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Intercellular interactions in skin tumors.
Kučera, Jan ; Smetana, Karel (advisor) ; Masařík, Michal (referee) ; Kovář, Marek (referee)
The dissertation is focused on the study of intercellular interactions in skin tumors. It is based on 5 original publications that cover several topics. We studied the origin of tumor-associated fibroblasts concerning the primary tumor population. We demonstrated using a mouse model that tumor-associated fibroblasts are produced from the host organism and thus did not arise from transformation directly from tumor cells. We also investigated the relationship between tumor-associated fibroblasts and keratinocytes. We have shown that tumor-associated melanoma fibroblasts affect keratinocytes which, under their influence, acquire the features typically observed in migrating cells and cells undergoing epithelial-mesenchymal transition. We studied the interactions between healthy fibroblasts and tumor cells. We have demonstrated that fibroblasts acquired from healthy skin from a patient suffering from melanoma are significantly different from control fibroblasts of healthy donors in the expression profile. Changes in distal fibroblasts support the view of melanoma as a systemic disease. We have further demonstrated that melanoma-associated fibroblasts do not carry a BRAF mutation, in contrast to BRAF positivity of melanoma cells. And therefore, they did not arise from the transition from melanoma. The...
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Dipeptidyl peptidase-IV and Fibroblast activation protein in gliomagenesis.
Trylčová, Jana ; Šedo, Aleksi (advisor) ; Mandys, Václav (referee) ; Mareš, Vladislav (referee)
"Dipeptidyl peptidase-IV Activity and/or Structure Homologues"(DASH) represent a newly defined group of multifunctional molecules, typically bearing dipeptidyl peptidase-IV- like hydrolytic activity. Dipeptidyl peptidase-IV (DPP-IV) cleaves out X-Pro dipeptides from the N-terminus of peptides. Other molecules carrying similar enzyme activity, such as Fibroblast activation protein (FAP), DPP-II, DPP8 and DPP9 or even DPP-IV structure-like but hydrolytically inactive molecules (DPP6 and DPP10) also belong to this group. Recent knowledge suggest a substantial role of DASH in cancer pathogenesis. The aim of this study is a preparation of a biological model and its use for understanding the mechanisms of interaction(s) between transformed glial cells and stroma in the processes of origin and development of tumors derived from neuroectoderm. Stable transfected human glioblastoma cell lines with inducible gene expression of DPP-IV, Fibroblast activation protein and their enzymatically inactive mutated forms, were prepared within the project. Prepared cell lines are used as a tool for studying not only the "autocrine" importance of DPP-IV and FAP for the expressing cells in in-vitro, but also for their potential "paracrine" effect(s) within the tumor microenvironment after homotopic implantation into the...
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Experimental verification of in silico predicted protein binder to FOXO4 transcription factor and transcriptome analysis of bladder cancer
Tauš, Petr ; Drbal, Karel (advisor) ; Převorovský, Martin (referee)
This diploma thesis includes an experimental and a bioinformatic part. The two parts are linked together through the subject of transcription factors of 'forkhead box O' (FOXO) family. FOXO transcription factors have a key role in many cellular processes including cell cycle regulation, apoptosis and metabolism. For a long time, they have been considered strictly as the tumor-suppressors yet a growing number of evidence is pointing out to their pro-tumorigenic role. In consequence FOXO transcription factors are studied intensively as potential therapeutic targets in cancer. In the past decade, in silico prediction of protein-protein interactions has become popular in basic research as well as in drug development. Nonetheless, the predicted structures are still far from fitting to the expected behavior of the respective biomolecules. In the experimental part of this thesis, I verified the interaction of four in silico predicted protein binders based on naturally occurring PDZ domain with FOXO4 using microscale thermophoresis. Non-invasive bladder tumors represent a heterogeneous disease where reliable prediction of tumor aggressiveness is still lacking despite an intensive research. In the bioinformatic part of this thesis, I described the cellular composition of the tumor microenvironment and demonstrated...
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Tumor microenvironment modulation and the impact on cancer immunotherapy
Musil, Jan
Modulation of the tumor microenvironment represents a possible way to inhibit cancer growth and enhance anti-cancer immune responses. In the presented work we employ two strategies for tumor microenvironment modulation. Firstly, we have constructed rVACV co-expressing the tumor suppressor gene insulin-like growth factor-binding protein-3 (IGFBP- 3) and the fusion gene encoding the immunogen SigE7LAMP. The expression of IGFBP-3 was regulated either by the early vaccinia virus H5 promoter or by the synthetic early/late (E/L) promoter. We have shown that expression of IGFBP-3 regulated by the H5 promoter yielded higher amounts of IGFBP-3 protein when compared with the E/L promoter. Immunization with P13-SigE7LAMP-H5-IGFBP-3 was more effective in inhibiting the growth of TC-1 tumors in mice and elicited a higher T-cell response against VACV-encoded antigens than the control virus P13-SigE7LAMP-TK- . We found that high-level production of IGFBP-3 enhanced virus replication both in vitro and in vivo, resulting in profound antigen stimulation. Production of IGFBP-3 was associated with a higher adsorption rate of P13-SigE7LAMP-H5-IGFBP-3 to CV-1 cells when compared with P13-SigE7LAMP-TK- . We have identified two structural differences between the IMVs of the IGFBP-3 expressing virus P13-SigE7LAMP-H5-IGFBP-3...
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Myeloid - Derived Suppressor Cell in the Context of Tumor Microenvironment
Košťálová, Monika ; Šírová, Milada (advisor) ; Indrová, Marie (referee)
Today, tumors are considered not only as a complex of genetically mutated cells with pathological function of excessive proliferation, invasiveness and increased viability, but increased attention is paid for the tumor microenvironment created by the tumor itself. This microenvironment generates conditions, which differ from the normal tissues - for example local hypoxia, lactic acidosis and tumor- induced immunosupression - all these abnormalities lead to increased viability of the tumor tissue. Myeloid-derived suppressor cells (MDSCs) seem to be one of the main mediators of the escape from immunosurveillance. MDSCs represent a heterogenous cell population of myeloid origin. In active state, MDSCs produce enhanced amount of reactive oxygen species, nitrogen compounds and arginase, which represent the mechanisms of the suppression of the anti-tumor immune response. That makes MDSCs a promising therapeutic target. However, recent studies also point out the physiological role of MDSCs, which seems to be essential to consider for succesfull MDSCs targeting. Key words: Tumor microenvironment, immunosurveillance theory, immunoediting, myeloid-derived suppressor cells, immunosuppresion in tumors, therapeutic targeting of MDSCs, physiological role of MDSCs Powered by TCPDF (www.tcpdf.org)
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Intercellular interactions in malignant melanoma
Nedvědová, Tereza ; Dvořánková, Barbora (advisor) ; Brábek, Jan (referee)
Melanomas are one of the most aggressive types of tumours, with increasing incidence, high mortality and high potential to metastasize to a variety of diverse locations. The aim of this thesis was to study the tumour as a complex structure consisting not only of tumour cells but also of tumour stroma. Stromal cells play a major role in cancer biology. This is well documented for example in squamous cell epithelium tumours of the head and neck. Similar mechanisms can be expected to occur in melanomas. In the first experiment, we simulated the conditions in vivo during the metastatic process and studied the influence of non-adhesive environment both with and without the influence of stromal fibroblasts. The presented data demonstrates a change of tumour cells' phenotype leading to increased plasticity of the melanoma cells in these conditions. It also indicates the crucial role of stromal fibroblasts in interactions with melanoma cells. Cancer cell lines show variability in their behaviour, which is in accordance with well-known melanoma heterogeneity in clinical practice. The previous experiments in our laboratory indicate that cancer associated fibroblasts are able to influence the phenotype of a tumour cell line and this effect is based on a tumour type-unspecific mechanism. In the second part of...
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Tumor microenvironment modulation and the impact on cancer immunotherapy
Musil, Jan ; Němečková, Šárka (advisor) ; Mikyšková, Romana (referee) ; Otáhal, Pavel (referee)
Modulation of the tumor microenvironment represents a possible way to inhibit cancer growth and enhance anti-cancer immune responses. In the presented work we employ two strategies for tumor microenvironment modulation. Firstly, we have constructed rVACV co-expressing the tumor suppressor gene insulin-like growth factor-binding protein-3 (IGFBP- 3) and the fusion gene encoding the immunogen SigE7LAMP. The expression of IGFBP-3 was regulated either by the early vaccinia virus H5 promoter or by the synthetic early/late (E/L) promoter. We have shown that expression of IGFBP-3 regulated by the H5 promoter yielded higher amounts of IGFBP-3 protein when compared with the E/L promoter. Immunization with P13-SigE7LAMP-H5-IGFBP-3 was more effective in inhibiting the growth of TC-1 tumors in mice and elicited a higher T-cell response against VACV-encoded antigens than the control virus P13-SigE7LAMP-TK- . We found that high-level production of IGFBP-3 enhanced virus replication both in vitro and in vivo, resulting in profound antigen stimulation. Production of IGFBP-3 was associated with a higher adsorption rate of P13-SigE7LAMP-H5-IGFBP-3 to CV-1 cells when compared with P13-SigE7LAMP-TK- . We have identified two structural differences between the IMVs of the IGFBP-3 expressing virus P13-SigE7LAMP-H5-IGFBP-3...
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New ferritin nanoparticles for specific targeting of experimental melanoma in mice: in vitro and in vivo tests.
Rajsiglová, Lenka ; Vannucci, Luca Ernesto (advisor) ; Šírová, Milada (referee)
Cancer diseases represent second most frequent cause of death after cardiovascular diseases in Europe. Nowadays used medical treatments like chemotherapy and radiotherapy are nonspecific and cause huge side effects. Various systems to deliver therapy directly inside the tumour microenvironment and reduce side effects are under development. Protein nanoparticles seem to be very promising strategy to achieve that goal. Our group in cooperation with CNR in Rome tested nanoparticles based on heavy chain of human ferritine. These constructs, modified to expose the tumor targeting molecule, were able to be specifically internalised by B16F10 melanoma cells in vitro. They also specifically target and localise at the sites of primary melanoma and lung metastases of different size in mouse in vivo model. These nanoparticles can carry either therapeutic or diagnostic molecules. Thus they represent a suitable candidate for further studies for potential use in clinical praxis as a diagnostic and/or therapeutic agents (theranostics). Powered by TCPDF (www.tcpdf.org)
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Cellular senescence escape mechanisms - anti-cancer barrier
Davidová, Eliška ; Hodný, Zdeněk (advisor) ; Horníková, Daniela (referee)
Cancer is one of the most dangerous diseases of the modern world. Therefore, many world laboratories engaged in research into the causes leading to the outbreak of this insidious disease. In this context, it has already been found that the normal animal cells do not divide indefinitely, but have a finite replicative life span. After this period, cells undergo either apoptotic processes or enter into so-called senescence, typical for proliferation arrest, but preserved metabolic processes. Further research has revealed that senescence serves as an effective anticancer program and currently is shed light on its significance in relation to various physiological or pathological processes associated with aging. In this work, the focus is on the role of senescence as a barrier for cancer development, and effectiveness. It can be assumed, that if the senescent cycle arrest functioned perfectly, the incidence of cancer among people would be recorded in much lower extent. The aim of this thesis is the current knowledge about the physiological and pathological roles of senescence and possible causes of overcoming this barrier, the result may be the uncontrolled cell division and tumorigenicity.
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