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Zabłocki, Krzysztof (1956- ) : Supervisor
Publisher:Instytut Biologii Doświadczalnej im. Marcelego Nenckiego PAN
Place of publishing: Date issued/created: Description:144 pages : illustrations ; 30 cm ; Bibliography ; Summary in English
Degree name: Degree discipline : Degree grantor:Nencki Institute of Experimental Biology PAS ; degree obtained: 17.04.2026
Type of object: Subject and Keywords:Endothekial cells ; Mitochondria ; Lipopolysaccharide ; N-methyltransferase
Abstract:
The vascular endothelium is a layer of specialized cells that lines the inner walls of all blood vessels. As a secretory organ, the endothelium modulates the inflammatory response, regulates blood flow, controls vascular permeability and the balance between coagulation and fibrinolysis, and also plays an important role in the processes of angiogenesis and in the regulation of blood pressure. Due to its location, it constitutes one of the first protective barriers against pathogenic factors circulating in the blood, including lipopolysaccharide (LPS). LPS is a strong inducer of the inflammatory response and is widely studied in the context of the sepsis it causes. An interesting, yet little discussed, factor that seems to influence inflammatory processes in the body is the enzyme nicotinamide N- methyltransferase (NNMT), which catalyzes the transfer of the methyl residue from S- adenosylmethionine (SAM) to nicotinic acid amide. The involvement of NNMT in the response to stress and inflammation associated with various disorders has been proven in numerous in vitro and in vivo models, but its role in the endothelium, especially in the context of sepsis, is poorly understood. This dissertation consists of two parts. The first one focused on characterizing the response of unchanged HAEC cells to LPS administered under conditions of concentration and treatment time mild enough to obtain the typical pro-inflammatory response of the cells, but not sufficient to reduce their survival. This approach allows for minimizing irreversible effects that make the interpretation of adaptation processes difficult. Some of the obtained results were consistent with expectations and confirmed previously known effects and the correctness of the established model, while some were new observations regarding the reversibility of changes in the organization of the mitochondrial network and the profile of glycolytic metabolites and the tricarboxylic acid cycle, as well as the rate of cell respiration. Of particular note is the finding that treatment of HAECs with LPS results in a significant increase in the amount of the NNMT. This completely new observation became the inspiration to undertake the research described in the second part of the dissertation. Their aim was to check whether the presence of the NNMT was related to the observed changes induced by LPS. The siRNA technique was used to silence the NNMT and it was shown that the response of cells with reduced levels of this enzyme to LPS was clearly attenuated. This concerned the reduction of oxidative stress, alleviation of changes in the architecture of the mitochondrial network, and the LPS-stimulated increase in the content of Opa1 and Mnf1 responsible for mitochondrial fusion, as well as the Fis1 involved in their fragmentation. Importantly, in cells with a silenced NNMT gene, the normalization of the level of glycolysis and the Krebs cycle metabolites in LPS-treated cells was significantly accelerated. Moreover, silencing the NNMT prevented the increased activation of the store operated calcium entry (SOCE) observed in cells with unchanged expression of this gene treated with LPS and appears to promote the normalization of the autophagy process and the activation of endoplasmic reticulum stress. It was not possible to link the changes caused by LPS with the expected and direct effects related to the reduction of protein levels and NNMT activity, such as increasing the level of SAM and reducing the amount of NAD+ in cells. In the first case, the effect of LPS itself masked the expected result, and in the second, the presence of nicotinamide in the medium did not allow for a possible NAD+ deficit. Therefore, elucidating the biochemical mechanism linking NNMT activity with the effects of LPS in HAEC cells requires further research. Nevertheless, this dissertation collects a number of new observations which, apart from the cognitive aspect, can provide the basis for application research.
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Copyright holder:Publication made available with the written permission of the author
Digitizing institution:Nencki Institute of Experimental Biology of the Polish Academy of Sciences
Original in:Library of the Nencki Institute of Experimental Biology PAS
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