|
|
Use of Hsp90 as a target of biological therapy of tumors
Bednárová, Kristína ; Bouchalová,, Pavla (referee) ; Müller, Petr (advisor)
Antibody-drug conjugates (ADCs) represent a relatively new class of highly potent anti-tumor drugs. Thanks to highly specific monoclonal antibodies, ADCs are able to deliver a cytotoxic payload directly to tumor cells and thus minimize damage to healthy cells. Therapeutic efficacy depends on the selection of an appropriate antigen that undergoes internalization upon conjugate binding. For this project, pro-oncogenic Hsp90 and c-Met were selected as potential targets. Hsp90 is a molecular chaperone that is overexpressed in tumor cells and, in addition, can be translocated to the membrane of these cells. Overexpressed Hsp90 contributes to angiogenesis, tumor cell motility or metastasis. C-Met is a receptor tyrosine kinase that plays a central role in epithelial morphogenesis and malignant transformation. Its increased activity induces pathways responsible for the proliferation, invasion and migration of malignant cells. The aim of the diploma thesis was to study the potential use of antibodies with anti-Hsp90 and anti-c-Met activities in anti-tumor therapy. The experimental part involved the purification of the EEV1-2.1 antibody with anti-Hsp90 activity and its subsequent characterization. Furthermore, it included the characterization and selection of anti-c-Met antibody clones. It was also focused on selection and optimization of the right conjugation strategy. The activity of the antibodies and their conjugates was examined by fluorescence microscopy and flow cytometry. In vivo experiments were further aimed at verifying the efficacy of ADC by monitoring the rate of inhibition of proliferation of selected tumor cell lines. The results revealed that the EEV1 antibody does not enter the cells specifically by antigen-mediated way, and is therefore not suitable for use in conjugation with a cytostatic drug. On the other hand, anti-c-Met antibody ADC conjugates exhibit high affinity for native antigen, internalization through antigen binding, and additionally inhibited proliferation of c-Met overexpressing OE33 cells.
|
|
|
Horizontální přenos genů rezistence mezi bakteriemi
Valach, Radek
Transfer of bacterial antibiotic resistence is highly discussed topic in past years. Its spreading is reason of creating super-resistant bacterial strains, that are resistant to a wide range of conventionally used drugs. Zinc oxide, used in pig farming as a feed supplement, has in recent years replaced antibiotics, given to prevent gastrointestinal disease and to promote growth. These substituent may increase transfer of genetic elements carying antibiotic resistence genes. This thesis is focussed on possible influence of zinc oxide and its nanoparts on Enterococcus faecalis bacteria. The aim of this work was to investigate whether the action of ZnO and ZnONPs would increase the transfer of a resistance gene plasmid between bacteria through the process of conjugation.
|
|
|
Surface functionalization of the biological gold nanoparticles for micro-rna targeting
Pourali, Parastoo ; Benada, Oldřich ; Benson, Veronika
Among non-viral gene carriers with low toxicity and high transfection efficiency, the use of gold nanoparticles (AuNPs) is of particular interest due to their biocompatibility and special properties. This is the first time we attempted to functionalize the surface of the biological AuNPs in order to conjugate them with antimiR-135b through electrostatic interactions and knockdown the microRNA-135b gene expression inside the cells. A fungal strain, Fusarium oxysporum, was cultured in Sabouraud Dextrose Broth (SDB), centrifuged, and the mycelium-free supernatant was challenged with 1 mmol final concentration of HAuCl4.3H2O and incubated for 24 h at 37°C in a shake flask. AuNPs were characterized by visible spectrophotometry, Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), Energy-Dispersive X-ray spectroscopy (EDS), and a zetasizer. The washed and sterilized AuNPs were used for cytotoxicity and conjugation assays. First transferrin (Tf) and then polyethylenimine (PEI) were used to functionalize and change the surface charge of the AuNPs and then antimiR-135b was conjugated to the AuNPs trough electrostatic interactions. Their association was confirmed by visible spectrophotometry and electrophoresis. Confocal microscopy was used to investigate the internalization of the AuNPs-antimiR-135b complex. The results proved the formation of AuNPs with a maximum absorption peak at 528 nm, round and oval shapes (15-20 nm), and average zeta potential of -21.02 mV. The AuNPs-antimiR-135b showed delayed electrophoresis unlike antimiR-135b or AuNPs alone. Functionalized AuNPs did not cause any toxicity in cell culture and confocal microscopy showed successful transfection of AuNPs-antimiR-135b into the vast majority of 4T1 cells. We concluded that the biological AuNPs were non-toxic and they could carry antimiR-135b to enable gene silencing
|
|
|
Redox Properties of Conjugated Phosphole Derivatives – Electrochemical Study
Koláčná, Lucie ; Polák, P. ; Tobrman, T. ; Ludvík, Jiří
The study describes pentasubstituted phospholes with a π-conjugated arms in positions 2 and 5 of the phosphole ring. According to the expectations, substituents with electron-donating groups led to lower oxidation potentials, the presence of electron-withdrawing groups shifts the reduction potential to less negative values. Because all studied phospholes have multiple substituents of both types, their resulting potentials represent the sum of their influences. Moreover, since many of the studied compounds have two redox centers (phosphole moiety itself and the pi-conjugated arm), their locations were determined and intramolecular interactions were followed, because substituents which can be conjugated with the π-system of phosphole are able to extend the delocalized π-system resulting in stabilization of primary redox intermediates. Chemical modification of phospholes using various combinations of substituents can set the desired electrochemical properties of studied molecules.
|
|
|
Use of Hsp90 as a target of biological therapy of tumors
Bednárová, Kristína ; Bouchalová,, Pavla (referee) ; Müller, Petr (advisor)
Antibody-drug conjugates (ADCs) represent a relatively new class of highly potent anti-tumor drugs. Thanks to highly specific monoclonal antibodies, ADCs are able to deliver a cytotoxic payload directly to tumor cells and thus minimize damage to healthy cells. Therapeutic efficacy depends on the selection of an appropriate antigen that undergoes internalization upon conjugate binding. For this project, pro-oncogenic Hsp90 and c-Met were selected as potential targets. Hsp90 is a molecular chaperone that is overexpressed in tumor cells and, in addition, can be translocated to the membrane of these cells. Overexpressed Hsp90 contributes to angiogenesis, tumor cell motility or metastasis. C-Met is a receptor tyrosine kinase that plays a central role in epithelial morphogenesis and malignant transformation. Its increased activity induces pathways responsible for the proliferation, invasion and migration of malignant cells. The aim of the diploma thesis was to study the potential use of antibodies with anti-Hsp90 and anti-c-Met activities in anti-tumor therapy. The experimental part involved the purification of the EEV1-2.1 antibody with anti-Hsp90 activity and its subsequent characterization. Furthermore, it included the characterization and selection of anti-c-Met antibody clones. It was also focused on selection and optimization of the right conjugation strategy. The activity of the antibodies and their conjugates was examined by fluorescence microscopy and flow cytometry. In vivo experiments were further aimed at verifying the efficacy of ADC by monitoring the rate of inhibition of proliferation of selected tumor cell lines. The results revealed that the EEV1 antibody does not enter the cells specifically by antigen-mediated way, and is therefore not suitable for use in conjugation with a cytostatic drug. On the other hand, anti-c-Met antibody ADC conjugates exhibit high affinity for native antigen, internalization through antigen binding, and additionally inhibited proliferation of c-Met overexpressing OE33 cells.
|
|
| |
|
|
Diversity and ecology of filamentous green conjugate algae
Strouhalová, Pavla ; Šťastný, Jan (advisor) ; Pichrtová, Martina (referee)
Filamentous conjugating algae have a cosmopolitan distribution. They often inhabit fragile freshwater habitats such as temporary hydrated ditches or puddles of melting snow. Occurrence in this environment entails having to deal with extreme conditions. That helps them to variously adaptation and also the formation of resistant stages. Algae belonging to this group have an important role in nature, because they are often the first species that inhabit newly created habitats and consequently also often become the dominant species. Diversity, as well as the ecological requirements of individual representatives, is unclear. Taxonomy of filamentous algae is conjugating is most based on morphological characters that are not very reliable. For filamentous conjugating algae is polyploidy really frequent. With changeable ploidy relate also morphological variability and different environmental requirements.
|
|
|
Cryptic and pseudocryptic diversity of conjugating green algae (Zygnematophyceae)
Kupčíková, Eva ; Šťastný, Jan (advisor) ; Škaloud, Pavel (referee)
This bachelor thesis summarizes the most commonly used species concepts in the class Zygnematophyceae. This group of algae and the molecular markers used in the zygnematophytes' molecular phylogeny will be presented in this thesis. Zygnematophytes have unicellular or multicellular forms and they generally occur in freshwater habitats. Desmids are useful as indicators of water quality and they are also used for scoring of conservation value. Therefore it is important to have the species well defined. The confusion in defining of desmid species was caused by often very variable morphology. Some authors had different opinions on the classifying of desmid species and theirs subunits. The combination of molecular phylogeny, electron microscopy and geometric morphometrics was used in revealing of (pseudo)cryptic species in the genera Micrasterias or Xanthidium. The geometic morphometrics was helpful for finding of important details for distinguishing of particular pseudocryptic species. Key words: cryptic and pseudocryptic diversity, conjugation, desmids, Micrasterias, Euastrum, Xanthidium, molecular phylogeny, taxonomy, geometric morphometrics, biogeography, green algae
|
|
|
Synthesis of core/shell quantum dots for diagnostic
Mihajlovic, A.
In this paper, synthesis of colloidal core and core/shell quantum dots (QDs) was described. First, CdTe QDs capped with glutathione, thioglycolic or mercaptopropionic acid were prepared in aqueous phase, and used for synthesis of colloidal core/shell CdTe/ZnS QDs. Core/shell QDs were used for conjugation with bovine serum albumin (BSA) or immunoglobulin G (IgG) via different crosslinkers (CDI, EDC/NHS, EDC). QDs as well as QDs-protein/antibody conjugates were characterized via UV-Vis spectroscopy and capillary electrophoresis (CE). Based on UV-Vis spectroscopy results it was found that, with increasing concentration of BSA, fluorescence intensity of QDs decreased. CE confirmed formation of QDs-BSA and QDs-IgG conjugates.
|
|
|
Quantum dot-based immunoprobe for optical and electrochemical detection
Dvořáková, V. ; Čadková, M. ; Datinská, Vladimíra ; Chałupniak, A. ; Korecká, L. ; Merkoci, A. ; Klepárník, Karel ; Foret, František ; Bílková, Z.
Three-stage labeling strategy allows the preparation of specific conjugates composed of required antibodies and quantum dots as an extremely sensitive bio-probe applicable in varied immunoassays with following optical and electrochemical detection. This approach is essentially based on carbodiimide chemistry however each single step of whole procedure is separated and controlled which makes this applied protocol oriented, highly efficient and it enables gain of pure antibody-conjugated quantum dots in ready-to-use condition.
|
guest ::
