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Optimization of proces for detection of free tumor DNA in plasma and its clinical utility for colorectal cancer, lung cancer and pancreatic cancer patients
Belšánová, Barbora ; Benešová, Lucie (advisor) ; Tachezy, Ruth (referee)
In current days, examination of circulating tumor DNA (ctDNA) finds new use across different cancers. It is directed at tumor-derived short fragments of DNA present in peripheral blood of patiens (mainly in advanced stages). Due to its minimal invasivity, almost 100 % specificity and relatively high sensitivity in stage IV patients, this approch found its main potential clinical utility especially in early detection of disease relapse or progression after tumor resection (i.e. post-operative follow-up), prediction and monitoring of therapy response and estimation of prognosis. As a result of minute levels of ctDNA on a high background of other non-tumor DNA fragments present in plasma, a suitable method exhibiting highest sensitivity is the key for proper detection of this marker. The approach is predominantly based on initial identification of a mutation in tumor tissue and its subsequent detection in plasma. The present work is aimed at optimization of ctDNA isolation and method of its detection based on PCR amplification followed by heteroduplex analysis by denaturing capillary electrophoresis (DCE) to achieve highest sensitivity for detection of mutated fraction in plasma sample. I have applied the optimized protocol to examine ctDNA in three types of cancers, namely colorectal cancer (122...
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Regulation of transcription by proteins of the Early growth response and Myb families
Čermák, Vladimír ; Dvořák, Michal (advisor) ; Vomastek, Tomáš (referee) ; Elleder, Daniel (referee)
The regulation of transcription of tens of thousands of genes in a vertebrate organism is an enormously complex phenomenon which entails the participation of thousands of various regulatory proteins. The largest functional category of these regulators is accounted for by sequence-specific DNA-binding proteins known as transcription factors. Proteins of the EGR and Myb families of transcription factors are long-studied regulators of a variety of physiological processes including cellular proliferation and differentiation. The structural and physical aspects of their function have been well characterized. Their cell-type specific participation in complex gene-regulatory networks, on the other hand, is still incompletely understood and represents a major challenge in the respective research areas. Preliminary analysis of gene expression data from metastasizing PR9692 and non- metastasizing PR9692-E9 chicken sarcoma cell lines revealed that the transcription factor EGR1 is expressed at a higher level in metastasizing cells and can thus take part in the regulatory processes that underlie the differences between the two cell lines. Further investigation demonstrated that the introduction of exogenous EGR1 into PR9692-E9 cells restored their metastatic potential to a level indistinguishable from PR9692...
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The use of CAM assay for characterization and study of cancer cell invasive properties
Vágnerová, Lenka ; Dvořák, Michal (advisor) ; Geryk, Josef (referee)
The chorioallantoic membrane (CAM) of chicken embryos belongs to the in vivo model systems frequently used for the study of angiogenesis and cell invasiveness. Using CAM assay we have tested selected chicken sarcoma cell lines characterized by different angiogenic properties and different ability to form metastasis. In addition to CAM assay, several other methods have been used to characterize the phenotype of these cell lines. We have selected a few proteins which could significantly influence the angiogenic and metastatic properties of investigated cell lines. We have established cell lines stably overexpressing these genes and compared their phenotypes with parental cell lines. We have shown that genes encoding ISL1, ARNT2, PROM1, HOXA11 proteins participate, in our experimental model, in activation of programes controlling angiogenesis and cell invasion.
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Perineural spread of pelvic tumors: mechanism and diagnosis
Čapek, Štěpán ; Sameš, Martin (advisor) ; Haninec, Pavel (referee) ; Hořínek, Daniel (referee)
Perineural spread of pelvic tumors mechanism and diagnosis Abstract Neoplastic lumbosacral plexopathies are infrequent affections of the lumbosacral plexus. Cases with minimal or non-specific finding on imaging can be particularly puzzling to diagnose. We describe a series of patients with perineural spread from the site of the primary tumor along the visceral autonomous nerves to the lumbosacral plexus and further proximally to the spinal nerves and even intradurally and also distally to the sciatic nerve. On series of 17 patients diagnosed with perineural spread of pelvic malignancy we describe characteristic clinical presentation and imaging finding. In many of these cases a tissue biopsy is necessary to finalize the diagnosis. We describe operative technique of targeted fascicular biopsy of the sciatic nerve and our experience with this procedure. On series of 117 patients, we report the outcome and complication: diagnostic yield was 84.8% and complication rate was 2.7 %. If a tissue sample is needed to conclude the diagnosis, targeted fascicular biopsy does increase the yield at an acceptable complication rate. Perineural spread of pelvic cancer is a new clinical-pathological entity with an unknown natural history or ideal treatment strategy. Based on the imaging finding in this group we present a...
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The analysis of cancer invasion plasticity in a 3D environment
Škarková, Aneta ; Brábek, Jan (advisor) ; Anděra, Ladislav (referee) ; Bryja, Vítězslav (referee)
iii Abstract Cells have evolved multiple mechanisms of cellular motility ranging from the migration of large cell cohorts to specialized migration of individual cells. The wide range of invasion modes has been exploited by cancer cells to their advantage, which has rendered the metastatic process so difficult to defeat. To allow for a better understanding of cancer invasion plasticity, we have employed studies on cancer cells that adopt the proteolytically active, adhesion-dependent, elongated mesenchymal invasion mode, the protease-independent, low adhesion, rounded amoeboid invasion mode, or combination of both. To study invasion plasticity directly, we have established two model systems of the mesenchymal- amoeboid transition (MAT) that allow for regulated induction of MAT in 3D in vitro environments. Using these systems, MAT was induced in HT1080 fibrosarcoma cells and the acquisition of a motile, invasive amoeboid phenotype was confirmed. We then observed the mesenchymal and amoeboid invasion strategies within 3D collagen in more detail using a digital holographic microscope. Further, HT1080 cells before and after MAT were subject to high throughput proteomic and transcriptomic studies. Comparison of gene expression and protein levels of mesenchymal and amoeboid cells disclosed an inflammatory-like...
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Cancerogenic and metastatic potential of cancer cells with non-functional CRL4 ubiquitination complex
Slámová, Monika ; Procházka, Jan (advisor) ; Grantz Šašková, Klára (referee)
Ubiquitination complex CRL4 (Cullin Ring Ligase) attracts a lot of attention due to its involvement in physiological and pathological processes, especially in the development of cancer. Cullin4 a/b proteins are reported to serve as oncoproteins in various malignancies. Due to their role in the regulation of cancer drugs targeting CRL4 have been identified, including thalidomide and its derivatives inhibiting one of the substrate receptors of the complex, the Cereblon protein. The adapter protein within the CRL4 complex - DDB1, which is involved i.a. in DNA repair, also has a role in cancer. However, the mechanism of this function has not yet been fully elucidated. The subject of this master thesis was to study the effects of elimination and suppression of CRL4 complex functions in the prostate cancer cell line LNCaP. Significantly variable changes in cell proliferation and migration have been observed if the complex functions were affected by thalidomide. The creation of the LNCaP cell line with conditionally suppressed DDB1 function was used to study tumor dynamics in a mouse model. Results show that suppression of DDB1 function has an inhibitory effect on tumor cell proliferation but increases their ability to invade adjacent tissues. Complete deletion of the DDB1 gene in the LNCaP cell line...
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The role of mitochondrial respiratory chain in invasiveness and metastasis of cancer cells and possible therapeutic interventions
Legátová, Anna ; Brábek, Jan (advisor) ; Truksa, Jaroslav (referee)
The mitochondrial respiratory chain, also called the electron transport chain (ETC), has a pivotal role in key features of cancer cells e.g., proliferation, the metabolic shift from oxidative phosphorylation (OXPHOS) to aerobic glycolysis, or the ability to form metastases. This review summarizes current knowledge about ETC and its relationship to cancer, especially to invasiveness and metastases formation. Firstly, it deals with a process called the Warburg effect and with metabolic complexity in the tumor microenvironment. Then it shows how OXPHOS activity affects invasiveness of cancer cells and metastases formation, and it points out the connection between invasiveness and increased levels of ETC-generated reactive oxygen species. At the end, the review deals with possible use of ETC inhibitors in anticancer therapy.
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Time development analysis of treated lesion in spinal CT data
Nohel, Michal ; Jan, Jiří (referee) ; Jakubíček, Roman (advisor)
This diploma thesis is focused on time-development analysis of treated lesion in CT data. The theoretical part of the thesis deals with the anatomy, physiology, and pathophysiology of the spine and vertebral bodies. It further describes diagnostic and therapeutic options for the detection and treatment of spinal lesions. It contains an overview of the current state of usage of time-development analysis in oncology. The problems of the available databases are discussed and new databases are created for subsequent analysis. Futhermore, the methodology of time-development analysis according to the shape characterization and the size of the vertebral involvement is proposed. The proposed methodological approaches to feature extraction are applied to the created databases. Their choice and suitability is discussed, including their potential for possible usege in clinical practice of monitoring the development and derivation of characteristic dependences of features on the patient's prognosis.
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Molecular mechanisms of fibroblastoid cell phenotype transitions:dedifferentiation of myofibroblasts and influencing of invasiveness and metastasis of sarcoma
Kosla, Jan
Fibroblasts are the principal cellular component of the connective tissue. They are a heterogeneous group of cells which contribute to the structure of connective tissue and wound healing by their ability to produce extracellular matrix (ECM). Fibroblasts and cells derived from them are involved in many pathological processes such as formation of malignant tumors and fibrosis. Tumor progression which finally leads to metastasis is a serious biomedical problem. There is a growing body of the recent evidence showing an important role of the tumor stroma and its interaction with cancer cells in cancer progression. Tumor stroma comprises mainly of myofibroblasts and their products, namely ECM, soluble factors, and enzymes. Myofibroblasts contribute more or less to all steps of cancer progression. Furthermore myofibroblasts play a key role in fibrosis, another serious human disease which is not efficiently treatable and which is associated with cancer progression. These facts made us to search for molecular means capable of eliminating the myofibroblastic phenotype. We succeeded to entirely dedifferentiate primary myofibroblasts by concomitant inhibition of TGFβ signaling and perturbation of MAPK signaling in a chick model that we have introduced. Malignant fibroblasts form sarcomas. ECM is the first...
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Methods of Detection, Segmentation and Classification of Difficult to Define Bone Tumor Lesions in 3D CT Data
Chmelík, Jiří ; Flusser,, Jan (referee) ; Kozubek, Michal (referee) ; Jan, Jiří (advisor)
The aim of this work was the development of algorithms for detection segmentation and classification of difficult to define bone metastatic cancerous lesions from spinal CT image data. For this purpose, the patient database was created and annotated by medical experts. Successively, three methods were proposed and developed; the first of them is based on the reworking and combination of methods developed during the preceding project phase, the second method is a fast variant based on the fuzzy k-means cluster analysis, the third method uses modern machine learning algorithms, specifically deep learning of convolutional neural networks. Further, an approach that elaborates the results by a subsequent random forest based meta-analysis of detected lesion candidates was proposed. The achieved results were objectively evaluated and compared with results achieved by algorithms published by other authors. The evaluation was done by two objective methodologies, technical voxel-based and clinical object-based ones. The achieved results were subsequently evaluated and discussed.
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