hydroxylase in poplar confirmed its function in enhancing B-ring hydroxylation. However, overexpression of cytochrome b5 in flavonoid 3’5’-hydroxylase- overexpressing plants did not further increase flavonoid B-ring hydroxylation. Thus its role in flavonoid B-ring hydroxylation remained unclear. These results show that flavonoid MYBs could also alter flavonoid structure. Together, these studies outline the complex regulatory network formed by flavonoid MYB activators and repressors, and bHLH cofactors controlling both flavonoid accumulation and structure. / Graduate / 2020-12-04

Extract from milk thistle (Silybum marianum (L.) Gaertn., synonym Carduus marianus L., Asteraceae) silymarin contains among others primarily bioactive flavonolignans. They have hepatoprotective and antioxidative effects and also anticancer, chemoprotective, dermatoprotective and hypocholesterolemic activity. This thesis focuses on the preparation of metabolites of the second phase of biotransformation unexplored flavonolignans 2,3-dehydrosilybin (DHSB), silychristin (SCH), 2,3-dehydrosilychristin (DHSCH). Pure sulfated derivatives were prepared using aryl sulfotransferase from Desulfitobacterium hafniense and p-nitrophenyl sulfate (p- NPS) as a donor. Flavonolignans yield exclusively monosulfates at the position C- 20 (C-19 in the case of silychristin and 2,3-dehydrosilychristin), except for 2,3- dehydrosilybin that gives also the 7,20-disulfated derivatives. For all samples were made antioxidant tests - DPPH assay (the highest activity had 2,3-dehydrosilychristin sulfate: IC50= 7,87 µM), Folin-Ciocalteau reduction assay (the highest activity had 2,3-dehydrosilychristin: 1,58 ekvivalents of gallic acid), ABTS+ scavenging (the highest activity had silychristin: 1,50 ekvivalents of vitamin C), inhibition of microsomal lipid peroxidation (the highest activity had 2,3-dehydrosilybin: IC50 = 10,6 µM),...

Flavonoids have multiple subclasses. A major subclass of flavonoids is flavonols. Flavonols are the most abundant subclass of flavonoids and are widely spread throughout nature. Flavonols are identified as having a hydroxyl group in the 3rd position of the C ring. The most prevalent modification to flavonols is glucosylation which adds glucose to an acceptor molecule. The flavonol specific 3-O glucosyltransferase (Cp3GT) enzyme from grapefruit (Citrus paradisi) is the topic of this research and specifically adds glucose to flavonols at the 3-OH position. The level of activity with Cp3GT and a flavonol varies depending on the flavonol structure. Since there is varying activity with Cp3GT, Cp3GT is an ideal model system for studying the structure/function relationship of Cp3GT site-directed mutants. Multiple mutants of Cp3GT were created by site directed mutagenesis. The mutant of study is S20G+T21S. As compared to the wild type Cp3GT, S20G+T21S has significantly higher activity with kaempferol, quercetin, dihydroquercetin, and naringenin. One of the more striking difference of S20G+T21S is its ability to add a glucose molecule to the 7-OH position of naringenin. Naringenin is a flavanone and indicates that S20G+T21S has a change in flavonoid class specificity as well as regiospecificity for the addition of glucose. The S20G+T21S mutant was first verified in E. coli via DNA sequencing. Next, S20G+T21S was transformed into Pichia pastoris by linearizing S20G+T21S DNA using Sac I, phenol chloroform purification, and electroporation. Transformation was verified by colony PCR and DNA sequencing. After verification, a time course analysis of expression conduction was completed. Optimal expression was concluded to be 24 hours and was verified by SDS-Page gel and western blot. In preparation for crystallization, S20G+T21S was purified using an IMAC infinity column. Purification was verified using a western blot, Coomassie blue, and silver stain. Progress on optimizing the crystallization conditions for S20G+T21S will be reported.

The organization of biological processes via protein-protein interactions and the subcellular localization of enzymes is believed to be fundamental to many aspects of metabolism. Although this organization has been demonstrated in several systems, the mechanisms by which it is established and regulated are still not well understood. The flavonoid biosynthetic pathway offers a unique system in which to study several important aspects of metabolism. Here we describe a novel toolset of mutant alleles within the flavonoid biosynthetic pathway. In addition, we discuss the use of several of these alleles together with a number of emerging technologies to probe the role of subcellular localization of chalcone synthase, the first committed flavonoid biosynthetic enzyme, on metabolic flux, and to characterize a novel chalcone synthase-interacting protein. The over-expression of this interacting protein induces novel phenotypes that are likely associated with the production or distribution of auxin. Further, interaction analyses between recombinant flavonoid biosynthetic enzymes point to the possibility that post-translational modifications play an important role in promoting interactions. / Ph. D.

München, Techn. Univ., Diss., 2001. / Computerdatei im Fernzugriff.

München, Techn. Universiẗat, Diss., 2001.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical botanic and ekology Candidate: Nikol Vavřichová Supervisor: Ing. Kateřina Macáková Ph.D. Title of diploma thesis: Iron - chelating effects of flavonoid metabolites - small polyphenolic substances (2015/2016) Iron is a trace element which is necessary for function of an organism. There is no specific excretion mechanism for iron, therefore any deflection causes deficiency or abundance. Iron abundance leads to hemochromatosis. An application of iron chelators is a treatment of choice for hemochromatosis. There are three clinically used chelators for treatment of iron overload: deferoxamine, deferasirox and deferiprone. These drugs are inappropriate for therapy (side effects, dosage form). Therefore there is a place for serching for new types of medicine for treatment of hemochromatosis. Flavonoids are natural substances with positive influence on a human organism. The most investigated effects are antioxidant and chelating activity. As flavonoids are metabolised in a human body, there is an arising question if flavonoid metabolites are also able to chelate iron ions. For this work we chose derivates of benzoic acid: benzoic acid, 3 - hydroxybenzoic acid, 4 - hydroxybenzoic acid, 2,4 - dihydroxybenzoic...