|
|
CRISPR/Cas genome editing in human disease models and the translation into therapies
Faltínová, Pavlína ; Krupková, Michaela (advisor) ; Šedová, Lucie (referee)
Rare diseases, as their name indicates, individually affect only a low number of people around the world. Due to their low prevalence, finding appropriate therapy is very difficult. Insufficient understanding of the molecular causalities and mechanisms accompanying these disorders and the inability to conduct clinical studies to the usual extent due to the low occurrence of rare diseases belong to the main problems hindering the development of proper treatment. The creation of mouse models is a promising way to solve these difficulties since mice have numerous qualities necessary for modelling human diseases. CRISPR/Cas9 enables scientists to make precise changes in the genome by employing Cas9 nuclease which creates double-strand breaks in the DNA after the specifically designed guide RNA leads it to the site of interest. If the sequence of the chosen site is known, the possible edits can be installed almost anywhere in the genome and, moreover, their repertoire is practically endless. The use of the CRISPR/Cas9 technology proved to be perfect for creating mouse models of rare diseases as most of these disorders are caused by genetic mutations that this method is fully capable of mimicking. This thesis focuses on strategies used in creating such mouse models with the CRISPR/Cas9 system and...
|
|
|
Role of tissue-specific expression of the ISWI protein Smarca5/Snf2h in murine hematopoiesis.
Turková, Tereza ; Stopka, Tomáš (advisor) ; Forejt, Jiří (referee) ; Mráz, Marek (referee)
The process of cell differentiation is dependent on the chromatin-remodeling activity of the ISWI ATPase SMARCA5 (SNF2H) and its complexes. A series of mouse models have been used to study the functions of this ATPase, starting with a model with constitutive deletion of the Smarca5 gene and followed by models with tissue-specific deletion of Smarca5 gene (especially at different stages of hematopoiesis). The severity of phenotypic manifestations in mouse models varies from early embryonic lethality in the constitutive deletion model, through fetal lethality associated with erythropoiesis failure (Vav1iCre model - deletion in the definitive hematopoietic progenitor) to lymphopoiesis defects that do not limit animal survival (hCD2iCre model - deletion in T and B lymphocytes) in conditional deletion models. This work builds on the observations obtained in the study of deletion models of this Smarca5 gene, i.e. that deletion leads to arrest of cell proliferation and their entry into apoptosis. Defects were observed already at the stem cell and hematopoietic progenitor stages, which were unable to enter the differentiation process in the absence of the SMARCA5 protein. In these models, it was not possible to study the progression of differentiation because of this reason. Therefore, we decided to create...
|
|
|
Mouse models for Angelman syndrome: generation and characterization
Syding, Linn Amanda ; Sedláček, Radislav (advisor) ; Vyklický, Ladislav (referee) ; Valeš, Karel (referee)
Angelman syndrome (AS) is a neurodevelopmental disease found in 1 to 10,000 to 40,000 births, exhibiting an equal gender ratio. Key characteristics of the disease include an ataxic gait with tremor, severe mental retardation, profound speech impairment and seizures. Behavioral deficits such as increased anxiety and autism spectrum disorder features is found in affected individuals as well. The disease stems from the imprinted region 15q11.2-13q where genes are either maternally or paternally expressed as a result of parent-of-origin specific expression of the alleles. There are four main genetic etiologies causing AS namely, i) a large deletion ranging from 4-6 Mb on the maternally inherited allele including imprinted and bi-allelically expressed genes, ii) maternal deletion of the Ubitiquin ligase E3 (UBE3A) gene, iii) paternal uniparental disomy and iv) imprinting defect leading to inappropriate methylation of the locus. So far, there is no cure for AS rather the symptoms are ameliorated using a multidisciplinary approach. The goal of the doctoral study was to further decipher the role of Ube3a and Gabra5 using two mouse models to gain more knowledge about the involvement of these two genes for future therapeutic interventions in for Angelman syndrome. One model generated was a full gene deletion...
|
|
|
Emotional transition and nonstructural changes in mouse models of autism
Nováková, Rozálie ; Kubik-Zahorodna, Agnieszka (advisor) ; Bendová, Zdeňka (referee)
Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterised by repetitive behaviour and impairments in social behaviour and social communication. The whole aetiological heterogeneity is still not fully elucidated. It is then very important to focus on experimental research, especially on animal models, to help with drug development and recovery. To broaden the variability of focus of behavioural tests on mouse sociability, a new modification of a test to assess transfer of emotional information was proposed. A similar test was published recently for the first time, but it is still not common to use it in mice. Results show that it is possible to measure transfer of fear between conspecifics only during their immediate direct encounter through behavioural evaluation, but not in further standardised anxiety-evaluating tests. Self-grooming behaviour was the only parameter significantly affected by transferred anxiety in the experimental setup used, and therefore should be considered as the most sensitive behavioural parameter describing animal emotional state. However, the variability in individual animal behaviour is still considerably large, which confounds the results to a great degree. Such a behavioural test for transfer of emotional information may be especially useful in...
|
|
|
Immune stimulation by lipid nanoparticles
Aulichová, Veronika ; Grantz Šašková, Klára (advisor) ; Pačesová, Andrea (referee)
Lipid nanoparticles are the most advanced non-viral delivery system of nucleic acids. They enable safe and efficient transport of nucleic acids to the targeted cells and represent a key enabling technology of RNA therapeutics. RNA therapeutics represent a rapidly emerging field of genetic medicine; however, due to the nature of RNA molecules and their susceptibility to nuclease degradation, the delivery system is crucial for its translation to the clinic. This thesis explores the ability of lipid nanoparticles based on the XMAN6 ionizable lipidoid to elicit an immune response against mRNA-encoded antigen. XMAN6 is a lead lipidoid from a new class of adamantane-based ionizable lipidoids developed in our team. The mRNA-LNP formulations were first tested in vitro to evaluate their functionality and cytokine production and then in vivo using a mouse model. LNPs were applied via two different routes and in different experimental set ups, and their safety was evaluated by analyzing mouse activity. The data showed that XMAN6 is a promising lipidoid for in vivo delivery of mRNA. The mRNA-LNPs successfully induced the antibody response against the encoded antigen by intraperitoneal and intramuscular application. XMAN6 LNPs showed to be suitable for in vivo use, as they were well tolerated and caused only...
|
|
|
Research of epigenetic aspects of hematopoietic and spermatogenesis stem cells.
Hybešová, Michaela ; Pimková, Kristýna (advisor) ; Děd, Lukáš (referee)
Stem cell differentiation is controlled by coordinated regulation of gene transcription. One of the regulatory factors is the loosening of chromatin and the accessibility of DNA to transcription factors. Chromatin remodeling is mediated by remodeling complexes. The ISWI chromatin remodeling ATPase Smarca5 (S5) is an important factor of remodeling complexes. It is a highly conserved chromatin-remodeling factor forming a catalytic subunit that can be found in several oligosubunit complexes. In these complexes, it actively regulates nucleosome structure and remodeling during DNA replication, repair and transcription. S5 has been identified as a key protein in embryonic development. Its deficiency leads to defects in hematopoiesis and male genital development. In the presented study, we focused on the role of S5 in hematopoiesis and spermatogenesis. Using a mouse model with transgenic expression of S5, co-immunoprecipitation and mass spectrometry, we identified S5 complexes in hematopoietic and testicular cells. We also studied the phenotypic consequences of S5 deficiency in mouse testes and found that it leads to impaired sperm development and male sterility. Using transcriptomic and proteomic analysis, we identified several molecular programs that could lead to reproductive disorders. Our work...
|
|
|
Hematopoietic stem and progenitor cell defects in transgenic model of Diamond-Blackfan anemia
Holečková, Markéta ; Kokavec, Juraj (advisor) ; Valášek, Leoš (referee)
Diamond-Blackfan anemia (DBA) is a rare congenital bone marrow failure syndrome characterized by deficient development of erythroid progenitors and accompanied by a variable set of developmental defects. About 25 % of patients have mutations of the small ribosomal subunit protein RPS19, and the precise mechanism of single aminoacidic mutations of RPS19 protein in the pathology of Diamond-Blackfan anemia remains largely unknown. To understand the interaction between of genotype and phenotypic variability we have created a mouse model with homozygous mutation in a highly conserved arginine 67 (Rps19R67Δ/R67Δ ). Mouse model with this mutation display many of the same phenotypical trades as patients with DBA. We decided to focus on hematopoiesis and erythropoiesis in this mouse model and tried to characterize those processes. We discovered that Rps19R67Δ/R67Δ mice similarly to DBA patients suffer from anemia and that the erythropoiesis process is disrupted at the stage of proerythroblasts. We also observed changes in hematopoiesis in stages as early as multipotent progenitors. The role of p53 protein as a modifier of DBA phenotype is well known. We created mouse model with p53 depletion to assess the role of p53 protein in relation with mutation in Rps19. Rps19R67Δ/R67Δ Trp53-/- mice show no signs of...
|
|
|
Role of NEUROD1 transcriptional network on the development and function of inner ear neurons
Merc, Veronika ; Pavlínková, Gabriela (advisor) ; Mašek, Jan (referee)
Identification of transcription factors involved in a complex network regulating the development of neurosensory cells in the inner ear is a key point for understanding the pathophysiology of hearing loss, development of new therapeutic tools, and for hearing loss prevention. The aim of this thesis was to elucidate the function of the transcription factor NEUROD1 in the development of the inner ear and sensory neurons. Using the Cre-loxP recombination system, a unique mouse model was created with tissue-specific deletion of Neurod1 in NEUROD1-Cre positive cells (Neurod1ST). In the inner ear, Neurod1 was deleted only in neurons permitting to identify the secondary effects of Neurod1 elimination in neurons on sensory cell development. We showed that neither the early development of the inner ear nor the formation of the statoacoustic ganglia was significantly affected by Neurod1 deletion. The primary consequence of the deletion was manifested by increased neuronal death due to apoptosis, which resulted in a reduced number of differentiated neurons in the inner ear. Spiral and vestibular ganglia were smaller in the mutants, and there was a number of neurons misplaced, indicating impaired migration. The cochlear sensory epithelium was shortened probably due to the reduced number of neurons within the...
|
|
|
Genotype influence on development of infections caused by Trypanosomatidae in mouse
Šíma, Matyáš ; Lipoldová, Marie (advisor) ; Krulová, Magdaléna (referee) ; Kolářová, Iva (referee)
Parasitic protists of genera Trypanosoma and Leishmania are members of Trypanosomatidae family. In our studies, we investigated genetic influence on infections caused by these parasites in a mouse model. These diseases are on genetic level controlled by quantitative trait loci (QTLs), when the resulting phenotype is controlled by set of genes with small individual effect. As a mouse model for mapping of QTLs controlling these infections, we used recombinant congenic strains (RCS). Each RCS carry unique set of 12.5% of the genome from donor parental strain on genetic background of other parental strain. For mapping of QTLs controlling infections caused by Trypanosoma brucei brucei (T. b. brucei) and Leishmania tropica (L. tropica) and eosinophil infiltration into inguinal lymph nodes after Leishmania major (L. major) infection, we used RCS from CcS/Dem series, where STS is donor strain and BALB/cHeA is strain of genetic background. First, it was necessary to find suitable model strains for mapping. In all three studies, we selected RCS, which exceeded range of monitored phenotype parameters in comparison with any other tested RCS or parental strains. Mice of RCS CcS-11 showed shorter survival after T. b. brucei infection and strain CcS-9 exhibited higher eosinophil infiltration after L. major infection. For...
|
|
|
Deciphering the biological role of Ddi1-like protein family
Sivá, Monika ; Grantz Šašková, Klára (advisor) ; Bařinka, Cyril (referee) ; Stopka, Pavel (referee)
Ddi1-like protein family has been recently raised into the spotlight by the scientific community due to its important roles in cellular homeostasis maintenance. It represents a specific group among shuttling proteins of the ubiquitin-proteasome system. When compared to other shuttles, Ddi1-like protein family members harbor a unique retroviral-protease like domain besides the conventional ubiquitin-like (UBL) domain and domains interacting with ubiquitin. In addition, a helical domain of Ddi (HDD) has been recently found in most of the orthologs. In this thesis, I focus on characterization of several members of Ddi1-like protein family, both on molecular level using NMR and in model mouse strains via a variety of biological methods. Solution structure of the UBL domain of Ddi1p of S. cerevisiae was solved and its characteristics were compared to those of the UBL domain of its human ortholog. Furthermore, we show that human DDI2 specifically binds to ubiquitin with its terminal domains, both the UBL and the UIM; however, with very low affinity in contrast to binding properties of its yeast counterpart. Our study also show that hDDI2 does not form a head-to-tail homodimer. Based on our structural studies, we hypothesize that human DDI2 might have evolved a different function compared to its yeast...
|
guest ::
