The regulation of metabolic and mitogenic cellular processes is a complex system, depending on the precise function of several signalling cascades. One example is the insulin pathway, which is mediated by a group of three sequentially and structurally highly similar hormones (insulin, insulin-like growth factor (IGF) -I and -II) and their homologous tyrosine kinase receptors (IR-A, IR-B, IGF-1R). Such a high degree of homology leads to crosstalk in receptor communication, with each of the ligands triggering different biological responses. The design of insulin analogues activating primarily metabolic effects or IGF antagonists suppressing an unfavourable mitogenic response is one of the main goals in this research field, which can be facilitated by identification of the regions responsible for receptor binding. The work included in this thesis is focused on the effects on receptor interactions following the introduction of selected elements from the IGF-I primary sequence into the IGF-II molecule. In particular, these include a point mutation of Ser29 to Asn, an insertion of Gly-Ser after Arg34 , an insertion of Pro-Gln after Ser39 , or combination of both insertions. Although the IGF-II modifications described here negatively affected binding affinity towards IR-A, they did not enhance the IGF-1R...

Hydrophilic interaction liquid chromatography is in the present time very often used for separation of polar compounds which are complicated separated in reverse phase HPLC or they are not sufficiently retained. Various stationary phases suitable for HILIC are commercially available and new stationary phases are still developing. In this work bare silica and isopropyl cyclofructan modified silica stationary phases were compared. Chromatographic behavior of sets of four pentapeptides and five nonapeptides was tested in detail under hydrophilic interaction liquid chromatography. Additionally, mobile phase composition changes the types of interaction and their strengths were studied using linear free energy relationship method. Obtained results denoted that the main role in the retention mechanism on the isopropyl cyclofructan stationary phases play hydrogen bond acidity and dipolarity/polarizibility, while dispersion interactions are preferred in the mobile phase. On the other hand, bare silica stationary phase provides only one interaction type - hydrogen bond acidity. Key words: peptides, cyclofructan, LFER, HILIC

Sand fly saliva contains proteins and peptides that have an important role in bloodfeeding. Some of those proteins are antigenic and repeated sand fly bites result in a specific antibody response of the bitten host. Antigenic salivary proteins of Phlebotomus orientalis, main vector of visceral leishmaniasis in Sudan and Ethiopia, were identified using immunoblot with dog sera. The 5 most promising antigens were expressed in an E. coli bacterial system. Subsequently, these proteins were tested in ELISA with sera of domestic animals from Ethiopia naturally exposed to P. orientalis, and with sera of mice bitten experimentally by this sand fly species. Salivary gland homogenate (SGH) was used as the positive control. The best antigenic properties were detected in two recombinant proteins, Yellow-related protein PorSP24 and ParSP25-like protein PorSP65, especially in tests with sheep and dog sera. However, nonspecific binding of dog sera was also detected using both antigens. In addition, we proved that sera of mice repeatedly bitten by P. papatasi and Sergentomyia schwetzi do not crossreact with SGH and the tested recombinant proteins of P. orientalis. In a second part of this thesis we designed peptides representing epitopes recognized by specific anti-saliva antibodies. Two peptides were derived from...

Leishmania is a protozoan parasite of vertebrates transmitted by the bite of infected phlebotomine sandflies. In humans, it causes a disease called leishmaniasis, which ranks as one of the most serious neglected tropical diseases. In the vectorial part of the life cycle, the crucial moment is when the flagellate forms (promastigotes) attach to the midgut epithelium of the sandfly. For most leishmania species, little is known about which types of phlebotomine receptors and leishmania surface antigens participate in the binding. Phage display was used to screen for Leishmania mexicana peptide ligands which may play a role in such binding. By affinity selection of phages incubated with promastigote cells, 16 unique peptides were identified. Fluorescent labelling of peptide-bearing phages indicated their putative binding sites on the leishmania surface. Based on the hypothesis that the identified peptides may be a part of receptors found in the phlebotomine midgut, experiments were performed where the sandflies were infected with promastigotes whose binding sites were blocked by two different peptide-bearing phages. The extent of the infection was different between the two cases. However, no statistically significant difference from the control group was observed. Despite unsuccessful attempts to identify a...

Master’s thesis deals with the design and testing of methods of increasing the transfection efficiency in mammalian cells. In this work a polyethylenimine (PEI) transfection method was used. The transfection efficiency was tested using MDA-MB-231 breast cancer cell line and pcDNA3.1-GFP-hMT-TOPO vector. We focused on optimization of PEI (1-6 µg) and plasmid DNA (1-3 µg) concentration, transfection time (0-48 h), number of cells per 9 cm2 (100,000-2 million) and medium composition. A complex containing linear penetration peptides (CPPs) KALA and GALA was assembled. Concentration of CPPs (7,5-45 µg) was optimized and a zeta potential and a size of the complexes were investigated. Due to the formation of large aggregates (over 1200 nm), modification with polyethyleneglycol (PEGylation) of PEI was performed. With the resulting complexes (~15 nm), transfection was performed. Transfection and apoptosis induction rates were compared between PEI and PEGylated PEI and as the best was chosen conjugate of PEGylated PEI (3 µg) with 45 µg of GALA and 3 µg pDNA with efficiency of 75%.

In English The thesis deals with research of novel antimicrobial peptides (AMP) Halictines (HAL-1, GMWSKILGHLIR-NH2 a HAL-2, GKWMSLLKHILK-NH2) and their structural analogs isolated from the venom of the wild bee Halictus sexcinctus. The structure and antimicrobial activity of these peptides had been described earlier [1]. The goal of this diploma thesis is to find peptide which is strongly toxic only for cancer cells and nontoxic for normal cells. Using of the fluorescent marked peptides we aimed to acquire the information about mechanism of action of the studied peptides on the cells. Using the MTT test (determination of valuation IC50), the toxicity of HAL-1 and HAL-2 and their analogs against 2 normal cell lines (Human umbilical vein endothelial cells, HUVEC, and normal rat intestinal cells, IEC) and against 2 cancer cell lines (cancer cells of suppository uterine, HeLa-S3 and cancer cells of human colorectal carcinoma, CRC SW 480) was determined. First we tested antimicrobial peptides with antimicrobial activity and low hemolytic activity. For verification the toxicity of less active analogs was also determined. We found out that the HeLa-S3 cells are the most sensitive to these peptides. The most toxic peptides (HAL-1/9, HAL-1/18, HAL-2/2) kill 50% of cells in the concentration 2,5 - 10 µM. To obtain...

Osteogenic differentiation of mesenchymal stem cells (MSCs) would be possible to induce by creating of a cell bioactive scaffold that mimic the properties of bone extracellular matrix (ECM). This induction will be not only due to the addition of osteogenic supplements, but also due to the addition of differentiation peptides. These peptides activate signaling pathways leading to cell differentiation. The aim of this study was to evaluate the effect of selected peptides on adhesion, metabolic activity, proliferation and osteogenic differentiation of porcine MSCs. Four peptides with amino acid sequences of DGEA, IAGVGGEKSGGF, GQGFSYPYKAVFSTQ and KIPKASSVPTELSAISTLYL were selected. These peptides were derived from receptor binding sequences of collagen I, collagen III, BMP-7 and BMP-2 respectively. Scaffolds were prepared from a biocompatible and biodegradable poly-ε-caprolactone (PCL) polymer, suitable for cell cultivation. Cells were cultured on scaffolds for three weeks. Various concentration of differentiation peptides were added to the culture medium. As observed in the experiment of cells cultured in basal medium supplemented with differentiation peptides no effect on adhesion, proliferation or metabolic activity of porcine MSCs was observed. In groups treated with peptides derived from BMP-2...

Apart from biological functions, peptides are of uttermost importance as models for un- folded, denatured or disordered state of the proteins. Similarly, miniproteins such as Trp-cage have proven their role as simple models of both experimental and theoretical studies of protein folding. Molecular dynamics and computer simulations can provide an unique insight on processes at atomic level. However, simulations of peptides and minipro- teins face two cardinal problems-inaccuracy of force fields and inadequate conformation sampling. Both principal issues were tackled in this theses. Firstly, the differences in several force field for peptides and proteins were questioned. We demonstrated the inability of the used force fields to predict consistently intrinsic conformational preferences of individual amino acids in the form of dipeptides and the source of the discrepancies was traced. In order to shed light on the nature of conformational ensembles under various denatur- ing conditions, we studied host-guest AAXAA peptides. The simulations revealed that thermal and chemical denaturation by urea produces qualitatively different ensembles and shift propensities of individual amino acids to particular conformers. The problem of insufficient conformation sampling was dealt by introducing gyration- and...

EN The growing emergence of bacteria resistant to conventional antibiotics is very alarming. This has prompted an intensive search for alternative antimicrobial agents which kill bacteria with different modes of action than do traditional antibiotics and do not develop drug resistance. Among these, antimicrobial peptides (AMPs) are considered as promising compounds against resistant pathogens. These positively charged peptides permeabilize or disrupt bacterial cell envelope which leads to leakage of cytoplasmic components and cell death. The aim of my dissertation thesis was the study of the action mechanism of novel antimicrobial peptides which I have isolated from the venom of different wild bees. I identified six novel AMPs which were named panurgines (PNG), codesane (COD) and antapines (ANTPs). These peptides were isolated from the venom of three different bee species (Panurgus calcaratus, Collete daviesanus and Anthophora plumipes). I was also involved in the structural studies of lasiocepsin (Las), the antimicrobial peptide identified in the venom earlier in our laboratory. All studied peptides possess activity against various strains of bacteria and low or moderate hemolytic activity. We prepared series of PNG, COD and ANTP analogs in order to study the effect of physicochemical properties...

Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of: Biophysics and Physical Chemistry Consultant: Doc. Ing. Alice Lázníčková, CSc. Student: Pavla Kosová Title of thesis: Radiolabeling of PCTA-Lys3-Bombesine and determination of stability of the radiolabeled product The dissertation deal with study of radiolabeling of new receptor-specific peptide - bombesin, which was modified with chelator PCTA, PCTA-Lys3-bombesin, with radionuclide 177 Lu, and determination of labelled product quality and its stability. For quality control we used method high-efficiency liquid chromatography (HPLC) and thin-layer chromatography (ITLC) on ITLC-SG. HPLC offer clear proof about pollution and possible dissociative outputs. Comparison of both methods for determination of purity of labelled peptide showed that HPLC is more accurate than TLC method.