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Cancer Immunotherapy exploiting engineered antibody fragments against prostate-specific membrane antigen
Das, Gargi ; Bařinka, Cyril (advisor) ; Vaněk, Ondřej (referee) ; Ormsby, Tereza (referee)
Prostate cancer (PCa) remains a leading cause of male cancer-related mortality, necessitating thus the development of novel therapeutic approaches as conventional treatments have limited efficacy. Prostate-specific membrane antigen (PSMA) is an established biomarker for both imaging and therapy of PCa, as it is highly upregulated in neoplastic PCa tissues and metastatic castration- resistant prostate cancer. Consequently, immunological targeting of PSMA has gained significant attention as a therapeutic platform for the management of the disease. The thesis is focused on engineering of antibody fragments and fusion proteins derived from the high affinity anti-PSMA 5D3 monoclonal antibody that can be used as immune cell engagers to target and eliminate PSMA-positive cells. To this end, we engineered 5D3 single chain variable fragments (scFv) that were subsequently fused to anti-CD3 scFv and CP33 sequences, creating thus immune cell engagers targeting T-cells (BiTE) and monocytes (5D3-CP33), respectively. The engagers were expressed in insect cells, purified to homogeneity and their biophysical and functional characteristics evaluated using size exclusion chromatography, differential scanning fluorimetry, ELISA and flow cytometry. Ensuing cell-based assays revealed that both BiTE and 5D3-CP33 can...
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Buněčné a molekulární charakteristiky hemolymfy u raků
KIFAYATULLAH, NA
The cellular and molecular components of the hemolymph are the major arm of the innate immune system in decapod crustaceans. In-depth knowledge of the hemolymph components, including hemocytes and hemolymph proteins, can enhance our understanding of innate immunity in crustaceans. We utilized transmission electron microscopy and quantitative proteomics to study the cellular and molecular aspects of coagulation and phagocytosis in the hemolymph. Chapter 2 reviews the cellular and molecular parameters of the innate immune system and the effects of environmental stressors and their abiotic and biotic stress mechanisms in decapod crustaceans. The innate immune system of decapod crustaceans heavily relies on hemocytes in the circulating hemolymph. Generally, three types of hemocytes are accepted based on their morphology, however, there is still a lack of consensus among researchers regarding the classification of hemocyte types. The key innate immune functions such as coagulation and phagocytosis are still poorly understood and require further investigation especially at a molecular level. Environmental stressors can adversely affect the immune responses of decapod crustaceans, increasing their susceptibility to diseases. However, the abiotic stress mechanism is poorly understood due to the lack of available literature and needs further investigation. In Chapter 3, transmission electron microscopy was used to investigate the ultrastructural behavior of hemocytes during coagulation and phagocytosis in the early stages of leg amputation injury in marbled crayfish Procambarus virginalis. The granular hemocytes were the first to be activated, and the morphology of cytoplasmic granules changed from electron-dense to electron-lucent forms in an expanding manner. The transformed granules containing amorphous electron-lucent materials merged and discharged their contents into the extracellular space for coagulation. We observed that the leftover nucleus from degranulated hemocytes participates in the process of coagulation, which could be confused with hyalinocytes in some previous studies. In addition, leg amputation caused massive muscle degeneration, followed by a significant influx of phagocytic hemocytes that removed a substantial amount of muscle fibers and organelles, such as mitochondria, generated from disintegrating and decaying muscle. Furthermore, we found that phagocytic hemocytes contained varying numbers of granules in their cytoplasm and, for the first time, discovered that these cells incorporate necrotic bodies resulting from degenerated muscles into their organelles, such as cytoplasmic granules and nucleus. The granular hemocytes were found to be the main cells that carry out phagocytic activity in the injury site. This study provides a comprehensive description of all the stages of morphological changes in hemocytes during coagulation and phagocytosis after injury in crayfish for the first time. In Chapter 4, proteomic analysis of non-clotted and clotted samples indicated that quantities of most proteins remained unchanged during the coagulation process, suggesting that necessary proteins for coagulation are pre-synthesized and stored before clot formation. Due to their open circulatory system, decapod crustaceans possess robust clotting mechanisms. Upon injury, pre-synthesized clotting factors are released, resulting in clot formation. Therefore, only a few proteins, such as C-type lectin domain-containing proteins, Laminin A chain, and Tropomyosin, were down-regulated during clotting, suggesting their possible roles in the structural integrity of cells. Their downregulation could facilitate degranulation, a crucial step for clot formation.
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Interaction of Borrelia sp. with HL-60 cells and monocytes and cultivation of Anaplasma phagocytophilum in HL-60 cell culture
Marková, Lucie ; Hulínská, Dagmar (advisor) ; Beranová, Jana (referee)
Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum are causative agents of Lyme disease and human granulocytic anaplasmosis. Their common vector in Europe are the ticks from the genus Ixodes. In our work, we focused on interaction of innate immune cells with the causative agent of Lyme diseases, that are insubstitutable in their function in the early phase of the disease. Anaplasma phagocytophilum is hard to cultivate, the only possibility is to cultivate it in cell cultures. Successful cultivation of Anaplasma phagocytophilum acquired from patients in our geographic area is crucial for following experiments and for diagnostics too. In our experiments, we used validated cell cultures of HL-60 cells, canine monocytes DH82 and murine monocytes P388D1. During our studies of interaction of the causative agent of Lyme diseases with cells, we used two strains of different species Borrelia. Borrelia garinii M192 and Borrelia burgdorferi sensu stricto B31. These strains vary in virulence. The strain M192 is virulent, but the strain B31 lost its virulence by passages. We specialised in study of morphological changes using light microscopy (observation of dyed and fixed preparates and observation in dark field), eventually by transmision electron microscopy. During our experiments, we concluded that HL-60...
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Exprese CD47 a jeho topologie na povrchu primárních buněk karcinomu močového měchýře při interakci s makrofágy
Rajtmajerová, Marie ; Drbal, Karel (advisor) ; Brdička, Tomáš (referee)
CD47 is a so-called "don't eat me" signal, which protects cells from phagocytosis. Its high expresion on tumor cells brings new perspective to the tumor therapy. Monoclonal antibodies, which are these days undergoing clinical trials, prevent CD47 binding to the SIRPA inhibitory receptor on macrophages, and so they enhance their phagocytic functional capacity. In this way they enable phagocytic removal of tumor cells. Overall expression, structural conformation and stoichiometry of CD47 on a particular cell predestine whether it will be phagocytised. The aim of the thesis is to develop and test methods to characterise expression parameters of CD47 via flow cytometry (FCM), quantitative PCR (qPCR) and microscopy. To achieve this goal I performed competition tests of commercially available antibodies in order to characterise their binding epitopes on cell lines. After performing tSNE analysis of primary BCa patient samples I correlated CD47 expression with other cell surface markers. I focused on CD47 expression in various differentiation stages of the tumor. To better understand the relationship between CD47 expression and differentiation status of cells I performed qPCR analysis of particular transcription factors. Using cell lines I examined method for phagocytosis quantification, which will be...
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Proportional and functional characteristics of particular neutrophil subpopulations in umbilical cord and peripheral blood
Miková, Eliška ; Hrdý, Jiří (advisor) ; Krulová, Magdaléna (referee)
Early postnatal period is characterised by generally immature phenotype of the newborn's immune system. The maturation of the immune system including setting appropriate regulatory responses is occurring during this period and encountering pioneering bacteria colonizing neonate plays an important role. In the early days after birth, the immune system of a newborn is very limited, and the adaptive part is mostly represented by antibodies transferred from the mother by cord blood (CB) in the womb and then by colostrum and mother's milk after labour. Therefore, innate immunity plays a key role in defence (against pathogens) in neonates and is largely represented by neutrophils. This study aims to better understand neutrophil biology and phenotype in umbilical CB, compared to neutrophils from peripheral blood (PB) of mothers and healthy non pregnant women (referred to as HC). The assessment of neutrophil phenotype based on surface markers was performed using flow cytometry. Expression of genes linked to antimicrobial function was measured using quantitative PCR. Functional properties of neutrophils, metabolic activity during activation and phagocytosis, and suppressive properties were assessed using the SeaHorse machine and flow cytometry, respectively. Here we confirm the presence of immature CD16low...
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Role apoptózy a nekrózy makrofágů mléčné žlázy skotu
Klimešová, Monika
This bachelor thesis is aimed to the role of apoptosis and necrosis of bovine mamma-ry gland macrophages provides an overview of macrophages that are part of nonspecific immunity. It describes their development from pluripotent bone marrow cells via monocytes to macrophages and their importance in the bovine mammary gland, which inc-ludes the phagocytosis of microorganisms and dead cells, the production of cytokines, and the stimulation of cells that regulate inflammatory responses. In the next part the bachelor thesis is focused on individual types of cell death (apoptosis and necrosis). They describe their structural, biochemical and genetic features and subsequent events that take place after cell death, ie. the removal of dead cells from the organism. The effects of cell death on the surrounding tissue are also of no signifi-cance. At the end of the work are summarized the basic differences between apoptosis and necrosis and their roles during the inflammation of the bovine mammary gland.
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Virulence factors of Entamoeba histolytica
Novotná, Monika ; Tachezy, Jan (advisor) ; Tůmová, Pavla (referee)
The parasitic protist Entamoeba histolytica causes intestinal disease called amoebiasis (amoebic dysentery), which is one of the most significant diseases worldwide, mainly in developing countries. The goal of this bachelor thesis is to summarize current knowledge about virulence factors of E. histolytica. It is primarly focused on adhesive lectin Gal/GalNAc, cysteine proteases, phagocytosis of apoptotic cells, amoebapore forming pores in the membranes of the target cells and trogocytosis. Keywords: virulence factors, Entamoeba histolytica, parasite, protist, amoebiasis, lectin Gal/GalNAc, cysteine proteinase, phagocytosis, trogocytosis, amoebapore
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Molecular interactions of Staphylococcus aureus with the host
Cabrnochová, Marie ; Melter, Oto (advisor) ; Vopálenský, Václav (referee)
The aim of this thesis is to summarize S. aureus interactions with selected mechanisms of innate host immunity especially interactions with neutrophils and processes on the cell level which lead to host colonization. S. aureus surface proteins MSCRAMM interact with host cell surface proteins such as fibrinogen, keratin and thereby mediate adhesion to the host cell, which is an essential point for colonization of the host cell. The central mechanism of innate immunity against any S. aureus infection is the interaction of the pathogen with neutrophils, which produce neutrophil extracellular traps and phagocytes S. aureus cells. A crucial role in the elimination of bacterial cells in the phagosome of neutrophils is lysis by the antimicrobial peptides and degradation of bacterial biomolecules by the oxygen radicals. S. aureus defence mechanisms against action of immune system are considered to be virulence factors, due to its contribution to the establishment of the infection. These mechanisms are based on cell wall modification, inhibition of neutrophil chemotaxis, and production of enzymes that inhibit the effect of antimicrobial peptides, lysozyme, oxygen and nitrogen radicals. Expression of virulence factors of a particular S. aureus strain and host-specific risk factors can lead through successful...
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Exprese CD47 a jeho topologie na povrchu primárních buněk karcinomu močového měchýře při interakci s makrofágy
Rajtmajerová, Marie ; Drbal, Karel (advisor) ; Brdička, Tomáš (referee)
CD47 is a so-called "don't eat me" signal, which protects cells from phagocytosis. Its high expresion on tumor cells brings new perspective to the tumor therapy. Monoclonal antibodies, which are these days undergoing clinical trials, prevent CD47 binding to the SIRPA inhibitory receptor on macrophages, and so they enhance their phagocytic functional capacity. In this way they enable phagocytic removal of tumor cells. Overall expression, structural conformation and stoichiometry of CD47 on a particular cell predestine whether it will be phagocytised. The aim of the thesis is to develop and test methods to characterise expression parameters of CD47 via flow cytometry (FCM), quantitative PCR (qPCR) and microscopy. To achieve this goal I performed competition tests of commercially available antibodies in order to characterise their binding epitopes on cell lines. After performing tSNE analysis of primary BCa patient samples I correlated CD47 expression with other cell surface markers. I focused on CD47 expression in various differentiation stages of the tumor. To better understand the relationship between CD47 expression and differentiation status of cells I performed qPCR analysis of particular transcription factors. Using cell lines I examined method for phagocytosis quantification, which will be...
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