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Functional analysis of posttranscriptional gene regulation by TENT5A in biomineralization and metabolism
Aranaz Novaliches, Goretti ; Sedláček, Radislav (advisor) ; Hovořáková, Mária (referee) ; Tencerová, Michaela (referee)
Non-canonical poly-A polymerases, such as TENT5A, belong to the Terminal nucleotidyl transferases (TENTs) family and are crucial for mRNA protection, stability, and translation. A Tent5a knock-out (KO) mouse model was generated in our laboratory, which exhibited a phenotype in teeth, skeleton structure, and metabolism. In my PhD project, I aimed to characterize the molecular mechanism underlying these phenotypes and explore their potential connection to rare human diseases. I focused on the biological function of Tent5a gene in enamel development (amelogenesis) and mRNA stabilization. Micro-computed tomography and scanning electron microscopy revealed that Tent5a KO mice displayed thin, hypomineralized enamel with disrupted microstructure, a condition known as Amelogenesis imperfecta. Direct mRNA sequencing demonstrated that TENT5A is responsible for polyadenylation of amelogenin (AmelX) and other secreted proteins, leading to a shortened poly-A tail in Tent5a KO ameloblasts. Moreover, Tent5a KO mice disclosed impaired self- assembly of enamel matrix proteins (EMPs) such as AMELX and ameloblastin (AMBN), leading to compromised hydroxyapatite deposition and enamel formation. In addition to its role in teeth, I investigated the physiological functions of EMPs in other tissues, considering that EMP...
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CX3CR1+ migratory dendritic cells in the mechanisms of central tolerance
Březina, Jiří ; Filipp, Dominik (advisor) ; Černý, Jan (referee)
Display of thousands of self-antigens in the thymus is fundamental for the establishment of central tolerance as its failure can lead to the development of autoimmunity. Medullary thymic epithelial cells (mTECs) and thymic dendritic cells (DCs) constitute essential populations of antigen presenting cells (APCs) which present these self-antigens to developing T cells. While mTECs produce and present antigens in self-autonomous manner, DCs can hijack mTEC-derived antigens by the process of cooperative antigen transfer (CAT). It is well found that CAT is essential for working central tolerance, however, the overall heterogeneity of thymic APCs participating in CAT remains unclear. Using transgenic mouse models and multicolor flow cytometry analysis, we determined that APCs involved in CAT are exclusively of CD11c+ phenotype. Within these cells, we identified previously unrecognized CX3CR1+ subset of migratory DCs (mDCs) exhibiting monocyte/macrophage markers. These CX3CR1+ mDCs are more efficient in CAT than their CX3CR1- counterparts and reveal robust antigen presenting properties with the capability to present CAT-acquired antigen. Genetic ablation of CX3CR1+ mDCs resulted in increased cellularity of CD8+ and CD4+ thymocytes, indicating importance of this mDC subset for negative selection of...
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Methodology of transgenic mouse preparation.
Reslová, Gabriela ; Komrsková, Kateřina (advisor) ; Pěknicová, Jana (referee)
The thesis deals with methods used for preparation of transgenic mice and with comparison of them in terms of efficiency and suitability of applications for research purposes. Technologies for the transfer of gene constructs involve microinjection of DNA into the pronukleus of fertilized oocyte, which seems to be the most reliable one. Another possibility is associated with the use of sperm, which carry the incorporated gene construct into the oocyte during in vitro fertilization, or with the use of retroviral vectors by which the oocytes are transfected. Somatic cell nuclear transfer into enucleated oocyte, the use of embryonic stem cells which are incorporated into strange embryo or transgenesis by liposomes are techniques far less explored and not so often used. The next section of this thesis introduces to the reader individual steps which are necessery for successful transgenesis. It is focused on ovarian stimulation which is necessery to obtain sufficient amount of oocytes, as well as on stimulation of recipient mother in embryo transfer as well as on capacitation of sperm required for in vitro fertilization. The thesis also deals with backward analysis confirming positivity of transgene expression by genotyping of pups and by crossbreeding of transgene positive and compares advantages and...
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Function of Zinc finger protein 644 (Zfp644) in mouse organism.
Szczerkowska, Katarzyna Izabela ; Sedláček, Radislav (advisor) ; Komrsková, Kateřina (referee) ; Blahoš, Jaroslav (referee)
ZNF644 (Zinc Finger Protein 644) is a C2H2 zinc finger gene encoding a putative transcription regulator, of which a point mutation (S672G) is associated with inherited high myopia in humans. It is also described to be a partner of the G9a/GLP (G9a- euchromatic histone- lysine N-methyltransferase 2, EHMT2; GLP - euchromatic histone-lysine N-methyltransferase 1, EHMT1) complex, known for its essential role in histone methylation, specifically H3K9me1and H3K9me2. It was reported that another transcription factor, WIZ (Widely-Interspaced Zinc Finger-Containing Protein), can bind to this complex and cooperate in gene silencing simultaneously. In order to study Zfp644 impact on myopia, we generated a mouse model, Zfp644S673G that mimics human mutation. In addition, a mouse with a persuasive truncated form of the protein, Zfp644Δ8 was created. Both mouse models went through an examination of retinal function and morphology. Moreover, with use of ultrasonography, different ocular parameters were examined. We conclude, that Zfp644 gene is causative for myopia in mice. Further examinations of Zfp644Δ8 animals show severe symptoms in metabolism and female fertility. To describe the impact of Zfp644 in mouse fertility we performed various experiments including analysis of expression of Zfp644 in reproductive...
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CX3CR1+ migratory dendritic cells in the mechanisms of central tolerance
Březina, Jiří ; Filipp, Dominik (advisor) ; Černý, Jan (referee)
Display of thousands of self-antigens in the thymus is fundamental for the establishment of central tolerance as its failure can lead to the development of autoimmunity. Medullary thymic epithelial cells (mTECs) and thymic dendritic cells (DCs) constitute essential populations of antigen presenting cells (APCs) which present these self-antigens to developing T cells. While mTECs produce and present antigens in self-autonomous manner, DCs can hijack mTEC-derived antigens by the process of cooperative antigen transfer (CAT). It is well found that CAT is essential for working central tolerance, however, the overall heterogeneity of thymic APCs participating in CAT remains unclear. Using transgenic mouse models and multicolor flow cytometry analysis, we determined that APCs involved in CAT are exclusively of CD11c+ phenotype. Within these cells, we identified previously unrecognized CX3CR1+ subset of migratory DCs (mDCs) exhibiting monocyte/macrophage markers. These CX3CR1+ mDCs are more efficient in CAT than their CX3CR1- counterparts and reveal robust antigen presenting properties with the capability to present CAT-acquired antigen. Genetic ablation of CX3CR1+ mDCs resulted in increased cellularity of CD8+ and CD4+ thymocytes, indicating importance of this mDC subset for negative selection of...
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Methodology of transgenic mouse preparation.
Reslová, Gabriela ; Komrsková, Kateřina (advisor) ; Pěknicová, Jana (referee)
The thesis deals with methods used for preparation of transgenic mice and with comparison of them in terms of efficiency and suitability of applications for research purposes. Technologies for the transfer of gene constructs involve microinjection of DNA into the pronukleus of fertilized oocyte, which seems to be the most reliable one. Another possibility is associated with the use of sperm, which carry the incorporated gene construct into the oocyte during in vitro fertilization, or with the use of retroviral vectors by which the oocytes are transfected. Somatic cell nuclear transfer into enucleated oocyte, the use of embryonic stem cells which are incorporated into strange embryo or transgenesis by liposomes are techniques far less explored and not so often used. The next section of this thesis introduces to the reader individual steps which are necessery for successful transgenesis. It is focused on ovarian stimulation which is necessery to obtain sufficient amount of oocytes, as well as on stimulation of recipient mother in embryo transfer as well as on capacitation of sperm required for in vitro fertilization. The thesis also deals with backward analysis confirming positivity of transgene expression by genotyping of pups and by crossbreeding of transgene positive and compares advantages and...
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Characterization of the role of SPINK 6 in the epidermis using transgenic models
Buryová, Halka ; Sedláček, Radislav (advisor) ; Entlicher, Gustav (referee)
Epidermal homeostasis, including proper turnover of keratinocytes, plays important role in the barrier function and serine proteases and their inhibitors are the key players. Activated proteases cleave desmosomes in uppermost layer and thus shed the cells from the epidermal surface. Therefore the serine protease inhibitors are secreted in lower epidermal layers to prevent premature activation of proteases and consequent disruption of epidermal barrier. The most studied inhibitors in epidermis belong to Serine proteases inhibitors Kazal-type family (SPINK). This diploma thesis is aimed to investigate function of murine SPINK6 in epidermal compartment in vivo. To achieve this, the transgenic mice overexpressing mSPINK6 under modified human involucrin promoter was generated. Two of five transgenic lines exhibited higher expression of mSPINK6 at mRNA and protein levels. The mSPINK6 transgenic mice are viable with no apparent phenotype. The small but in most cases not significant differences were observed on microscopic level among mSPINK6 transgenic and wild type animals In conclusion, this work showed that mSPINK6 does not play major role in skin homeostasis but gains significant importance under specific challenges of epidermal barrier. Therefore mSPINK6 transgenic mice, in combination with other deletion or...
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