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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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Immunoscore in 3D tissue
Novák, Jaromír ; Drbal, Karel (advisor) ; Procházka, Jan (referee)
Solid tumors are complex structures comprising besides the cancer cells vasculature, extracellular matrix (ECM), soluble molecules and a plethora of various other cell types. These components form a so-called tumour microenvironment. From the numerous cell types that are part of tumor microenvironment, tumor infiltrating lymphocytes (TILs) play a major role in patient prognosis. Their presence is also of major importance with regard to new biological therapies based on immune checkpoint inhibitors. Crucial role of TILs is also reflected by the new approaches in cancer diagnostics namely by Immunoscore method (currently used in clinical settings). Immunoscore is based on localization and quantification of CD3+ and CD8+ TILs in thin histological sections of tumor tissue. The question remains to which extent the information obtained from 2D slices reflects the situation in tumor microenvironment considering its spatial heterogeneity. The development of new methodological approaches allowing evaluation of histological information in 3D is the key to answer this question. The theoretical part of this work first describes the heterogeneity of the tumor microenvironment and the role of immune cells within it. Then, the role of spatial heterogeneity and its possible influence on the histopathological...
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Tumor microenvironment and the importance of anti-tumor immunity for clinical course of human cancers
Partlová, Simona ; Špíšek, Radek (advisor) ; Drbal, Karel (referee) ; Kovář, Marek (referee)
Cancer development and progression vary depending on tumor type, localization, invasion, immunogenicity and the ability of immune system to become activated. There are frequent interactions between tumor cells and immune cells, occuring locally at the site of primary tumor or distally through paracrine signalling of various mediators and cytokines. The main subject of this PhD thesis is to study key factors and aspects of immune response in cancer patients. In the first part, we analyzed immune cells infiltrating tumor tissues of ovarian cancer patients at different stages of disease. We focused on the dynamics of immune response, primarily on frequency of individual T lymphocyte populations in peripheral blood and tumor infiltrating T lymphocytes in tumors of early and advanced stages of ovarian cancer. We found that during disease progression there is a gradual decrease of proinflammatory Th17 and Th1 immune responses and a specific recruitment of regulatory T cells to the tumor site, which results in a significant immune suppression in the tumor microenvironment. In the second part, we demonstrated that the character of immune response in HPV-positive head and neck cancer patients is very different from the patients with tumors not associated with HPV infection. In HPV-positive patients, significantly...
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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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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 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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Role of fibroblasts in wound healing and cancer
Mateu Sanz, Rosana ; Smetana, Karel (advisor) ; Masařík, Michal (referee) ; Mokrý, Jaroslav (referee)
Fibroblasts are stromal cells ubiquitously present in the human body. They often appear in a quiescent state and can become activated in response to tissue remodeling signals. Activated fibroblasts acquire biosynthetic, pro-inflammatory and contractile properties, key functions for wound healing. In addition, the presence of permanently activated fibroblasts is one of the hallmarks of cancer. The purpose of this work is to investigate the differences between newborn and adult fibroblasts and keratinocytes in their implication in scarless wound healing, the origin of cancer associated fibroblasts (CAF)s and the influence of fibroblasts in melanoma invasion. Evidence suggests that wounds heal almost without scar in newborns. To understand the mechanisms that contribute to scarless wound healing we focused on the differences between newborn and adult fibroblasts and keratinocytes, which are cells present in human skin and participating in wound healing process. A comparison of the expression profile between newborn and adult fibroblasts showed differentially regulated genes related to the acute phase of the inflammatory response and ECM organization, traits involved in wound healing. We also found that newborn fibroblast showed higher differentiation potential, exhibited markers of pluripotency and...
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Pathologic STAT3 signalling pathway activation in cancer and viral diseases.
Podestátová, Barbora ; Reiniš, Milan (advisor) ; Škarková, Aneta (referee)
STAT3, one of the seven members of STAT protein family, is able to transduce signal into the nucleus, where it binds to specific DNA sequences and acts as a transcription factor. Under physiological conditions, STAT3 regulates genes associated with number of functions such as cell proliferation, differentiation, apoptosis or immune response. In the case of pathological conditions, STAT3 can be dysregulated or constitutively activated, which may result in cancerogenesis. During this process, STAT3 is frequently activated directly in tumor cells where it acts tumorigenically. STAT3 is also associated with inflammatory reactions mediated by immune cells, which along with tumor and stromal cells are involved in the formation of the tumor microenvironment. The role of STAT3 is also important in the fight against viral infections, and when STAT3 activated aberrantly, it can lead to chronic diseases, including cancer. Due to these serious roles during pathogenesis, STAT3 is the subject of research of various inhibitors that either directly inhibit the STAT3 molecule function or indirectly any of the components of its signaling pathway.
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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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Detection and characterization of macrophages in the tumors of viral and non-viral etiology
Dalewská, Natálie ; Tachezy, Ruth (advisor) ; Krulová, Magdaléna (referee)
Head and neck cancers are etiologically associated with smoking and alcohol consumption. Part of these tumors is induced by HPV and their incidence is increasing in the last decade. Patients with virally induced tumors have better prognosis even though they are usually diagnosed with tumors in advanced stage. One of the possible explanations may be better stimulation of the immune system by viral antigens. Macrophages are cells of the innate immune system which belong to professional phagocytes. They are called TAM upon infiltration to the tumor where they represent heterogeneous group of cells. Two main phenotypes are antitumor M1 and protumor M2 macrophages. TAMs are a major component of tumor microenvironment of many types of tumors, one of them are also head and neck cancers. In my thesis I focused on the immunohistochemical detection of M1 and M2 macrophages in the head and neck tumors of viral and non-viral etiology and at the same time RT-qPCR analyses of gene expression of macrophage-associated and/or immunosuppressive genes IDO1, ARG1, CD163, NOS2 a PTGS2 was performed. My data showed that HPV- negative tumors had higher number of M2 macrophages with typical markers CD163, ARG1 and PTGS2. It is known that patients with these tumors have worse prognosis of the disease. Due to high...
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