Rola neuromedyny U (NMU) w regulacji potencjału migracyjnego komórek raka jelita grubego

Rak jelita grubego (RJG) jest jednym z najczęściej diagnozowanych nowotworów na świecie. Pomimo poznania mechanizmów molekularnych prowadzących do powstania oraz rozwoju RJG, nie opracowano dotychczas precyzyjnych metod diagnostycznych oraz efektywnych schematów terapii zapobiegających metastazie. Dlatego wciąż niezbędna jest identyfikację czynników, które są aktywne we wczesnych etapach powstawania zmian prowadzących do powstania przerzutów. Neuromedyna U, małe białko wydzielnicze, w ostatnich latach zyskał zainteresowanie z uwagi na jego potencjalną rolę w rozwoju chorób nowotworowych. Udział NMU w progresji RJG, nie został dotychczas opisany, dlatego w niniejszej pracy doktorskiej, pt.: „Rola neuromedyny U (NMU) w regulacji potencjału migracyjnego komórek raka jelita grubego” postanowiłam zweryfikować hipotezę, że NMU może regulować potencjał migracyjny oraz inwazyjny komórek RJG. Do badań wykorzystano panel 6 linii komórkowych RJG wyizolowanych od pacjentów na różnych etapach zaawansowania choroby. Wybrane linie zbadano pod kątem ekspresji NMU oraz receptorów, NMUR1 i NMUR2, a także receptora alternatywnego NTSR1/GHSR1b. Następnie sprawdzono obecność NMU w lizatach komórkowych, medium pohodowlanym oraz w lizatach z mikropęcherzyków zewnątrzkomórkowych. Dodatkowo zbadano, czy poziom ekspresji NMUR1 lub NMUR2 zależy od procesu metylacji. Kolejno sprawdzono aktywację ścieżek sygnałowych charakterystycznych dla receptorów GPCR, które mogą być uruchamiane pod wpływem NMU. Przeprowadzone badania pokazały, że komórki RJG charakteryzują się zróżnicowaną ekspresją NMU oraz receptorów dla NMU. Dodatkowo pokazano, że ekspresja NMUR1 i NMUR2 może być regulowana przez proces metylacji. NMU jest wydzielana na zewnątrz komórek w postaci różnych form pre-pro-peptydów oraz jako zawartość mikropęcherzyków zewnątrzkomórkowych. W komórkach RJG z ekspresją receptora NMUR2 dochodzi do mobilizacji wewnątrzkomórkowych jonów wapnia oraz fosforylacji kinaz białkowych ERK1/2 pod wpływem działania SBL-NMU-17 (agonista NMUR2). Drugim etapem pracy była ocena zmian potencjału migracyjnego oraz inwazyjnego linii komórkowych RJG pod wpływem NMU. Do oceny zmian fenotypu komórek oraz badań funkcjonalnych zostały wykorzystane linie komórkowe z ekspresją NMUR2 i stabilną nadekspresją NMU. Otrzymane wyniki wskazują, że pod wpływem NMU dochodzi do zmian fenotypu sublinii HT29 (fenotyp epitelialny) i rozluźnienia połączeń między komórkami, jednocześnie obniżyła się ich zdolność do formowania kolonii. W przypadku sublinii Caco-2 (fenotyp mezenchymalny) nie zaobserwowano zmian w morfologii, ale nastąpił wzrost ich zdolności do formowania kolonii. Następnie udowodniono, że niezależnie od fenotypu, NMU powoduje zwiększenie potencjału migracyjnego oraz inwazyjnego komórek, poprzez wiązanie z NMUR2 orazfosforylację kinaz białkowych ERK1/2. Przedstawione badania potwierdzają również, że NMU jest zaangażowana w regulację poziomu powierzchniowych podjednostek integrynowych istotnych w progresji RJG. Otrzymane wyniki sugerują, że NMU poprzez interakcje z NMUR2, aktywuje ścieżkę sygnałową MEK/ERK. NMU wydzielana do środowiska przez komórki RJG, powoduje zwiększenie ich potencjału migracyjnego oraz inwazyjnego w zastosowanym modelu. Wydaje się, że transdukcja sygnału NMU-NMUR2 jest jednym z istotnych mechanizmów komórkowych prowadzących do zmiany fenotypu komórek RJG na bardziej inwazyjny.Colorectal cancer (CRC) is one of the most frequently diagnosed cancers in the world. Despite the knowledge of the molecular mechanisms leading to the formation and development of CRC, no precise diagnostic methods and effective treatment regimens to prevent metastasis have been developed so far. Therefore, it is still necessary to identify the factors that are active in the early stages of the formation of lesions leading to the formation of metastases. Neuromedin U, a small secretory peptide, has gained interest in recent years due to its potential role in the development of cancer. The role of NMU in the progression of CRC has not been described so far, therefore in this Ph.D. thesis entitled "The role of neuromedin U (NMU) in the regulation of the migration potential of colorectal cancer cells", I decided to verify the hypothesis that NMU can regulate the migration and invasive potential of CRC cells. A panel of 6 CRC cell lines isolated from patients at various stages of the disease was used for the study. Selected lines were tested for the expression of NMU and the receptors, NMUR1 and NMUR2, as well as the alternative receptor NTSR1/GHSR1b. Next, the presence of NMU in cell lysates, post-culture medium and lysates from extracellular microvesicles was checked. Additionally, it was investigated whether the expression level of NMUR1 or NMUR2 depends on the methylation process. Subsequently, the activation of signaling pathways characteristic of GPCR receptors, which can be activated under the influence of NMU, was checked. The performed research showed that CRC cells are characterized by differential expression of NMU and its receptors. Additionally, it has been shown that the expression of NMUR1 and NMUR2 can be regulated by the methylation process. NMU is secreted outside the cells in the form of various forms of pre-pro-peptides and as the content of extracellular microvesicles. In CRC cells expressing the NMUR2 receptor, intracellular calcium ions are mobilized and ERK1/2 protein kinases are phosphorylated influence of SBL-NMU-17 (NMUR2 agonist). The second part of my Ph.D. thesis was the assessment of changes in the migration and invasive potential of CRC cell lines under the influence of NMU. Cell lines with NMUR2 expression and stable NMU overexpression were used to assess changes in cell phenotype and functional studies. The obtained results indicate that the phenotype of the HT29 subline (the epithelial phenotype) is changed under the influence of NMU, and the connections between cells are loosened, while their ability to form colonies is reduced. In the case of the Caco-2 subline (mesenchymal phenotype), no change in morphology was observed, but there was an increase in their ability to form colonies. Subsequently, it was proved that, regardless of the phenotype, NMU increases the migration and invasive potential of cells by binding to NMUR2 and phosphorylation of ERK1/2 protein kinases. The presented studies also confirm that NMU is involved in regulating the level of surface integrin subunits important in CRC progression. The obtained results suggest that NMU, by interacting with NMUR2, activates the MEK/ERK signaling pathway. NMU secreted into the environment by CRC cells increases their migration and invasive potential in our model. Our study suggests, the transduction of the NMU-NMUR2 signal is one of the most important cellular mechanisms leading to the change of the phenotype of CRC cells to a more invasive.Badania stanowiące podstawę niniejszej rozprawy doktorskiej zostały przeprowadzone w ramach projektu Sonata Bis pt. „Neuromedyna U jako nowy potencjalny regulator przerzutowania w raku jelita grubego i odbytnicy” (2016/22/E/NZ3/00341), kierownik dr Patrycja Przygodzka

Antibody response to DNA vaccine against H5N1 avian influenza virus in broilers immunized according to three schedules.

Broiler type chickens were immunized intramuscularly with a DNA vaccine encoding hemagglutinin (HA) from H5N1 avian influenza virus. The chickens were divided into four groups: control group which was not immunized, a group which obtained only one dose, and two groups which were immunized twice, one group with a boost two weeks after the priming and the other four weeks. Blood samples were collected at several time points and the dynamics of the humoral response to the vaccine was studied. High level of anti-HA antibodies was detected only in the last two groups, that is in chickens immunized according to the prime-boost strategy, regardless of the schedule. An additional interesting observation of this study was detection of the cross-reactivity of an anti-H5 HA positive serum with H5N2 and H1N1 viruses, suggesting that the DNA vaccine tested can induce antibodies of a broad specificity

„Idée sur les romans” Donatiena Alphonse’a Françoisa de Sade’a. Rozważania na temat utworu

The article shows the wider literary context of the epoch and defines the creative inspirations of the author in past and historic events. An Essay on Novels, servings as the introduction to the collection of short stories The Crimes of Love (1800), is discussed in a broader, holistic context of a publication containing 11 titles. Sade’s definition of the novel is presented and analyzed, from an outline developed by him of the history of literature from ancient Egypt to the present and his prediction of the future, and the most important motives and attributes of fictional works through-out history were enumerated in accordance with his guidelines. The rules of writing that Sade formulated in relation to the works of Samuel Richardson and Henry Fielding are another subject discussed here. De Sade’assessment of the work by contemporary writers and his literary forecasts were juxtaposed with the romantic features of the coming era: the desire for freneticism and horror caused by the French Revolution. The text is presented in a broader historical context of the 1800s (M. Milewska, Vinegar and Tears) and a comparative one with references to the works and literary thought of Polish writers (M. Janion, Romantyzm, rewolucja, marksizm. Colloquia gdańskie). The article also draws attention to footnotes penned by de Sade and his reflections on the contemporary book market. The aim here is to gain a deeper understanding of the writings of the Marquis and show his deep knowledge of literature.The article shows the wider literary context of the epoch and defines the creative inspirations of the author in past and historic events. An Essay on Novels, servings as the introduction to the collection of short stories The Crimes of Love (1800), is discussed in a broader, holistic context of a publication containing 11 titles. Sade’s definition of the novel is presented and analyzed, from an outline developed by him of the history of literature from ancient Egypt to the present and his prediction of the future, and the most important motives and attributes of fictional works through-out history were enumerated in accordance with his guidelines. The rules of writing that Sade formulated in relation to the works of Samuel Richardson and Henry Fielding are another subject discussed here. De Sade’assessment of the work by contemporary writers and his literary forecasts were juxtaposed with the romantic features of the coming era: the desire for freneticism and horror caused by the French Revolution. The text is presented in a broader historical context of the 1800s (M. Milewska, Vinegar and Tears) and a comparative one with references to the works and literary thought of Polish writers (M. Janion, Romantyzm, rewolucja, marksizm. Colloquia gdańskie). The article also draws attention to footnotes penned by de Sade and his reflections on the contemporary book market. The aim here is to gain a deeper understanding of the writings of the Marquis and show his deep knowledge of literature

Neuromedin U: A Small Peptide in the Big World of Cancer

Neuromedin U (NMU), a neuropeptide isolated from porcine spinal cord and named because of its activity as a rat uterus smooth muscle contraction inducer, is emerging as a new player in the tumorigenesis and/or metastasis of many types of cancers. Expressed in a variety of tissues, NMU has been shown to possess many important activities in the central nervous system as well as on the periphery. Along with the main structural and functional features of NMU and its currently known receptors, we summarized a growing number of recently published data from different tissues and cells that associate NMU activity with cancer development and progression. We ask if, based on current reports, NMU can be included as a marker of these processes and/or considered as a therapeutic target

Different Oxidation Pathways of 2-Selenouracil and 2-Thiouracil, Natural Components of Transfer RNA

Sulfur- and selenium-modified uridines present in the wobble position of transfer RNAs (tRNAs) play an important role in the precise reading of genetic information and tuning of protein biosynthesis in all three domains of life. Both sulfur and selenium chalcogens functionally operate as key elements of biological molecules involved in the protection of cells against oxidative damage. In this work, 2-thiouracil (S2Ura) and 2-selenouracil (Se2Ura) were treated with hydrogen peroxide at 1:0.5, 1:1, and 1:10 molar ratios and at selected pH values ranging from 5 to 8. It was found that Se2Ura was more prone to oxidation than its sulfur analog, and if reacted with H2O2 at a 1:1 or lower molar ratio, it predominantly produced diselenide Ura-Se-Se-Ura, which spontaneously transformed to a previously unknown Se-containing two-ring compound. Its deselenation furnished the major reaction product, a structure not related to any known biological species. Under the same conditions, only a small amount of S2Ura was oxidized to form Ura-SO2H and uracil (Ura). In contrast, 10-fold excess hydrogen peroxide converted Se2Ura and S2Ura into corresponding Ura-SeOnH and Ura-SOnH intermediates, which decomposed with the release of selenium and sulfur oxide(s) to yield Ura as either a predominant or exclusive product, respectively. Our results confirmed significantly different oxidation pathways of 2-selenouracil and 2-thiouracil

MicroRNA composition of plasma extracellular vesicles : a harbinger of late cardiotoxicity of doxorubicin

Abstract Background The use of doxorubicin is associated with an increased risk of acute and long-term cardiomyopathy. Despite the constantly growing number of cancer survivors, little is known about the transcriptional mechanisms which progress in the time leading to a severe cardiac outcome. It is also unclear whether long-term transcriptomic alterations related to doxorubicin use are similar to transcriptomic patterns present in patients suffering from other cardiomyopathies. Methods We have sequenced miRNA from total plasma and extracellular vesicles (EVs) from 66 acute lymphoblastic leukemia (ALL) survivors and 61 healthy controls (254 samples in total). We then analyzed processes regulated by differentially expressed circulating miRNAs and cross-validated results with the data of patients with clinically manifested cardiomyopathies. Results We found that especially miRNAs contained within EVs may be informative in terms of cardiomyopathy development and may regulate pathways related to neurotrophin signaling, transforming growth factor beta (TGFβ) or epidermal growth factor receptors (ErbB). We identified vesicular miR-144-3p and miR-423-3p as the most variable between groups and significantly correlated with echocardiographic parameters and, respectively, for plasma: let-7g-5p and miR-16-2-3p. Moreover, vesicular miR-144-3p correlates with the highest number of echocardiographic parameters and is differentially expressed in the circulation of patients with dilated cardiomyopathy. We also found that distribution of particular miRNAs between of plasma and EVs (proportion between compartments) e.g., miR-184 in ALL, is altered, suggesting changes within secretory and miRNA sorting mechanisms. Conclusions Our results show that transcriptomic changes resulting from doxorubicin induced myocardial injury are reflected in circulating miRNA levels and precede development of the late onset cardiomyopathy phenotype. Among miRNAs related to cardiac function, we found vesicular miR-144-3p and miR-423-3p, as well as let-7g-5p and miR-16-2-3p contained in the total plasma. Selection of source for such studies (plasma or EVs) is of critical importance, as distribution of some miRNA between plasma and EVs is altered in ALL survivors, in comparison to healthy people, which suggests that doxorubicin-induced changes include miRNA sorting and export to extracellular space