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Analysis of quantitative and qualitative genetic features in the pathogenesis of hereditary solid tumors.
Zemánková, Petra ; Kleibl, Zdeněk (advisor) ; Živný, Jan (referee) ; Tichý, Boris (referee)
Cancer the second most common causes of death in the Czech Republic. Carriers of mutations in genes predisposing to hereditary cancers represent a small but clinically significant group of high risk individuals. Today, dozens of predisposing genes for hereditary tumor syndromes are known and targeted next generation sequencing (NGS) has become a standard approach for their analysis. NGS allows rapid acceleration diagnostics of causal mutation in high-risk individuals. To identify mutations in genes predisposing to hereditary cancers, we designed a panel NGS analysis including subsequent bioinformatics analysis allowing a reliable identification of single nucleotide variants, insertions/deletions, and large intragenic rearrangements. The bioinformatics procedures described in this thesis were used for panel NGS validation, but also for identification of alterations associating with so far undescribed hereditary tumor types. Bioinformatics analyzes have become the basis for the unified processing of large datasets from the CZECANCA consortium and enable the construction of a population-specific database of genotypes that serve to improve clinical diagnostics of cancer predisposition in Czech patients. The versatility of NGS also allows its use for RNA (cDNA-based) analyzes of splicing variants in the...
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Identification and Characterization of Genetic Aberrations in Acute Childhood Leukemia
Lukeš, Julius ; Kubričanová Žaliová, Markéta (advisor) ; Machová Poláková, Kateřina (referee) ; Živný, Jan (referee)
Childhood acute leukemias are genetically complex disorders, with recurrent or random aberrations found in most patients. Their proper functional characterization is crucial for understanding the role they play in the process of leukemogenesis. We aimed to identify and characterize the genetic background of two leukemic entities. The transient myeloproliferative disorder (TMD) is a preleukemic condition that occurs in 10% of newborns with Down syndrome. Trisomy 21 together with in-utero gained mutations in the GATA1 gene are essential in TMD and represent an ideal "multi-hit" model to study leukemogenesis. We investigated an alternative pathogenic mechanism enabling TMD development in a confirmed absence of trisomy 21. Novel deletions in the GATA1 and JAK1 genes were described as potential drivers of this TMD. The deletion D65_C228 in GATA1 results in the expression of an aberrant isoform, which is predicted to lose transactivation potential and, more importantly, to partially lose the ability of recognizing physiological DNA binding sites, possibly triggering TMD alone. Our thorough characterization of JAK1 F636del questions its role in TMD development. Analysis of JAK/STAT signaling suggested decrease of kinase activity upon F636 loss. Cells harboring the aberrant JAK1 did not obtain cytokine-...
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Nutritional recommendations during cancer chemotherapy
Balcarová, Kateřina ; Maruna, Pavel (advisor) ; Živný, Jan (referee)
This bachelor thesis investigated the nutritional recommendations during cancer chemotherapy. The aim of this study was to determine whether the side effects of chemotherapy affect the dietary intake of a cancer patient. Apart from that, several goals were set. Firstly, to determine the awareness of oncologic patients treated with chemotherapy about nutritional recommendations that could influence the side effects of this therapy. Secondly, to investigate whether they act in accordance with these nutritional recommendations and to detect the source which is commonly used. Finally, to summarize the information about cancer treatment focusing on chemotherapy, its side effects and nutritional recommendations for them. The research was conducted in the oncology department of the Jablonec nad Nisou Hospital lasting from January to February in 2021. The data were collected anonymously by the non-standardized questionnaire. This questionnaire was given to the patient in the outpatient clinic and collected the next day when the patient attended the treatment. The research group involved adult oncology patients undergoing chemotherapy within the last 3 months. The total research group contained 37 patients, 20 women and 17 men were involved. The research focused on the relationship between chemotherapy and...
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Expression and function of cellular prion protein in blood cells
Glier, Hana ; Holada, Karel (advisor) ; Živný, Jan (referee) ; Rusina, Robert (referee)
The cellular prion protein (PrPc) is essential for pathogenesis of fatal neurodegenerative prion diseases. Recently reported four cases of vCJD transmission by blood transfusion raise concerns about the safety of blood products. Proper understanding of PrPc in blood is necessary for development of currently unavailable blood screening tests for prion diseases. Flow cytometry is an attractive method for prion detection, however, the reports on the quantity of PrPc on human blood cells are contradictory. We showed that the majority of PrPc in resting platelets is present in the intracellular pool and is localized in α-granules. We demostrated that both, human platelets and red blood cells (RBC) express significant amount of PrPc and thus may play an important role in the transmission of prions by blood transfusion. Our results suggest a unique modification of PrPc on human RBC. Such modification of pathological prion protein could distort the results of blood screening tests for prions. Further we showed that the storage of blood prior to analysis and the choice of anti-prion antibody greatly affect the detection of PrPc by flow cytometry and we identified platelet satellitism as a factor contributing to the heterogeneity of PrPc detection in blood cells. Moreover, we demonstrated existence of...
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The relationship between selected inflammation markers and markers of the endothelial dysfunction to preterm labor and fetal inflammatory response
Koucký, Michal ; Hájek, Zdeněk (advisor) ; Krofta, Ladislav (referee) ; Živný, Jan (referee)
The doctoral dissertacion is focused on the role of inflammation in the pathogenesis of preterm labor. In the first part, we describe the current view on pathophysiology of preterm labor. In the second part, we evaluated the relationship of specific markers of inflammation and endothelial dysfunction to preterm birth and fetal inflammatory response. The most important findings of our study was that we found decreased levels of MMP-2 and decreased levels of sRAGE in women with preterm labor in comparison with the control group of pregnant women. Similarly, we found decreased levels of MMP-2 in women with subsequent diagnosed fetal inflammatory response. sRAGE is currently ranked among patttern recognition receptors. In the case of sRAGE we followed the results of our pilot project, it can be assumed that the its low level are connected with tissue damage. We confirmed that it can play an important role in the pathogenesis of preterm labor. We assume abnormal regulatory mechanisms of the production of MMP-2. In both cases, however, further studies are required to elucidate the functional significance of our results.
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Th1/Th2 cytokine gene polymorphisms in patients with urine fibroid
Sosna, Ondřej ; Kužel, David (advisor) ; Živný, Jan (referee) ; Novotný, Zdeněk (referee)
Background: Uterine fibriod (UF) or leiomyoma is the most frequent benign tumour upon lower genital tract and represents the most frequent indication for hysterectomy. The aetiology remains still unknown. The genetic factors contributing for the development of UF are being intensively investigated. The aim of our study was to look for possible genetic markers which could be used as prognostic tools for evaluation of an increased risk for development of UF. Methods: The study group enrolled 102 patients diagnosed with UF and 145 healthy controls. Ultrasonographic examination of the pelvis was performed and a single blood sample was taken in all women. Histological verification followed the surgery in the patient group. The principal of the cytokine gene polymorphisms detection is based on PCR reaction with sequence-specific primers. Results: A large spectrum of Th1/Th2 cytokine gene polymorphisms in patients with uterine fibroid was compared with control group. The frequencies of the majority of tested cytokine gene SNP in the patient cohort were not statistically different from the cytokine SNP in the control group. However, an intriguing association between polymorphisms of the IL-4 gene promotor at positions -590 C/T and -33 C/T, and the risk of leiomyoma was observed. The CC genotype of IL-4 at position...
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Markers of transplantation tolerance in kidney transplantation
Krepsová, Eva ; Viklický, Ondřej (advisor) ; Krejčí, Karel (referee) ; Živný, Jan (referee)
Long-term renal graft acceptance still requires long-term immunosuppressive therapy, which is accompanied by many adverse effects. Contrarily insufficient immunosuppression could lead to graft rejection and its failure. Therefore, research continues for biomarkers that reflect a patient's immunological status and thus allowing for individualized immunosuppressive therapy. In our study we showed lower incidence of acute rejection in kidney transplant recipients treated with rabbit anti-thymocyte globulin (rATG) or basiliximab induction within the first three months after transplantation. The rATG induction caused profound decrease of recipient's peripheral blood T and NK cells, as well as transcripts that are exclusively expressed by these cell types together with expansion of regulatory T cells (Tregs) among CD4+ T cells. In rATG group the increase of two transcripts associated with rejection (MAN1A1 and TLR5) was also observed in early post-transplant period. After the basiliximab induction we transiently detected CD4+CD25low/-FoxP3+ cell population along with disappearance of CD4+CD25+FoxP3+ Tregs. Basiliximab induction resulted in a transient increase in CD4+FoxP3+ Tregs, accompanied by the highest peripheral expression levels of markers associated with operational tolerance (FOXP3 and TCAIM)....
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Specific prion protein antibodies characterisation and use in diagnostic
Šafaříková, Eva ; Holada, Karel (advisor) ; Mareš, Michael (referee) ; Živný, Jan (referee)
Transmissive spongiform encephalopathies (TSEs) are neurodegenerative diseases characterized by depositions of abnormally folded prion protein (PrPTSE ) in brain. PrPTSE is at present the only specific biochemical marker of human and animal TSEs. Diagnostic tests are based on the detection of PrPres after proteinase K digestion of brain homogenate using Western blot or on the immunohistochemistry of fixed brain tissue, which are both difficult and time consuming. In this work we focused on development of a new type of tests based on PrP detection without need of proteinase K digestion. As deposits of PrPTSE remain in the body for a long time, there is a substantial chance of them being nonenzymatically modified by glycation. The detection of glycated PrPTSE may have a potential to serve as a diagnostic marker. We prepared monoclonal antibodies specific for carboxymethyl lysine/arginine modified prion protein. Bacterially expressed and purified recombinant human prion protein (rhPrP) was modified by glyoxylic acid that introduces carboxymethyl groups on lysine and arginine residues present within the molecule of the protein. Modified rhPrP (rhPrP-CML) was used for immunization of laboratory mice and hybridoma cells were prepared. Screening of cell supernatants resulted in the selection of 4...
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The Role of Cellular Prion Protein in Erythroid Differentiation
Panigaj, Martin ; Holada, Karel (advisor) ; Živný, Jan (referee) ; Rusina, Robert (referee)
4 Abstract The cellular prion protein (PrPC ) is evolutionary conserved protein expressed in cells of various origins. Although PrPC plays a basic role in the pathogenesis of the fatal neurodegenerative prion disorders, its physiological role remains enigmatic. Prion diseases are characteristic by long latency period during which they are not identifiable by any conventional methods. Although the blood is an ideal material for developing of screening tests, little is known about traits of PrPC and its role in blood cells. We showed that human erythrocytes express low amounts of PrPC per cell, but due to the high numbers of erythrocytes, they are major contributors to the pool of blood cell-associated PrPC . Based on our biochemical characterization we propose that PrPC on human erythrocytes is uniquely modified. Such a modification in abnormal prion protein may complicate screening tests for prion diseases in blood. It was reported that prion diseases deregulate the transcription of erythroid genes, and PrP-/- mice demonstrate a defective response to experimental anemia. To investigate the role of the PrPC in erythropoiesis, we studied the protein's expression on mouse erythroid precursors in vivo and in vitro. We showed that surface expression of PrPC on erythroid precursors in bone marrow and spleen...
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Identification and Characterization of Genetic Aberrations in Acute Childhood Leukemia
Lukeš, Julius ; Kubričanová Žaliová, Markéta (advisor) ; Machová Poláková, Kateřina (referee) ; Živný, Jan (referee)
Childhood acute leukemias are genetically complex disorders, with recurrent or random aberrations found in most patients. Their proper functional characterization is crucial for understanding the role they play in the process of leukemogenesis. We aimed to identify and characterize the genetic background of two leukemic entities. The transient myeloproliferative disorder (TMD) is a preleukemic condition that occurs in 10% of newborns with Down syndrome. Trisomy 21 together with in-utero gained mutations in the GATA1 gene are essential in TMD and represent an ideal "multi-hit" model to study leukemogenesis. We investigated an alternative pathogenic mechanism enabling TMD development in a confirmed absence of trisomy 21. Novel deletions in the GATA1 and JAK1 genes were described as potential drivers of this TMD. The deletion D65_C228 in GATA1 results in the expression of an aberrant isoform, which is predicted to lose transactivation potential and, more importantly, to partially lose the ability of recognizing physiological DNA binding sites, possibly triggering TMD alone. Our thorough characterization of JAK1 F636del questions its role in TMD development. Analysis of JAK/STAT signaling suggested decrease of kinase activity upon F636 loss. Cells harboring the aberrant JAK1 did not obtain cytokine-...
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