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Sledování výskytu patogenních mikroorganismů v potravinovém odpadu po přídavku různých aditiv
Tomková, Lenka
Food waste and food waste management is a global problem. It is necessary to reduce waste and, when food waste is generated, to choose environmentally friendly solutions for its disposal, such as composting. The composting process is a natural decomposition process whose final product is valuable compost that can be used as fertiliser. In this work, the effect of additives on the suppression of pathogenic microorganisms potentially present in food waste is investigated to make the food entering the composting process safe for health. Classical culture methods (selective media for bacterial strains Salmonella spp., Escherichia coli, Helicobacter pylori and enterococci) were used for microbiological analyses and MALDI-TOF mass spectrometry and Sanger sequencing were performed for accurate identification of the grown colonies. The results showed that waste without the addition of additive contained more pathogenic microorganisms than waste with the addition of additive. Serratia spp. and the genus Pseudomonas were the most common in the samples. Tea tree oil appears to be the best additive, as it effectively suppressed the odour and also the waste with its addition did not contain significant pathogens such as Pseudomonas fluorescens compared to the control sample without its addition.
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Molecular genetic analysis of rare ocular disorders in Roma population from the Czech Republic
Rysková, Natálie ; Lišková, Petra (advisor) ; Klímová, Aneta (referee)
The Roma are the largest and most widespread transnational ethnic community. The Czech Republic estimates their 2% representation in its population. Due to the high level of endogamy, the spectrum and frequency of hereditary diseases in the Roma population differ from the majority population. Hereditary eye diseases are one of the most common causes of blindness in younger adults and thus represent a real socio-economic burden. The aim of the thesis was to perform molecular genetic analysis in individuals of Roma origin suffering from hereditary diseases affecting vision, including dual impairments and syndromes, and to determinate the frequency of the detected pathogenic variants in this population. Molecular genetic analysis of 17 families was performed using direct and whole exome sequencing. Within the framework of an international collaboration, the degree of their mutual kinship was calculated using the PLINK program. The frequency of selected variants was determined in a control dataset comprising 156 Roma exomes and genomes. The spectrum of analyzed diseases included various retinal dystrophies, primary congenital glaucoma, Usher syndrome, neuronal ceroid lipofuscinosis, Noonan syndrome, nanophthalmos and congenital cataract, facial dysmorphism and neuropathy. The results of the thesis...
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Analysis of mutations in p53 gene by sequencing
BUŘIČOVÁ, Tereza
The task of this work is a detailed literature search to get acquainted with the methodology of the research part in the faculty laboratory of the University of South Bohemia at the Faculty of Health and Social Studies in České Budějovice. The tumor suppressor gene TP53 plays a key role in protection against tumor growth. This gene is mutated in more than 50 % of malignant tumors. In the first part, this bachelor thesis deals with cancer and tumor suppressor genes as a whole, and then in the next part it focuses on the above-mentioned tumor suppressor gene TP53 and mutations in it. The practical part of the work focuses on the analysis of mutations in exon 11. The analysis was performed on thirty samples, which were provided by anonymous probands. These were two groups of probands. Half of the samples, which are numbered 1-15, were provided by probands who had a family history of cancer. The other half consisted of samples, described by letters from A to O, from probands who were unaware that the cancer was in their family. These samples were isolated, amplified by PCR and sequenced by GenSeq s.r.o. in České Budějovice. In the "ocometric" evaluation, it was found that not a single heterozygous mutation was present in the samples. . After inserting the sequence into the NCBI Blast program, I thought that some samples might contain a homozygous mutation. However, after a proper examination, it was found that this was only an illegible part of the sequence. Thus, no mutation was present in any of the examined samples.
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Sequencing of the ZNF341 gene
POSPÍCHALOVÁ, Kateřina
In my bachelor thesis I dealt with the issue of the ZNF341 gene, which is associated with a defect in the STAT3 protein, causing the syndrome of hyperimmunoglobulinemia E. Hyper IgE syndrome is a multiorgan disease causing eczema, allergies, scoliosis and many other diseases. In the theoretical part, I focused on the ZNF341 gene, its characteristics, its location on the chromosome and the disease that causes its deficiency. Subsequently, I focused on the already mentioned disease of hyperimmunoglobulinemia E. In this disease, I investigated in what forms it occurs and what are its causes and consequences. I also mentioned in the theoretical part about the PCR method and DNA sequencing methods. In the practical part I processed 20 anonymized samples. For all samples I performed DNA isolation, PCR, electrophoresis, purification of PCR products. This was followed by preparation for Sanger sequencing. I sent the samples to GenSeq s.r.o. for sequencing because there was no appropriate sequencer in the school lab. In the final part of the bachelor thesis I evaluated the obtained sequences. I used the BioEdit program and the NCBI internet database to evaluate the sequences. I also received several sequences from children from the České Budějovice and Karviná regions. I then compared the incidence of gene mutations in these samples. Out of a total of 40 samples evaluated, I found a mutation in two of them. It was the same mutation that has not yet been described in the NCBI database.
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Sequencing of the ORMDL3 gene
KRUPILOVÁ, Kristýna
The main topic of the presented bachelor thesis is a sequence analysis of the ORMDL3 gene, which is identified as a potential risk factor for many diseases, the most discussed of which is childhood asthma. This gene is a member of the ORMDL gene family, which encodes transmembrane proteins anchored in the endoplasmic reticulum. In humans, ORMDL3 is located on chromosome 17, in region 17q21.1. In the theoretical part of the thesis the autoimmune diseases, on which origin and development the mutations in the ORMDL3 gene participate to a certain extent, are specified. These diseases include the aforementioned asthma, Crohn's disease, rheumatoid arthritis, insulin-dependent diabetes mellitus and primary biliary cirrhosis. Also, the Sanger sequencing method by which the samples were sequenced, and the modern NGS method are described here. The practical part of the thesis is focused on preparation of samples for Sanger sequencing, and evaluation of sequencing data. To amplify the desired region for the sequencing reaction, the PCR method was used. 20 anonymized DNA samples were prepared this way. For sequencing by Genseq s.r.o. 15 samples were sent. Furthermore, 20 provided samples were evaluated, 10 of which were from children from the České Budějovice region and 10 from children from the Karviná region. These samples were also anonymized. To analyze the sequences of individual DNA samples, freely available BioEdit program and the NCBI database was used. The sequences were read from both sides (forward and reverse), the found mutations were identified and the resulting data were plotted in graphs. After the sequences were read, a heterozygous mutation was present in 5 of the 15 samples. The same mutation was also present in the provided sequences, specifically in 8 out of 10 from České Budějovice and 3 out of 10 from Karviná. This mutation has not yet been described in the NCBI database.
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ALAS2 gene sequencing
HOLOUBKOVÁ, Renata
In my bachelor thesis I dealt with the detection of mutations in the ALAS2. In the theoretical part, I dealt with the introduction to genetic concepts and the field of genetics itself. The work begins from the cell itself, to the chromosomes, through mutations. I also dealt directly with the ALAS2 gene. In the following chapters, I focused on heme, because the ALAS2 gene encodes an enzyme that is responsible for heme production. Last but not least, I have described the disease erythropoietic protoporphyria and sideroblastic anemia, which arise precisely due to mutations in this gene. In the practical part, I examined the ALAS2 gene of exon 11. I examined 25 anonymized samples, from which I isolated DNA. I then amplified the desired section of the gene by PCR. I prepared the samples for the sequencing method, which took place at GenSeq s.r.o. In the last part of my bachelor's thesis, I dealt with the evaluation of sequences and processing of results, but also with the evaluation of sequences from children from Karviná and České Budějovice, which I received. No mutations were found from the whole examined group and the group to be evaluated. For evaluation from a larger data set, the results of the mutations found in the research were added.
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Analysis of gene polymorphisms in the MBL2 gene and its diagnostic significance
PEŠKOVÁ, Jana
MBL2 gene codes a protein called mannose binding lectin, a component of our immune system. This protein takes a part in a nonspecific humoral immune response, opsonizing pathogenic microorganisms and providing activation of the lectin pathway in the complement system, leading to elimination of pathogens and simultaneously inducing the inflammation. MBL deficiencies are widely researched in cases of recurrent infections, autoimmune diseases and others. MBL-protein serum level is affected by polymorphisms in MBL2 gene, located in three codones marked as '52', '54' and '57'. A mutation of the '52' codon is referred to as an allele D, for the '54' codon as an allele B and for the '57' codon as an allele C. In many cases the alleles might be referred to as the alleles zero (0), or as an 'A' in the case of wildtype alleles. Other polymorphisms are located in non-translated locations. The first one may be found in the promotor 1, in a position -550 (variants H/L); the second one in a position -221 (variants X/Y); and the third one in a non translated 5'-end-part of a locus in a position +4 (variants P/Q). The practical part of this bachelor thesis was executed in the genetic lab in the company GENLABS s.r.o., České Budějovice. I focused on the analysis of MBL2 polymorphisms located in exon 1 (alleles B, C, D). I examined 30 patient samples, 25 of them were provided by patients with ongoing dementia and 5 of the samples were taken from patients with no signs of dementia. The protocol of the analysis consisted of an isolation of DNA, a measurement of DNA concentration, a preparation and an execution of a PCR method followed by a control of the products in a gel electrophoresis. These PCR products were then purified and sequenced. A precise description of the analysis including an overview of the obtained results are summed in the practical part of this bachelor thesis.
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Detection of mutations and intragenic rearrangements in BRCA1 and BRCA2 genes by sequencing and MLPA method
PECHOVÁ, Kristýna
In my bachelor thesis I dealt with the detection of mutations in BRCA genes and their effect on the development of breast cancer. Nowadays, this topic is very important, because breast cancer is the most common cancer among women in the Czech Republic (Weinberger a Zikán, 2016). In the theoretical part I focused on breast cancer, particularly on its diagnosis and consequent treatment. I also dedicated genetic counselling and examination, which is very important for the diagnosis of hereditary forms of cancer. I mentioned basic information about BRCA genes, and their protein products and I summarized the issue of mutations in these genes and their relationship to cancer, particularly breast and ovarian. In the practical part, I focused on the examination of selected areas of BRCA genes by the Sanger sequencing method. At present, BRCA genes are examined by the NGS method, because it allows for the analysis of all exons of one particular gene at once, but this testing wasn't possible to realize in my laboratory conditions, therefore I have chosen the method of Sanger sequencing, which does, however, have certain capacity limitations. By using the PCR method, I prepared samples for testing by Sanger sequencing. This testing was conducted by the firm Genseq s.r.o. In the last part of my thesis I processed the obtained sequences and I evaluated the results. I examined a total of 20 anonymized samples using the sequencing method. Mutation was present in 4 samples of the total amount - one was pathogenic, one benign and two of uncertain significance.
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Gene sequencing in patients with cancer anamnesis
MARKOVÁ, Iveta
Cancer is the second most common cause of death in the Czech Republic, of which 5-10% are occupied by hereditary cancer syndromes. They are caused by a congenital - hereditary mutation in one of the alleles of the genes, when after the second random intervention in the other allele hereditary cancer develops. It is important to distinguish between hereditary and sporadic carcinomas due to the high risk of inheritance of mutated alleles in the family. The indication may be, for example, the onset of the disease at a young age or the recurrence of the cancer in the family In my work I focused on the analysis and evaluation of data obtained by Sanger sequencing. The aim was to find mutations in the genes mentioned below and to evaluate their pathogenicity by comparison with databases. In the theoretical part of the bachelor thesis I deal with cancer in general and hereditary cancer. I specify the hereditary breast and ovarian cancer syndrome, including genes, that may cause this syndrome - BRCA1, BRCA2, TP53, PTEN, ATM, PALB2, I also deal with Lynch syndrome and the MMR gene system. Last but not least, I describe a familial adenomatous polyposis associated with the APC gene. In the research part I focused on the examination of selected areas of 18 anonymized samples in the gene PALB2 - exon 13 and in the gene BRCA2 - exon 10/4 and exon 11/12. Using the PCR method, I prepared the samples for Sanger sequencing, which then took place in GenSeq s.r.o. In the last part of my work I deal with the analysis and evaluation of the results using the BioEdit program and the NCBI database. I found a mutation in 5 samples - in 4 it was a deletion of one nucleotide with a conflicting interpretation of pathogenicity, the last mutation was pathogenic - causes hereditary breast and ovarian cancer syndrome, it was a nucleotide duplication.
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Examination of the genes DNM2, GARS, MORC2, TRPV4 and SOD1 among Czech patients with hereditary neuropathy axonal type
Neupauerová, Jana ; Laššuthová, Petra (advisor) ; Vlčková, Eva (referee) ; Vasovčák, Peter (referee)
Examination of the genes DNM2, GARS, MORC2, TRPV4 and SOD1 among Czech patients with hereditary neuropathy axonal type For my PhD thesis I chose to work with patients with axonal form of CMT, because at that time axonal forms were less likely to be clarified by classical methods of molecular genetics. For further examination in patients with unclear cause of the axonal CMT, the genes DNM2, GARS and TRPV4 were selected. The aim was to determine the significance of pathogenic mutations in these genes as the cause of CMT2 in Czech patients. In the course, we identified causal variants in the genes MORC2 and SOD1 with WES. Therefore, we have tested additional CMT2 patients for the presence of these variants. Using Sanger sequencing, I examined a representative set of patients for the DNM2 (37), GARS (10) and TRPV4 (24) genes without finding a causal mutation, then we investigated genes SOD1 (43 patients) and MORC2 (161 patients). The cohort (50 patients) was also subjected to MLPA analysis using a P406-A1 CMT2 duplication and deletion detection kit for genes RAB7A, GARS, HSPB1, HSBP8 and SPTLC1 (kit P406-A1 CMT2). At that time, massively parallel sequencing (MPS) was becoming important. We compared the cost of classical sequencing versus MPS, and accordingly, we decided that the genes DNM2, GARS, MORC2, TRPV4...
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