The Proteomics Facility at IMBB was established in 2009 thanks to a competitive grant from Capacities research funding schemes under the FP7 programme of the European Union. The idea behind ProFI is to spark off integration of mass spectrometry based proteomics and metabolomics approaches with the locally and nationally existing research tools and to establish successful experimental flows and collaborative projects. ProFI is equipped with state-of-the-art proteomic and bioinformatic tools and is operated by highly experienced staff as an excellent research and service module within IMBB. In that framework, ProFI scientists have successfully developed and integrated at IMBB research tools for bottom-up and top-down shotgun proteomic and metabolomics analysis, interaction and comparative proteomics, study of protein post-translational modifications, protein quantification, for a plethora of biological systems and questions.

p53 is the core of an evolutionary conserved master tumor suppressor pathway. The central role of p53 in cancer development reflects the importance and multitude of cellular functions regulated by p53 (1). Upon stimulation by a variety of cellular stress conditions, p53 orchestrates a wide spectrum of cellular response, with aim to prevent any critical cell damage from propagating to the next cellular generation (1, 2). p53 function is largely regulated by alterations of p53 protein levels. The degradation of p53 is mainly regulated by the murine double minute 2 product (MDM2) acting as a p53-specific E3 ubiquitin ligase, targeting p53 protein for proteosome-mediated degradation. Lymphomas represent a frequent type of cancer that harbor in their great majority wt-p53 (6). To investigate the incompletely understood signaling pathways involved in lymphomatogenesis, we activated p53 pathway using the small-molecule and MDM2-antagonist, Nutlin-3a (N3a), that has been shown to induce apoptotic cell death in various types of tumor cells harboring wt-p53 (including lymphoma), inducing minimal and, mostly, reversible effects on normal cells, verifying the principle that sustained inactivation of the master tumor suppressor gene p53 is essential for perpetuating the cancer phenotype (4, 5). Subsequently, we applied an integrated high-throughput comparative transcriptomics, proteomics and phosphoproteomics approach in Hodgkin and Non-Hodgkin lymphomas before and after N3a-induced p53 activation. Overall, our study reveals the utility of hypothesis-free systems biology approach in characterizing signaling networks that may provide insights into pathogenesis mechanisms in different lymphomas and suggest novel targeted diagnostic, prognostic and therapeutic strategies.

Protein-energy wasting (PEW) is the clinical condition describing the depletion of protein and energy stores, a common feature in patients with advanced stage of chronic kidney disease (CKD stage 5/end-stage renal disease, ESRD). PEW is associated with adverse clinical outcome, particularly in patients undergoing renal replacement therapy [1]. Even though, several studies have been conducted investigating urinary proteins of PEW in ESRD-patients, in search of potential biomarkers [2-4], blood remains the predominant sample-type in diagnostic analyses in clinical practice. Recent advances in proteomic technology have allowed the in-depth identification of >1000 proteins in blood plasma [5]. Deciphering the concentration and modifications of these proteins in the serum may directly correlate with pathological changes in the ESRD-related PEW patients, assisting in the discovery of potential blood biomarkers for the prevention/diagnosis/progression and treatment of the disease. To gain better insight into the complex mechanisms underlying loss of muscle protein and body fat in ESRD-patients, we employed a comparative high-throughput MS-based plasma proteome profiling of various patients receiving hemodialysis. Following a set of critical steps for the handling and processing of the patients' blood samples, a shotgun “bottom-up” proteomics approach was applied, involving in-solution protein tryptic digestion coupled on line with high resolution, accuracy and sensitivity nLC-MS/MS analysis for protein identification and relative quantitation. Further bioinformatics analysis was employed to translate our complex into biological knowledge. Our first results successfully demonstrate that utilizing an MS-based quantitative plasma proteomics approach for the relative quantification of protein blood biomarkers is very informative. Future analyses will include the integration of MS-data with telomere-associated details and information deriving from commonly used clinical methods on the nutritional status of patients with ESRD, potentiating a linkage between particular risk factor protein abnormalities, nutritional status and tailored therapeutic interventions in ESRD-related PEW patients.

Lymphomas constitute  a haematological disorder of the cells of lymphatic system, well known for their high diversity in both clinical and biological terms. At molecular level, inactivated  wild type (wt) p53 protein by its main negative overexpressed regulator, MDM2 is considered one of the major characteristic of this blood malignancy. This  deregulation, affects directly the p53-dependent signaling pathways, like DNA repair, cellular senescence, resulting in an uncontrolled proliferation [2].Nutlin-3a (N3a),an MDM2 antagonist which has recently entered clinical trials, has proven to efficiently re-activate non-functional wtp53 in lymphomas, exhibited by cell cycle arrest and apoptosis[3,4]. The aim of this work is to  understand lymphomas’ pathobiology and progression  under the prism of a systemic approach, targeting the identification of the key mal-functioning p53-centered network of interactions. Geared towards this goal, microarray transcriptomic and mass-spectrometry-based proteomic analyses on three model lymphoma cell lines (ALCL, HL, MCL) treated with N3a were integrated and interrogated using specific network inference methods and algorithms [5]. Development and application of two distinct approaches (Hypothesis Free and Targeted) led us to the identification of novel protein-protein interactions, delineating specific and overlapping molecular signatures among the different lymphoma subtypes. Using Cytoscape and its available  tools possible correlation between differentially regulated transcript-protein and specific lymphoma phenotypes was revealed. To conclude, our systems biology analysis investigated the regulatory pathways and protein interaction networks playing crucial role in cellular processes of lymphoma pathophysiology. Such findings may shed light into the identification of unique and common molecular characteristics both between different lymphoma entities as well as in other diseases, leading future research to new drug development and/or drug repurposing.

The radiomimetic drug zeocin is widely used as DNA damaging agent, antitumor drug and antibiotic due to its wide range of toxicity. Specifically its genotoxicity is caused, mostly, by the reduction and removal of the chelated Cu2+ when the drug enters the cells. Our research group revealed that, zeocin affects distinct biological processes in the cells in addition to inducing DNA damage response.
Using a variety of molecular and biochemical approaches, we have evidence for a functional interference of the drug with the copper-modulated transcription factor Mac1, inhibiting its DNA binding ability. This protein-drug interaction results in the deregulation of copper/iron homeostasis in yeast.
Exploring the transcriptional profile of S. cerevisiae in the presence of the anticancer drug using RNA sequencing technology, it was verified that the most transcriptionally repressed genes were Mac1-regulated. Furthermore, analysis of all downregulated genes, suggested functional interference of zeocin not only with Mac1 but also with other zinc-regulated transcription factors. In addition, zeocin specifically resulted in RNA reduction of ribosome biogenesis and assembly genes while upregulated genes were those mostly involved in mitochondrial functions such as energy production.
The role of pharmacological substances is to modify (inhibit or restore) the structure and functional response of proteins associated with a disease or other dysfunctions of an organism. However, most used drugs have multiple effects deviating from their original aim. Our system provides an opportunity to study such cellular responses unique to zeocin.

Endothelial dysfunction is implicated in the most life-threatening diseases, such as cardiovascular, inflammatory diseases and cancer-angiogenesis. However, the exact mechanisms that govern the properties of healthy and dysfunctional endothelium are only poorly understood. Critical endothelial bioactive molecules that are essential to haemostasis, inflammation, vascular tonicity and angiogenesis are stored in Weibel Palade bodies (WPBs), which are are specialized secretory organelles. The main constituent protein of WPBs is the haemostatic protein von Willebrand Factor (VWF). Upon activation of endothelial cells, WPBs fuse with plasma membrane and release their cargo molecules in the blood stream. To date, only a part of the WPBs-released proteins have been identified.
To understand better the mechanisms responsible for the diversity of functions of endothelial cells, we employed unbiased proteomic analysis of WPBs. Large-scale cultures of primary human umbilical vein endothelial cells were stimulated with VEGF/FGF/ATP, to trigger WPB exocytosis, and the secreted material was collected and concentrated. The complete secretome profile was analysed by high resolution mass spectrometry coupled on line with a nano-liquid chromatography system (nLC-MS/MS). Bioinformatic analysis of the MS data led to the identification of 9 proteins that have not been linked until now with secretion of WPBs. Among these proteins, galectin 1 (Gal-1) was the most intriguing, since it is implicated in cancer, cell adhesion, metastasis and immunoregulation. Interestingly, Gal-1 is a cytoplasmic protein that is secreted despite the lack of a typical secretory signal sequence. Thus, until now the mechanism of secretion of this protein remains enigmatic. Here, we verified that Gal-1 is targeted to secretory WPBs, which suggests that exocytosis of WPBs contributes to the extracellular release of Gal-1. Yet, the mechanism that directs Gal-1 to WPBs remains puzzling. Our working hypothesis is that "lingering-kiss" or “kiss and run” exocytosis is a possible way of entrance of Gal-1 into WPBs. Taken together, we show here that Gal-1 is novel cargo molecule of WBPs and propose a new mechanism that may allow cytoplasmic proteins to enter into exocytic organelles.

The inner wall of blood vessels is covered by a cell monolayer that consists of endothelial cells, which play key role in blood vessel homeostasis. Dysfunction of these cells is implicated in the most life-threatening diseases, such as cardiovascular disorders and angiogenesis in cancer. Critical molecules in the above pathophysiological processes are stored in secretory organelles, called Weibel Palade bodies (WPBs), in endothelial cells. To accomplish their function, WPBs undergo maturation, communication with other intracellular organelles, transport and fusion with the plasma membrane. Key players to these processes are members of the Rab GTPase family. Rabs undergo continuous cycles of cytoplasm-to-membrane recruitment and release to the cytosol, which are coupled to GDP-to-GTP nucleotide exchange and GTP hydrolysis, respectively. In a previous study we found that, among the 60 members of the human genome, there are 5 Rabs (Rab3, 15, 27, 33 and 3) that are specifically localised at the limiting membrane of WPBs. These Rabs are therefore called WPB-Rabs.
To get insights into the role of the WPB-Rabs in WPB dynamics, here we studied the spatiotemporal organization of these Rabs. More specifically, we studied the order and timing of their recruitment to the membrane of WPBs, from the time that WPBs are formed at the Golgi, until they mature and fuse with the plasma membrane. We found that the above 5 Rabs are recruited to the membrane of WPBs at about the same time, i.e. 1 hour after these organelles are generated. Moreover, FRAP experiments revealed that the kinetics of recycling between cytoplasm and membrane of the 5 WPB-Rabs are Rab3a>Rab15=Rab33>Rab27a=Rab37, which is suggestive to the relative rate of GTP hydrolysis and exchange or/and to the stability of the complexes formed by these Rabs at the membrane. Finally, ongoing experiments using siRNAs against the individual Rabs, suggest that Rab27 regulates the timing of the recruitment to the membrane of Rab37 and Rab3a.
All in all, the present study provides the first insights into the dynamics of individual Rabs on WPBs. Further ongoing experiments will shed light into the mechanisms orchestrating the recruitment and activation of the 5 WPB-Rabs and their impact on WPB maturation, communication with other organelles and exocytosis.

Mural cells (MCs), such as the pericytes (PCs) that reside at the capillary walls and the smooth muscle cells (SMCs) that exist in larger arteries are essential components of blood vessels. They interact with endothelial cells (ECs), which form the inner lining of the vessel wall, and are essential for vascular maturation and stability. Lack of MCs leads to embryonic lethality, whereas alterations in MC density and phenotype is associated with several human diseases such as diabetic retinopathy and atherosclerosis. However, the molecular mechanisms underlying their phenotypic plasticity and their interaction with ECs have not been studied extensively, partially due to limitations concerning their isolation and expansion.
We developed a simple, efficient and quick method, using feeder free and low serum conditions to induce the differentiation of human embryonic stem cells (hESCs) and human induced Pluripotent Stem Cells (hiPSCs) to defined MC populations (human pluripotent stem cell derived MCs/hPSCs-MCs). Briefly, after nine days of culturing PSCs in a specific for the expansion of adipose derived stem cell basal medium supplemented with 2,5% FBS and glutamine, cells expressed MC markers and were re-plated on gelatin coated dishes using the same medium. Cells were extensively characterised regarding their phenotype and function, which were stable for at least 8 passages. They expressed the typical surface mural cell markers CD29, CD44, CD90, CD73, CD105, CD140B, NG2 and the contractible markers aSMA, calponin, and SM22α. hPSC-MCs stabilised vascular tube formation, when co-cultured with ECs and  their phenotypic plasticity (contractile, synthetic) was elucidated using defined culture conditions.
Finally, in order to confirm their functionality, we carried out co-implantation of distinct populations of hPSCs-MCs with ECs on an in vivo matrigel assay. We implanted the co-implants as 3D spheroids, which allow complex interactions between the co-implanted cells reconstituting in a superior manner the physiological situation providing valuable information with regard to regulation of cell growth, migration, differentiation and survival being also adaptable for multiple applications in cell-based angiogenic therapeutic strategies. Each spheroid consists of 20% methycellulose and 80% of cells (1000 cells in a ratio 9 HUVEC to 1 hPSC-MCs).

The research project is focused on the downstream effects of PI3KCA mutations in colon cancer. The phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PI3KCA) has been described to be commonly mutated in various cancers, such as glioblastoma, gastric, breast, ovary, lung, and colorectal cancer (CRC) [1]. Mutations of PI3KCA have been reported in 10–20% of CRC, about 80% of mutations found in two hot spots in exon 9 and exon 20 [1]. To study the functional effects of these alterations, we used DLD1 colon cancer cell line in which the E545 mutation in helical domain of exon 9 was inactivated by gene targeting, generating cell lines harboring either the wild type (WT) or the mutated (MUT) allele [2]. This system has the advantage that one can investigate the mutation of interest in an isogenic background. Based on the observation that DLD1 MUT presented a growing advantage in suboptimal cultured conditions (0,5% serum) compared DLD1 WT cell lines [2], we carried out microarray analysis of both cell lines in these serum conditions. Microarray analysis of DLD1 WT and DLD1 MUT cell lines yielded some candidate genes which, according to the literature, may play a role in tumorigenesis and/or angiogenesis. One of the most important genes upregulated in DLD1 MUT cell line is the Chemokine (C-X-C motif) ligand 5 (CXCL5). CXCL5 is a low molecular weight chemotactic chemokine, which binds the G-protein coupled receptor CXCR2 and acts as a proangiogenic agent, an inflammatory mediator and a powerful attractant for neutrophils [3]. Indeed, we could show the high secretion of CXCL5 by DLD1 MUT cells compared to DLD1 WT. To further investigate the role of CXCL5 in our system we knocked out CXCL5 gene in DLD1 MUT cells by CRISPR/Cas9 Double nickase system and we generated two knockout clones (CXCL5 -/- CL.) We studied the CXCL5 knockout through a full characterization of our cell lines by in vitro techniques such as proliferation, migration and invasion assays using the IncuCyte ZOOM System. The first results of the analysis of wound healing experiments showed that the ability of migration and invasion through matrigel of the two knockout clones (CXCL5 -/- CL.) is reduced compare to the DLD1 MUT cells. To determine the impact of CXCL5 knockout in angiogenesis and metastasis we performed Matrigel plug angiogenesis assay and metastasis experiments in SCID mice. First results showed that matrigel plugs containing the knockout clones (CXCL5 -/- CL.) appeared smaller with lower angiogenesis.

1 Cathomas G (2014) PIK3CA in Colorectal Cancer. Front. Oncol. 4, 35.
2 Samuels Y, Diaz L a, Schmidt-Kittler O, Cummins JM, DeLong L, Cheong I, Rago C, Huso DL, Lengauer C, Kinzler KW, Vogelstein B & Velculescu VE (2005) Mutant PIK3CA promotes cell growth and invasion of human cancer cells. Cancer Cell 7, 561–573.
3 Zheng J, Zhu X & Zhang J (2014) CXCL5 knockdown expression inhibits human bladder cancer T24 cells proliferation and migration. Biochem. Biophys. Res. Commun. 446, 18–24.

Numerous epidemiological and clinical studies have shown an inverse correlation between plasma High-Density Lipoprotein (HDL) cholesterol levels and the risk of cardiovascular diseases in humans. Apolipoprotein A-I (apoA-I) is the main protein constituent of HDL and plays a key role in HDL metabolism by interacting with several enzymes and receptors. HDL has various atheroprotective functions including cholesterol efflux and maintenance of endothelial integrity as well as anti-inflammatory, anti-apoptotic and anti-oxidative functions. Recent studies suggest that those activities could be compromised as a result of the myeloperoxidase (MPO)-mediated modification of apoA-I. MPO can modify multiple residues of apoA-I through several mechanisms which involve oxidation of Met148 and chlorination of Tyr192. However, the effect of these modifications on HDL structure and functions in vivo have not been determined. In this study, we aimed to investigate the functional properties of two human apoA-I mutants bearing substitutions at amino acid residues Met148 and Tyr192 in vivo. For this purpose, we generated recombinant adenoviruses carrying either wild-type (apoA-I WT) or mutant forms of apoA-I [apoΑ-Ι(Met148Ala) and apoΑ-Ι(Tyr192Ala)] and used them to infect apoA-I knockout C57BL/6 mice. We analyzed the infected mice for human apoA-I mRNA levels in the liver and apoA-I protein in the plasma as well as for plasma lipid levels and HDL structure (size, shape and apolipoprotein content) and functions (cholesterol efflux, anti-oxidant and anti-inflammatory properties). We anticipate that the HDL from the mice expressing the two mutant apoA-I forms will be resistant to MPO-induced oxidation and as a consequence, the mice will be better protected from the development of atherosclerosis compared to mice expressing wild type apoA-I.  This study will advance our knowledge underlying the biogenesis of dysfunctional HDL in vivo and may provide potential therapeutic approaches for prevention of cardiovascular diseases.

Background: Epidemiological studies have shown an inverse correlation between the risk for Coronary Artery Disease (CAD) and the plasma levels of High Density Lipoprotein cholesterol (HDLc). HDL has numerous atheroprotective functions including the reverse cholesterol transport as well as antioxidant, antiapoptotic and anti-inflammatory properties mainly on the endothelium. Recent studies show that HDL functionality can be compromised in patients with CAD or chronic inflammatory diseases. ANGPTL4 is a secreted protein, produced in  many tissues including endothelial cells and accumulating evidence indicates a protective role of ANGPTL4 against the development of atherosclerosis.
Objective: Τhe present study aims to investigate the effect of HDL on global gene expression changes in cultured endothelial cells in order to identify novel HDL-target genes and to understand the mechanisms that are involved in this regulation.
Methods/ Results: DNA microarray analysis on Human Aortic Endothelial Cells (HAECs) after 12hr treatment with reconstituted HDL containing the apolipoprotein A-I (rHDL-AI), revealed 188 differentially expressed genes (84 upregulated and 104 downregulated, at least 2.0 fold change and ≤ 0.05 FDR ). Expression levels of genes involved in lipid metabolism, cell growth, cell death, cell movement, cell development, cell cycle and cell signaling were validated with high throughput qRT-PCR screening. ANGPTL4 was one of the genes that showed the most robust upregulation upon HDL treatment. The endothelial cell line EA.hy 926 was utilized to study the regulation of ANGPTL4 by HDL. Not only reconstituted HDL but also natural HDL, which was purified from transgenic mice expressing human apolipoprotein A-I (HDL-Tg), was able to induce ANGPTL4 mRNA levels indicating that ANGTPL4 is a novel endothelial HDL-target gene. HDL-Tg induced ANGPTL4 mRNA levels both in a dose-dependent and in a time-dependent manner. HDL-Tg could increase ANGPTL4 mRNA levels in the presence of cyclohexamide, a protein synthesis inhibitor, indicating that HDL regulates ANGPTL4 expression directly. To clarify the signaling cascades that are participating in this regulation, endothelial cells were treated with HDL-Tg in the presence or absence of known kinase inhibitors. We observed that both PI3K/AKT and p38-MAPK signaling pathways were involved in this effect, while inhibition of ERKs and JNKs had no effect. Interestingly, an inhibitor of the transcription factor forkhead box 1 (FOXO1) mimicked HDL function. Treatment of EA cells with HDL for different time points showed that HDL induces FOXO1 phosphorylation and translocation to the cytoplasm. This result indicates that FOXO1 functions as an inhibitor of ANGPTL4 gene expression, while HDL, through the activation of AKT, blocks FOXO1 activity and induces ANGPTL4 gene expression.
Conclusion: Our data reveal for the first time gene signatures that could mediate the numerous atheroprotective functions of HDL in endothelial cells and could be used as a marker of HDL functionality. We also reveal for the first time the role of the PI3K/AKT/FOXO1 signaling cascade in HDL-mediated gene changes in endothelial gene expression including the ANGPTL4 gene which could be utilized further for the development of novel HDL-based therapies for patients with CAD and other diseases that are characterized by HDL dysfunction.

Objective: The metabolic syndrome (MetS) is a cluster of clinical disorders such as dyslipidemia, diabetes and obesity which are associated with increased risk for cardiovascular disease. The etiology of MetS is poorly understood and effective therapies are urgently needed. Our main goal was to monitor global changes in the expression of hepatic genes and circulating miRNAs in ApoE3L.CETP mice that were used as a model of MetS.
Methods: Male mice were fed either a High (HFD) or a Low (LFD) Fat Diet for different time periods. Liver RNA was analyzed on Affymetrix Mouse Gene 2.0 ST arrays followed by bioinformatical analysis. Mice were monitored for body weight, blood glucose levels, insulin levels and total plasma cholesterol. Total miRNAs were isolated from serum and quantitated by RT-qPCR.
Results: Significant changes at specific clinical parameters imply progressive impairment of glucose tolerance, hyperlipidemia, hyperinsulinemia and obesity within 12 weeks of diet-induced MetS. Furthermore, the microarray analysis identified an increasing number of differentially expressed transcripts during MetS development in the liver. A comparison of the HFD vs the LFD fed mice revealed 114, 200 and 358 differentially expressed transcripts upon 4, 8 and 12 weeks feeding by applying conservative thresholds (absolute fold change 1.5 and Two-Way Anova p-value ˂ 0.05). Functional analysis of the differentially expressed genes revealed a number of biological processes, and networks related mostly to lipid metabolism, steatosis and inflammation. The top list of affected pathways includes Ppar signaling, AMPK signaling and cholesterol biosynthesis that probably mediate the observed metabolic dysregulation of the MetS. Particularly, Pparg is predicted to regulate 85 target-genes that were found to be differentially expressed through its network of interactions in response to a 12 week HFD. Clustering of the genes based on similar expression profiles across all the time points identified gene signatures associated with the progression of the MetS pathology. Finally, miRNAs previously correlated to metabolic diseases were detected in the serum of ApoE3L.CETP mice and their levels were increased in response to a 12 week HFD administration.
Conclusions: Our findings are indicative of characteristic hepatic gene and plasma miRNA signatures during the MetS development in ApoE3L.CETP mice which could be exploited further for diagnostic or therapeutic purposes.

Aim: Our aim was to investigate the role of Hepatocyte Nuclear Factor 4α (HNF-4α) in lipoprotein metabolism using mice with genetic ablation of Hnf-4α in the liver or the intestine.
Methods: Plasma and tissue samples were obtained from livHNF-4α-/- or intHNF-4α-/-mice and their littermate controls (HNF-4αF/F). Global gene expression changes were analysed using Affymetrix Mouse Gene 2.0 ST arrays and validated by Real-Time qPCRs. Plasma was used for protein and lipid measurements as well as for lipoprotein profiling. DNA-protein interactions were studied by chromatin immunoprecipitation (ChIP).
Results:  LivHNF-4α-/- mice developed liver steatosis and had significantly reduced total and HDL cholesterol levels in plasma whereas 2D-gel electrophoresis revealed a defect in the maturation of HDL. To investigate further the molecular causes of this dyslipidemia, we performed microarray analyses which revealed 1043 differentially expressed transcripts in the liver of livHNF-4α-/- versus HNF-4αF/F mice. RT-qPCR confirmed that liver-specific ablation of Hnf-4α was associated with diminished expression of all major apolipoprotein genes (Αpoa4, Αpoc3, Apoe, Apob, Apoc2 and Apom) with the exception of Apoa1 as well as with a significant decrease in the expression levels of Lcat, Ldlr and of the bile acid transporters Abcg5 and Abcg8. ChIP assays confirmed that HNF-4α directly targets the promoters of all of the above genes in vivo. Interestingly, Hnf-4α ablation in the small intestine did not affect plasma total and HDL cholesterol levels and RT-qPCR analysis showed that in the absence of HNF-4α many of its major targets were not affected possibly due to the high levels of expression of the HNF-4γ isoform which could compensate for the loss of HNF-4α.
Conclusion: Hnf-4α ablation in mouse liver causes significant changes in gene expression, hepatic steatosis and dyslipidemia. In contrast, Hnf-4α ablation in small intestine does not affect plasma lipid levels possibly due to the highly expressed Hnf-4γ isoform in this tissue.

The full extent of proline (Pro) hydroxylation has yet to be established, as it is largely unexplored in bacteria. We describe here a so far unknown Pro hydroxylation activity which occurs in active sites of polysaccharide deacetylases (PDAs) from bacterial pathogens, modifying the protein backbone at the Cα atom of a Pro residue to produce 2-hydroxyproline (2-Hyp). This process modifies with high specificity a conserved Pro, shares with the deacetylation reaction the same active site and one catalytic residue, and utilizes molecular oxygen as source for the hydroxyl group oxygen of 2-Hyp. By providing additional hydrogen-bonding capacity, the Pro→2-Hyp conversion alters the active site and enhances significantly deacetylase activity, probably by creating a more favorable environment for transition-state stabilization. Our results classify this process as an active-site“maturation”, which is highly atypical in being a protein backbone-modifying activity, rather than a side-chain-modifying one.

Type III Secretion Systems (T3SS) are employed by Gram‑negative bacterial pathogens to inject “effector” proteins into their host cytoplasm. The pathogens, in order to avoid wasting valuable resources for the energetically costly T3SS unless a host is present, need to tightly control the activation of the T3SS. This is achieved by the precise and complicated interplay of several T3SS-specific proteins encoded by genes usually clustered together in “pathogenicity islands”. Control is exerted on two fronts; transcription and secretion regulation. Usually, we regard these processes as distinct, facilitated by different sets of proteins. We will present the results of our recent work on economically important plant pathogenic bacteria, such as Pseudomonas syringae and Erwinia Amylovora. A direct link between these processes is revealed where the same proteins are employed in both transcription control and gatekeeping of the T3SS pore to control secretion.

The genome of B.anthracis contains ten putative polysaccharide deacetylase (PDA) genes, with high sequence homologies. This family of enzymes catalyses the removal of an acetyl group from the peptidoglycan layer that surrounds the gram-positive bacteria, rendering the cell invisible to the host’s immune system. Crystallographic studies complemented by mass-spectrometry and mutagenesis, reveal an unusual hydroxylation which targets the α-carbon of a conserved Proline, in the active site of the NodB catalytic domain.  The presence of this post-translational modification correlates with the activity of the de-N-acetylation reaction, suggesting a contribution to catalysis via a maturation step. One of the PDA’s namely BA3943, has lost its ability to perform catalysis, due to the absence of key residues. Mass spectrometry and enzymatic activity experiments on BA3943, confirmed the absence of both hydroxylation and de-N-acetylation reactions. Structure solution at 1.5A (unpublished), allowed for the careful re-construction of the active site, to the extent that it would be able to perform catalysis. Enzymatic activity experiments confirmed the resurrection of the enzyme’s activity, and masspec analysis reported an increase in the α-carbon hydroxylation level, consistent with previous results. Further experiments are being performed, to elucidate the exact mechanism by which this hydroxylation event contributes to the proposed model of catalysis. To conclude, the role of this dead enzyme inside the cell remains a mystery, with few data supporting its prominence as a sporulation agent.

Η χρήση και ο χειρισμός ποντικών για επιστημονικούς και ερευνητικούς σκοπούς αποτελεί πρακτική που έχει συμβάλει ουσιαστικά στην προαγωγή της βιοϊατρικής επιστήμης. Η μονάδα ζωικών προτύπων Βιοϊατρικής Έρευνας του ΙΜΒΒ – ΙΤΕ διαθέτει σημαντική εμπειρία στη δημιουργία γενετικά τροποποιημένων ποντικών καθώς και στην στέγαση, εκτροφή και την αναπαραγωγή τους για βασική και μεταφραστική έρευνα. Η μονάδα παρέχει υπηρεσίες υψηλών προδιαγραφών που εκτελούνται από εξειδικευμένο και έμπειρο προσωπικό σε σύγχρονες υποδομές υψηλών προδιαγραφών. Για τα επόμενα χρόνια, σκοπός είναι η περαιτέρω ενίσχυση της ήδη υπάρχουσας τεχνογνωσίας και εξειδίκευσης με έμφαση στην δημιουργία νέων μοντέλων ανθρώπινων ασθενειών με πολλαπλά οφέλη τόσο για τον άνθρωπο, το ΙΜΒΒ και στο σύνολο της Ελληνικής και Διεθνές ερευνητικής κοινότητας.

M-phase surveillance in Mitosis and Meiosis I (MI) is regulated by the Spindle Assembly Checkpoint (SAC). The SAC blocks M-phase exit, in response to spindle or genomic damage, by inhibiting the Anaphase Promoting Complex/Cyclosome (APC/C) ligase and therefore preventing Cyclin B-Cdk1 downregulation. Unlike Mitosis and MI, Metaphase II (MII) arrest occurs naturally in mammalian oocytes awaiting fertilization. MII arrest is maintained through the action of the APC/C inhibitor, Early Mitotic Inhibitor 2 (Emi2). We have found that Emi2 also participates in spindle or chromatin damage-induced MI arrest. Emi2-depleted mouse oocytes incubated with Taxol, Nocodazole or Etoposide fail to launch a MI arrest response. Therefore, the SAC is not sufficient to maintain MI arrest without the support of Emi2. Interestingly, we find that during prolonged exposure to spindle poisons, SAC components fail to maintain their localization at the kinetochores. Thus, Emi2 is the major regulator of prolonged MI arrest. By using a specific MAPK kinase inhibitor and a constitutively active form of CaMKII, we find that Emi2 action during MI arrest is enabled by MAPK and can be inhibited by CaMKII. With this work we identify a novel mechanism controlling M-phase arrest which functions through the Emi2-dependent inhibition of APC/C activation and Cyclin B – CDK1 downregulation.

Neuroblastoma (NB) is one of the most common childhood solid tumors. It originates from the embryonic neural crest and can arise anywhere along the sympathetic chain. It is a highly heterogeneous tumor, and this is reflected in the NB cell lines which are comprised of the so-called N- (neuroblastic) and S- (substrate-adherent) types. Neuronal tumors such as neuroblastoma display genetic changes including N-myc amplification which has been associated with a poor prognosis. N-myc is a transcription factor with different biological functions and a key molecule in neuroblastoma tumorigenesis. Targeting the N-myc network might be a promising strategy to treat aggressive neuroblastoma. Trim32 is a positive regulator of asymmetric cell division that has been shown to act against N-myc, and hence can be considered as a tumor-suppressor gene. Trim32 is a member of the Trim-NHL protein family with an E3 ligase activity but until now, only few targets have been identified. Importantly, distinct mutations in Trim32 have been associated with certain developmental disorders, including ciliopathies. We are investigating the role of Trim32 both in vivo (zebrafish) and in vitro (neuroblastoma cells). We have generated polyclonal antibodies in rats utilizing as antigens synthetic peptides unique to Trim32. In gain- and loss-of-function experiments we observed abnormalities in neural tube development as well as in the formation of the Kupffer’s vesicle, a transient ciliated organ. To acquire a thorough and detailed understanding of these mechanisms we have also isolated the human ortholog of the zebrafish gene, in order to analyze the influence of its overexpression both on N-myc turnover as well as on molecules regulating cilia formation and cell cycle, in N-myc amplified and N-Myc nonamplified neuroblastoma cells.

Το νευροβλάστωμα (neuroblastoma, NB) είναι ο πιο συνήθης όγκος του συμπαθητικού νευρικού συστήματος και εμφανίζεται κυρίως πριν την ηλικία των δύο ετών. Ένα από τα θεμελιώδη σηματοδοτικά μονοπάτια που έχει βρεθεί υπερενεργοποιημένο στο ΝΒ είναι το PI3K/Akt/mTOR. Στην έρευνα μας, χρησιμοποιούμε τους αναστολείς RAPAMYCIN και LY294002, για να αναστείλουμε το σύμπλοκο mTORC1 και την κινάση PI3K, αντίστοιχα. Έχουμε δείξει πως η χρήση του αναστολέα LY294002 σε νευροβλαστωματικά κύτταρα ποντικού οδηγεί σε αντιστρεπτή αναστολή του πολλαπλασιασμού τους, ενώ η χρήση της RAPAMYCIN έχει ως αποτέλεσμα μία μη αντιστρεπτή αλλαγή των ιδιοτήτων των κυττάρων. Η χρήση του αναστολέα RAPAMYCIN σε συνδυασμό με την αναστολή της p38 MAPK, μιας κινάσης σημαντικής για την ομοιόσταση των κυττάρων, μειώνει την ικανότητα πολλαπλασιασμού των κυττάρων, οδηγεί σε έξοδο από τον κυτταρικό κύκλο και επάγει τον σχηματισμό cilium. Για να κατανοήσουμε καλύτερα την αλληλεπίδραση των παραπάνω μονοπατιών στην αναδιοργάνωση του κυτταρικού σκελετού, επεκτείναμε τη μελέτη μας στην ανθρώπινη κυτταρική σειρά SK-N-BE(2)C, χαρακτηριστικά της οποίας είναι η ενίσχυση του γονιδίου Ν-myc, το μεγάλο ποσοστό καρκινικών βλαστικών κυττάρων, και το υψηλό ογκογόνο δυναμικό. Παρότι η υπερέκφραση του N-myc αποτελεί σημαντικό παράγοντα της κακοήθειας των νευροβλαστωματικών κυττάρων, οι μηχανισμοί μέσω των οποίων συμβάλλει στην επιθετικότητα του νευροβλαστώματος είναι ακόμη άγνωστοι. Παρατηρήσαμε ότι στα κύτταρα SK-N-BE(2)C η αναστολή του mTORC1 επηρεάζει επιλεκτικά την έκφραση των γονιδίων της οικογένειας μεταγραφικών παραγόντων RFX (regulatory factor X) και οδηγεί σε έντονες αλλαγές στην πολικότητα και την κινητικότητα των κυττάρων. Ένα χαρακτηριστικό των κυττάρων της νευρικής ακρολοφίας από τα οποία προέρχονται οι νευροβλαστωματικοί όγκοι είναι η ικανότητά τους να υφίστανται τη διαδικασία της Επιθήλιο-Μεσεγχυματικής Μετάπτωσης (ΕΜΜ), μια σειρά γεγονότων στη διάρκεια της οποίας ένα επιθηλιακό κύτταρο χάνει την επαφή του με τα γειτονικά, και αποκτά μεσεγχυματικά χαρακτηριστικά. Η διεργασία της EMM φαίνεται ότι επαναλαμβάνεται κατά τη διάρκεια της εξέλιξης όγκων όπως το νευροβλάστωμα, αποτελώντας ένα σημαντικό βήμα κατά την μετάσταση από την αρχική τους θέση. Στη μελέτη μας λοιπόν αυτή, στις παραπάνω συνθήκες, γίνεται μία προσπάθεια διερεύνησης των γεγονότων που χαρακτηρίζουν την ΕΜΜ,  των μορίων που ελέγχουν το φαινόμενο αυτό,  καθώς και του ρόλου των στοιχείων του μονοπατιού hedgehog.

Pyramidal neurons receive inputs in two anatomically and functional distinct domains, the apical and the basal tree. Inputs to the basal tree, due to their proximity to the soma, greatly influence neuronal output, whereas the more remote apical tree has less potential to influence somatic activity. How these inputs co-operate to form the functional output of the neurons is currently unknown. In this work we focused on how inputs to the apical and basal trees shape orientation tuning in L2/3 V1 neurons. In particular, we investigated how dendritic integration of orientation tuned inputs to the apical versus basal trees allows for the emergence of stable neuronal orientation preference. Towards this goal a model L2/3 V1 pyramidal neuron was implemented in the NEURON simulation environment. The passive and active properties of the model neuron were extensively validated against experimental data. Synaptic properties, number and distribution were also constrained according to available data. Using this model neuron we investigated a) the differences in the mean orientation preferences of the two trees and b) the distribution of orientation preferences to individual synapses that allow for the emergence of orientation tuning. Given the parameter combinations that allow for the emergence of orientation tuning, we found that neuronal orientation tuning follows in large part the orientation tuning of the basal tree. In addition, we have further identified how apical versus basal dendritic tree ablation would affect neuronal tuning in the different conditions implemented. Model results provide insights regarding the ‘tolerance’ to different input properties at the apical and basal tree in order to achieve stable orientation preference.

Malaria is an infectious disease caused by the Plasmodium parasites, which are transmitted by mosquitoes. Malaria has a huge impact on human health and economic development with about half a million lethal cases every year. Malaria is controlled by drugs targeting the parasite in the human host and insecticides to limit the exposure to the mosquito. However, drug resistance threatens progress against malaria. Consequently, methods to block transmission of the parasites through the mosquito are urgently needed. However, the development of such methods is hampered by the fact that these parasite stages are not well understood.The long term goal of our studies is to understand the biology of the parasite and so to contribute to the development of new intervention strategies targeting the mosquito stages. We use a combination of techniques such as reverse genetics together with cell and molecular biology to investigate key events during the development of the parasite within the mosquito and discover targets for malaria transmission blocking.
One of our research interests is the study of actin 2, a divergent actin and a potential target for malaria transmission blocking strategies. Our studies revealed two essential functions of actin 2 in mosquito parasite stages, where the protein is specifically expressed. Generating different actin 2 mutant parasite lines, we aim to understand the molecular function during parasite development in the mosquito vector.
We are also involved in the development of methods to directly target the parasite in the vector using nanoparticles. Preliminary data from this project will be presented.

Schizophrenia is a very complex, psychotic disease with still an unknown neurobiological etiology.  However, the leading theory of its etiopathology is the neurodevelopmental hypothesis, which supports the notion that genetic and/or toxic events during gestation disturb the normal development of the nervous system. The goal of this study was to establish the neurodevelopmental MAM model of schizophrenia in mice and study sex differences in the ‘schizotypic-like’ phenotype. This animal model is based on the neurodevelopmental hypothesis of the disease and has been established in rats. Pregnant mice were injected with the DNA methylating mitotoxin, methylazoxymethanol acetate (MAM) (26mg/kg) or saline on embryonic day 16 (MAM-E16) or 17 (MAM-E17). Male and female offspring (MAM- or saline-treated) were examined during adulthood (> 90-days old). Most experiments used MAM-E16 mice, because they had a stronger phenotype. Both male and female mice were tested in the following experiments to establish schizotypic-like characteristics: a) locomotor activity in response to systemic administration of the NMDA receptor antagonist, dizocilpine (MK-801) (0.2mg/kg), b) pre-pulse inhibition (PPI) of the acoustic startle reflex, c) analysis of gross anatomical features of the prefrontal cortex (PFC) and the hippocampus (HPC) using the Nissl staining technique, and d) parvalbumin (PV) protein expression, using fluorescent immunohistochemistry. Cognitive function was assessed with the contextual fear-conditioning paradigm and long-term potentiation (LTP) in the CA3-CA1 synapses of HPC, and in layer II of Prelimbic cortex, by recording field excitatory postsynaptic potentials (fEPSPs). Additionally, trait anxiety levels were measured using the elevated plus maze (EPM) task. Our results demonstrate that both male and female mice, prenatally treated with MAM on E16, display several core schizophrenia-like deficits, suggesting that the MAM model could be used as a neurodevelopmental model of schizophrenia in mice.  The histological adaptations are more evident in male mice, compared to female mice.  We further reveal cognitive deficits in both male and female mice treated with MAM, but only female mice exhibit comorbidity with increased trait anxiety, whereas male mice exposed to MAM show decreased trait anxiety.

The present study aims to investigate the effect of working memory training on cognitive flexibility in both humans and mice. The human study included forty healthy male participants, who were divided in: control group, partially adapted group (administration of an executive working memory task, (LNS), up to the strings with three digits, for six consecutive days) and fully adapted group (for six consecutive days participants were administered the entire LNS test). Following training, all participants were tested in another cognitive flexibility task, the ID/EDS. Results showed that the fully adapted group had improved performance on the ID/EDS test, since they made fewer errors and fewer attempts to complete the stages of the test. Using a similar experimental design, the animal study examined the effect of WMT (utilizing the delayed alternation task in the T- maze) on cognitive flexibility. Male (B6) mice (8 months of age) are divided into the naïve, the non-adaptive (mice learned to alternate arms but without any delays) and the adaptive group (mice performed the alternation procedure with increasing delays). WMT lasted for 9 days, and 2 days later, all mice undergo the Attentional Set - Shifting Task (AST), a test cognitive flexibility. Our preliminary results show that the adaptive group had a better performance at specific stages of the test (the reversal stages and the extradimentional shift).

In addition to serving fat and energy storage functions, the adipose tissue is a highly active metabolic and endocrine organ which exerts its systemic effects through secretion of adipose derived hormones (adipokines). The adipokine adiponectin affects organismal homeostasis through binding to its cognate receptors. Impairment of this signaling axis has been associated with human disease, particularly with type II diabetes and obesity. Manipulation of hormonal signaling pathways may influence lifespan by regulating survival under stress conditions. We examined the involvement of adiponectin signaling in the ER unfolded protein response (UPRER) and the regulation of lifespan in C. elegans. We find that animals with lesions in PAQR-1, one of the three adiponectin receptor homologues in C. elegans, are resistant to ER stressors. Resistance depends on the XBP-1 and PERK UPRER branches. paqr-1(tm3262) mutant animals exhibit elevated HSP-4/BiP baseline levels, and robustly induce chaperone expression and canonical UPRER upon ER stress, in a manner reminiscent of hormesis. PAQR-1 deficiency differentially affects lipid content in ER-stressed animals in an age-dependent manner. Our findings indicate that ER stress triggers lipid macroautophagy (i.e lipophagy) and that PAQR-1-mediated signaling negatively regulates the expression of the ATGL-1 lipase. The selective ability of paqr-1 mutant animals to degrade lipids through lipophagy and ATGL-1-mediated lipolysis may underlie their enhanced survival when challenged with ER stressors. Elucidation of the molecular circuitry that links adiponectin signaling with lipid turnover may contribute to the development of effective therapeutic strategies for the treatment of obesity.

Mitochondrial metabolism is crucial for the modulation and maintenance of cellular homeostasis. Mitochondrial number and functionality largely determine intracellular ATP, Ca2+ and ROS levels, which in turn regulate cellular redox state, and may initiate signaling cascades involved in cell death, ageing and disease. Various stressors affect mitochondrial function and trigger retrograde and anterograde responses that enable cells to adapt their metabolism under stress conditions. Cellular metabolic state, upon stress and during ageing, is also determined by the fraction of mRNAs undergoing translation, storage or degradation. Recent studies have shown that tight control of mRNA turnover mechanisms is needed for normal cell physiology, highlighting their global regulatory capacity. The significance of these mechanisms is further supported by their evolutionary conservation from lower to higher eukaryotes. Emerging findings indicating that mitochondrial metabolism is altered in response to cytoplasmic stress, prompted us to examine the functional crosstalk between the two pathways. We are currently investigating this interplay in vivo by combining state of the art optical imaging methodologies with the power of genetic analysis in C. elegans. We find that reduced expression of key mRNA metabolism components causes alterations in mitochondrial morphology and function. Downregulation of the decapping protein DCAP-2 perturbs mitochondrial content and network integrity. In addition, mitochondrial impairment caused by pharmacological treatment, either with paraquat or CCCP, alters the expression of mRNA metabolism factors. Our results indicate that the two mechanisms are coupled and regulate energy metabolism, stress resistance and longevity. Elucidation of this complex association is important towards understanding how metabolic alterations trigger (re)adjustments in mitochondrial physiology that ultimately influence healthspan and ageing.

Oxygen deprivation (hypoxia) is a key feature of several common and devastating disorders including stroke, ischemic heart disease and cancer. Survival under hypoxia requires activation of various hypoxia-responsive genes, involved in mitochondrial function, glucose metabolism, glycolysis, autophagy, the unfolded protein response (UPR) and apoptosis. Although the hypoxia-inducible factor-1 (HIF-1) is an essential transcription factor coordinating many of these transcriptional responses, HIF-1-independent hypoxia-responses also occur. The complex regulatory network engaged upon hypoxia, independently of HIF-1, is not fully understood. We uncovered a HIF-1-independent hypoxia response mechanism which involves the C. elegans homolog of the mammalian TRIAP1/p53CSV, T09A5.7/TRIAP-1, and mitochondrial metabolic adaptation. Notably, knock-down of TRIAP-1 promotes organismal survival under conditions of prolonged hypoxia, in the absence of HIF-1. In addition, TRIAP-1 depletion induces mitochondrial metabolic adaptation upon hypoxia. We find that TRIAP-1 is required for the proper function of key stress response pathways including autophagy, UPR and the intrinsic apoptotic pathway. Taken together, our findings indicate that TRIAP-1 plays an important role for in organismal adaptation to hypoxia that is independent of HIF-1.

Mitochondrial function is a key modulator of the ageing process. Abnormal mitochondria accumulate during ageing and their efficient elimination ensures longevity and stress response in C. elegans. The precise mechanisms through which mitochondrial biogenesis and function impinge on lifespan are currently under intensive investigation. Mitochondrial biogenesis is coordinated at distinct points; a) at the transcriptional level, through the action of specific transcription factors, b) at the organellar level, where conserved mitochondrial protein import translocases assist targeting of nuclear encoded mitochondrial proteins to their final destination within the organelle, and c) at the quality control level, where the mitochondrial pool is maintained through fusion/fission events and autophagic elimination of damaged mitochondria. We are investigating the cause-and-effect relationships between distinct mitochondrial protein import machineries and lifespan in the nematode. Specifically, we targeted channel-forming subunits of the general mitochondrial import pore (tomm-40) and the translocase of the inner mitochondrial membrane complex (timm-23) that mediate transport across the outer and inner membranes respectively. We find that RNAi depletion of tomm-40 or timm-23 alters mitochondrial morphology, lowers mitochondrial content, ATP and ROS levels, and promotes longevity. Tissue-specific knock-down of tomm-40 reveals the critical role of the intestine and body wall muscles in mediating the longevity effects. Finally, lowering mitochondrial content confers benefits in a mitochondrial disease model background.

Mitochondria are essential for energy production and have vital roles in calcium signalling and storage, metabolite synthesis and apoptosis, among others, in eukaryotic cells. Neuronal cells are particularly dependent on proper mitochondrial function. Thus, maintenance of neuronal homeostasis necessitates a tight regulation of mitochondrial biogenesis, as well as, the elimination of damaged or superfluous mitochondria. Mitochondrial impairment has been implicated in several age-related neurodegenerative diseases. Mitophagy is a selective type of autophagy mediating elimination of damaged mitochondria, and the major degradation pathway, by which cells regulate mitochondrial number in response to metabolic state. However, little is known about the effects of mitophagy deficiency in neuronal physiology. To address this question, we developed two composite, in vivo imaging approaches to monitor mitophagy in neurons. Neuronal mitophagy is induced in response to oxidative stress. Mitochondrial dysfunction leads to transportation of axonal mitochondria towards the neuronal cell body, in calcium- and an AMPK-dependent manner. Autophagy deficiency increases mitochondrial number in neurons of age-matched nematodes and abolishes mitochondrial axonal transport upon stress. Additionally, impairment of mitophagy results in enhanced cell death in C. elegans models of neurodegeneration. Our results indicate that mitophagy contributes to preserve mitochondrial homeostasis and neuronal health.

Accumulation of DNA damage is a key determinant of ageing and has been implicated in neurodegeneration. Although it is well known that ultraviolet (UV) radiation induces apoptosis, the contribution of necrotic cell death to DNA damage-related pathology remains elusive. To address this question, we developed a nematode model for DNA damage-induced neurodegeneration by using UV-C irradiation to trigger DNA damage in C. elegans neurons. Initial observations using this model show a marked increase of cytoplasmic calcium concentration upon UV irradiation. To examine whether this acute cytoplasmic calcium elevation triggers necrosis in neurons, we exposed DNA repair-defective mutants to UV light. These mutant animals are hypersensitive to UV irradiation and exhibit widespread necrotic cell death in somatic tissues upon exposure, while neurons are particularly affected. Runaway autophagy has previously been implicated in necrotic neurodegeneration. In this context, we investigated the contribution of autophagy in DNA damage-induced cellular pathology and nuclear dynamics. Notably, we found that DNA damage induces autophagic flux and alters nuclear dynamics both in nematodes and mouse cells. We are currently dissecting the interplay between DNA damage-induced autophagy, nuclear membrane alterations and necrotic cell death, aiming to identify evolutionarily conserved molecular mechanisms interfacing these processes.

The integration of sensory stimuli to appropriately modulate behavioral responses to environmental signals is critical for organismal survival. The molecular mechanisms that underlie such responses are not fully understood. Dopamine signaling is involved in several forms of behavioral plasticity. In Caenorhabtitis elegans the functionality of the dopamine and serotonin pathways can be easily assessed by monitoring specific locomotory responses to environmental food availability cues, termed basal and enhanced slowing. We found that degenerin ion channel proteins expressed in dopaminergic and other sensory neurons modulate basal and/or enhanced slowing responses. Degenerin effects are largely influenced by stress conditions, such as heat and starvation. Notably, the stress response transcription factors DAF-16/FOXO and SKN-1/Nrf couple degenerin ion channel function to environmental conditions and behavioral output.

NEET family comprises a special type of Iron Sulfur Cluster (ISC) binding proteins implicated in various human pathologies ranging from neurodegeneration to cancer and age-related diseases. Despite the well known structural properties of the mammalian NEETs, the mechanisms by which they affect longevity remain largely enigmatic.  The C.elegans gene cisd-1 (W02B12.15) encodes a functional homolog of the mammalian CISD1 (CDGSH Iron Sulfur Domain 1) and CISD2 (CDGSH Iron Sulfur Domain 2) proteins, based on sequence analysis and the presence of the conserved domain CDGSH for binding to ISCs. Like its mammalian homologs, CISD-1 is an outer mitochondrial membrane protein, ubiquitously expressed in neuronal, intestinal and muscle cells.  Downregulation of cisd-1 expression through RNA interference shortens animal lifespan, boosts mitochondrial activity and induces germline apoptosis.  Based on our results, CISD-1 exerts differential effects on ageing through its involvement in both the intrinsic apoptosis pathway and the autophagy process. By elucidating the mechanisms through which a mitochondrial protein such as CISD-1 modulates ageing, our work will provide critical insights into the functional interplay between autophagy and apoptosis and how these processes interact to affect longevity.

Posttranslational modifications have pivotal roles in cellular processes. SUMOylation, the attachment of SUMO (small ubiquitin-related modifier) to a protein, is implicated in the regulation of DNA damage response, cell division, sub-cellular protein localization and protein-protein interactions, among others. Protein SUMOylation levels increase progressively during ageing. Nevertheless, whether elevated SUMOylation is only an unrelated consequence of the ageing process or it serves a causative, regulatory role in senescent decline is not understood. The C. elegans genome contains a single gene encoding SUMO (smo-1) and the post-mitotic feature of adult animals rendering the nematode a convenient model in which to genetically dissect the role of SUMOylation in organismal physiology and ageing. The insulin/IGF-1 signaling pathway is a well-characterized and conserved pathway which has a major role in determining the lifespan of animals, mainly through the DAF-16/FOXO transcription factor and the stress response-related transcription factor SKN-1/NRF2. Interestingly, these two key transcription factors contain putative SUMOylation sites. Deletion of smo-1 causes embryonic lethality. However, we find that RNAi knockdown of smo-1 initiated at the L4 stage shortens the lifespan of both wild type and long-lived animals. Notably, knockdown of a SUMO protease gene (ulp-1), extends the lifespan of long-lived mutants (clk-1, daf-2, ife-2), but not wild type animals. The lifespan changing effect of SUMOylation is tissue-specific. In addition, we observed that manipulation of SUMOylation levels by either knockdown of smo-1 or ulp-1 influences the activity of DAF-16 and SKN-1, as well as, stress resistance, energy metabolism and mitochondrial homeostasis, in a genetic background- and age-dependent manner.

MINOTECH biotechnology is the in-house biotechnology production facility of the IMBB-FORTH and has over 30 years of experience specialized in production of bacterial-derived proteins. MINOTECH biotechnology remains the sole microbial biotechnology unit in Greece active in the production of Molecular Biology tools and is highlighted as one of the 16 international suppliers of Restriction Enzymes (REs) in the largest database on REs (REBASE, rebase.neb.com). We produce a wide array of high purity and superior quality Restriction Endonucleases, DNA Modifying Enzymes and DNA Molecular Weight Markers that aim to meet the needs of scientists engaged in Research, the Biotechnology Industry and the Clinical Laboratory. Our products are cited in more than 200 peer-reviewed publications and are supplied to customers directly or under OEM agreements through major European, Asian and North American distributors.

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The three main pillars of MINOTECH biotechnology future growth are:
1. Participation in research programs with partners from Academia or Industry.
2. Training of Young Scientists.
3. Expanding of OEM Agreements and distribution network.