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The evolution of SARS-CoV-2 virus during the pandemic of COVID-19
Karban, Šimon ; Tachezy, Ruth (advisor) ; Pačes, Jan (referee)
Coronaviruses are animal and human viruses which, in the case of humans, cause respiratory diseases. The genome of coronaviruses is non-segmented and encodes several structural and several non-structural proteins. As their genome consists of single-stranded RNA in a positive sense, they encode RNA-dependent RNA polymerase. The origin of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is uncertain and may never be known. However, this thesis covers up-to-date knowledge and the arguments for the main theories on the emergence of this virus. The pandemic of disease COVID-19 caused by the SARS-CoV-2 had an enormous impact on the health and lives of people worldwide. The length and severity of the pandemic were caused by the characteristic of the virus, transmissibility and asymptomatic type of infection with severe symptoms in elderly and chronically ill individuals and the fast evolution of the virus after its appearance in humans. This thesis will describe important characteristics of the most important variants of the virus and changes which gave them a selection advantage. In the end, trends in the evolution of SARS-CoV-2 will be discussed. Keywords: SARS-CoV-2, Coronaviridae, variants, phylogeny, origin, evolution
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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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The differences in the virome of different populations of honey bee (Apis mellifera)
Kadlečková, Dominika ; Tachezy, Ruth (advisor) ; Votýpka, Jan (referee)
European honey bee (Apis mellifera) is major pollinator for agriculture and vital for food production. Large number of viruses infecting A. mellifera have been discovered over the years, but it isn't yet known if they are pathogenic for their host. However, presence of non-viral pathogens like Varroa destructor can greatly increase their virulence and have fatal consequences for the colony. The aim of this study was to test and verify robustness of the method for virome detection on healthy honey bees from the Czech Republic. Last but not least we aimed to detect non-viral parasites and correlate their presence with detected viruses. We have successfully identified large number of viral sequences from different viral families. Viral composition was found to be influenced mainly by colony from where the honey bees were collected. That was mainly given by a large amount of bacteriophages in the samples. However, analysis of individual viruses, known to infect honey bee, indicated that viral prevalence and viral loads of specific viruses is quite different among individual honey bees from the same colony. Interestingly we were able to find highly diverse Lake Sinai viruses. We were able to observe correlations either between individual viruses or viral other non-viral pathogens. Further analysis is...
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The expression of miRNA in HPV-associated and HPV-independent head and neck tumors
Vojtěchová, Zuzana ; Tachezy, Ruth (advisor) ; Hejnar, Jiří (referee) ; Slabý, Ondřej (referee)
Head and neck cancers represent a group of tumors with two different etiologies. The first type is associated with the viral HPV infection, the second one is virus-independent and it is associated with smoking and alcohol consumption as two main risk factors. Numerous studies show that HPV-positive tumors are more frequent in younger patients, as well as that the prognosis and overall survival of these patients is remarkably better. Therefore, the modification of the treatment is considered. For this, however, specific, sensitive and clinically relevant biomarkers for accurate identification of tumor etiology is needed. Suitable candidates for such biomarkers are miRNAs, small non-coding regulatory molecules stable in archived samples, that have been shown as differentially expressed in human cancers and the expression pattern seems specific for tumors of different origin. The submitted thesis focuses on miRNA profiling in HPV-positive and HPV-negative tonsillar tumors and cervical carcinomas with the aim to find out the differences between regulation of important carcinogenetic pathways of tumors of viral and non-viral etiology. Our data have shown very large heterogeneity of the miRNA expression profiles of these tumors. Despite the well characterized and uniform samples collection, we have found...
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Studium celogenomové variability lidského cytomegaloviru.
Dvořák, Jan ; Tachezy, Ruth (advisor) ; Roubalová, Kateřina (referee)
This work is part of a project focused on the study of the variability of human cytomegalovirus (HCMV) among clinical isolates with the aim to map the geographical distribution of HCMV genotypes, reveal the relationships between genotypes and the severity of HCMV-associated diseases, and identify regions in the HCMV genome with a potential for use as diagnostic and therapeutic targets. Attention was paid to the development of the methodology for the preparation of the material for next-generation sequencing (NGS) from HCMV clinical isolates and evaluation of the obtained sequencing data. Blood and urine samples collected from hematopoietic stem cell transplantat recipients and congenitally infected children were analyzed. Samples suitable for NGS were sequenced by the Illumina platform and sequences were created by de novo assembly followed by mapping assembly. Urine samples in comparison to blood samples had higher yield of material for NGS. Of the samples positive for HCMV DNA (7 of 50) after amplification in the cell cultures, only one sample had high purity of the viral DNA (98%) while six samples had purity of less than 7%. The sample containing 98% of the viral DNA was fully sequenced and the sequence was compared to the sequences of other clinical isolates from Belgium in 11 polymorphic...
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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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Analysis of miRNAs in HPV-associated carcinomas
Pagáčová, Lucie ; Tachezy, Ruth (advisor) ; Vopálenský, Václav (referee)
Papillomaviruses are small DNA viruses that are associated with the induction of epithelial tumors. HPV is an important infectious agent causing almost 100 % of cervical tumors but it can also cause tumors in other anogenital and head and neck locations in both men and women. Active HPV infection induces changes in miRNA expression that contribute to the tumor formation and progression. It is already known that papillomaviruses do not encode their own viral miRNAs but they affect the expression of cellular miRNAs. In my thesis I have in selected epithelial tumors (vulva, cervix, anus and tonsils) determined their etiology and analyzed the presence of miRNAs in tissues by next generation sequencing. From these data I determined the expression profiles of deregulated miRNAs in tumors relation to healthy tissues of corresponding location. Even though, sufficient number of samples was analyzed, it was not possible to detect HPV-core miRNA common to all analyzed HPV-induced tumors due to the absence of statistically relevant differentially expressed miRNAs in HPV positive vulvar tumors. Among the tumors of the other sites I found an overlap in three miRNAs. One of these miRNAs (miR-139-5p) and another one (miR-9-5p) which I have selected based on the study of other published data, were used for...
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Cytomegalovirus infection in transplant patients
Dvořák, Jan ; Tachezy, Ruth (advisor) ; Harant, Karel (referee)
Cytomegalovirus (HCMV) is a ubiquitous human -herpesvirus highly prevalent in the population. HCMV is transmitted by close contact between individuals. In infected person this virus causes mainly asymptomatic primary infection, after which the latency is established. In pregnant women HCMV infection can lead to abortions, defects of the fetus and congenital abnormalities of newborn babies. Even more serious complications are caused by this virus in the immunocompromised patients, especially those infected by HIV and in patients who undergo solid organ transplantation and hematopoietic stem cell transplantation. This work is a complex report about HCMV biology with emphasis on complications which HCMV causes in patients after solid organ transplantation and hematopoietic stem cell transplantation. This article also contains summary of the methods used for diagnostic of HCMV infection and monitoring and prevention of HCMV associated diseases. Keywords: Cytomegalovirus, hematopoietic stem cell transplantation, solid organ transplantation, detection, monitoring, polymerase chain reaction, cellular immunity, humoral immunity
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Methylation of the regulatory sequences of integrated and extrachromosomal form of HPV 16 in tonsillar tumors
Pokrývková, Barbora ; Tachezy, Ruth (advisor) ; Hubáček, Petr (referee)
Human papillomavirus is connected with induction of cervical carcinoma as well as for some other anogenital carcinomas and subset of carcinomas of head and neck. Presence of viral E6 and E7 oncoproteins may induce cell transformation, higher load of oncoproteins is caused by the regulatory E2 protein inactivation. Aims of recent study are mechanisms of E2 protein inactivation. One option is integration of viral DNA into the host genome, which is located into the E2 gene region. Some carcinomas, where virus with extrachromosomal form was presented, were found. It appears that epigenetic changes can play the role in the development of this type of tumors, especially DNA methylation or mutation in the regulatory region of the virus. The methylation degree analysis on samples of tonsillar carcinomas with extrachromosomal and integrated form of the virus was conduced, as well as viral load of both groups was compared and the expression of E6 and E7 gene was confirmed. The results of methylation analysis showed increased methylation of the virus with integrated DNA. Mutations in the E2 protein binding sites are not revealed. The expression of the viral oncogenes were confirmed in all tumors regardless of the form of the viral genome. The mechanism of tumors induction, especially for virus with...
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