Bc.Veronika Hrabcová BRAF mutations in metastatic malignant melanoma. Diploma thesis Charles University in Prague, Faculty of Pharmacy in Hradec Králové Healthcare bioanalytics - Specialist in Laboratory Methods Backround: Melanoma is malignant disease with increasing incidency. Treatment of advanced stage of melanoma is still limited. With a progress of knowledge in genetics and tumorigenesis, the incidence of mutated BRAF protein was observed at 50 % of melanomas. In 80-90 % mutated melanomas contain BRAF V600E mutation. The aim of study was to establish a suitable molecular biological method for the diagnosis of mutations in codon V600 BRAF. Methods: Cobas 4800 BRAF V600 mutation test and BRAF StripAssay test were used to analyze DNA. Cobas 4800 BRAF V600 mutation test is based on PCR using TaqMan probes designed for the wild-type and mutant BRAF V600E sequence. BRAF StripAssay test is based on PCR amplification with biotinylated primers and subsequent hybridization of the stripped with allele-specific oligonucleotide probes. Examined DNA samples were derived from 35 patients with advanced malignant melanoma or from archive of laboratory. Results: BRAF V600 mutation was detected in approximately half of the tumors, consistent with the results of other studies. In comparison methods Cobas test...

The main objective of this master thesis entitled The study of polyneuropathy in Leonberger dogs (LPN) was to determine the variability of ortholog of CMT2 gene in a selected population of this breed. The number of individual samples studied was 211. The entire set of DNA samples was divided to the cases (110 samples) and controls (101 samples). Partial objectives were the study of information available on the hereditary neuropathies, the selection of candidate gene polymorphisms and their association analysis with the disease. The candidate gene studied is associated with the autosomal recessive form of axonal neuropathy in humans with early onset of symptoms. Compound mutation was discovered in the gene (substitution and 1 bp deletion). The normal allele predominated in healthy individuals. In contrast, both mutant alleles always predominated in diseased individuals. Statistical comparison of genotypes depending on the health status did not show a clear correlation with disease (p = 0,1562).

In domesticated animals there are many different coat colours and mutations, often connected with pleiotropic effects. The aim of this thesis named The study of colour genes sequences in chinchilla based on homology of human and mice sequences was describe molecular genetic principles of pigmentation, introduce genes involved in melanogenesis and influencing a melanin function, their structure, mutations and mention other mutations which change the phenotype. Informations about alleles TYR, TYRP1, TYRP2/DCT, agouti, AGRP, gene group MCR (MC1R-MC5R) and more are focused on human (Homo sapiens), mouse (Mus musculus) and chinchilla (Chinchilla lanigera). In these three species was compared selected genes sequences TYR and TYRP2/DCT, results were studied, commented and mutations, which are very specific, but there are some analogs between human and mouse, were highlighted.

Trombophilia means an increased disposition to creation of trombs. Health complication incurred as a consequence of hypercoagulation can be very serious. When a trombophilic mutation is found at a patient, it brings necassity of thrombosis - control in risk situations (e.g. pregnancy, operation) for the rest of the patient's life. There were filed 300 people (206 women and 94 men) with trombophilic mutations into my study of clinical signification of trombophilic mutations. These people were examinated in years 2008 - 2010. Most of positive medical findings - 266 people, were recorded in the area of MTHFR (C677T i A1298C) mutations. There were less findings in the field of FV Leiden and FII prothrombin mutations. Multipath trombophilic mutations were found at 99 patients. I accordance with foreign literature, our results advert to clinical consequences of trombophilic mutations like: repeated spontanious aborts, cerebrovascular akcident (CA), ischaemic heart disease (IHD), thrombosis, flebothrombosis, pulmonary embolism, varicose veins, aseptic necrosis of hip bone, arterial sclerosis and aortic stenosis. Mutations MTHFR C677T and MTHFR A1298C we found mainly at patients with CA, IM and IHD. Leiden mutation was most often found at patients with thrombosis, flebothrombosis and pulmonary embolism. We...

The aim of this thesis is to summarize the available data concerning the influence of trombophilic states on the process of pregnancy and to present the results obtained by investigating the prevalence of congenital trombophilia in infertile women and its influence on the success rate of assisted reproduction. It is aimed at those interested in the problematics - be it professionals, medical students or women planning pregnancy or cannot get pregnant. The theoretical part draws data from literature written by leading Czech experts in the field and from scientific journals, included are also foreign sources. The practical part was executed with the kind permission of the PRONATAL s.r.o. centre for assisted reproduction. Key words: venous thromboembolism, trombophilia, trombophylaxis, gravidity, mutation, screening

Hypochondroplasia (MIM 146000) is a skeletal dysplasia characterized by disproportional dwarfism with rhizomelic or mesomelic shortening of the upper and lower extremities, with variable severity. Patients often have macrocephaly with normal facial features. Hypochondroplasia is a disease with autosomal dominant inheritance. In some patients it is caused by germline mutations in the FGFR3 gene, in others the cause of the disease remains unknown . The FGFR3 gene encodes a tyrosine kinase receptor. This receptor negatively regulates the conversion of cartilage to bone. FGFR3 gene mutations that cause hypochondroplasia lead to constitutive activation of the receptor and inhibit the growth of long bones. In this study, we analysed selected regions (exons) of the FGFR3 gene in 98 patients with disproportional dwarfism and clinical diagnosis of hypochondroplasia. Eighteen patients from 12 families had familial and 80 patients had sporadic form of the disease. All patients were previously tested negative for frequent germline mutations in exon 13 (codon 540) and exon 15 (codon 650). Genomic DNA was isolated from patient's peripheral blood leukocytes. The examination was conducted with the informed consent of the patient or his legal representative. We performed mutational analysis by direct sequencing of...

Protein p130Cas is the major tyrosine phosphorylated protein in cells transformed by v-crk and v-src oncogenes. P130Cas plays an important role in invasiveness and metastasis of Src-transformed cells. In breast cancer patients, high p130Cas levels are associated with higher recurrence of disease, poor response to tamoxifen treatment and lower overall survival. In non-transformed cells, after the stimulation of integrins, protein p130Cas is phosphorylated in substrate domain affecting cell migration and cytoskeletal dynamics. For this signalling is the SH3 domain of p130Cas indispensable. In this thesis, was for the first time using the Phage display method analysed and subsequently characterized the binding motif of SH3 domain of p130Cas. Based on this high-affinity motif [AP]-P-[APMS]-K-P-[LPST]-[LR]- [LPST], we predicted new interaction partners of protein p130Cas and subsequently confirmed the interaction with the Ser/Thr kinase PKN3. This kinase colocalizes with p130Cas in the nucleus and perinuclear region and could phosphorylate p130Cas. In this thesis, we also analysed the effect of phosphomimicking mutation of tyrosine from sequence ALYD, which is conserved in the sequence of SH3 domains, on ability of these domains to bind ligands. This mutation reduced binding by about 3 orders of...

The thesis The analysis of genetic factors of breast cancer by NGS deals with the current serious problematics of breast cancer from the perspective of genetic predisposition. Breast cancer is one of the most common tumors in women. Every year more than 7000 women are diagnosed with this disease and the mortality rate in the Czech Republic is nearly 2000 cases. Of the total number of patients diagnosed with breast cancer, approximately ten percent of patients have congenital mutations in one of the predisposing genes that cause a significantly increased risk of developing a cancer. More than half of these mutations occur in germline mutations of the BRCA1 or BRCA2 genes, others include a number of other genes, eg tp53, CDH1, PTEN, STK11, ATM, PALB2, CHEK2.Early diagnosis and identification of persons with increased risk of developing breast cancer is of key importance for their inclusion in preventive programs. Therefore, the thesis aims to testing genes that can cause a breast cancer. In the thesis, 219 known and candidate predisposition genes were analyzed in a group of 263 non-selected breast cancer patients using a targeted panel NGS, the Illumina platform. Selected identified suspect variants were further confirmed by Sanger sequencing. The aim of this work was also a mutational analysis of...

The main topic of this thesis are changes in Circadian rhythms caused by cerebellar disorders. Mice with Lurcher mutation, which have specifically degenerated Purkinje cells layer, were choosen as animal model. Our results show that mutation of the glutamate receptor GluRδ2, which causes gradual degeneration of Purkinje cells, leads to damage of Circadian system. Mice with this mutation have reduced capability to adapt to external conditions in different light modes. They are also showing increased variability in endogenous cycle. The mice are also unable to show anticipatory behavior in time-restricted feeding. Compared to control group, affected mice do not show significant rhythm in levels of protein of Bmal1 gene in suprachiasmatic nuclei, paraventricular nuclei nor in habenula. Phosphorylated kinases ERK1/2 and GSK3ß also had distorted rhythms in suprachiasmatic nuclei. Because Circadian oscillations in locomotor activity are partly preserved, Circadian system is likely not damaged on molecular level. Cerebellar mutation hampers synchronization between suprachiasmatic nuclei of neurons and can also affect processes in the ventromedial hypothalamus regulating food intake. Our findings are the first to suggest functional interactions between cerebellum and Circadian pacemaker in suprachiasmatic...

Myelodysplastic syndrome (MDS) is a heterogeneous group of diseases characterized by ineffective hematopoiesis which is caused by damage of differentiation of pluripotent haematopoietic stem cells. TP53 gene mutations are identified approximately in 10% of MDS and represent a negative prognostic factor. Altered TP53 gene expression may have similar effect as the mutation. Mutations or deregulated expression of this gene have an impact on many cellular processes including apoptosis, DNA repair, cell growth and angiogenesis. In this work, the expression mRNA levels of genes involved in p53 signalling pathway were studied in CD34+ pluripotent haematopoietic cells from bone marrow of patients with low- risk MDS. MDS patients showed increased expression of genes involved in apoptosis induction, regulation of cell cycle and DNA repair (BAX, BBC3, CCNE1, CDC25A, CDKN1A, FAS, GADD45A) as compared to healthy subjects. The patients with TP53 mutation had decreased expression of apoptotic genes (BAX, PIDD, TRAF2) and increased gene expression of apoptotic inhibitor (BCL2A1), indicating a reduced activity of apoptotic pathways and that way the pathological cell clone may gain a growth advantage. Deregulation of 21 genes (BAX, BBC3, EGR1, KAT2B, MDM2 etc.) was observed in patients with del (5q) compared to...