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Laboratory diagnostics of Hepatitis B
JANŮ, Denisa
Hepatitis B is an inflammatory viral disease of the liver. The etiological agent is a DNA virus from the Hepadnaviridae family. It is transmitted through the blood and secretions of an infected person. The greatest danger of the disease lies in the transition to chronicity. Chronic infection carries a high risk of developing liver cirrhosis and hepatocellular carcinoma. Hepatitis B is widespread throughout the world. The most reliable prevention is vaccination and avoidance of risky behavior and contact with persons with risky behavior, especially drug users and promiscuous persons. The aim of this bachelor thesis is to get acquainted with the disease of viral hepatitis B and to describe the current possibilities of diagnostics of hepatitis B markers using the COBAS e601 device from ROCHE by electrochemiluminescent immunoassay and quantification of viral DNA on the COBAS 4800 from ROCHE by real time PCR and evaluation of results using both methods. The set of examined samples comes from the laboratory of infectious serology and virology of the company VIDIA-DIAGNOSTIKA. The results show that HBsAg is the most investigated marker of hepatitis B. Positive findings make up only 0.67%. The ratio of HBsAg positive men and women in 2019 was almost balanced. Most HBsAg positive patients were aged 30-40 years. I also examined the prevalence of anti-HBc Ig total antibodies. The positivity of anti-HBc Ig total was confirmed in 4.27% of examinations. The ratio of anti-HBc Ig total positive men and women in 2019 was also almost balanced and the most positive were at the age of 30-40 years.
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The role of Hepatitis B virus capsid protein in the host ubiquitin proteasome pathway
Eliáš, Vratislav ; Weber, Jan (advisor) ; Šmahel, Michal (referee)
Hepatitis B virus (HBV) is a Hepadnaviridae virus infecting mammals. Its infection can result in an acute or chronic infection. Chronic infection can result in hepatocellular carcinoma and liver cirrhosis, potentially leading to death of the patient. HBV is a small 42 nm virus with a genome length of 3.2 kb encoding seven viral proteins. HBV Core protein (HBc) is a capsid forming protein which is pleiotropic in function. We have identified two ubiquitin ligases which could interact with this protein: F-box only protein 3 (FBXO3; E3 ubiquitin ligase) and Ubiquitin conjugating enzyme E2 O (UBE2O; E2/E3 ubiquitin ligase). By employing multiple methods we have confirmed these interactions. Co- immunoprecipitation and further western blot analysis unveiled multiple new insights into the ligases′ impact on HBc: FBXO3-mediated HBc polyubiquitination stimulation and UBE2O-mediated HBc monoubiquitination promotion. FBXO3's and UBE2O's role in HBV life cycle was investigated as well. By silencing the expression of FBXO3 and UBE2O respectively, we have observed changes in HBV replication levels: FBXO3 serves as an inhibitor of HBV replication, while UBE2O stimulates the course of HBV life cycle. Further investigation of these newly-discovered understandings may lead to a whole new HBV - host interplay...
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The role of capsid protein in hepatitis B virus life cycle
Eliáš, Vratislav ; Weber, Jan (advisor) ; Drda Morávková, Alena (referee)
Hepatitis B virus (HBV) can cause either an acute or a chronic infection of hepatocytes, often leading to a hepatocellular carcinoma or a liver cirrhosis. HBV encodes seven proteins in its 3.2kb genome. Two of these proteins are transcribed from the same ORF (HBcAg and HBeAg). HBeAg, as a soluble variant of the core protein, plays a crucial role in virus immunogenicity. HBcAg, apart from building viral capsid, represents a pleiotropic protein engaging in multiple viral stages. Its primary structure is divided into two domains: an assembly domain and a C-terminal domain (CTD). The latter, containing four arginine clusters, is an important life cycle regulation element. By interaction with multiple viral and cellular factors, it directs the virus through a successful replication. In this thesis, we have collected the available data about the various roles of HBcAg in viral life cycle. We believe that further investigation of this field could lead to therapeutic advances resulting in the decrease of HBV infections worldwide, subsequently to a lower lethality connected with the diseases caused by HBV. Powered by TCPDF (www.tcpdf.org)
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Interference of selected DNA viruses with apoptotic processes
Sauerová, Pavla ; Forstová, Jitka (advisor) ; Štěpánek, Luděk (referee)
This work is focused on selected DNA viruses and some of their mechanisms used for inhibition or induction of the apoptotic processes. The selected DNA viruses are Hepatitis B virus, polyomaviruses, papillomaviruses and herpesviruses. Viruses developed different strategies for fighting the host defense mechanism during their evolution. One of the host defense mechanisms that reacts against virus infection is apoptosis. In case of viruses we can observe the phenomenon of inhibition or induction of apoptosis (which both depend on the life cycle phase of the virus). The purpose of these "fighting" strategies is to ensure successful replication, virus releasing from the cell and finally to let it spread in an organism or among them. Some "fighting" strategies are similar e.g. targeting and manipulation on p53 oncosupresor level or production of Bcl-2 homologs; other strategies are very specific. Certain viruses have mechanisms which allow them to survive in a host organism for a long time.
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Preparation of nanoparticles for hepatitis B viral therapy
Kružíková, Zuzana ; Grantz Šašková, Klára (advisor) ; Žáčková Suchanová, Jiřina (referee)
Hepatitis B virus (HBV) represents one of the hot topics of current basic and pharmaceutical research. Although an effective vaccine against HBV exists since 1982, the world prevalence of chronic infection is still alarming. The infection can lead to significant liver damage, often resulting in hepatocellular carcinoma. Chronic HBV infection cannot be cured due to the fact that the viral genome persists in the infected hepatocyte hidden from the host immune response as well as from the antiviral treatment. One of the novel approaches aiming for HBV cure suggests that this cccDNA pool could be destroyed using gene editing tools such as CRISPR/Cas9 system. In order to shift this gene editing system to possible medicinal application, CRISPR/Cas9 has to be specifically delivered into the target cell in order to minimize its putative off-target activity. This thesis focuses at first on the design and efficacy testing of new sgRNAs targeting HBV cccDNA and secondly, it describes modular lipid nanoparticles developed specially for delivery of the CRISPR/Cas9 system in the form of RNA. Keywords: hepatitis B virus, CRISPR/Cas9, gene editing, lipid nanoparticles, mRNA delivery, targeted delivery
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The role of Hepatitis B virus capsid protein in the host ubiquitin proteasome pathway
Eliáš, Vratislav ; Weber, Jan (advisor) ; Šmahel, Michal (referee)
Hepatitis B virus (HBV) is a Hepadnaviridae virus infecting mammals. Its infection can result in an acute or chronic infection. Chronic infection can result in hepatocellular carcinoma and liver cirrhosis, potentially leading to death of the patient. HBV is a small 42 nm virus with a genome length of 3.2 kb encoding seven viral proteins. HBV Core protein (HBc) is a capsid forming protein which is pleiotropic in function. We have identified two ubiquitin ligases which could interact with this protein: F-box only protein 3 (FBXO3; E3 ubiquitin ligase) and Ubiquitin conjugating enzyme E2 O (UBE2O; E2/E3 ubiquitin ligase). By employing multiple methods we have confirmed these interactions. Co- immunoprecipitation and further western blot analysis unveiled multiple new insights into the ligases′ impact on HBc: FBXO3-mediated HBc polyubiquitination stimulation and UBE2O-mediated HBc monoubiquitination promotion. FBXO3's and UBE2O's role in HBV life cycle was investigated as well. By silencing the expression of FBXO3 and UBE2O respectively, we have observed changes in HBV replication levels: FBXO3 serves as an inhibitor of HBV replication, while UBE2O stimulates the course of HBV life cycle. Further investigation of these newly-discovered understandings may lead to a whole new HBV - host interplay...
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Preparation of nanoparticles for hepatitis B viral therapy
Kružíková, Zuzana ; Grantz Šašková, Klára (advisor) ; Žáčková Suchanová, Jiřina (referee)
Hepatitis B virus (HBV) represents one of the hot topics of current basic and pharmaceutical research. Although an effective vaccine against HBV exists since 1982, the world prevalence of chronic infection is still alarming. The infection can lead to significant liver damage, often resulting in hepatocellular carcinoma. Chronic HBV infection cannot be cured due to the fact that the viral genome persists in the infected hepatocyte hidden from the host immune response as well as from the antiviral treatment. One of the novel approaches aiming for HBV cure suggests that this cccDNA pool could be destroyed using gene editing tools such as CRISPR/Cas9 system. In order to shift this gene editing system to possible medicinal application, CRISPR/Cas9 has to be specifically delivered into the target cell in order to minimize its putative off-target activity. This thesis focuses at first on the design and efficacy testing of new sgRNAs targeting HBV cccDNA and secondly, it describes modular lipid nanoparticles developed specially for delivery of the CRISPR/Cas9 system in the form of RNA. Keywords: hepatitis B virus, CRISPR/Cas9, gene editing, lipid nanoparticles, mRNA delivery, targeted delivery
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The role of capsid protein in hepatitis B virus life cycle
Eliáš, Vratislav ; Weber, Jan (advisor) ; Drda Morávková, Alena (referee)
Hepatitis B virus (HBV) can cause either an acute or a chronic infection of hepatocytes, often leading to a hepatocellular carcinoma or a liver cirrhosis. HBV encodes seven proteins in its 3.2kb genome. Two of these proteins are transcribed from the same ORF (HBcAg and HBeAg). HBeAg, as a soluble variant of the core protein, plays a crucial role in virus immunogenicity. HBcAg, apart from building viral capsid, represents a pleiotropic protein engaging in multiple viral stages. Its primary structure is divided into two domains: an assembly domain and a C-terminal domain (CTD). The latter, containing four arginine clusters, is an important life cycle regulation element. By interaction with multiple viral and cellular factors, it directs the virus through a successful replication. In this thesis, we have collected the available data about the various roles of HBcAg in viral life cycle. We believe that further investigation of this field could lead to therapeutic advances resulting in the decrease of HBV infections worldwide, subsequently to a lower lethality connected with the diseases caused by HBV. Powered by TCPDF (www.tcpdf.org)
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Interference of selected DNA viruses with apoptotic processes
Sauerová, Pavla ; Forstová, Jitka (advisor) ; Štěpánek, Luděk (referee)
This work is focused on selected DNA viruses and some of their mechanisms used for inhibition or induction of the apoptotic processes. The selected DNA viruses are Hepatitis B virus, polyomaviruses, papillomaviruses and herpesviruses. Viruses developed different strategies for fighting the host defense mechanism during their evolution. One of the host defense mechanisms that reacts against virus infection is apoptosis. In case of viruses we can observe the phenomenon of inhibition or induction of apoptosis (which both depend on the life cycle phase of the virus). The purpose of these "fighting" strategies is to ensure successful replication, virus releasing from the cell and finally to let it spread in an organism or among them. Some "fighting" strategies are similar e.g. targeting and manipulation on p53 oncosupresor level or production of Bcl-2 homologs; other strategies are very specific. Certain viruses have mechanisms which allow them to survive in a host organism for a long time.
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