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Functional assessment of Bcl-2 family proteins in mitochondrial metabolism and beyond
Sovilj, Dana ; Anděra, Ladislav (advisor) ; Mráček, Tomáš (referee) ; Živný, Jan (referee)
(CZ) Od jejich prvotní identifikace v háďátku C. elegans a také v lidských buňkách před více než 30 lety jsou proteiny z rodiny Bcl-2 spojovány s indukcí, regulací a potlačením mitochondriální apoptotické signalizace, ale také se mohou uplatňovat při modulaci neapoptotických signálních dráh. V této studii jsme si stanovili za hlavní cíl rozšířit stávající znalosti o neapoptotických rolích hlavních proapoptotických proteinů z Bcl-2 rodiny, BAX a BAK, zejména pak na jejich roli v buněčném metabolismu. Pomocí genové editace využitím CRISPR/Cas9 jsme eliminovali expresi těchto proteinů v lidských nádorových buňkách různého tkáňového původu a v těchto Bax/Bak- deficitních buňkách jsme primárně analyzovali mitochondriální respiraci a buněčnou glykolýzu. Zatímco eliminace exprese Bax a Bak neměla žádný patrný vliv na glykolýzu ve všech testovaných buněčných liniích, v závislosti na buněčném typu modulovala mitochondriální respiraci. Eliminace exprese Bax a Bak v buňkách rakoviny tlustého střeva HCT-116 neměla vliv na mitochondriální respiraci, ale zjevně ovlivnila mitochondriální respiraci v Bax/Bak-deficitních buňkách odvozených od glioblastomu (U87) a lymfomů (HBL-2, UPF1H, UPF1G). Bax/Bak -/- buňky U87 významně upregulovaly mitochondriální respiraci a akcelerovaly svou proliferaci a také nádorový růst...
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Modulation of mitochondrial transfer by influencing mesenchymal stem cells
Fráňová, Markéta ; Krulová, Magdaléna (advisor) ; Rohlenová, Kateřina (referee)
Mesenchymal stem cells (MSCs) have the ability to modulate the immune response. They use several mechanisms to affect the function of immune cells, and mitochondrial transfer is one of them. Recieving mitochondria from MSCs induces metabolic changes in immune cells, thereby promoting their shift to an anti-inflammatory phenotype. Due to their properties, MSCs have a potencial to be used in therapies, for example in a treatment of autoimmune diseases. The problem of MSCs-based therapies is their low efficacy, mainly due to the high mortality of stem cells after transplantation. In order to achieve at least some effect, the large number of cells is needed for application. The required number of cells can be obtained only by in vitro expansion. However, a long-term culture has a negative impact on MSCs and their immunomodulatory properties. Enhancing MSCs function could increase the efficacy of MSCs-based therapies. The aim of this thesis was to determine whether mitochondrial transfer can be modulated by stimulation of MSCs with selected factors. MSCs were treated with rapamycin, insulin-like growth factor 1 (IGF-1), interferon gamma, or oligomycin. Then the effect of these factors on mitochondria and their transfer to immune cells, metabolism, and immunomodulatory properties of MSCs was analyzed. We...
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Glucose effect on lipid metabolism homestasis in pancreatic β-cells
Stokičová, Linda ; Holendová, Blanka (advisor) ; Horáková, Olga (referee)
Lipids create an essential part of pancreatic β-cells. Not only they are the principal structural components and energy source, but they also play an indispensable role in β-cell physiology. Their metabolism is tightly interconnected with the metabolism of glucose, the fundamental β-cell molecule. The presence of lipids is critical for glucose-stimulated insulin secretion and their turnover is inevitable for correct β-cell function. Lipids in the form of triacylglycerols, retinyl esters and cholesterol esters are stored in lipid droplets. These dynamic cellular structures are important for lipid metabolism and the protection of the cells against various types of stress. However, chronic exposure of β-cells to glucose and lipids can lead to disrupted glycerol/non-esterified fatty acid (GL/NEFA) cycle function, glucolipotoxicity and further dysfunction of β-cells, their dedifferentiation, insulin resistance, and finally type 2 diabetes. The experimental part focused on lipid metabolism in pancreatic β-cells in connection with glucose metabolism and redox environment. Glucose-induced expression of proteins involved in lipid metabolism (fatty acid activation, lipolysis, lipogenesis, etc.) and the effect of modulated redox environment was investigated in β-cell line INS1E and isolated mouse pancreatic...
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The role of glutamine in leukemia cell metabolism
Sitdikova, Karina ; Hložková, Kateřina (advisor) ; Zelenka, Jaroslav (referee)
Leukemia is a heterogeneous group of hematological malignancies that result from the abnormal proliferation of immature blood cells. One of the hallmarks of tumor cells is their altered metabolism. Therefore, therapy targeting deregulated metabolic processes is an attractive strategy for the treatment of malignancies, including hematological ones. Amino acid metabolism is an important part of cellular metabolism, and targeting it appears to be a key attractive strategy in the treatment of leukemia. Glutamine, a conditionally essential amino acid, plays a crucial role in energy metabolism and maintaing the redox balance of leukemia cells, thereby contributing to their growth and proliferation. Strategies to treat leukemia by targeting glutamine metabolism include glutamine depletion, the use of glutamine transporter inhibitors and glutaminase enzyme inhibitors. To ensure the effectiveness of leukemia treatment, it is essential to recognize that glutamine is involved in numerous metabolic pathways, each of which is regulated by multiple factors. As a result, therapies targeting glutamine metabolism should be carefully designed to avoid affecting healthy cells and patient immunity. This thesis describes leukemia, including its types and treatments, and glutamine metabolism and its potential targeting...
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Zinc in food suplements
Sauchanka, Katsiaryna ; Suková, Petra (referee) ; Řezáčová, Veronika (advisor)
The aim of this bachelor thesis was to determine zinc in the dietary supplements by the voltammetric method. The general properties of zinc, its occurrence, toxicity and influence on human health are described in the first part. Next the methods, including a detailed description of the voltammetric analysis, are summarized. The experimental part is focused on the process of optimization of the conditions for zinc voltammetric analysis and the application of the optimised method on real samples.
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Investigation of the effect of creatine in combination with magnesium and vitamin C on the performance of the individual person
Vlasák, Jan ; Němcová, Andrea (referee) ; Diviš, Pavel (advisor)
Creatine is nitrogen-containing organic acid which naturally occurs in the human body. The aim of this work was to determine the optimal dose of creatine in combination with vitamin C and magnesium for male respondents aged 18-26 years. They were divided into two groups differing in the creatine dosage. Group 1 took smaller dose of creatine (3 g per day) and group 2 higher dose of creatine (10 g per day). Both groups took both magnesium and vitamin C at constant doses throughout the study. The effects of significantly different dose of creatine in the individual groups were compared with each other in terms of the performance of individuals in the powerlifting, the anthropological changes and the overal metabolism of the intakes. In all disciplines of powerlifting, group 1 recorded higher average weight gains, which were not found to be statistically significant at a significance level of alpha 0,05. Anthropological changes were measured using the InBody 160 and a diagnostic measuring tape. In both cases, group 1 recorded better results than group 2, but these results were not statistically significant at a significance level of alpha 0,05. The total metabolism of the accepted dietary supplements was investigated through analytical methods. The urine of each respondent was regularly collected and subsequently analyzed during the research. Determination of creatinine, a creatine waste product, was performed by UV-VIS spectrophotometry using the Jaffe reaction. Vitamin C was analyzed by RP-HPLC. Magnesium was determined by the ICP-OES method. After creatine suplemantion of 3 per day, group 1 showed a slight increase in creatinine in the urine, but still in the physiological range. At the significance level alpha 0,05 there was no statistically significant difference. Group 2 showed an increase above the physiological limit which was already a statistically significant difference. Overall, creatine supplementation of 3 g per day has been found as a sufficient intake of creatine needed to build up muscle mass, increase energy metabolism and overall physical performance. The metabolization itself works very well and within the physiological values.
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Metabolic setup of Drosophila macrophages during the immune response
KREJČOVÁ, Gabriela
Adjustment of cellular metabolism is a key function that allows macrophages to fulfill their roles in the body. While the pro-inflammatory polarization of macrophages has been extensively studied in mammalian models, it has not yet been satisfactorily investigated in insects. The study presented in this thesis therefore attempts to elucidate the metabolic setup of macrophages during the immune response in Drosophila melanogaster.
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The role of macrophages in the regulation of systemic metabolism in Drosophila
KREJČOVÁ, Gabriela
Macrophages are immensely versatile cells in the mammalian body, fulfilling roles ranging from protection against pathogenic intruders and engulfing apoptotic cells to morphogenesis and maintenance of tissue homeostasis. This impressive functional versatility may be achieved due to plasticity of macrophage cellular metabolism called metabolic polarization. The adoption of different polarization phenotypes by macrophages determines their function and is essential for the health of the organism. Nonetheless, if the cells lose their metabolic plasticity or polarize inadequately to a particular situation, it can lead to the development of chronic pathological states such as metabolic syndrome. Metabolic polarization of immune cells is thus a key factor in determining whether macrophage function within the organism will be adaptive or pathological. Despite Drosophila melanogaster represents a major model organism for immunological studies, the metabolic setup of activated immune cells has not been addressed up to now. The results of this thesis document that Drosophila immune cells undergo metabolic polarization toward aerobic glycolysis when challenged by extracellular bacteria. Mammals alike, this cellular metabolic switch is regulated by the transcription factor HIF1, thus documenting the conservation of this process between insects and vertebrates. Furthermore, we show that the adoption of aerobic glycolysis is directly linked to the production of the signaling factor IMPL2, which induces the mobilization of lipid stores from the fat body via the silencing of insulin signaling. By this mechanism, immune cells secure sufficient nutrients for successful elimination of the pathogen. Moreover, the mammalian ImpL2 homolog IGFBP7 appears to act analogously in the mammalian liver not only during severe infectious states but also in the liver of obese individuals. While such macrophage activity in regulating systemic metabolism is beneficial to the host during bacterial infection, it becomes maladaptive when chronically activated. Further evidence for a metabolism-regulatory role of immune cells has been found during insect metamorphosis and early post-metamorphic development. This thesis documents that during this period, macrophages infiltrate and engulf the histolyzing larval fat body and convert nutrients into storage peptides and lipoproteins. Subsequently, these nutrients are exploited by the maturing adult structures.
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Kontrola výživy a metabolismu dojnic v chovu s automatizovaným systémem krmení a dojení
Záboj, Dominik
The aim of the thesis was to evaluate the effect of changing the technology of feed loading (conventional feed wagon loading vs. automated feed loading) on the content of milk components, especially on the concentration of urea as an indicator of feed balance over time. For the analysis of the milk constituent content in relation to ration balance, individual samples from the performance check, pool samples analysed by the dairy and herd averages from the Lely Astronaut A4 milking robot were used, which were collected from 1st January 2021 to 31st December 2022. The results of the study document that the change in feeding technology had neither a positive nor a negative effect on the milk constituent content. Statistical evaluation revealed only a significant difference in the number of somatic cells in milk (p < 0.01). Other evaluated milk composition parameters (protein, fat, lactose, urea) were not significantly affected by the change in feeding technology (p > 0.05). In the correlation test between pool samples and individual samples, statistically significant correlations were found for urea (r = 0.66), fat (r = 0.45) and somatic cell count (r = 0.63) at a significance level of p < 0.05. However, correlation analysis of milk testing results from robotic milking with data from pool samples and individual performance control samples yielded inconsistent data, with a positive correlation found only between the mean somatic cell count from the robotic milking parlor and individual KU samples (r = 0.22). In a set of examinations of individual milk samples from KU (n = 2112), correlation analysis was performed between all determined components. Statistically significant correlations (p < 0.05) were found between protein and fat (r = 0.82), lactose and fat (r = 0.68), somatic cell count and fat (r = 0.13), urea and fat (r = 0.45), lactose and protein (r = 0.80), somatic cell count and protein (r = 0.13), urea and protein (r = 0.47) and for urea and lactose (r = 0.45). It is clear from the results that the results of the analysis of milk from the robotic milking parlour differ from the standard values found in performance monitoring and in the examination of pool milk samples, which thus appear to be more suitable for monitoring nutrition and metabolism.
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