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Detection of mutation HSF4 causing hereditary cataract in sheep dogs
VOKÁLKOVÁ, Lenka
Hereditary cataract belongs amongs the most common diseases of the eye with the shepherd dog breeds. It is a very serious disease, which causes the deterioration of sight of the dog. With the neglection can the illness cause even blindness. Heteritary cataract manifests itself with bilateral turbidity of the eye. Course of the illness differs and the beginning of the sickness can start at any age of the dog. The formation of the cataract can be the result of another eye disease or the in jury of the eye. In this case it is a secondary cataract.Primary cataract is directed by genes. Candidate gene for the formation of the hereditary cataract is the HSF4 gene. This gene include an important role in the immunity system. It encodes proteins of the thermal shock, that protects the cell from adverse effects of the outer environment, like high temperatures or UV rays. Besides that it also cantributes to the development of the eye lens fibers. Mutation in the exon of the gene HSF4 causes the degradation of these fibers, hence cataract. Hereditary cataract is a serious illness, which must be cured by surgical implantation of the eye lens. Therefore, timely diagnosis and decommission of affected individuals from the breed, that the transfer of the ilness to the next generation will be prevented. I deal with the detection of a mutation in the HSF4 gene causing cataracts in my diploma thesis. I have chosen the fragment analysis as the most suitable method for detection of the mutation in the gene HSF4. The mutation is in the insertion of one nucleotid C. Fragment analysis can determine very precisely the mass of the DNA molecule and permits to differ even a fragment, which differs only in one base. In the practical part of the thesis were examined 100 samples of the dogs of breeds australian shepperd, border collie, german shepherd and long ans short haired collie. Only one of the dogs was evaluated as the carrier of the hereditary cataract. It was the dog of the australian shepperd breed. Three of the samples were non evaluable, caused by wrong sampling or degradation of the DNA. The rest of the evaluated dogs (96 samples) were healty.
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Genetic markers for monitoring post-transplant chimerism
Řehounková, Michaela ; Beránek, Martin (advisor) ; Matoušková, Petra (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Michaela Řehounková Supervisor: doc. PharmDr. Martin Beránek, Ph.D. Title of diploma thesis: Genetic markers for monitoring post-transplant chimerism The aims of the thesis: Data processing of patients, who underwent allogenic hematopoietic stem cell transplantation in a period from 2010 to 2014 in University Hospital Hradec Kralove and whose state of chimerism was monitored at the Section of Molecular Biology at the Institute of Clinical Biochemistry and Diagnostics. Consequently, analysis of the possible relationship between selected clinical parameters and used genetic markers for chimerism quantification was carried out after the processing of acquired data. Finally, the possible influence of treatment success and mortality by chosen clinical parameters was evaluated. Methods: Analysis of short tandem repeat loci, which uses genetic variability between donor and recipient of transplanted graft, was employed for quantification of post- transplant chimerism. DNA of donor and recipient was isolated by QIAmp DNA Blood Mini Kit (QIAGEN, Germany), amplified by AmpFlSTR Identifier Kit (Applied Biosystems, USA) and separated by capillary electrophoresis (analyzer ABI 3130-4, Applied...
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Ověření dědičnosti markerů mikrosatelitového panelu u velbloudů
Grygarová, Tereza
The diploma thesis is focused on the verification of inheritance of selected microsatellites from the microsatellite panel of the MHC locus in camels. The theoretical part deals with the characteristics of the genus Camelus. Special focus is given to adaptation mechanisms allowing camels to survive in conditions that are inhospitable for other livestock species. Microsatellites which are also described became an excellent choice not only in parentage analysis. The practical part was based on fragment analysis in genetic analyser ABI PRISM 3500, which preceded DNA isolation, multiplex PCR amplification and gel electrophoresis. Using GeneMapper 5 software, amplified fragment sizes were calculated and mendelian segregation verified. For assembly microsatellite panel four microsatellite markers were selected, M29_I, M35_II, M41_III a M42_III. The least polymorphic was microsatellite M29_I of the MHC I region and the most polymorphic was microsatellite M42_III of the MHC III region. In general, microsatellites from the microsatellite panel can be considered reliable for verifying the origin of the alleles and can be further used to reveal polymorphism of the MHC region of camels.
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Early cancerogenesis in head and neck squamous cell carcinoma.
Kastner, Jan ; Betka, Jan (advisor) ; Salzman, Richard (referee) ; Smilek, Pavel (referee)
Squamous cell carcinoma is the most frequent malignant tumour of head and neck. Prognostic and predictive information as an individual imprint of molecular-genetic analysis of HNSCC will help to determine the best indivicual treatment. And in case of surgical appraoch the optimal resection with adequate quality of life and long-term survival. Study of early cancerogenesis in our project is based on knowledge, that histological normal mucosa next to tumor shows preneoplastic molecular alterations. Molecular genetic changes in a histological normal mucosa harbouring a tumor may play a principal role in revealing of early cancerogenesis process. Molecular-genetic analysis of cancerogenesis in HNSCC reveals prognostic and predictive factors, which are necessary for evaluation and decission for the best individual treatment. This is the concept of tailored medicine The text summarizes current knowledge of early cancerogenesis in HNSCC and presents molecular-biological trends, which are necessary to discover details of early cancerogenesis and thus to get a tool for better detection as well as treatment of malignant disease. The study is based on fragment analysis of microsatelites lesions in tumor tissue in comparison to adjacent mucosa and the healthy mucosa. Results show significant molecular-biological...
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Genetic markers for monitoring post-transplant chimerism
Řehounková, Michaela ; Beránek, Martin (advisor) ; Matoušková, Petra (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Michaela Řehounková Supervisor: doc. PharmDr. Martin Beránek, Ph.D. Title of diploma thesis: Genetic markers for monitoring post-transplant chimerism The aims of the thesis: Data processing of patients, who underwent allogenic hematopoietic stem cell transplantation in a period from 2010 to 2014 in University Hospital Hradec Kralove and whose state of chimerism was monitored at the Section of Molecular Biology at the Institute of Clinical Biochemistry and Diagnostics. Consequently, analysis of the possible relationship between selected clinical parameters and used genetic markers for chimerism quantification was carried out after the processing of acquired data. Finally, the possible influence of treatment success and mortality by chosen clinical parameters was evaluated. Methods: Analysis of short tandem repeat loci, which uses genetic variability between donor and recipient of transplanted graft, was employed for quantification of post- transplant chimerism. DNA of donor and recipient was isolated by QIAmp DNA Blood Mini Kit (QIAGEN, Germany), amplified by AmpFlSTR Identifier Kit (Applied Biosystems, USA) and separated by capillary electrophoresis (analyzer ABI 3130-4, Applied...
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Genetic markers for monitoring post-transplant chimerism
Řehounková, Michaela ; Beránek, Martin (advisor) ; Matoušková, Petra (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Michaela Řehounková Supervisor: doc. PharmDr. Martin Beránek, Ph.D. Title of diploma thesis: Genetic markers for monitoring post-transplant chimerism The aims of the thesis: Data processing of patients, who underwent allogenic hematopoietic stem cell transplantation in a period from 2010 to 2014 in University Hospital Hradec Kralove and whose state of chimerism was monitored at the Section of Molecular Biology at the Institute of Clinical Biochemistry and Diagnostics. Consequently, analysis of the possible relationship between selected clinical parameters and used genetic markers for chimerism quantification was carried out after the processing of acquired data. Finally, the possible influence of treatment success and mortality by chosen clinical parameters was evaluated. Methods: Analysis of short tandem repeat loci, which uses genetic variability between donor and recipient of transplanted graft, was employed for quantification of post- transplant chimerism. DNA of donor and recipient was isolated by QIAmp DNA Blood Mini Kit (QIAGEN, Germany), amplified by AmpFlSTR Identifier Kit (Applied Biosystems, USA) and separated by capillary electrophoresis (analyzer ABI 3130-4, Applied...
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Morphologic plasticity of \kur{Centaurea jacea.}
KARÁSEK, Jakub
Morphological plasticity of brown knapweed (Centaurea jacea L.) was examined. South bohemian populations of subsp. jacea were compared to plasticity in larger area. The plasticity of local population overlaps with both subspecies. The correlation between abiotical factors and determination characteristics were found. Molecular survey using ISSR method shows no difference between subspecies. The final resolution of subspecies existence will be questioned in following study.
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Possibility of utilization of molecular methods for study of population genetics of noble crayfish Astacus astacus
ŠABATA, Jakub
The noble crayfish (Astacus astacus) is one of two native species of crayfish living in this country who are all over our country strictly protecte, because they are identified as critically endangered species. In Europe it is one of five native species of crayfish, reported by IUCN as an endangered species that needs protection management. Its population was dramatically reduced due to crayfish plagues, which carry non-native crayfish species from North America, who were introduced in the past in Europe and later to the Czech Republic. In the past, have been isolated and described microsatellite markers for crayfish (Koiv et al., 2008a). As part of this work was tested using the eight microsatellite markers on samples obtained from the czech population of crayfish. Testing was performed on 53 samples from six populations of crayfish. Test samples were subjected to isolation of DNA from tissues of the third walking legs using DNA Lego Kit. Then test isolate DNA electrophoresis on agarose gel. Testing the temperature cycle of PCR amplification based on the original publication Koiv et al., (2008a), followed by PCR cycles were adjusted according to the quality of PCR products obtained in our laboratory. Primer annealing temperature 60°C was chosen as the best for six tested loci i.e. Aas 3666, Aas 3115, Aas 790, Aas 1198, Aas 3950 and Aas 766, for two other loci Aas 2489 and Aas 3040 was chosen 55°C annealing temperature. The resulting PCR products were tested on agarose gel and subsequently fragment analysis on an automatic sequencer Beckman Coulter CEQ8000 determining the lenght of PCR products in multiplex consisting of several loci. In the individual loci were in our 53 samples found from 1 to 13 alleles ? one of the loci was monomorphic in all samples analyzed. The moravian population in the Boskovice tank showed the greatest variability, the average number of alleles per locus 3.86, then the north bohemian population from Jaroměř 3.43 alleles per locus. Zelenohorská population of 3 alleles at a locus and Světlohorská population of 2.57 alleles per locus. The lowest average number of alleles per locus had a population from Landštejn 2.43. The aim of this bachelor thesis was to develop a literature search of methods use in molecular biology studies of crayfish and also validate the use of eight microsatellite markers have described for the noble crayfish. In laboratory conditions were succesfully optimized using seven microsatellite markers from the eight described. These microsatellite markers should be used for population studies, or for determining parentage (paternity) in breeding experiments. The verification of the applicability of microsatellite markers have been evaluate and some fundamental characteristics of the population, usability testing and optimization of microsatellite markers, however, was done only on a very small number of samples, because these characteristics have only a very limited explanatory power.
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