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Chemically modified Murine Polyomavirus-like particles and their interaction with Prostate-Specific Membrane Antigen (PSMA)
Blažková, Kristýna ; Konvalinka, Jan (advisor) ; Horníková, Lenka (referee)
Prostate cancer is one of the most abundant types of cancer among men and the demand for a specific treatment is very high. In this thesis, I have focused on using Glutamate Carboxypepti- dase II (GCPII), as a target for a proof-of-principle delivery system. GCPII is a transmembrane protein that internalizes after a binding of a ligand and is overexpressed in prostate cancer. Virus-like particles from Murine polyomavirus (VLPs) are a suitable nanocarrier for the delivery of imaging agents and drugs. Here I describe modifying these VLPs with inhibitors of GCPII and fluorescent dyes and characterize their binding to GCPII on surface plasmon resonance and to cells expressing GCPII on confocal microscopy. VLPs carrying a GCPII inhibitor show specific binding to GCPII on surface plasmon reso- nance, however they bind non-specifically to cells that don't express GCPII. Several approaches have been tried to avoid that. The substitution of BC loop on the exterior surface of VLPs that is partially responsible for the binding of sialic acid did not seem to affect specificity on cells. Another approach tested was coating of the wild-type VLPs with large polymer carrying a flu- orescent label and a GCPII inhibitor. After the conjugation of the polymer to the VLP, specific binding and internalization in GCPII-positive...
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Characterization of Glutamate Carboxypeptidase II, its Close Homologs and their Interaction with Ligands
Tykvart, Jan ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee) ; Pavlíček, Jiří (referee)
Cancer, group of diseases characterized by an uncontrolled cell growth, represents one of the great challenges of modern clinical research. Currently, the standard treatment of the cancer disease relies mainly on the whole body exposition to various factors, which targets the dividing cells, combined with surgical resection of the tumor. Unfortunately, this treatment is sometimes accompanied by numerous severe side-effects (e.g., nausea, loss of hair, infertility etc.). Therefore, in the past 40 years enormous resources and effort have been invested into finding a way how to specifically target and destroy the cancerous cells. This goal has been primarily addressed by the search for molecules, mainly proteins, which are predominantly expressed in the cancerous tissues compared to the healthy cells. Glutamate carboxypeptidase II (GCPII), also known as prostate specific membrane antigen (PSMA), represents such a target since it is highly expressed in a prostate carcinoma as well as in a solid tumor neovasculature. Additionally, GCPII is widely used as a model target molecule for proof-of-principle studies on targeted drug delivery. GCPII thorough biochemical characterization is essential for its appropriate use. Therefore, our laboratory has been investigating GCPII from various perspectives for more...
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Protease Inhibitors as a Research Tool: Design, Synthesis and Evaluation of HIV PR and GCPII Inhibitors
Schimer, Jiří ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee) ; Ruml, Tomáš (referee)
This dissertation thesis focuses on creating tools for the analysis and potential therapeutic intervention in the biological processes regulated by proteolysis. I focus on two important proteolytic enzymes: HIV-1 protease, which is indispensable for the polyprotein processing of the nascent virus and thus for the development of infectious viral particle, and glutamate carboxypeptidase II, a tumor marker and a neuropeptidase from the prostate and central nervous system. Rational design of inhibitors of these therapeutically relevant enzymes serves two purposes: firstly, protease inhibitors were shown to be powerful drugs (HIV protease is in fact the example of successful drug development driven by structural biology). Secondly, and in the context of this thesis perhaps more importantly, inhibitors of medicinally relevant proteases might serve as tools for the elucidation of basic biological questions concerning regulation, timing and spatiotemporal control of such key processes as virus maturation or cancer development. The experimental work described in this thesis summarizes my results in both these areas. Human Immunodeficiency Virus Protease Human immunodeficiency virus (HIV), a causative agent of AIDS, has been estimated to kill close to 40 million people during the past four decades with 1.5...
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Evaluation of the properties of polymer conjugates which specifically bind proteins and can be used in molecular biology
Parolek, Jan ; Konvalinka, Jan (advisor) ; Liberda, Jiří (referee)
During last three decades, a great effort was invested to the development of polymer conjugates of low molecular drugs with the aim to improve the specific targeting of drugs to diseased tissues, cells and organs. The main reason for this effort was the fact that high molecular weight copolymers have a favourite distribution profile in tissues and organisms. A linker between a polymer backbone and drug has very important role: it is possible to synthesize a biodegradable linker, which can be enzymatically hydrolyzed. Conversely, there is a possibility to synthesize an inert linker, resistant to the hydrolysis. Proper choice of the suitable precursor- polymer is also essential, hence it has to accomplish all of the stringent demands for biocompatibility. Macromolecular polymer-drug conjugates tend to accumulate in solid tumors because of the so called enhanced permeability and retention (EPR) effect. There is a whole range of possible applications of high molecular polymer-drug conjugates. In the introduction part of this thesis, I summarize potential use of drugs based on poly(N-(2-hydroxypropyl)methacrylamide) (HPMA) copolymers. Moreover, I introduce some therapeutically important proteins used in experimental drug discovery. In our laboratory, we have developed a concept of HPMA copolymers...
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Production of recombinant cathepsin C from human blood fluke
Illichová, Hana ; Konvalinka, Jan (advisor) ; Martínková, Markéta (referee)
Blood flukes of the genus Schistosoma cause schistosomiasis, a serious parasitic disease occurring in tropical and subtropical areas. Cathepsin C (EC 3.4.14.1) is a digestive enzyme of the blood flukes which participates in the degradation of hemoglobin through its dipeptidyl aminopeptidase activity. This enzyme is critical for metabolism of the parasite and represents a potential target for the development of antischistosomal drugs. Cathepsin C has not yet been studied in detail. This bachelor thesis is focused on the development of expression systems for production of recombinant cathepsin C of Schistosoma mansoni (SmCC). The yeast Pichia pastoris system was used for the expression of an inactive SmCC precursor (zymogen) whose proteolytic stability was analysed. Furthermore, the expression system for SmCC in the protozoan Leishmania tarentolae was employed, and four different SmCC constructs were prepared to optimize production.
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Role of the 14-3-3 protein in the regulation of G-protein signaling
Řežábková, Lenka ; Obšil, Tomáš (advisor) ; Konvalinka, Jan (referee) ; Bařinka, Cyril (referee)
Univerzita Karlova v Praze Přírodovědecká fakulta Studijní program: Fyzikální chemie Mgr. Lenka Řežábková Studium úlohy proteinů 14-3-3 v regulaci G-proteinové signalizace Role of the 14-3-3 proteins in the regulation of G-protein signaling Disertační práce Školitel: doc. RNDr. Tomáš Obšil, Ph.D. Konzultanti: doc. RNDr. Petr Heřman, CSc. doc. RNDr. Jaroslav Večeř, CSc. Praha, 2012 Abstract The 14-3-3 family of phosphoserine/phosphothreonine-binding proteins dynamically regulates the activity of their binding partners in various signaling pathways that control diverse physiological and pathological processes such as signal transduction, metabolic pathways, cell cycle and apoptosis. More than 300 different cellular proteins from diverse eukaryotic organisms have been described as binding partners for the 14-3-3 proteins. During my Ph.D., I was particularly interested in the role of 14-3-3 proteins in the regulation of G protein signaling pathway. The 14-3-3 proteins affect the G protein signaling via the interaction with negative regulators of G protein cascade - the RGS proteins and phosducin. I employed both biochemical and biophysical approaches to understand how the activity and function of RGS3/14-3-3 and phosducin/14-3-3 complexes are regulated. I solved the low-resolution solution structure of...
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Splice variants of the gene coding for GCPII and their role in cancer development
Jindrová, Helena ; Konvalinka, Jan (advisor) ; Liberda, Jiří (referee)
Alternative splicing is a mechanism of generating distinct proteins that are encoded by the same gene. These proteins differ in amino acid sequence, overall structure and function. Splicing dysregulations have been shown to be implicated in several pathologic processes including cancer. For example, non-physiological splicing of osteopontin was proved to play a key role in cell progression of breast cancer. Glutamate carboxypeptidase II (also called prostate specific membrane antigen, PSMA) is present in both normal prostate and prostate cancer. Several splice variants of PSMA have been described and it has been suggested that the overexpression of some of them could be involved in the progression of prostate cancer. Nevertheless, more detailed investigation of each of the PSMA splice variant in terms of their occurrence in prostate cancer cells remains to be performed. This thesis focuses on the exploration of the expression of PSMA splice variants with deleted exons 6 and 18 in samples of a cell line derived from human prostate cancer, benign prostate hyperplasia and prostate cancer. For this purpose, RT-PCR was utilized to determine the ratio of deletions of exons 6 and 18 in cDNA of the prostate specific membrane antigen. Furthermore, the ratio of deletions of exon 6 and 18 was determined in...
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Microcalorimetry as a method to analyze protein interactions with ligands
Durčák, Jindřich ; Konvalinka, Jan (advisor) ; Vaněk, Ondřej (referee)
The interactions of proteins with their binding partners occur in every living organisms, in almost every cell process. Therefore the exploration of protein interactions forms significant part of biochemical research. It appears that even more valuable information than the value of equilibrium constants of these interactions is determination of individual energy components - changes in enthalpy and entropy. Thermodynamic analysis by isothermal titration calorimetry (ITC) can determine the changes in entropy and enthalpy caused by formation of complex of binding partners. Microcalorimetry is also an important optimization technique in development of new drugs, for example antiretrovirotics. Despite HIV is a virus known for over 30 years, intensive research has neither brought vaccine nor drug that would permanently cure patients. Already 26 drugs were approved, most of them target viral enzymes reverse transcriptase and protease. Antiretroviral treatment prevents the propagation of HIV and maintains immune system, but long - term use leads to resistance against drugs, which is caused by mutations in the target proteins. One of relatively new targets of therapeutic intervention is capsid core formation of during assembly of new virions. During the assembly many protein - protein interactions take...
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