Transdifferentiation induces chromatin reconstructions and epigenetic changes that affect gene expression spectum and cause cell remodeling in general. Direct conversion of mature somatic cell line into another mature cell type occures during the transdifferentiation thereby differences betweeen individual germ layers are eliminated. The aim of the master thesis is transdifferentation of mesenchymal cells - mouse embryonic fibroblast into endodermal cells - hepatocytes in vitro, using combination of transcripion factors Hnf4α and Foxa1. Detection of fibroblasts transformation has been initiated immediately after retroviral transduction and final generation of induced hepatocyte culture was confirmed by morphological and function analysis. The population of mouse induced hepatocytes served as a possible model for human liver disease in case of a pacient whose liver proteins could not be detected immunohistochemically. Genome editing of induced hepatocytes was realized by CRISPR/Cas9 technology which is based on cooperation of guideRNA and Cas9 nuclease followed in addition to generation of DNA-specific double strand breaks. These specific breaks in the Tight junction protein 2 gene were repaired via homologous recombination that induced a missense mutation with amino acid changes in the target...

This work documents the first case of idiopathic AA amyloidosis in humans caused by mutation in the promoter region of SAA1 gene. Knowledge of the mechanism of the disease may be an indication for targeted treatment in the future. Mutations in the SAA1 promoter should be considered in all cases of idiopathic forms of AA amyloidosis in which neither the immune nor the inflammatory component of the disease are clearly present.

N-methyl-D-aspartate (NMDA) receptors are glutamate-gated calcium permeable ion channels that play a key role in excitatory synaptic transmission and plasticity, and their dysfunction underlies several neuropsychiatric disorders. The overactivation of NMDA receptors by tonically increased ambient glutamate can lead to excitotoxicity, associated with various acute and chronic neurological disorders, such as ischemia, Alzheimer and Parkinson's disease, epilepsy or depression. On the opposite, NMDA receptor hypofunction is thought to be implicated in autism, schizophrenia, or intellectual disability. Recent DNA screening for neurological and psychiatric patients revealed numerous mutations in genes encoding for NMDA receptor subunits. The activity of NMDA receptors is influenced by a wide variety of allosteric modulators, including neurosteroids that could both inhibit and potentiate the activity of NMDA receptors, which makes them promising therapeutic targets. In this thesis, we describe new classes of neurosteroid analogues which possess structural modifications at carbons C3 and C17 of the steroidal core, and analogues without D-ring region (perhydrophenanthrenes). We evaluated the structure-activity relationship (SAR) for their modulatory effect on recombinant GluN1/GluN2B receptors. Our results...

In Introduction, the author of this dissertation deals with postoperative venous thromboembolism (VTE), hereditary and acquired risk factors, prophylaxis regimens and recent recommendation of VTE prevention in surgery. In Practical part of this work the author assesses the risk of VTE in surgical patients according to risk assessment model. Genetic testing is carried out in all patients to determine the incidence of hereditary thrombophilia and coagulation markers are measured in 28-days postoperative period. Prevalence of VTE in 1-year postoperative period is observed. The results are analysed in group of patients with benign disease (hernia and gallstone disease) and group of patients with malignancy (colorectal cancer and pancreatic cancer) separately. The objective of this work is to determine the incidence of the most frequent thrombophilic mutations (factor V Leiden mutation and protrombin G20210A mutation) and assess the impact of hereditary thrombophilia on incidence of postoperative venous thromboembolism in general surgery. Validation of venous thrombosis risk assessment model recommended by Czech Society for Thrombosis and Hemostasis is further objective.

Summary: Background: An increased risk for development of hereditary breast cancer is associated with germline mutations in BRCA1/2 and the influence of NBN mutations is also supposed. The aim of this study is to specify the frequency of recurrent mutations in BRCA1/2 in unselected breast cancer patients and the frequency of most common pathogenic mutations in NBN in Czech republic, to assess current criteria for genetic testing and to consider the addition of NBN to the tested genes. Methods: Screening for recurrent mutations 5382insC and 300T>G in BRCA1 was performed by RFLP, screening for mutations in exon 11 of BRCA1 was performed by PTT, screening for mutations in a selected region of exon 11 of BRCA2 by DHPLC, and screening for mutations in exon 6 of NBN by HRMA. All the mutations were confirmed by direct sequencing. Results: In 679 unselected breast cancer patients 7 carriers of 5382insC, 3 of 300T>G, and 4 of other mutations in BRCA1 were identified. 2 locally prevalent mutations were found in BRCA2. In 730 controls only one 5382insC BRCA1 mutation was identified. Out of 5 NBN mutations found in 600 high-risk patients two were 657del5 and one R215W. A total of 8 NBN mutation carriers were identified among 703 breast cancer patients, 2 of them 657del5 carriers and three R215W carriers. In 915...

Objective: Mutations in the parkin (PARK2) gene have been associated with autosomal recessive early-onset Parkinson's disease (EOPD) with various frequencies in different populations. The aim of the study is to describe phenotypic characteristics of Czech EOPD patients, to evaluate the influence of environmental risk factors, and to determine the frequency of parkin allelic variants in patients and healthy controls. Methods: A total of 70 EOPD patients (age at onset ≤ 40 years) and 75 controls were phenotyped and screened for the sequence variants and exon rearrangements in the parkin gene. Results: The main features in the phenotype of the patients' sample were: the absence of cognitive deficit, high occurrence of dystonia, depression, hyperhidrosis, an excellent response to dopaminergic therapy, early onset of dyskinesia and motor fluctuation. Patients with mutations in the parkin gene had significantly lower age at onset. The agricultural occupation and work with chemicals increased the risk of EOPD, however the coffee drinking appeared to be a protective factor. Parkin mutations were identified in five patients (7.1%): the p.R334C point mutation was present in one patient, four patients had exon deletions. The detected mutations were observed in the heterozygous state except one homozygous...

Stabilní proteiny nacházejí široké uplatnění v řadě medicínských a biotechnologických aplikacích. Přírodní proteiny se vyvinuly tak, aby fungovaly převážně v mírných podmínkách uvnitř buněk. V důsledku toho vzniká zájem o stabilizaci proteinů za účelem jejich širšího uplatnění také v průmyslovém prostředí. Obor proteinového inženýrství se v posledních letech rozvinul do úrovně umožňující modifikovat proteiny pro různá využití, ačkoliv identifikace stabilních mutací je stále zatížená drahou a časově náročnou experimentální prací. Výpočetní metody se proto uplatňují jako atraktivní alternativa, která dovoluje prioritizovat potenciálně stabilizující mutace pro laboratorní práci. Během posledních let bylo vyvinuto velké množství výpočetních strategií: i) výpočty energie pomocí silových polí, ii) evoluční metody, iii) strojové učení a iv) kombinace více přístupů. Spolehlivost a využití nástrojů jsou často limitovány predikcí pouze jednobodových mutací, které mají malý dopad na stabilitu proteinů, zatímco sofistikovanější metody pro predikci multibodových mutací vyžadují větší množství práce na straně uživatele. Hlavním záměrem této práce je poskytnout uživatelům plně automatizované metody, umožňující návrh vysoce stabilních vícebodových mutantů bez potřeby pokročilých znalostí bioinformatických nástrojů a zkoumaného proteinu. V této práci jsou prezentovány následující nástroje a databáze:  FireProt je plně automatizovaná metoda pro návrh stabilních vícebodových mutantů z kategorie tzv. hybridních přístupů. Ve svém výpočetním jádře spojuje jak energetické tak i evoluční metody, přičemž evoluční informace jsou užívány především jako filtry pro časově náročné výpočty energií. Kromě detekce potenciálně stabilizujících mutací se FireProt rovněž snaží spojit tyto mutace do jednoho vícebodového mutanta s minimalizací rizika vzniku antagonistických efektů. FireProt-ASR je plně automatizovaná platforma pro rekonstrukci ancestrálních sekvencí, která dovoluje uživatelům využít tuto strategii bez nutnosti velkého objemu manuální práce a hluboké znalosti zkoumaného proteinu. FireProt-ASR řeší všechny kroky ancestrální rekonstrukce, včetně sběru biologicky relevantních sekvencí, konstrukce zakořeněného fylogenetického stromu a rekonstrukce ancestrálních sekvencí.HotSpotWizard je nástroj pro návrh mutací a mutačních knihoven za účelem zlepšení stability a aktivity zkoumaných proteinů. Nástroj dovoluje provést i širší analýzu za využití čtyř různých strategií běžně používaných v oboru proteinového inženýrství: i) identifikace evolučně variabilních pozic v blízkosti katalytických kapes a tunelů, ii) identifikace pohyblivých regionů, iii) výpočet sekvenčního konsensu a iv) identifikace korelovaných pozic.FireProt-DB je databáze dostupných experimentálních dat popisujících stabilitu proteinů. Hlavním účelem této databáze je standardizovat data v oblasti proteinové stability, poskytnout uživatelům platformu k jejich snadnému ukládání a umožnit intuitivní vyhledávání, které by mohly být využité k trénování nových nástrojů s využitím technik strojového učení.

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...

Osteogenesis imperfecta, innate brittle bone disease, is a very serious disease. It is inheritable disease of connective tissue, which shows by abnormal fragility of bones. The occurrence of this disease is one case in 10 000 30 000 births. The theoretical part of the thesis deals with the disease itself, also the psychical impact on children suffering from Osteogenesis imperfecta and the impact on their families as well. At the beginning of the research, three goals of this thesis were set: map out (on the basis of theoretical and practical backgrounds) the pitfalls of life of children with the disease Osteogenesis imperfecta, find out what are the most common difficulties by children with the disease Osteogenesis imperfecta and also find out the experiences of nurses with the care for children with disease Osteogenesis imperfecta. The empirical part of the thesis was processed by means of qualitative research conducted by the technique of semi-structured interview and narrative biographical interview. The research set were nurses working at the child departments in hospitals, parents of ill children and also an adult woman with the diagnosis of Osteogenesis imperfecta and two doctors. From the research emerged that among the most common difficulties of children is pain, which decreases the quality of their life. Small children can't engage in typical activities of children, such as going to a playground, older children can't attend for example music festivals. Children feel fear from fractures and are therefore limited in sports. Because of injuries and their treatments, the children have more absences at schools and therefore are isolated from peers. Nevertheless, the children with this disease can live a happy life. From the results of the research also emerges, that nurses working at the child departments of the hospitals attended by children with this illness have a good experiences with their treatment. They are able to give parents important information and know the specifics of application of the treatment. The results of the diploma thesis were presented at a national student conference and will be further published.

Cbf11 and Cbf12 proteins, the members of the CSL transcription factors family, are involved in a wide range of cellular processes in the fission yeast Schizosaccharomyces pombe - among other things they regulate cell adhesion and they have also been implicated in maintenance of genome integrity. At the level of the whole genome we previously identified target loci bound by CSL proteins in vivo. Many of them do not contain any consensus CSL-binding element. There are probably different DNA binding modes of the Cbf11/12 proteins and it has not been known what specific biological function is associated with the particular way of DNA binding. For the purpose of studying CSL DNA binding modes we have worked in this project on the implementation of the DNA binding mutation (DBM), which prevents direct DNA binding of CSL proteins to canonical motif in vitro, into the chromosomal locus of the cbf11 and cbf12 genes. Using the "ura4 selection system" we have successfully constructed the scar-less Cbf12-TAP and Cbf12DBM-TAP knock-ins, i.e. the strains without/with DBM in the open reading frame of Cbf12 where Cbf12 is C- terminally TAP-tagged and contains the intact 3'UTR. In our laboratory we have established the CRISPR/Cas9 system by which we have been able to prepare the Cbf11- TAP strain. We have failed to...