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New trends in the use of probiotics in cosmetic products and their characterization
Dvořáková, Monika ; Němcová, Andrea (referee) ; Trachtová, Štěpánka (advisor)
Probiotic products have always been, in the form of various dairy products, a part of our diet. In recent years, they also became prominent in the pharmaceutical sector, where they can often be found as food supplements, and in the cosmetic sector, where they are being added to intimate hygiene and skin care products. The first part of the thesis consists of conducting a theoretical research in the field of probiotics, microbiome and other possibilities of where we can find probiotic organisms in cosmetic products. Further, a part of the thesis is devoted to methods of cultivating and isolating bacterial DNA from the following products: Benton, Mechnikov, La Rosche-Posay creams and Benton and Neogen masks, in sufficient quality for subsequent PCR amplification. DNA was isolated using magnetic carriers, a commercial kit, and phenol extraction. The presence of bacterial DNA was proved by a q-PCR analysis using genus-specified primers of selected bacteria. Genus identification corresponded with the information declared by the manufacturer. To conclude, a PCR and propidium monoazide dye were used in combination for quantification of viable and dead cells, and a HRM analysis was performed.
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Use of advanced molecular biology techniques for detailed characterization of probiotic bacteria from a dietary supplement
Folwarczná, Tereza ; Trachtová, Štěpánka (referee) ; Smetana, Jan (advisor)
The aim of the diploma thesis was the identification of probiotic bacteria from a probiotic food supplement in the form of syrup. DNA was isolated from the complex matrix of the product in sufficient purity and quality suitable for the real-time PCR method followed by PCR-HRM. Specific primers for the genera Lactobacillus, Bifidobacterium and Streptococcus and at the species level for the species Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus reuteri, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium infantis and Streptococcus thermophilus were used for amplification by real-time PCR and PCR-HRM. After optimizing the conditions for specific primers, all declared bacteria were detected in the product.
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Optimization of real-time PCR method for quantification of human mtDNA in clinical samples
LOSKOT, Martin
This bachelor thesis dealing with measuring mitochondrial DNA (mtDNA) and how is it important to pathological conditions. Mitochondria are one of the cell organelles in eukaryotic cells. They are important to energy metabolism, aging process, and apoptosis. In current studies mitochondrial dysfunctions cause mitochondrial diseases and common illnesses, for example cardiovascular diseases, brain pathology or cancer. Studies show, that copy number of mtDNA correlates with state of health and aging process. Copy number of mtDNA is often measured by ratio of mtDNA to nuclear DNA. Measuring mtDNA of peripheral blood is appropriate indicators for mitochondrial functions because decreased copy number of mtDNA correlates with decreased function of mitochondria. We don't know, how copy number of mtDNA influence on illnesses, because it doesn't still clarify. However, studies show, that copy number of mtDNA can be biomarker of control of state of health, when we take real time measurement. In the practical part of this bachelor thesis describe preparation of samples from buccal swabs and from peripheral blood. It also describes method of measuring mtDNA by real-time PCR. There is calculation of copy number of mtDNA and evaluation of our results too. As part of the measurement optimization are described differences between two methods for diagnostic of relative copy number of mtDNA.
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Molekulárně-biologická diagnostika lidských polyomavirů
Ryšavá, Markéta
Most of the human population encounters human polyomaviruses during childhood, when the first infection is asymptomatic or with mild symptoms. However, these viruses persist in the human body and most often, they reactivate during immunosuppression. Together with JC virus reactivation, the progressive multifocal encephalopathy is associated. Hemorrhagic cystitis is associated with BK virus, resulting in the loss of allograft during kidney transplantation. Accurate diagnostics can detect viruses in a timely manner and mitigate tissue damage. This diploma thesis deals with biotechnologies, which can be used in virus detection with a focus on real time PCR, which is the gold standard in virus diagnostics. The literary research summarizes basic information about polyomaviruses with a focus on human BKV and JCV polyomaviruses. It summarizes the biotechnological methods used for detection of polyomaviruses, both in routine diagnosis and in alternative approaches involving biosensors and CRISPR. The experimental part of the work includes the design of a detection system for polyomaviruses from in silico genome analysis, through the design of potential primers, to theoretical specificity analysis. The practical part of the work compares the efficiency and sensitivity of amplification of two reaction mixtures, where one is intended for simple systems and the other for multiplexes. It also includes testing of selected additives of PCR and determining the sensitivity and validity parameters of the reaction mixture test selected for PCR system development. The results showed that detection using a multiplex reaction mixture is sufficiently sensitive, as it meets the conditions and requi-rements of clinical recommendations and at the same time shows very good values of sensitivity and specificity of the test comparable to published technologies. This reaction mixture appears to be relevant to the use of the development and optimization of a PCR system for the detection of polyomaviruses.
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Molecular detection of selected gene polymorphisms related to nutrition (nutrichip validation)
TURKOVÁ, Kateřina
Lactose intolerance is the most common food intolerance in the world. Individuals with lactose intolerance are unable to produce the enzyme lactase in the small intestine, which makes it possible to break down the lactose contained in dairy products. Insufficient lactase production may be genetically determined. Two single nucleotide polymorphisms responsible for the persistence of lactase activity in adulthood have been found in the European population. Celiac disease is one of the autoimmune diseases that mainly affects the mucous membrane of the small intestine. The disease is characterized by intolerance to gliadin, which is part of gluten. Intolerance leads to chronic inflammation of the small intestinal mucosa, leading to chronic diarrhea, fatty stools, vomiting and fatigue. The development of celiac disease is conditioned by the presence of a genetic predisposition. Genetic predisposition is linked to HLA system alleles. Specifically, these are the HLA-DQ2 and HLA-DQ8 haplotypes.
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Detection of human adenoviruses from the respiratory samples from the patients of Motol University Hospital
NOVÁKOVÁ, Veronika
Human adenoviruses are world-wide pathogens causing endemic and epidemic outbre-aks of disease. Adenoviral infections are related with high morbidity and mortality especially among the paediatric allogeneic haematopoietic stem cell recipients. Presented work is comparing the commercial and in-house assay for detection of the adenoviruses to improve the diagnostics of these viruses in Motol University Hospital, especially among the immunocompromissed patients, in which is the rapid and precise detection highly important. Theoretical part of work presents the characteristics of adenoviruses, clinical symptoms and diseases of the adenoviral infection, their use in the experimental therapy. Practical part is aiming the screening of the respiratory tract samples with quantitative in-house test and comparing of the results with previously performed detection by commercial Anyplex TM II RV16 test. Detection of adenoviruses was performed in 1,881 samples from 1,420 patients with symptoms of respiratory tract infection by real-time PCR. In total, adenovirus was detected in 187 samples (9.9%) from 169 patients (11.9%). One hundred samples (53.5%) was positive by both methods, 26 (13.9%) with commercial detection only and 61 (32.6%) was positive only with in-house assay. Most frequently, adenoviruses were detected among the patients from Dept. of Paediatrics, Dept. of Paediatric Haematology and Oncology and from adult patients from 3rd Dept. of Surgery and Dept. of Anaestesiology, resuscitation and intensive medicine. In all positive samples, subsequent sequence analysis of hypervariable part of 1-6 hexon gene was performed and representative sequence for identification was obtained in 70 samples. Most frequently detected adenovirus genotypes were C2 (25.7 %), A31 (24.3 %), B3 (15.7 %) a C1 (11.4 %). From 26 samples positive by Anyplex TM II RV16, only 3 samples were identified (all as genotype B3). Contrary, among 61 positive samples detected only with in-house assay, 15 samples were identified as genotype A31 and 3 as AdV from group C. Our results shows the lower sensitivity of the commercial test in detection of adenoviru-ses.
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Molecular diagnostics of hepatitis C in a selected group of patients
KALINAYOVÁ, Daniela
This bachelor thesis deals with the occurrence of hepatitis C and its detection focused on direct evidence of the virus. The theoretical part describes and divides hepatitis C into acute and chronic, focuses on its genetic variability and explains how this virus behaves, is transmitted and mutated. Mutation creates other variants of it, which must be distinguished from each other if we want to set up an effective treatment. The practical part gives us information about the incidence of HCV among men and women, which are clearly divided in tables according to the year of birth, positivity and negativity. It also deals with the determination of a given genotype / subtype.
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Výskyt laktózové intolerance v české populaci
CHÁNOVÁ, Jiřina
The aim of this diploma thesis is to summarize the current knowledge on the issue of very common gastrointestinal disorder - lactose intolerance. In the experimental part, the occurrence of genotypic frequencies in the MCM6 gene was screened. Specifically, the occurrence of two single nucleotide polymorphisms C/T-13910 and G/A-22018, which are associated with primary hypolactasia. A further aim of the work was to evaluate the possible association between lactose intolerance and irritable bowel syndrome.
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Development of methods for genetic analysis of plant foods
Fialová, Lenka ; Brázda, Václav (referee) ; Doškař, Jiří (referee) ; Márová, Ivana (advisor)
Multiplex real-time PCR-HRM is an approach which has gained some attention in recent years. It has already found applications in clinical diagnostics and food authenticity and safety control. Compared to its corresponding singleplex PCR assays, an optimized multiplex PCR assay provides the same information in a fraction of time. First part of this work dealt with isolation of DNA from both fresh fruits and processed commercial products. Six different DNA isolation protocols were tested with fresh fruits – three silica column-based kits, two magnetic carrier-based kits and one conventional protocol. One method was chosen as the most suitable and was applied to DNA isolation from commercial products. These experiments also involved optimisation of the chosen method. The second part of this work was focused on the development of a triplex real-time PCR assay for simultaneous detection of blueberry, strawberry and raspberry, and its application on DNA isolated from commercial products. During DNA isolation, calcium chloride was shown to be a promising agent for removal of pectin from samples. In several samples, presence of raspberry DNA was confirmed by singleplex PCR. We found out that for accurate results of food analysis by this assay, further optimization of its parameters would be needed.
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Splice variants of the gene coding for GCPII and their role in cancer development
Jindrová, Helena ; Konvalinka, Jan (advisor) ; Liberda, Jiří (referee)
Alternative splicing is a mechanism of generating distinct proteins that are encoded by the same gene. These proteins differ in amino acid sequence, overall structure and function. Splicing dysregulations have been shown to be implicated in several pathologic processes including cancer. For example, non-physiological splicing of osteopontin was proved to play a key role in cell progression of breast cancer. Glutamate carboxypeptidase II (also called prostate specific membrane antigen, PSMA) is present in both normal prostate and prostate cancer. Several splice variants of PSMA have been described and it has been suggested that the overexpression of some of them could be involved in the progression of prostate cancer. Nevertheless, more detailed investigation of each of the PSMA splice variant in terms of their occurrence in prostate cancer cells remains to be performed. This thesis focuses on the exploration of the expression of PSMA splice variants with deleted exons 6 and 18 in samples of a cell line derived from human prostate cancer, benign prostate hyperplasia and prostate cancer. For this purpose, RT-PCR was utilized to determine the ratio of deletions of exons 6 and 18 in cDNA of the prostate specific membrane antigen. Furthermore, the ratio of deletions of exon 6 and 18 was determined in...
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