Poszukiwanie w płytkach krwi molekularnych markerów określających predyspozycje człowieka do wystąpienia Ostrych Zespołów Wieńcowych

Ostre zespoły wieńcowe (OZW) należą do grupy schorzeń kardiologicznych spowodowanych nagłym lub narastającym zmniejszeniem drożności w tętnicach wieńcowych, a za jedną z głównych przyczyn patofizjologii OZW uznaje się patologiczną aktywację płytek krwi. Celem niniejszej rozprawy doktorskiej było określenie zaburzeń w transkryptomie i proteomie płytek krwi, które mogłyby stanowić molekularne podłoże ich wzmożonej aktywności pro-zakrzepowej w OZW oraz identyfikacja cząsteczek o potencjale wysokoczułych molekularnych markerów płytkowych, określających predyspozycje człowieka do wystąpienia OZW. Przeprowadzone badania pozwoliły na zidentyfikowanie 8 cząsteczek miRNA których poziom ekspresji różnicował pacjentów z OZW od grupy kontrolnej. Modelowanie statystyczne, przeprowadzone w celu identyfikacji potencjalnego biomarkera OZW, pozwoliło wytypować cząsteczkę hsa-miR-142-3p wraz z AST do łączonego modelu diagnostycznego, który różnicował porównywane grupy z 82% czułością oraz 88% specyficznością. Co więcej, w niniejszej pracy doktorskiej wykazano, iż wśród pacjentów z OZW obserwowany jest istotnie statystyczny wzrost ekspresji (na poziomie mRNA oraz białka) receptorów powierzchniowych dla ADP (P2Y12) i fibrynogenu (GP IIb/IIIa). Krzywa ROC wykonana dla modelu opartego na ekspresji receptora P2Y12 na poziomie mRNA oraz białka wraz z ekspresją hsa-miR-223-3p wykazała 97% czułość oraz 74% specyficzność w różnicowaniu pacjentów z OZW od grupy kontrolnej. Ponadto, wyniki uzyskane z przesiewowej analizy płytkowego proteomu wykazały zwiększoną ekspresję transgeliny-2. Podsumowując, zmiany proteomiczne oraz transkryptomiczne (na poziomie mRNA oraz miRNA) zidentyfikowane w płytkach krwi wskazują na molekularne podłoże ich wzmożonej aktywności pro-zakrzepowej w OZW. Ponadto, wykryte zaburzenia w ekspresji płytkowych cząsteczek miRNA pomiędzy badanymi grupami wykazują zdolność do różnicowania pacjentów z OZW od zdrowych dawców.Praca doktorska byłą finansowania ze źródeł: 1) Projektu "Diamentowy Grant", który był finansowany przez Ministerstwo Edukacji i Nauki nr DI2016 022046 w latach 2017 - 2021 nt.: "Płytkowe mikroRNA jako marker predykcyjny Ostrego Zespołu Wieńcowego. Kierownik projektu: mgr Rafał Szelenberger, opiekun: Prof. dr hab. Joanna Saluk-Bijak. 2) Dotacji celowej przyznawanej na działalność związaną z prowadzeniem badań naukowych lub prac rozwojowych oraz zadań z nimi związanych, służących rozwojowi młodych naukowców oraz uczestników studiów doktoranckich w roku 2019

Ochratoxin A—The Current Knowledge Concerning Hepatotoxicity, Mode of Action and Possible Prevention

Ochratoxin A (OTA) is considered as the most toxic of the other ochratoxins synthesized by various fungal species belonging to the Aspergillus and Penicillium families. OTA commonly contaminates food and beverages, resulting in animal and human health issues. The toxicity of OTA is known to cause liver damage and is still being researched. However, current findings do not provide clear insights into the toxin mechanism of action. The current studies focusing on the use of potentially protective compounds against the effects of the toxin are insufficient as they are mainly conducted on animals. Further research is required to fill the existing gaps in both fields (namely the exact OTA molecular mechanism and the prevention of its toxicity in the human liver). This review article is a summary of the so far obtained results of studies focusing on the OTA hepatotoxicity, its mode of action, and the known approaches of liver cells protection, which may be the base for expanding other research in near future

Increased O-GlcNAcylation by Upregulation of Mitochondrial O-GlcNAc Transferase (mOGT) Inhibits the Activity of Respiratory Chain Complexes and Controls Cellular Bioenergetics

O-linked β-N-acetylglucosamine (O-GlcNAc) is a reversible post-translational modification involved in the regulation of cytosolic, nuclear, and mitochondrial proteins. The interplay between O-GlcNAcylation and phosphorylation is critical to control signaling pathways and maintain cellular homeostasis. The addition of O-GlcNAc moieties to target proteins is catalyzed by O-linked N-acetylglucosamine transferase (OGT). Of the three splice variants of OGT described, one is destined for the mitochondria (mOGT). Although the effects of O-GlcNAcylation on the biology of normal and cancer cells are well documented, the role of mOGT remains poorly understood. In this manuscript, the effects of mOGT on mitochondrial protein phosphorylation, electron transport chain (ETC) complex activity, and the expression of VDAC porins were investigated. We performed studies using normal and breast cancer cells with upregulated mOGT or its catalytically inactive mutant. Proteomic approaches included the isolation of O-GlcNAc-modified proteins of the electron transport chain, followed by their analysis using mass spectrometry. We found that mitochondrial OGT regulates the activity of complexes I-V of the respiratory chain and identified a group of 19 ETC components as mOGT substrates in mammary cells. Furthermore, we observed that the upregulation of mOGT inhibited the interaction of VDAC1 with hexokinase II. Our results suggest that the deregulation of mOGT reprograms cellular energy metabolism via interaction with and O-GlcNAcylation of proteins involved in ATP production in mitochondria and its exchange between mitochondria and the cytosol

Variations in Blood Platelet Proteome and Transcriptome Revealed Altered Expression of Transgelin-2 in Acute Coronary Syndrome Patients

Proteomic analyses based on mass spectrometry provide a powerful tool for the simultaneous identification of proteins and their signatures. Disorders detection at the molecular level delivers an immense impact for a better understanding of the pathogenesis and etiology of various diseases. Acute coronary syndrome (ACS) refers to a group of heart diseases generally associated with rupture of an atherosclerotic plaque and partial or complete thrombotic obstruction of the blood flow in the infarct-related coronary artery. The essential role in the pathogenesis of ACS is related to the abnormal, pathological activation of blood platelets. The multifactorial and complex character of ACS indicates the need to explain the molecular mechanisms responsible for thrombosis. In our study, we performed screening and comparative analysis of platelet proteome from ACS patients and healthy donors. Two-dimensional fluorescence difference gel electrophoresis and nanoscale liquid chromatography coupled to tandem mass spectrometry showed altered expressions of six proteins (i.e., vinculin, transgelin-2, fibrinogen β and γ chains, apolipoprotein a1, and tubulin β), with the overlapping increased expression at the mRNA level for transgelin-2. Dysregulation in protein expression identified in our study may be associated with an increased risk of thrombotic events, correlated with a higher aggregability of blood platelets and induced shape change, thus explaining the phenomenon of the hyperreactivity of blood platelets in ACS

Dysregulation in the Expression of Platelet Surface Receptors in Acute Coronary Syndrome Patients—Emphasis on P2Y12

The pathological conditions caused by blood platelet activation constitute a fundamental core in the pathogenesis of Acute Coronary Syndrome (ACS). The hyperactivity of platelets in ACS is well-documented, but there is still little research into the molecular basis of phenotypic changes in platelet functionality. To expand the knowledge of this phenomenon, we analyzed the disturbances in the expression of several key platelet receptors and the aspect of regulating potential abnormalities. Platelet surface receptors are responsible for maintaining the hemostatic balance, platelet interaction with immune cells, and support of the coagulation cascade leading to occlusion of the vessel lumen. Due to their prominent role, platelet receptors constitute a major target in pharmacological treatment. Our work aimed to identify the molecular alteration of platelet surface receptors, which showed augmented mRNA expression of P2Y12, GP1BB, ITGA2B, and ITGB3 and increased protein concentrations of P2Y12 and GP IIb/IIIa in ACS. The upregulation of the P2Y12 level was also confirmed by confocal and cytometric visualization. Furthermore, we evaluated the expression of two microRNAs: miR-223-3p and miR-126-3p, which were suggested to regulate platelet P2Y12 expression. Results of our study present new insight into the molecular background of ACS

Screening Analysis of Platelet miRNA Profile Revealed miR-142-3p as a Potential Biomarker in Modeling the Risk of Acute Coronary Syndrome

Transcriptome analysis constitutes one of the major methods of elucidation of the genetic basis underlying the pathogenesis of various diseases. The post-transcriptional regulation of gene expression is mainly provided by microRNAs. Their remarkable stability in biological fluids and their high sensitivity to disease alteration indicates their potential role as biomarkers. Given the high mortality and morbidity of cardiovascular diseases, novel predictive biomarkers are sorely needed. Our study focuses for the first time on assessing potential biomarkers of acute coronary syndrome (ACS) based on the microRNA profiles of platelets. The study showed the overexpression of eight platelet microRNAs in ACS (miR-142-3p; miR-107; miR-338-3p, miR-223-3p, miR-21-5p, miR-130b-3p, miR-301a-3p, miR-221-3p) associated with platelet reactivity and functionality. Our results show that the combined model based on miR-142-3p and aspartate transaminase reached 82% sensitivity and 88% specificity in the differentiation of the studied groups. Furthermore, the analyzed miRNAs were shown to cluster into two orthogonal groups, regulated by two different biological factors. Bioinformatic analysis demonstrated that one group of microRNAs may be associated with the physiological processes of platelets, whereas the other group may be linked to platelet–vascular environment interactions. This analysis paves the way towards a better understanding of the role of platelet microRNAs in ACS pathophysiology and better modeling of the risk of ACS