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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.
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Terapeutické monoklonální protilátky v nádorovém léčbě
Danišová, Terezie
In recent years, therapy with monoclonal antibodies has been increasingly used in clinical practice. Treatment based on these antibodies has a wide range of advantages over classical therapeutic procedures such as chemotherapy, including reduced side effects. Their greatest advantage is their specificity towards cancer cells, which does not damage surrounding healthy cells. The aim of this compilation work is to summarize and present the molecular basis of tu-mour development and the processes that monoclonal antibodies suppress, stimulate, and contribute to stopping tumour progression. This compilation work provides a basic literary over-view of carcinogenesis at the molecular level, which is necessary for understanding the princi-ples on which monoclonal antibodies function. This work later focuses on monoclonal antibodies, their mechanism of action, and production techniques.
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NMDA receptors outside CNS and their subunit composition
Hotovec, Matěj ; Kolcheva, Marharyta (advisor) ; Horáková, Olga (referee)
N-Methyl-D-Aspartate receptors (NMDARs) are a type of ionotropic glutamate receptor that is widely present in the central nervous system and in lesser numbers in other parts of the body, including the gastrointestinal tract. NMDARs play a crucial role in various physiological and pathological processes in the CNS, including synaptic plasticity, learning and memory, and excitotoxic damage. The role of NMDARs outside the CNS is still under investigation. This thesis aims to confirm the presence of NMDARs in the gastrointestinal tract and their subunit composition. Subunit specific NMDAR modulators showed effect on spontaneous phasic activity of ileum, indirectly confirming the presence of GluN1, GluN2A-D and GluN3 subunits. Attempt at direct evidence of protein expression by immunolabeling with monoclonal antibodies against NMDAR subunits was unsuccessful.
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Follow-up of patiens after COVID-19 monoclonal antibodies administration
Minaříková, Jiřina ; Zimčíková, Eva (advisor) ; Hendrychová, Tereza (referee)
Follow-up of patients after COVID-19 monoclonal antibodies administration Author: Jiřina Minaříková Supervisor: PharmDr. Eva Zimčíková, Ph.D. Consultant: PharmDr. Petra Rozsívalová Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of Social and Clinical Pharmacy Keywords: COVID-19, coronavirus, monoclonal antibodies, casirivimab and imdevimab, REGN-COV2 Introduction: At height of COVID-19 pandemic surge of Delta variant, monoclonal antibodies became a vital treatment option for SARS-CoV-2 positive outpatients at high risk of severe disease progression. Casirivimab and imdevimab (C/I) were used as an unauthorised medicinal product REGN-COV2 under European Medicines Agency emergency use authorisation (EUA). There was paucity of real-world data on safety and effectiveness. Objective: The study aimed to describe REGN-COV2 drug safety, self-reported symptom burden in SARS-CoV-2 positive outpatients within 90 days post C/I infusion. Methods: Prospective multicentric study of SARS-CoV-2 positive outpatients with mild symptoms at high-risk of severe COVID-19 progression (defined criteria under EUA authorization for C/I ambulatory administration) was conducted from September 2021 till April 2022 in three teaching hospitals in Czech Republic and Slovakia. The data collected...
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Structural analysis of the interaction interface between an antibody and a protein epitope
Moravec, Jan ; Krejčíř, Radovan (referee) ; Müller,, Petr (advisor)
The diploma thesis deals with the study of the mouse monoclonal antibody EEV1-2.1, which was created in the RECAMO laboratories by immunization of mice with the Hsp90 protein and subsequent fusion of splenocytes with a mouse myeloma line. The theoretical part of this work focuses mainly on the study of antibodies, especially their structure, genetics and the description of monoclonal antibodies. In this part of the work, you can also find a cross-section of modern biotechnological trends that are currently used for the production of antibodies. CHO cells and tetracycline inducible systems are also described. The aim of the thesis was to characterize the mentioned antibody. Using bioinformatics methods and prediction software to predict and describe its structure, especially then to analyze hypervariable CDR regions. Another goal was to clone the light and heavy chains of the antibody using the Gateway method. The final step was the creation of a tetracycline inducible system for efficient production of the antibody itself. The experimental part of the work initially deals with the isolation of the RNA sequence of the antibody using commercial kits. Next, the method of amplification of the given sequence using PCR with reverse transcription is described. The following is a description of the Gateway cloning method, which allows convenient insertion of the selected gene into the target vector. The basic bioinformatics methods used to study the structure of antibodies are also explained, as well as the prediction of a more complex 3D structure of the antibody, including possible interactions with the HSP90 protein. This section describes the methods of transfection of ExpiCHO cells and inducible antibody production using the tetracycline inducible system. The thesis led to the successful cloning of the light and heavy chain of the antibody and the subsequent insertion of the expression vector into the production ExpiCHO cells. The expression system based on induction by the addition of doxycycline was then successfully tested by several methods and the results show that the inducible system is not only functional, but can even lead to increased production of monoclonal antibody compared to conventional methods.
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Protilátky v terapii klíšťové encefalitidy
NEDVĚDOVÁ, Lenka
Tick-borne encephalitis (TBE) is a serious live threatening disease caused by tick-borne encephalitis virus (TBEV). The therapy is based on administration of non-specific treatment with anti-inflammatory drugs or IVIg (including non-specific antibodies). Application of non-specific antibodies may present a certain level of risk - maily of developing antibody dependent enhancement. We have developed and characterized monoclonal antibodies targetting non-structural protein 1 (NS1) of TBEV in order to assess its potential in TBEtreatment and diagnostics.
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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.
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Diversity of methods used for characterization of molluscan hemocytes
Jindrová, Zuzana ; Horák, Petr (advisor) ; Skála, Vladimír (referee)
Hemocytes are the main immune cells of invertebrates; therefore they can be found in molluscs, too. They differ both in morphology and function. The two generally accep- ted morphological types, granulocytes and hyalinocytes, vary in the level of phagocy- tosis and encapsulation, production of reactive oxygen species and nitrogen oxide, and presence of some enzymes. There is an array of methods by means of which hemocy- tes can be characterized. Microscopy serves particularly for study of morphology. An- tigens localized on the surface can be determined by monoclonal antibodies or lectin probes. Hemocytes can be divided on the basis of cell size and granularity using gra- dient centrifugation or flow cytometry. Production of nitrogen oxide and reactive oxy- gen species is monitored by adding appropriate substrate which changes its proper- ties after reaction with the radical. It may become fluorescent, change absorbance of the solution or form a visible precipitate. Another possibility is the use of chemilu- miniscence. The objective of hemocyte research is to explain mollusc-pathogen inter- action. 1
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New approaches in vaccination against HIV
Dobiášová, Julie ; Drda Morávková, Alena (advisor) ; Strachotová, Dita (referee)
A B S T R A C T Most current vaccines are based on using whole-inactivated viruses. After creating the immune response and immune memory is organism able to cope with infection create by patogens. In the case of HIV, however, fail to produce the vaccine, which would have been able vaccinated individual from subsequent infections protect. Virus HIV attacks CD4+ cells and destroys the immune system. Rate of his replication is high and virus HIV is resistant to existed antivirotics. And he is resistant before cells, which conveying the immune response. Moreover, the virus persists in cell in the form proviral DNA. For a successful vaccine against HIV is developed a lot of new vaccines and vaccination procedures. One way is the using recombinat viral glycoproteins, which are incorporated into the membrane of virus HIV, which should produce in the vaccinated organism production of neutralizing antibodies. Some modern models of vaccines strategies don't target the virus itself, but they target the restriction of HIV infection by destroying infected cells via apoptosis, or cytokine secretion. Using plasmid DNA cobination with recombinant vectors appear as the most perepective opportinity to develop HIV vaccine. Unfortunately, traditional models or new models of vaccine against HIV are failing to provide a...
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