|
|
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.
|
|
|
Identification of new regulators of proinflammatory signaling pathways
Dráberová, Helena ; Štěpánek, Ondřej (advisor) ; Krulová, Magdaléna (referee) ; Funda, David (referee)
Identification of new regulators of proinflammatory signaling pathways Helena Dráberová Protein 4.1R has been described in immune system as regulator of migration and cell adhesion, but was also shown to play a role in activation of T lymphocytes. Polymorphism in gene ORMDL-3 is associated with asthma risk in children and correlates with increased ORMDL-3 expression. This disertation thesis describes the function of proteins 4.1R and ORMDL-3 in activation of mast cells after stimulation of FcεRI receptor. IL-17 is a proinflammatory cytokine that plays a role in immune response against fungal and yeast infections. IL-17 however also plays a role in the pathology of autoimmune diseases such as reumatoid arthritis, psoriasis and multiple sclerosis. IL-17 signaling is tightly regulated, however the exact mechanism has not been described. This disertation thesis describes the IL-17R complex by mass spectrometry and analyze the function of its known and newly discovered components in cells deficient in individual proteins by method CRISPR-Cas9. Last part focuses on the discovery of new subunit of IL-17RC protein CMTM4, which role in IL-17 signaling has not been described so far. CMTM4 stabilizes IL-17RC and is required for its surface expression. In vitro data are supported by data from autoimmune model of...
|
|
|
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.
|
|
|
Zdravotní a produkční účinek imunomodulačního preparátu Imrestoru u vysokoužitkových dojnic
NEZNÍKOVÁ, Kateřina
This thesis deals with evaluation of the impact of immunomodulatory preparation Imrestor (Elanco Animal Health) on the health and production of high-producing dairy cows in the transition period. The experiment was done in the agricultural company Košetice in the district of Pelhřimov on dairy cows of the Holstein breed. The animals selected for the experiment were divided into two groups - control and experimental. Imrestor was applied subcutaneously to the dairy cows of the experimental group approximately one week before calving and on the day of calving. The active component of this preparation is a commercial form of a naturally occurring cytokine (bG - CSF), which specifically affects on the neutrophil precursor cells and increases their production. Blood samples were taken from all cows included in the experiment to evaluate the effect of the preparation Imrestor. At the same time was monitored their clinical health and the incidence of the most common diseases in the transition period. 81 blood samples were taken from 31 dairy cows from the control group and from 18 dairy cows from the experimental group. A biochemical and hematological examination was performed in the laboratory, where the parameters of red blood cells, trombocyte count, leukocyte count, leukocyte percentage, biochemical indicators of blood plasma and mineral content were determined. By statistical evaluation was found, that Imrestor statistically highly significantly (p <0.01) affected leukocyte counts and the percentage of neutrophil granulocytes and lymphocytes. Statistically significant differences (p <0.05) between the observed groups of dairy cows were found in the trombocyte count, percentage of monocytes and alkaline phosphatase activity.
|
|
|
The effect of nanoparticles on functional and regenerative properties of mesenchymal stem cells
Dostálová, Dominika ; Rössner, Pavel (advisor) ; Grobárová, Valéria (referee)
Mesenchymal stem cells (MSCs) represent a type of stem cells, localized in various tissues, where they are involved in the regeneration process. Because of their properties, MSC are tested for cell-based therapy. However, the treatment of injuries is often associated with bacterial infections, which complicate the process of healing. To establish a sterile environment, metallic nanoparticles (NPs) with proven antibacterial properties can be applied, for improving the healing process. Mechanisms of the antibacterial effect of NPs are non-specific and may cause the damage of eukaryotic cells. The aim of this thesis was to evaluate the effect of three antibacterial metallic NPs (silver, copper oxide and titanium dioxide) on the functional and regenerative properties of MSCs. The effect of NPs on the metabolic activity, differentiational potential, expression of genes for immunoregulatory molecules and production of cytokines by MSCs was determined. Additionally, the immunoregulatory properties of MSCs after cultivation with NPs were tested. The results showed that the metabolic activity was reduced in the presence of silver and copper oxide NPs, with the decrease dependent on the type of the tested NPs. The expression of selected immunoregulatory molecules was inhibited and the immunoregulatory...
|
|
|
Targeting IRAK4 kinase in autoimmune diseases and cancer
Synáčková, Alžběta ; Dráber, Peter (advisor) ; Brdička, Tomáš (referee)
Immune system provides host protection against invading pathogens. However, aberrant activation can lead to development of autoimmune diseases or cancer. Understanding the mechanisms of inflammation and immune responses is crucial for treatment of such conditions and reestablishing immune balance. Toll-like receptors and interleukin-1 family receptors are a key component of the innate immune system. Their downstream molecules, MyD88 and IRAK4, are essential for receptor signaling as their deficiency causes host susceptibility to infection. On the other hand, overactivation of this pathway was shown to be able to promote autoimmunity and cancer. The main focus of this text will be to summarize current knowledge about the mechanism of IRAK4 signaling and how it can be exploited in the development of therapeutics. Keywords IRAK4, MyD88, Toll-like receptors, IL-1 receptor, cytokines, autoimmunity, cancer
|
|
|
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...
|
|
|
Immunological profile of patients with multiple sclerosis
Šubjak, David ; Fišerová, Anna (advisor) ; Růžičková, Šárka (referee)
Immunological profile of multiple sclerosis patients Abstrakt Multiple sclerosis is an autoimmune neurodegenerative disease affecting predominantly the white matter of the CNS and the spinal cord. The mechanism of disease progression is not yet fully understood. In this study we focused on a comparison of selected immunological markers between patients with multiple sclerosis who were naïve newly diagnosed, subsequently treated with Avonex (IFNβ1a) and healthy donors. The T cells (particulary cytotoxic CD8+ T cells) are the major population involved in pathogenesis of MS causing the demyelization of axons. Subpopulation of CD161+ Th cells has a potential to be very important in this process. We focused on the role of NK cells phenotype and function in autoimmune response of patients and their changes during the therapeutic intervention. Using flow cytometry we analyzed the distribution of NK, NKT, T cells and monocytes with special regard to the expression of CD161 and NKG2D molecules on their surface. We observed increase counts of CD161+ cells in subpopulations NK CD56bright , NK CD56dim , Th, Tc CD8bright , Tc CD8dim and decrease counts of NKG2D+ cells in subpopulations NK CD56bright , NK CD56dim , NKT, Th, Tc CD8bright , Tc CD8dim and monocytes. The decreased cytotoxic activity of NK cells in naïve MS...
|
|
|
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)
|
|
|
The role of innate lymphoid cells in influenza virus infection
Mouyabi, Flaviancia ; Hrdý, Jiří (advisor) ; Kössl, Jan (referee)
Innate lymphoid cells (ILCs) are recently discovered group of innate immune cells. They do not have antigen-specific receptors but they can be activated by cytokines similarly to T lymphocytes. ILCs have a crucial role in the regulation of inflammation, tissue repair, containment of commensals, anti-infection immunity and regulation of tissue homeostasis. The presence of mouse and human ILCs can be detected in the lung during and after influenza virus infection when ILCs contribute to the restoration of damaged lung parenchyma. ILCs directly or indirectly provide protection against viral infections by secretion of various cytokines and co-operation with other cells (e.g. T cells, macrophages). Overall, lung ILCs are important in immune responses and tissue homeostasis, but further studies on this topic are needed to fully understand their role. The aim of this thesis was to specifically characterize these cells, focus on their function in the lung, and describe their role in the course of influenza virus infection.
|
guest ::
