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Specificita interakcí protein-protein a jejich modulace
Pham, Phuong Ngoc ; Schneider, Bohdan (advisor) ; Damborský, Jiří (referee) ; Vaněk, Ondřej (referee)
(EN) Protein-protein interactions (PPI) have essential roles in life processes, and abnormal PPI are associated with many human diseases. Given their importance, PPI have received increasing attention and became drug targets. However, the design of specific PPI and their modulation is challenging. Cytokine-receptor interactions are especially important in the regulation of the immune system. Interleukin-10 (IL-10) over-production results in excessive immunosuppressive effects, tumor growth and infection. The interaction between interferon gamma receptor 2 (IFN- γR2) and interferon gamma (IFN-γ) leads to activation of downstream signaling pathways but the mechanism of such interaction is elusive. Interleukin-24 (IL-24) is another cytokine that signals through receptors sharing the interleukin-20 receptor two (IL-20R2) subunit and has important roles in autoimmunity and cancer. The aims of this Ph.D. thesis are to study PPI from several aspects emphasizing their specificity. The first goal is to develop a novel protein scaffold and subsequently evolve it into a high-affinity binder specific for human IL-10. The second goal is to understand the structural basis for receptor specificity of human IFN-γ. The third goal is to modulate the binding affinity between human IL- 24 and its receptor IL-20R2 by...
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Engineering and selection of protein binders recognising medically important cytokines
Huličiak, Maroš
Protein engineering attracts more attention as a powerful tool of biotechnology and medicine. Small, engineered proteins derived from protein molecules of stable fold, the so called scaffolds, are potential replacements of supplements of more widely used antibodies. In this thesis, I introduce utilization of two scaffold molecules designed in our laboratory for development of stable and specific protein binders of high affinity. This thesis discusses the development of binders interacting with medically important human cytokines and their cellular receptors, interleukin-10, interleukin-28 receptor, and interleukin-9 receptor alpha. Recombinant cytokine and receptor proteins were expressed in eukaryotic cells in high yields and quality and served as molecular targets for selections using various display methods of directed evolution. We demonstrated that application of ribosome and yeast display methods or their unconventional combination in a newly developed integrated pipeline leads to successful generation of high affinity and specificity binders based on newly designed protein scaffolds called 57aBi and 57bBi.
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Engineering and selection of protein binders recognising medically important cytokines
Huličiak, Maroš ; Schneider, Bohdan (advisor) ; Pichová, Iva (referee) ; Kukačka, Zdeněk (referee)
Protein engineering attracts more attention as a powerful tool of biotechnology and medicine. Small, engineered proteins derived from protein molecules of stable fold, the so called scaffolds, are potential replacements of supplements of more widely used antibodies. In this thesis, I introduce utilization of two scaffold molecules designed in our laboratory for development of stable and specific protein binders of high affinity. This thesis discusses the development of binders interacting with medically important human cytokines and their cellular receptors, interleukin-10, interleukin-28 receptor, and interleukin-9 receptor alpha. Recombinant cytokine and receptor proteins were expressed in eukaryotic cells in high yields and quality and served as molecular targets for selections using various display methods of directed evolution. We demonstrated that application of ribosome and yeast display methods or their unconventional combination in a newly developed integrated pipeline leads to successful generation of high affinity and specificity binders based on newly designed protein scaffolds called 57aBi and 57bBi.
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Modulation of interactions of cytokines and their receptors
Kolářová, Lucie ; Schneider, Bohdan (advisor) ; Rozbeský, Daniel (referee) ; Osička, Radim (referee)
Protein-protein interactions and interactions with other molecules including DNA and RNA, play an important role in a range of biological activities and processes in all living cells. Understanding of protein-protein interactions, new approaches, and tools for their modulations are valuable for medicine, biotechnology, and drug development. We used the interleukin-10 family of cytokines as a model system for our research of biological interactions and modulation of their functions. A key prerequisite to study biological processes and a detailed understanding of biomolecular interactions is a recombinant protein that is stable under a broad range of conditions. Recombinant protein expression in sufficient yield and quality is often a challenging task. Therefore, we aimed at developing new approaches in protein design and production. In the first part of our study, we modified IL-24, a member of the IL-10 family to increase its expression and stability. We demonstrated that protein engineering is a powerful tool in research of difficult protein targets. In the second part of our study, we adopted new approaches in designing new protein scaffolds suitable for use in the ribosome and yeast display techniques. Protein scaffolds have become promising alternatives to antibodies in protein drug...
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Vliv chemoterapie a genotoxického stresu na imunologické vlastnosti nádorových buněk.
Horňáková, Michaela ; Reiniš, Milan (advisor) ; Drbal, Karel (referee)
Cancer treatment includes the use of chemotherapeutic agents, which have various effects on tumour cells, such as direct toxicity to cancer cells, immunogenic cell death induction and changes in cancer cells phenotype. Throughout the last decade many researchers have been focusing on the induction of genotoxic stress and cellular senescence, which chemotherapy can trigger. Even though induction of senescence in cancer cells represents an important mechanism for tumour suppression, there has been increasing evidence that shifting cancer cells into a senescent state by chemotherapy is not always beneficial. Senescent cells are associated with a specific secretory phenotype, which allows such cells to alter their microenvironment, modulate anti-tumour immunity, induce tumour suppression and even promote cancer development. Therefore, senescent cells elimination by innate or specific immunity, which can be boosted by immunotherapy, can be an important barrier preventing tumour growth. Powered by TCPDF (www.tcpdf.org)
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Bacterial components in experimental intestinal inflammation prevention and therapy
Kverka, Miloslav ; Tlaskalová - Hogenová, Helena (advisor) ; Šedivá, Anna (referee) ; Stříž, Ilja (referee)
Although strong protective immune response is essential for preventing invasion by pathogens, equivalent responses against antigens originating from commensal bacteria can lead to chronic inflammatory diseases, such as inflammatory bowel disease (IBD). Manipulating the mucosal immune responses with microbial antigens might be an excellent tool to IBD therapy or prevention. Our aim was to gain some insight into the regulation of the intestinal inflammation and to isolate bacterial immunomodulatory components that could be used in intestinal inflammation therapy and prevention. One particular mechanism of how healthy colon tissue regulates the inflammation during acute experimental colitis is through modulation of bioavailability of glucocorticoids (GCs) in gut mucosa. Here, we show that intestinal inflammation changes the local GC metabolism, which ultimately leads to decrease in inflammatory readiness of cells in the gut mucosa and in mesenteric lymph nodes. This pre-receptor regulation of GC function could represent an important homeostatic function of the gut mucosa. The actual triggers of intestinal inflammation in IBD seem to be either microbial dysbiosis or microbes with special "pathogenic" abilities, which both could be rectified by feeding with probiotics. Here, we report that oral feeding with live...
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Link between inflammation and cancer
Schierl, Jaroslav ; Poljaková, Jitka (advisor) ; Miarková, Eva (referee)
Chronic inflammation caused by many initiators can lead to a development of a tumor disease. Among these initiators, we found chronic infections as well as other biological, chemical or physical factors which have endogenous and exogenous origins as for example tobacco smoke, alcohol, radiation, obesity and others. The inflammatory response is orchestrated by immune system cells which contribute to a tumorigenesis by producing reactive oxygen and nitrogen species which harm cell structures, and by releasing cytokines - important mediators of inflammation - which increase cell proliferation and angiogenesis. But apart from higher risk of tumourigenesis due to chronic inflammation, the immune system cells also participate in tumor microenvironment formation. The main contributors are tumor associated macrophages, dendritic cells and T-cells. Besides other things, the complex tumor microenvironment is characterized by the presence of many inflammation mediators which assist in malignant cell proliferation, tumour progression and metastasis and angiogenesis. This bachelor thesis describes the key protumor and antitumor factors which are also involved in the inflammation process. These factors include proinflammatory cytokines, enzymes and transcription factors. The transcription factor NF-κB plays an...
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