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Mechanisms of immune dysregulation leading to inflammatory bowel disease
Horáčková, Klára ; Froňková, Eva (advisor) ; Filipp, Dominik (referee)
Bc. Klára Horáčková DIPLOMA THESIS Mechanisms of immune dysregulation leading to inflammatory bowel disease Abstract Inflammatory bowel disease (IBD) is a complex disorder characterized by chronic inflammation of the gastrointestinal tract. Classical IBD is a multifactorial disease with adulthood or later-childhood onset. However, children with very early onset IBD (VEO-IBD, before 6 years of age) are a specific cohort, whose pathology can be caused by severe genetic defects in genes connected to immune homeostasis in the gut. We aimed to identify the causal genetic variants in 20 pediatric patients diagnosed with IBD (age of onset from 3 to 154 months) using whole exome sequencing (WES). We evaluated several bioinformatical approaches for WES data analysis. This included a comparison of two methods of variant identification using VarScan2 or GATK4-based tools. Furthermore, we compared 4 gene lists ("virtual panels") for variant filtering, one of which was compiled purposefully for this thesis. We identified and validated via segregation analysis 5 causal variants in 4 genes (DUOX2 compound heterozygote, FOXP3, NLRP3 and NOD2) accounting for 20 % of the cohort. NOD2 (p.A755V) variant has already been reported in IBD cases, while DUOX2 (p.R1216W + p.A1131T), FOXP3 (p.H400L) and NLRP3 (p.V200M) were newly...
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Identification and characterization of novel regulators of hematopoietic stem cell function
Grušanović, Srđan ; Alberich Jorda, Meritxell (advisor) ; Balounová, Jana (referee) ; Froňková, Eva (referee)
Hematopoietic stem cells (HSCs) are sitting atop a carefully orchestrated system, called the hematopoietic system. HSCs maintain the constant production of mature blood cells throughout our life, and dysregulated HSC function may lead to severe health problems including bone marrow aplasia and leukemia. Thus, HSC activity needs to be tightly controlled by a complex network of cell intrinsic and cell extrinsic factors. In this thesis, we focus on two previously uncharacterized extrinsic mechanisms that might be involved in the regulation of HSC function. A common aspect of these mechanisms is the production of pro-inflammatory cytokines. First, we investigated the impact of donor NK cells on donor HSC function upon bone marrow transplantation, a frequent clinical intervention. We observed that NK cells negatively affect HSC engraftment and reconstitution during transplantation. To address the potential mechanism, we employed Cebpg knockout mice, which produce non-functional NK cells, and determined that NK cells affect HSC activity through the production of IFNγ. Remarkably, IFNγ-blocking antibodies improved murine and human HSC activity upon bone marrow transplantation. Altogether, we concluded that suppression of inflammatory signals generated by donor innate immune cells can have a significant...
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Single-cell RNA sequencing in leukemia
Brodská, Johana ; Froňková, Eva (advisor) ; Obr, Adam (referee)
Leukemia is a cancer of hematopoietic cells affecting the whole organism. Currently, there are many treatment options for all disease types, but it is still not always possible to fully cure the patient. The single-cell RNA sequencing method offers a new insight into the heterogeneity of both cancerous and non-cancerous cells in the leukemic environment. This thesis aims to briefly present the method and its history and to highlight current findings about leukemia obtained with the help of it. Keywords leukemia (AML, CML, ALL, CLL), sequencing, scRNA-seq, cells, transcriptome, treatment Okomentoval(a): [o1]: To the reader se v odborné literatuře moc nepoužívá
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Pathogenetic mechanisms of immune dysregulation and hematopoietic disorders
Svatoň, Michael ; Froňková, Eva (advisor) ; Filipp, Dominik (referee) ; Čermák, Jaroslav (referee)
Pathogenetic mechanisms of immune dysregulation and hematopoietic disorders Abstract The aim of my work was to introduce whole exome sequencing (WES) and a routine algorithm for data analysis in pediatric patients with suspected inborn error of immunity (IEI) or hematopoiesis. A clear molecular diagnosis based on genetic testingwas achieved in 25% of patients. Additionally, novel mutations not previously described in the pathogenesis of IEI or hematopoietic failure were revealed in 9% of the patients. Validation experiments were designed to confirm the causality of the identified mutations in the pathogenesis of the disease, based on literature review and known mechanisms of immune system development and regulation in these patients. Thanks to WES, we were able to elucidate the cause of two new congenital diseases - megaloblastic anemia due to a homozygous mutation of the SLC19A1 gene and immune dysregulation syndrome with autoimmune and autoinflammatory manifestations due to an X-linked mutation of the TLR8 gene in monozygotic twins with chronic autoimmune hemolytic anemia (AIHA). In five additional patients, novel mutations in the STAT3, SPTA1, SAMD9 and XIAP genes were identified and their causality in the pathogenesis of hematopoietic failure or IEI was demonstrated. In one patient with an atypical...
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Mechanisms of the tolerance and homeostasis of immune cells
Tsyklauri, Oksana ; Štěpánek, Ondřej (advisor) ; Černý, Jan (referee) ; Froňková, Eva (referee)
The ability of the immune system to tolerate self-antigens while mounting appropriate responses to pathogens is indispensable for the survival of the organism. Despite years of research, many details of the mechanisms of self-tolerance are still not well understood. The objective of this thesis is to extend our knowledge of the mechanisms of immune tolerance. The core of the PhD thesis consists of five publications related to two main research directions. The first one addresses the mechanisms of peripheral immune tolerance established by regulatory T cells (Tregs). We showed that Tregs increase the quorum of self-reactive CD8+ T cells required for the induction of autoimmunity. In addition, we identified a novel subset of antigen-stimulated CD8+ T cells, which expand in the absence of Tregs. We called them super-effector T cells. We revealed that the administration of IL-2 phenocopies the absence of Tregs, i.e., it induces super- effector T cells, and enhances CD8+ T cell response in autoimmunity and cancer. Our results provide strong evidence that the major suppressive mechanism of Tregs is limiting IL-2 availability for CD8+ T cells. Furthermore, in a collaborative project, we have shown that MyD88 signaling in thymic epithelial cells contributes to the development of Tregs and thus to the...
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Lineage plasticity in normal and malignant lymphocyte precursors
Rezková Řezníčková, Leona ; Froňková, Eva (advisor) ; Otáhal, Pavel (referee)
Klasické schéma vývoje hematopoetických buněk předpokládá časné oddělení lymfoidního a myeloidního prekurzoru. V poslední době jsou navrhovány složitější modely, které předpokládají větší flexibilitu hematopoezy a navrhují existenci progenitorů s lymfoidním i myeloidním potenciálem. Akutní hybridní leukémie jsou malignity, které podle různých kritérií nelze jednoznačně zařadit k lymfoidní nebo k myeloidní linii a jejichž chování spíše dává za pravdu novým modelům hematopoezy. Předkládaná práce se zabývala především výzkumem dětských leukémií s přesmykem z lymfoidní do myeloidní linie během indukční léčby. Jedná se o rozsáhlý projekt, v jehož rámci si diplomová práce si kladla za úkol určit liniové zařazení leukemických blastů pomocí detekce přestaveb genů pro imunoglobuliny a T-buněčné receptory (TCR). Potvrdili jsme, že myeloidní buňky derivované v průběhu léčby pochází u všech pacientů z původního lymfoidního klonu. Dále jsme u těchto případů zkoumali expresi vytipovaných genů ve srovnání s běžnými druhy leukémií. Třetí částí práce byl výzkum prognostického významu přítomnosti přestaveb TCR (a tedy příslušnosti k lymfoidní linii) u leukémií z T-lymfoidní řady.
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Study of HLA antigens and KIR genes in a donors and recipients of bone marrow
Kroulíková, Zuzana ; Vraná, Milena (advisor) ; Froňková, Eva (referee)
HLA and KIR genes are highly polymorphic regions within the human genome. Protein products of these genes play a critical role in hematopoietic stem cell transplantation. Genetic HLA match is a major barrier to engraftment and influences the outcome of this therapy. Therefore it is necessary to genotype donors and recipients selected for hematopoietic stem cell transplantation. Today HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 genes are tested by modifications of polymerase chain reaction or by sequence-based typing methods on the level of high- or low-resolution. Donors registered in bone marrow registries are selected on the basis of a 10/10 match. Donors KIR genotype leads to a better outcome, to relapse-free survival and overall survival in treatment of patients with acute myeloid leukemia. A better protection against relapse is achieved by Cen-B/B donor haplotype. Therefore KIR typing by polymerase chain reaction is used and the genotype is compared with the IMGT/KIR database by an online B- content calculator. Donors are divided in groups according to their genotype and their influence on the success of treatment for acute myeloid leukemia. The study of polymorphic systems and the development of genotyping donors and recipients significantly improve the outcome of hematopoietic stem cell...
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The analysis of immunoglobulin and T-cell receptor gene rearrangements using next generation sequencing
Hašek, Daniel ; Froňková, Eva (advisor) ; Javorková, Eliška (referee)
DNA sequencing is a molecular genetic method that results in data about sequence and type of nucleotides present in a given sample of deoxyribonucleic acid (DNA), a molecular carrier of genetic information. These data are frequently of a crucial value for many fields; research, medicine, industry, criminalistics or others. During a long period of time almost all the sequencing was performed using a method invented by Frederick Sanger in the 70's, a technique that uses modified nucleotides that once incorporated into a DNA strand prevent this from further elongation. DNA synthesis in presence of such nucleotides leads to a formation of a mixture of fragments of different lenght that are electrophoretically separated by lenght and the sequence is read from the resulting gel. Since the principle of this method entails some inherent drawbacks (e.g. low throughput and coverage) a significant effort is made lately to develop alternative sequencing approaches. These methods colectively refered to as next-generation sequencing (NGS) use several technologies in order to overcome the limitations of the Sanger sequencing. This thesis discusses the most important NGS methods and focuses on their possible application for sequencing of immunoglobulin and T-cell receptor gene rearrangements, an area of undisputable...
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