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Influence of storage on the microbial composition of French Saint-nectaire cheese
Šislerová, Lucie ; Mikulíková, Renata (referee) ; Brázda, Václav (advisor)
The aim of my work is the comparison of microbial composition between farmtype and dairytype of Saint-nectaire cheese and the influence of storage time and temperature on the development of microbial composition, content of fatty acids and aromatic substances. Selected microorganisms were identified by RT-PCR. In addition, Penicillium roqueforti and fuscoglaucum have been identified in the Saint-nectaire farm type compared to the dairy type. In both types of cheese, the highest amount of selected microorganisms was detected in fresh cheese. When stored at 20 °C, an increase over fresh cheese occurred in the following microorganisms: Streptococcus thermophilus, Lactobacillus bulgaricus, Cladosporium herbarum and Penicillium commune and camemberti, and the presence of contaminants and pathogens was noted. After one week of storage at 20 °C, they were Micrococcus luteus, Salmonella enterica and Staphylococcus aureus, and after another two weeks of storage, Listeria monocytogenes was identified. The fatty acid and volatile compounds were compared for five samples: fresh cheese, cheese stored in the refrigerator for one week and three weeks and cheese stored at 20 °C for one week and three weeks. The content of bound and free fatty acids was measured, both by GC-FID. The content of bound fatty acids was comparable in all measured samples. The highest content of free fatty acids was in the cheese after three weeks of storage at 20 °C. The most common fatty acid is palmitic acid. Volatiles were determined by HS-SPME-GC-MS. The most volatiles were identified in the cheese after three weeks at 20 °C and in the cheese after one week in the refrigerator. The most represented groups were alcohols, ketones and acids.
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Vytvoření a charakterizace transgenních linií drozofil manifestující neurodegenerativní onemocnění spinocerebelární ataxi typu 1 (SCA1)
JIČÍNSKÁ, Veronika
The aim of this thesis was to generate and characterize transgenic lines of Drosophila melanogaster manifesting spinocerebellar ataxia type 1 (SCA1), using GAL4/UAS system. Specific driver lines were used to express ataxin gene in pan neuronal, eye and motor neurons. Adult males from different crosses expressing abnormal ataxin gene with parallel controls aged 5 day and 30 day were used for all experiments. The effects of such expression on mobility, longevity and neurodegenerative changes in the brain was studied. Moreover, specific techniques such are RT-PCR, and western blotting were used to test expression of specific genes and to quantify level of specific proteins. Neurodegenerative changes in transgenic lines were documented using confocal microscopy. The expression of specific genes such as ataxin and matrix metalloproteinase 1 and 2 (dMMP1, dMMP2) was studied in all used fly lines. The results showed that gene expression of hAtXN1 was higher in lines manifesting SCA1 compared to controls. However, the trend of MMP expression was not completely clear and will be the subject of future research.
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Influence of storage on the microbial composition of French Saint-nectaire cheese
Šislerová, Lucie ; Mikulíková, Renata (referee) ; Brázda, Václav (advisor)
The aim of my work is the comparison of microbial composition between farmtype and dairytype of Saint-nectaire cheese and the influence of storage time and temperature on the development of microbial composition, content of fatty acids and aromatic substances. Selected microorganisms were identified by RT-PCR. In addition, Penicillium roqueforti and fuscoglaucum have been identified in the Saint-nectaire farm type compared to the dairy type. In both types of cheese, the highest amount of selected microorganisms was detected in fresh cheese. When stored at 20 °C, an increase over fresh cheese occurred in the following microorganisms: Streptococcus thermophilus, Lactobacillus bulgaricus, Cladosporium herbarum and Penicillium commune and camemberti, and the presence of contaminants and pathogens was noted. After one week of storage at 20 °C, they were Micrococcus luteus, Salmonella enterica and Staphylococcus aureus, and after another two weeks of storage, Listeria monocytogenes was identified. The fatty acid and volatile compounds were compared for five samples: fresh cheese, cheese stored in the refrigerator for one week and three weeks and cheese stored at 20 °C for one week and three weeks. The content of bound and free fatty acids was measured, both by GC-FID. The content of bound fatty acids was comparable in all measured samples. The highest content of free fatty acids was in the cheese after three weeks of storage at 20 °C. The most common fatty acid is palmitic acid. Volatiles were determined by HS-SPME-GC-MS. The most volatiles were identified in the cheese after three weeks at 20 °C and in the cheese after one week in the refrigerator. The most represented groups were alcohols, ketones and acids.
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Synthesis of the Var14 variant of 5'-UTR of GCPII gene as a standard for RT-PCR
Petrovová, Gabriela ; Ingr, Marek (advisor) ; Černá, Věra (referee)
The aim of this work was to prepare the synthetic 5'UTR sequence of splicing variant Var 14 of glutamate carboxypeptidase II (GCPII). A method of two-step PCA was used to this purpose. The sequence was divided into 8 overlapping oligonucleotides that were combined into a single dsDNA by two consecutive PCR. Product of the synthesis was cloned into the auxilliary cloning vector pUC19. After the sequencing analysis detected mutations were corrected. The product was subcloned into the target vector pcDNA4 His Var 14 which already contained the sequence GCPII gene. This construct was then used for the construction of the calibration curve, which will serve as a standard for RT-PCR for quantitative detection of this variant of GCP II in patients with prostate cancer. Construct will be further used as an expression vector to produce of the variants Var 14 GCPII in eucaryotic baculovirus expression system. Keywords: 5'UTR, two-step PCA, pUC19, RT-PCR, GCPII
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Heat shock proteins - - their role in diagnosis and prognosis of pregnancy related complications
Dvořáková, Lenka ; Hromadníková, Ilona (advisor) ; Růžičková, Šárka (referee)
Heat shock proteins increase their gene expression after exposure of cells or organisms to some forms of stress, which may be high temperature, infection, inflammation, hypoxia, lack of nutrients and water. Stressful situations for the body are also pregnancy-related complications associated with placental insufficiency - preeclampsia and IUGR, as well as other pregnancy-related complications such as fetal growth restriction and gestational hypertension. Therefore, I examined whether the occurrence of pregnancy-related complications (preeclampsia, fetal growth retardation, gestational hypertension) affect the gene expression of heat shock proteins. Five hsp systems was detected, namely Hsp27, Hsp60, Hsp70, Hsp90 and HspBP1 in placental tissue samples and whole maternal peripheral blood. Samples came from women with physiological pregnancy and from women with certain pregnancy-related complications (PE, FGR, GH). RNA was isolated from the samples. Detection of hsp expression was performed by using real-time RT-PCR and the comparative Ct method. Changes in gene expression between the test samples and reference sample were examined. To assess the difference between physiological pregnancies and pregnancies with selected pregnancy- related complications, an analysis of variance (ANOVA) was used....
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The comparison of properties of cell lines resistant to ellipticine, doxorubicin, and cisplatin
Černá, Tereza ; Poljaková, Jitka (advisor) ; Eckschlager, Tomáš (referee)
7 Abstract Neuroblastoma is the most common extracranial solid tumor of childhood. Despite advances in cancer diagnosis and therapy, the treatment of some forms of neuroblastoma is still complicated. One of the major complications of the chemotherapy is a developed drug resistance. This master thesis deals with the effect of cytostatics on protein and gene expression of selected proteins, which may contribute to chemoresistance of the human neuroblastoma cell line UKF-NB-4. The sensitive line UKF-NB-4 and the resistant line UKF-NB-4CDDP , UKF-NB-4DOXO and UKF-NB-4ELLI were exposed to cisplatin, doxorubicin, ellipticine for 24, 48 and 72 hours. The Western blot analysis showed that cytostatic agents cisplatin, doxorubicin or ellipticine added to the sensitive neuroblastoma cell line UKF-NB-4 in amounts which are added to resistant neuroblastoma cell lines in order to maintain resistance induced expression of p53 and reduced expression of retinoblastoma protein pRb after 72 hours of cultivation. Differences in the expression of RAS protein, cytochrome P450 1A1, 3A4 and cytochrome b5 has not been shown. Changes in the expression of the studied proteins in resistant lines UKF-NB-4CDDP , UKF-NB-4DOXO and UKF-NB-4ELLI cultured with and without cytostatic agents were not detected by the Western blot analysis....
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Identifikace genů vázaných na pohlavní chromozomy u Silene latifolia a Silene dioica
Machálková, Zuzana
Silene latifolia and Silene dioica are closely related dioecious plant species where sex is determined by the presence of sex chromosomes, X and Y. The sex chromosomes of S. latifolia and S. dioica are younger (10-20 million years) than in mammals (about 200 million years). These two species are thus important models for the study of early stages of sex chromosomes evolution. In this work a group of genes linked to sex chromosomes (Sl3, Sl7, DD44) was analyzed. These genes were de-novo isolated from S. dioica by screening of the BAC library. The expression of these genes in different tissues was compared using Real-Time PCR and HRM in S. latifolia and S. dioica. Using the low-copy FISH (fluorescence in situ hybridization) method, a physical map for individual genes was created. This map is the most detailed physical map created for closely related Silene species.
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Molecular characterization of Grapevine Pinot gris virus in Poland
Pieczonka, Karolina Katarzyna
Samples of different varieties grapevine from Małopolska and Podkarpacie collected on spring 2016 were tested for Grapevine Pinot gris virus (GPGV) by RT-PCR. 16 out of 65 plants were found positive for GPGV. Two set of primers were used for detection, targeting partial movement protein and coat protein, and RdRp domain, both of those regions were sequenced. Phylogenetic analyses settled the Polish isolates to described before asymptotic group. Also GPGV positive samples were tested by multiplex and simplex RT- PCR for multiple infections, and 18,5% of all samples were GPGV and GFkV positive. This was first survey of GPGV in Poland.
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The role of miR-150 in the physiopathology of oligoarticular juvenile idiopathic arthritis
Diviš, Daniel ; Pávek, Petr (advisor) ; Soukup, Tomáš (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Daniel Diviš Supervisors: Prof. Dr. Florence Apparailly, Directrice de Recherche Prof. PharmDr. Petr Pávek, Ph.D. (formal tutor) Title of diploma thesis: The role of miR-150 in the physiopathology of oligoarticular juvenile idiopathic arthritis Juvenile idiopathic arthritis (JIA) is the most common chronic rheumatoid disease affecting children, and its pathological mechanisms are still poorly understood. Innate and adaptive immunity including myeloid cells play a major role in these processes. Epigenetic deregulations along with non-coding microRNAs have been reported in many inflammatory diseases. Moreover, preliminary results obtained by the research group of Prof. Florence Apparailly showed accumulation of intermediate monocytes along with the high expression of miR-150 in the synovial fluid of children affected by oligoarticular JIA. Based on these findings a hypothesis has been postulated suggesting that miR-150 could have a role in the pathogenesis of this disease and in the regulation of monocyte differentiation and function. To study the impact of miR-150 on monocytes from the peripheral blood of healthy donors, transfection experiments were performed to neutralize miR-150. The...
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