Identification and functional characterization of proteins involved in hepatic triglyceride metabolism

Triglycerides are the main form of energy in the tissues and liver, along with the adipose tissue, is the main organ of triglyceride metabolism and storage in the lipid-droplet organelles. A number of proteins are involved in the regulation of the triglyceride metabolism in human liver, however their specific role is still not thoroughly known. The aim of this thesis is to evaluate the functional role of three proteins in triglyceride regulation in an experimental model of human liver. In Paper I we identified the gene Transmembrane 6 superfamily member 2 (TM6SF2) as the putative cause for the association between the 19p12 locus with plasma triglyceride levels and non-alcoholic fatty liver disease, by employing expression studies and expression quantitative trait locus analysis in 206 human liver samples. TM6SF2 encodes a protein of 351 amino acids localized in the Endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment, as investigated in human hepatoma cells. Functional studies showed that TM6SF2 siRNA inhibition led to reduced secretion of triglyceride-rich lipoproteins (TRLs) and increased cellular triglyceride concentration and number of lipid-droplets, however the putative pathophysiological mechanism of these observations is still unclear. In Paper II we investigated the physiological functions of Patatin-like phospholipase domain containing proteins 2, 3 and 4 (PNPLA2, PNPLA3 and PNPLA4), as potential triglyceride hydrolases in Huh7 and HepG2 human hepatomas. We found that siRNA inhibition of PNPLA3 or PNPLA4 is not associated with changes in triglyceride hydrolysis, TRL secretion or cellular triglyceride accumulation. However, PNPLA2 siRNA inhibition reduced intracellular triglyceride hydrolysis and decreased TRL secretion, both in the absence or presence of oleate-containing medium or of the PNPLA2 inhibitor Atglistatin. In contrast, we found no effects of PNPLA2 inhibition on lipid-droplet homeostasis. Visualization analysis with confocal microscopy found significant co-localization of PNPLA2 with the ER, but no clear evidence for PNPLA2 localization around the lipid-droplets. This data indicates that PNPLA2 hydrolyses a triglyceride compartment comprising of very small lipid-droplets that are involved in the regulation of TRL secretion, but are not detectable by confocal microscopy. In Paper III we studied the likely role of Abhydrolase domain-containing 5 (ABHD5) as the co-activator of PNPLA2 in the regulation of hepatic triglyceride metabolism. We employed siRNA inhibition techniques in Huh7 hepatoma cells and showed that ABHD5 siRNA inhibition reduced triglyceride hydrolysis and decreased TRL secretion while there was no effect on cellular triglyceride content. These results are similar to the effects of PNPLA2 siRNA inhibition on triglyceride metabolism as examined in Paper II. We also found no additive effects of combined ABHD5-PNPLA2 siRNA inhibition in hepatic triglyceride metabolism. We employed confocal microscopy analysis and observed localization of ABHD5 in the ER, but not in Golgi or around the lipid-droplets, while a significant colocalization of ABHD5 and PNPLA2 was observed. These observations suggest that ABHD5 is a co-activator of PNPLA2 with no separate triglyceride hydrolysis activity in human hepatocytes. Overall, this Thesis identifies TM6SF2 as a membrane protein regulating the TRL secretion in Huh7 and HepG2 hepatoma cells. It also demonstrates the role of triglyceride hydrolysis in the regulation of TRL secretion where PNPLA2 is the main triglyceride hydrolase activated by ABHD5. Finally, it suggests the existence of very small lipid-droplets containing the substrate compartment of the PNPLA2- and ABHD5-mediated triglyceride hydrolysis

Interplay between inflammation and calcification in cardiovascular diseases

Cardiovascular calcification has been linked to all-cause mortality and is a broadly adopted predictor of cardiovascular (CV) events. Rather than a mere by-product of the changing disease environment, calcification impacts actively the disease progression and pathogenesis as it predominates both in early- and late-stages, through mediating tissue biomechanical destabilisation and directly impacting tissue inflammation. However, its clinical contribution to the fate of the disease remains to be elucidated. Emerging body of evidence from both basic and clinical research has demonstrated the significance of the innate immune system in cardiovascular diseases (CVDs). Here, inflammation and calcification are engaged in a vicious cycle particularly at early-stages, whereas in advanced-lesions, large calcifications linked with suppressed inflammation and plaque stability. However, this interaction during disease progression remains largely elusive. The aim of this thesis is to investigate the interplay between inflammation and calcification in advanced atherosclerosis and calcific aortic valve disease (CAVD). Study I explores gene and protein expression signatures and biological pathways of advanced CAVD lesions in order to characterise the underlining mechanisms associated with the disease pathology. Multi-omics integration of overlapping transcriptome/proteome molecules with miRNAs, identified a unique CAVD-related protein-protein 3D layered interaction network. After addition of a metabolite layer, Alzheimer's disease (AD) was identified in the core of the gene-disease network. This study suggests a novel molecular CAVD network potentially linked to amyloid-like structures formation. Study II characterises osteomodulin (OMD) in the context of atherosclerosis, chronic kidney disease (CKD) and CAVD. Plasma OMD levels were correlated with markers of inflammation and bone turnover, with the protein being present in the calcified arterial media of patients with CKD stage 5. Circulating OMD levels were also associated with cardiac valve calcification in the same patients and its positive signal was detected in calcified valve leaflets by immunohistochemistry. In patients with carotid atherosclerosis, plasma OMD levels were increased in association with plaque calcification as assessed by computed tomography. Transcriptomic and proteomic data analysis showed that OMD expression was upregulated in atherosclerotic compared to non-atherosclerotic control arteries, and particularly in highly calcified plaques, where its expression correlated positively with markers of vascular smooth muscle cells (VSMCs) and osteoblasts. In vivo, OMD was enriched in VSMCs around calcified nodules in aortic media of nephrectomised rats and in plaques from ApoE-/- mice on warfarin. In vitro experiments revealed that exogenous administration of recombinant human OMD protein repressed the calcification process of VSMCs treated with phosphate by maintaining the VSMC contractile phenotype along with enriched extracellular matrix (ECM) organisation, thereby attenuating VSMC osteoblastic transformation. Study III analyses OMD expression in human carotid plaques and particularly its link with future CV events. Transcriptomic analysis revealed that OMD levels were increased in plaques from asymptomatic patients compared to symptomatic ones, with high levels being associated with fewer CV events in a follow-up analysis. Study IV investigates the link between mast cell (MC) activation and key features of human plaque vulnerability, and the role of MC in VSMC-mediated calcification. Integrative analyses from a large biobank of human plaques showed that MC activation is inversely associated with macrocalcification and positively with morphological parameters of plaque vulnerability. Bioinformatic analyses revealed associations of MCs with NK cells and other immune cells in plaques. Mechanistic in vitro experiments showed that calcification attenuated MC activation, while both active and resting MCs induced VSMC calcification and triggered their dedifferentiation towards a pro-inflammatory- and osteochondrocyte-like phenotype. Overall, this thesis demonstrates that the underlying mechanisms of CVD related to inflammation and calcification can be comprehensively characterised by integration of largescale multi-omics datasets along with cellular and molecular assays on one side, and disease specific biomarkers and advanced diagnostic imaging tools on the other. In summary, these studies not only indicate that advanced-calcification is a stabilising factor for plaque and disease progression but also, unveil novel insights into the cardiovascular calcification pathobiology, and offer promising biomarkers and new therapeutic avenues for further exploration

Εκτίμηση ασφάλειας έναντι πυρκαγιάς σε πλοίο τύπου φρεγάτας

Εθνικό Μετσόβιο Πολυτεχνείο--Μεταπτυχιακή Εργασία. Διεπιστημονικό-Διατμηματικό Πρόγραμμα Μεταπτυχιακών Σπουδών (Δ.Π.Μ.Σ.) “Ναυτική και Θαλάσσια Τεχνολογία και Επιστήμη

An overview of the armed conflicts in Late Byzantium.Theoretical Foundations and Current Research

Η μελέτη αποτελεί μία πρώιμη σύνοψη των συμπερασμάτων που προκύπτουν από το υλικό που συγκεντρώθηκε στο πλαίσιο του ερευνητικού προγράμματος «Ευρετήριο Πολεμικών Συγκρούσεων της Ύστερης Βυζαντινής Περιόδου». Οι παλαιότεροι μελετητές της στρατιωτικής ιστορίας είχαν υιοθετήσει ένα θεωρητικό υπόβαθρο το οποίο βασιζόταν στην έννοια της «αποφασιστικής μάχης», όπως την είχαν οραματιστεί οι θεωρητικοί του πολέμου τον 19ο αι. Μετά από μία σύντομη αναδρομή στην ιστορία της έρευνας, ιδίως των τελευταίων δεκαετιών, αναλύονται ορισμένα χαρακτηριστικά παραδείγματα εκστρατειών που χρονολογούνται στην υστεροβυζαντινή περίοδο. Το συμπέρασμα το οποίο συνάγεται από την ανάλυση αυτή είναι ότι οι εκ παρατάξεως μάχες ήταν κατά πολύ σπανιότερες σε σχέση με άλλου τύπου συγκρούσεις (κυρίως πολιορκίες και επιδρομές) που στόχο είχαν να φθείρουν τον αντίπαλο και όχι να καταστρέψουν τον στρατό του

Identification of cis-acting determinants mediating the unconventional secretion of tau.

The deposition of tau aggregates throughout the brain is a pathological characteristic within a group of neurodegenerative diseases collectively termed tauopathies, which includes Alzheimer's disease. While recent findings suggest the involvement of unconventional secretory pathways driving tau into the extracellular space and mediating the propagation of the disease-associated pathology, many of the mechanistic details governing this process remain elusive. In the current study, we provide an in-depth characterization of the unconventional secretory pathway of tau and identify novel molecular determinants that are required for this process. Here, using Drosophila models of tauopathy, we correlate the hyperphosphorylation and aggregation state of tau with the disease-related neurotoxicity. These newly established systems recapitulate all the previously identified hallmarks of tau secretion, including the contribution of tau hyperphosphorylation as well as the requirement for PI(4,5)P2 triggering the direct translocation of tau. Using a series of cellular assays, we demonstrate that both the sulfated proteoglycans on the cell surface and the correct orientation of the protein at the inner plasma membrane leaflet are critical determinants of this process. Finally, we identify two cysteine residues within the microtubule binding repeat domain as novel cis-elements that are important for both unconventional secretion and trans-cellular propagation of tau

Financial Assessment of Greece's Top 10 Energy Enterprises Amid the Covid-19 Pandemic

This study conducts a comprehensive financial assessment of Greece's ten largest energy companies during the tumultuous period of 2019 to 2022, amidst the unprecedented challenges posed by the COVID-19 pandemic. Employing a meticulous analysis of crucial profitability ratios, I delve into the financial resilience and adaptability of these firms within the ever-evolving energy sector landscape. My findings reveal a diverse spectrum of financial performances during the early pandemic years (2019-2020), with notable discrepancies in net profit margins. As the world transitioned to the post-pandemic era (2021-2022), I witnessed varying degrees of adaptability, with certain companies demonstrating impressive resilience while others grappled with shifting market dynamics. This research underscores the imperative of tailored financial strategies and adept cost management practices within the energy sector to effectively weather the challenges of an uncertain world. The insights gleaned offer valuable guidance to industry stakeholders and decision-makers in navigating the complex terrain of Greece's energy landscape

Economic Factors Influencing Homicide Rates: A European Perspective

Intentional homicide rates represent a critical societal issue, impacting public safety and social stability across Europe. Understanding the socio-economic factors underlying these crimes is paramount for effective policy intervention. This research aims to investigate the socio-economic determinants of intentional homicides in 15 European countries over the period 2010-2021, providing insights into the complex relationship between economic indicators and violent crime rates. The study hypothesizes that economic prosperity, government debt, and access to financial services significantly influence intentional homicide rates, with countries exhibiting higher levels of economic development and financial inclusion experiencing lower homicide rates. Utilizing robust statistical and econometric techniques, including regression analysis and correlation matrices, the research examines the relationships between various socio-economic indicators and intentional homicide rates. Data spanning from national tax authorities, statistical agencies, and international organizations are meticulously analyzed to uncover meaningful patterns and associations. The findings reveal compelling associations between economic indicators and intentional homicide rates. Higher GDP per capita and greater financial inclusion are correlated with lower homicide rates, while elevated levels of government debt exhibit a negative association with homicide rates. These results underscore the multifaceted nature of crime dynamics and highlight the importance of considering broader socio-economic factors in understanding violent crime patterns. The study contributes to both theoretical knowledge and practical policymaking by offering insights into the socio-economic determinants of intentional homicides. These findings can inform evidence-based policy interventions aimed at promoting social stability and enhancing public safety across Europe, emphasizing the importance of addressing underlying economic factors in crime prevention strategies