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Function of adaptor protein NDFIP1 in T lymphocytes
Dvořáček, Tomáš ; Brdička, Tomáš (advisor) ; Macůrková, Marie (referee)
T lymphocytes specifically recognize and promote immune response against pathogenic agents, which endanger health of host organism. However, this ability must be tightly controlled to prevent response against innocuous or self-tissue antigens, which would unnecessarily damage an organism. For a maintenance of this tolerance, there is a number of molecular mechanisms regulating T lymphocytes. The protein NDFIP1 is important component of these mechanisms. This transmembrane adaptor protein binds to E3 ubiquitin ligases from the NEDD4 family, which are under normal circumstances in inactive state. Binding of NDFIP1 leads to their activation and facilitates regulation of T cell behaviour by these ligases. NDFIP1 deficiency in mice causes the development of inflammatory disease affecting the skin, lungs and gastrointestinal tract. It is characterized by increased numbers of activated CD4+ T lymphocytes and eosinophils, which infiltrate the affected tissues. The disease is ultimately fatal. The aim of this work is to describe the function of the NDFIP1 protein in T lymphocytes, which are essential for the development of this pathology. The absence of NDFIP1 in T cells disrupts several molecular mechanisms, including the degradation of the transcription factors JUNB and RORγT and the regulation of mTOR...
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The role of Wnt signaling in interaction between circadian clock and cell cycle
Herrmannová, Terezie ; Sumová, Alena (advisor) ; Macůrková, Marie (referee)
The Wnt signaling represents a highly conserved signal transduction cascade that regulates stem cell proliferation and differentiation. It plays an irreplaceable role not only during embryonic development, but also in maintaining homeostasis of adult tissues. The cell division is also influenced by the circadian clock. The clock can interact with the cell cycle either directly within a single cell or regulate it intercellularly. In order to impact surrounding cells, it uses the Wnt signaling pathway that mediates signal transduction through the extracellular space. Both Wnt signaling and the circadian clock are essential for the physiological functioning of the mammalian organism, and their disruption can lead to the development of cancer. Keywords: circadian clock, clock genes, cell cycle, Wnt signaling, cell proliferation, cancer
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Characterization of the Caenorhabditis elegans pop-1 gene
Jakšová, Soňa ; Vacík, Tomáš (advisor) ; Macůrková, Marie (referee)
The TCF/LEF transcriptional factors regulate the target genes of the Wnt signalling pathway - one of the key signalling mechanisms involved in development of multicellular organisms. The TCF/LEF genes produce a number of various protein isoforms, which consequently leads to a great functional diversity of the TCF/LEF proteins. In this diploma project we focused on the Caenorhabditis elegans gene pop-1, the ortholog of the TCF/LEF genes, whose isoforms have not been studied yet. Using the Northern blot analysis we tried to identify alternative isoforms of the pop-1 mRNA in C. elegans. Using quantitative RT-PCR we also analyzed the pop-1 mRNA levels during seven developmental stages of C. elegans. Further, we also determined the expression profile of two important partners of pop-1, the bar-1 and sys-1 genes, whose protein products function as transcriptional co-activators. Key words: canonical Wnt signaling pathway, TCF/LEF transcription factors, Caenorhabditis elegans, pop-1
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The role of Fam208a during mouse embryogenesis
Bhargava, Shohag ; Chawengsaksophak, Kallayanee (advisor) ; Machoň, Ondřej (referee) ; Macůrková, Marie (referee)
(in English) Post-implantation embryo development proceeds through several key morphogenetic events that are fine-tuned by epigenetic modifications. Gastrulation is one of the most crucial developmental event that occurs during early post-implantation stage resulting in the formation of the three germ layers together with the establishment of the anterior-posterior (A-P) axis. It requires a highly coordinated interaction between the embryonic and extra-embryonic regions of the developing embryo. Gastrulation initiates with the formation of the primitive streak and, during which, cells of the epiblast delaminate and ingress through the primitive streak to form the mesoderm and definitive endoderm. During early post-implantation stages, the pluripotent cell population of the epiblast undergoes very rapid cellular proliferation and extensive epigenetic programming. There are numerous studies emphasizing various signaling molecules, transcription factors and epigenetic machinery maintaining the genomic stability that drive successful gastrulation and any discrepancy or defects almost always results in embryonic lethality. One such newly highlighted silencing machinery is that of the Human silencing hub (HUSH) complex comprising of the core members; FAM208A; H3K9me3-reader, MPP8 and PPHLN that together...
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Mitophagy in Huntington's Disease
Šonský, Ivan ; Hansíková, Hana (advisor) ; Macůrková, Marie (referee)
Mitochondrial dysfunctions contribute to the progression of many neurodegenerative diseases, including Huntington's disease (HD). In HD, mutation in the huntingtin gene (HTT) results in the expansion of CAG repeats, causing the growth of the polyglutamine tract. This growth is responsible for the gain of toxicity function of the protein. The turnover of dysfunctional and damaged mitochondria is mediated via mitophagy - a selective form of autophagy. Additionally, mitophagy impairments have recently been described to play a key role not only in neurodegenerative diseases. The protrusion of mitophagy results in the clustering of defective mitochondria, organelles which are responsible for fulfilling the energetic demands of neural cells. The most distinctive impact of the impairment is on the striatal medium spiny neurons and results in the development of motor and cognitive dysfunctions. This thesis describes how HD affects mitophagy and reveals the biggest obstacle of mitophagy - disruption of mitochondria targeting into emerging autophagosomes caused by the abnormal interaction of mHTT and p62. Induction of mitophagy at this stage could be crucial for the future therapeutic research of HD. Generally, initiation of mitophagy could become a relevant therapeutic target for many other...
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The role of dynamin in human pathologies
Zavadilová, Kristýna ; Macůrková, Marie (advisor) ; Voleman, Luboš (referee)
Dynamin (DNM) belongs to the family of large GTPases, which are characterized by their ability to hydrolyse GTP. Although DNM participates in many cell processes such as synaptic recycling, Golgi transport or regulation of cytoskeletal dynamics, its most imporant function is membrane scission during vesicle budding from the plasma during clathrin-mediated endocytosis. Human DNM is encoded by three different genes. DNM1 and DNM3 are most expressed in the nervous system, particularly in the brain, whereas DNM2 is expressed ubiquitously. Mutations in this gene cause two congenital neuromuscular diseases. One of them is autosomal dominant type of Centronuclear myopathy, manifested by skeletal muscle atrophy, the other is dominant intermediate type of Charcot-Marie-Tooth neuropathy, which affects myelination and neurotransmission in the peripheral nervous system. Myopathy mutations may increase GTPase activity and higher DNM2 oligomerization, while neuropathy mutations may cause decreased GTPase activity and loss of phospholipid binding properties of DNM2. Despite these findings, the mechanism by which DNM2 cause these two diseases is still unknown. By understanding the function of DNM in cell processes can help in outlining therapeutic approaches for the aforementioned diseases.
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Wnt/β-catenin signalling in the development of the marine annelid Platynereis dumerilii
Žídek, Radim ; Kozmik, Zbyněk (advisor) ; Janečková, Lucie (referee) ; Macůrková, Marie (referee)
Radim Žídek "Wnt/β-catenin signalling in the development of the marine annelid Platynereis dumerilii" (dissertation) Abstract: Wnt/β-catenin signalling is absolutely crucial for the early embryonic development of metazoan animals from the establishment of body axes, through the specification of germ layers and tissues to the development of organ systems. I used pharmacological manipulations of the Wnt/β-catenin pathway activity in the planktonic larvae of the marine polychaete annelid Platynereis dumerilii, the representative of the clade Spiralia, to investigate the role of Wnt/β- catenin signalling in the development and evolution of three hallmarks of Bilateria: the central nervous system, the body segmentation and the digestive tube. Wnt proteins are produced in all three aforementioned systems in Platynereis where they trigger the Wnt/β-catenin pathway in neighbouring cells. I describe here, for the first time in Platynereis, a homologue of the endpoint transcription factor of the entire pathway, Pdu-Tcf, which is subjected to an alternative splicing and along with a Wnt target gene Pdu-Axin is expressed in tissues with the active Wnt signalling - in the brain ganglia, in the neuroectoderm along the ventral midline, in segments, in the posterior growth zone and in the gut. Pharmacological manipulations...
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The role of TCF4 transcription factor in intestinal epithelial stem cells and tumors
Hrčkulák, Dušan ; Kořínek, Vladimír (advisor) ; Machoň, Ondřej (referee) ; Macůrková, Marie (referee)
For more than 20 years, T-cell specific factor 4 (Tcf4) is the most intensively studied member of the conserved Tcf/Lymphoid enhancer-binding factor (Lef) family of transcription factors. Together with β-catenin coactivator, Tcf4 represents the prominent nuclear effector of canonical Wnt signaling in the intestinal epithelium. Regulation of Wnt-β-catenin signaling in intestinal stem cells is crucial for tissue homeostasis and tumor formation initiation. Up to date, several mouse models were generated to manipulate Tcf4 abundance or activity in vivo and dissect its function. Moreover, mutational screens and expression profiling of human colorectal tumors were carried out to disclose a contribution of TCF4 to tumor progression. However, subsequent studies brought conflicting results in relation to the potential of Tcf4 to activate or repress Wnt target genes and drive or inhibit cell proliferation. Here in this study, we analyze publicly available datasets for global expression of TCF4 and its paralogs in human tissues and colorectal cancer (CRC) samples. Notably, we present newly generated Tcf4flox5 mouse with a conditional Tcf4 allele that can be used to eliminate expression of Tcf4 from two alternative promoters of the gene. Using this mouse strain we documented that Tcf4 loss led to the demise of...
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Regulation of gene expression at posttranscriptional levels.
Kollárová, Johana ; Kostrouch, Zdeněk (advisor) ; Macůrková, Marie (referee) ; Jindra, Marek (referee)
Regulation of gene expression in response to cellular and organismal needs is essential for sustaining organisms' survival and successful competition in the evolution of life forms. This regulation is executed at multiple levels starting with regulation of gene transcription, followed by regulation at multiple posttranscriptional levels. In this thesis, I focused on posttranscriptional mechanisms that contribute to gene expression regulation in the model organism Caenorhabditis elegans which enables powerful genetic and genomic techniques and allows the visualization of experimental genetic manipulations in toto, on the level of the complete organism during its life span. For this, we analysed the function of the orthologue of mammalian transcriptional corepressor NCOR, GEI-8. We used a functionally defective mutant gei-8(ok1671). I analysed the whole genome expression of homozygous gei- 8(ok1671) mutant and its link with observed mutant phenotype that includes defective gonad development and sterility and performed experiments leading to the proposition that disbalances in 21-U RNAs of piRNA class present in the most derepressed gene, the predicted mitochondrial sulfide:quinine reductase encoded by Y9C9A.16, are associated with the gonadal phenotype. In the second part of the thesis, I focused on...
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Function of nuclear phosphoinositides and their binding partners in gene expression
Uličná, Lívia ; Hozák, Pavel (advisor) ; Šolc, Petr (referee) ; Macůrková, Marie (referee)
(ENGLISH) Phosphoinositides (PIs) are negatively charged glycerol-based phospholipids with inositol head (ring) which can be phosphorylated. Inositol ring phosphorylation yields in seven different PIs species which can be mono-, bis,- or tris-phosphorylated. Roles of cytoplasmic PIs have been extensively studied in for membrane and cytoskeletal dynamics, vesicular trafficking, ion channels and transporters and generating of second messengers. Nuclear PIs have been implicated in posttranscriptional processing of pre-mRNA, DNA transcription and chromatin remodelling. While cytoplasmic functions are very well described, the molecular mechanism of their nuclear functions are still poorly understood. In this study we focus on description of localization of nuclear PIs in particular functional nuclear compartments, which enable us to reveal PIs involvement in nuclear processes. We also focused on identification of nuclear PIs involved in the regulation of genes transcription and revealed detailed mechanism of PI(4,5)P2 a PHF8 interaction in the regulation of ribosomal genes transcription. By two independent approaches, we have described PIs localization to the nuclear membrane, nuclear speckles, small foci in the nucleoplasm, and the nucleolus. This spread nuclear localization suggests and confirms PI's...
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