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The field of proteomics studies the structure and function of proteins in a large scale and high throughput manner. My work in the field of proteomics focuses on identifying interactions between proteins and discovering novel interactions. The identification of these interactions provides new information on metabolic and disease pathways and the working proteome of a cell. Cells are lysed and purified using antibody based affinity purification followed by digestion and identification using an HPLC coupled to a mass spectrometer. In my studies, I looked at the interaction networks of several AD related genes (Apolipoprotein E, Clusterin variant 1 and 2, Low-density lipoprotein receptor, Phosphatidylinositol binding clathrin assembly protein, Alpha-synuclein and Platelet-activating factor receptor) and an endosomal recycling pathway involved in cholesterol metabolism (Eps15 homology domain 1,2 and 4, Proprotein convertase subtilisin/kexin type 9 and Low-density lipoprotein receptor). Several novel and existing interactors were identified and these interactions were validated using co-immunopurification, which could be the basis for future research.

<p>In the case of injury to the CNS, different proteins act to repair and protect cells in the brain and spinal cord. In the present study, we looked at dorsal root injury and hypoglossal nerve avulsion and transection. Here we studied for the first time the expression of Parkin in these types of injuries. However the antibodies against Parkin used here have not been able to detect Parkin in the injuries examined, neither with fluorescence or using DAB. The roles of Mts1, GFAP, and clusterin after injury have been investigated earlier, but their co-localization in the same cells was first shown in this study in the hypoglossal nucleus with immunohisto-chemical methods. These results may also be of value in the process of finding an effective treatment for neurodegenerative disorders such as ALS.</p>

The field of proteomics studies the structure and function of proteins in a large scale and high throughput manner. My work in the field of proteomics focuses on identifying interactions between proteins and discovering novel interactions. The identification of these interactions provides new information on metabolic and disease pathways and the working proteome of a cell. Cells are lysed and purified using antibody based affinity purification followed by digestion and identification using an HPLC coupled to a mass spectrometer. In my studies, I looked at the interaction networks of several AD related genes (Apolipoprotein E, Clusterin variant 1 and 2, Low-density lipoprotein receptor, Phosphatidylinositol binding clathrin assembly protein, Alpha-synuclein and Platelet-activating factor receptor) and an endosomal recycling pathway involved in cholesterol metabolism (Eps15 homology domain 1,2 and 4, Proprotein convertase subtilisin/kexin type 9 and Low-density lipoprotein receptor). Several novel and existing interactors were identified and these interactions were validated using co-immunopurification, which could be the basis for future research.

In the case of injury to the CNS, different proteins act to repair and protect cells in the brain and spinal cord. In the present study, we looked at dorsal root injury and hypoglossal nerve avulsion and transection. Here we studied for the first time the expression of Parkin in these types of injuries. However the antibodies against Parkin used here have not been able to detect Parkin in the injuries examined, neither with fluorescence or using DAB. The roles of Mts1, GFAP, and clusterin after injury have been investigated earlier, but their co-localization in the same cells was first shown in this study in the hypoglossal nucleus with immunohisto-chemical methods. These results may also be of value in the process of finding an effective treatment for neurodegenerative disorders such as ALS.

The field of proteomics studies the structure and function of proteins in a large scale and high throughput manner. My work in the field of proteomics focuses on identifying interactions between proteins and discovering novel interactions. The identification of these interactions provides new information on metabolic and disease pathways and the working proteome of a cell. Cells are lysed and purified using antibody based affinity purification followed by digestion and identification using an HPLC coupled to a mass spectrometer. In my studies, I looked at the interaction networks of several AD related genes (Apolipoprotein E, Clusterin variant 1 and 2, Low-density lipoprotein receptor, Phosphatidylinositol binding clathrin assembly protein, Alpha-synuclein and Platelet-activating factor receptor) and an endosomal recycling pathway involved in cholesterol metabolism (Eps15 homology domain 1,2 and 4, Proprotein convertase subtilisin/kexin type 9 and Low-density lipoprotein receptor). Several novel and existing interactors were identified and these interactions were validated using co-immunopurification, which could be the basis for future research.

Une des difficultés majeures dans le traitement des cancers est l’acquisition de résistance par les cellules tumorales vis-à-vis de la mort induite par les différentes chimiothérapies. Au sein du laboratoire, nous nous intéressons aux propriétés anti-tumorales d’un donneur de monoxyde d’azote (NO), le Glycéryl TriNitrate (GTN), fréquemment utilisé dans le traitement de l’angine de poitrine. Au cours de ce travail, nous avons étudié les mécanismes moléculaires par lesquels le GTN sensibilise les cellules tumorales de plusieurs types de cancer (colique, mammaire, prostatique) à la mort impliquant des voies de signalisation régulées par des cytokines telles que le TNFα, l’IL-6 ou encore le GDF-15.Une meilleure compréhension des mécanismes sous-jacents à l’action anti-tumorale du GTN permettrait de potentialiser son utilisation comme nouvelle thérapie anti-cancéreuse.Modèle colique : le GTN, en présence de la cytokine pro-inflammatoire TNFα, sensibilise les cellules cancéreuses coliques et mammaires à l’apoptose. Du point de vue mécanistique, le GTN induit la S-nitrosylation de cIAP1, inhibant ainsi son activité ubiquitine E3 ligase. Ce qui abroge la voie de signalisation classique NF-кB de survie cellulaire activée par la voie TNFα/TNFR1 en faveur d’une voie de signalisation pro-apoptotique.Modèle mammaire : le GTN intervient au niveau de la migration cellulaire en altérant la voie de signalisation Jak2/STAT3 activée par la cytokine pro-inflammatoire IL-6, dans un modèle de cancer du sein triple négatif. En présence de dérivés du platine (carboplatine) générant de l’IL-6, le GTN freine la migration des cellules en induisant la S-nitrosylation, et probablement l’inactivation, de la kinase Jak-2, indispensable pour l’activation de la voie.Modèle prostatique : le GTN sensibilise à la mort les cellules cancéreuses prostatiques résistantes au docétaxel en modulant le taux de deux marqueurs de résistance à cette chimiothérapie : la clusterine (CLU) et le growth differentiation factor 15 (GDF-15). Au niveau moléculaire, le GTN diminue le taux de l'isoforme cytoprotectrice soluble de la CLU (sCLU) et augmente le taux de l'isoforme cytotoxique nucléaire (nCLU) dans les cellules prostatiques résistantes au docétaxel. Plus particulièrement, en présence de GTN, nous avons établi un lien entre le GDF-15 et la modulation du taux des isoformes de la CLU. / One of the main difficulties in the treatment of cancers is the acquisition of resistance by the tumor cells vis-à-vis the death induced by the different chemotherapies. In the laboratory, we are interested in the anti-tumor properties of a nitric oxide (NO) donor, Glyceryl TriNitrate (GTN), frequently used in the treatment of angina pectoris. In this work, we investigated the molecular mechanisms by which GTN sensitizes tumor cells of several types of cancer (colonic, mammary, prostate) to death involving signaling pathways regulated by cytokines such as TNFα, IL-6 or GDF-15.A better understanding of the mechanisms underlying the GTN's anti-tumor action would make it possible to use it as a new anti-cancer therapy.Colon model: GTN, in the presence of the pro-inflammatory cytokine TNFα, sensitizes colon and mammary cancer cells to apoptosis. From a mechanistic point of view, GTN induces S-nitrosylation of cIAP1, thus inhibiting its ubiquitin E3 ligase activity. This abrogates the classical NF-κB signaling pathway of TNFα / TNFR1 activated cell survival in favor of a pro-apoptotic signaling pathway.Mammary model: GTN intervenes at the level of cell migration by altering the Jak2 / STAT3 signaling pathway activated by the pro-inflammatory cytokine IL-6, in a model of triple negative breast cancer. In the presence of platinum (carboplatin) derivatives generating IL-6, GTN inhibits cell migration by inducing S-nitrosylation, and probably inactivation, of Jak-2 kinase, essential for the activation of the way.Prostate model: GTN sensitizes prostatic prostate cancer cells to death by modulating the level of two markers of resistance to this chemotherapy: clusterin (CLU) and growth differentiation factor 15 (GDF-15). At the molecular level, GTN decreases the level of the soluble cytoprotective isoform of CLU (sCLU) and increases the level of nuclear cytotoxic isoform (nCLU) in prostatic cells resistant to docetaxel. More particularly, in the presence of GTN, we have established a link between GDF-15 and the modulation of the isoform rate of the CLU.

Background: The secretory epithelial cells of the prostate gland use sophisticated vehicles named prostasomes to relay important information to sperm cells in semen. This prostasome-forming and secretory ability of the epithelial cells is also preserved in poorly differentiated prostate cancer cells. Aim: The aim of this thesis was to examine different characteristics of prostasomes, especially those derived from malignant prostate cells, linked to their potential role in diagnosis and prognostication of prostate cancer. Results: Serum samples of prostate cancer patients contained autoantibodies against seminal prostasomes in a higher concentration than did control sera. These autoantibodies were most frequently directed against 25 prostasome-associated proteins, but no one was prostate specific. Clusterin was one of the most frequently occurring prostasomal proteins. Elevated titers were however seen in both patients´ and control sera. Clusterin turned out to be a major antigen of seminal prostasomes. No prostate specific or prostate cancer specific protein was discovered upon proteomic analysis of prostasomes deriving from malignant cells of vertebral metastases of prostate cancer patients. Human chromosomal DNA was identified in both seminal prostasomes and PC-3 cell prostasomes and strong evidence existed that the DNA was localized inside the prostasomes. Four out of 13 DNA clones of seminal prostasomes featured gene sequences (31%). The corresponding figures for PC-3 cell prostasomes were 4 out of 16 clones (25%). Conclusions: Prostasomes are immunogenic and give rise to serum autoantibodies. The most frequently occurring autoantibodies were directed against 25 prostasomal proteins but none of these was exclusively prostate specific. Thirty different proteins were identified in prostate cancer metastasis-derived prostasomes but none of these proteins was prostate cancer specific. Human chromosomal DNA was identified in prostasomes of both normal and malignant cell origin.

Το σύστημα του Συμπληρώματος απαρτίζεται από μια ομάδα πρωτεϊνών του πλάσματος και κυτταρικών υποδοχέων που παίζουν σημαντικό ρόλο στην άμυνα του ξενιστή μέσω της αλληλεπίδρασής τους τόσο με συστατικά της φυσικής, όσο και της προσαρμοστικής ανοσίας. Παράλληλα, μια ομάδα από ρυθμιστικές πρωτεΐνες του πλάσματος και της κυτταρικής μεμβράνης προστατεύει τα κύτταρα του ξενιστή από την αυτόλογη δράση του Συμπληρώματος. Η σύνθεση του τελικού λυτικού συμπλόκου MAC, που οδηγεί σε κυτταρόλυση, ρυθμίζεται από τις ρυθμιστικές πρωτεΐνες : CD59, vitronectin και clusterin. Μέχρι σήμερα οι ρυθμιστικές πρωτεΐνες της λυτικής οδού του Συμλπηρώματος έχουν ελάχιστα χαρακτηριστεί σε σπονδυλωτά πέραν των θηλαστικών. Οι οστεϊχθύες αποτελούν ένα σημαντικό μοντέλο, καθώς είναι οι μόνοι οργανισμοί που εμφανίζουν ένα πλήρως αναπτυγμένο και διευρυμένο σύστημα συμπληρώματος, μέσω διπλασιασμού πολλών γονιδίων του Συμπληρώματος. Προκειμένου να μελετηθεί η παρουσία και η φυλογενετική εξέλιξη του ρυθμιστικού μορίου της clusterin, το οποίο παρεμποδίζει την πρόσδεση του συμπλόκου C5b-7 στην μεμβράνη του κυττάρου-στόχου, με αποτέλεσμα την αναστολή της κυτταρόλυσης, έγινε κλωνοποίηση και χαρακτηρισμός του γονιδίου της clusterin στην ιριδίζουσα πέστροφα (Oncorhynchus mykiss irideus). Απομονώθηκαν δύο ισομορφές του γονιδίου της clusterin, clusterin-1 (TCLU-1) και clusterin-2 (TCLU-2) από cDNA βιβλιοθήκη ήπατος πέστροφας, με χρήση ειδικών ολιγονουκλεοτιδίων. Οι προκύπτουσες αμινοξικές αλληλουχίες των ισομορφών clusterin-1 και clusterin-2 παρουσιάζουν 89% ταυτοσημία μεταξύ τους, καθώς και 40 και 38% ταυτοσημία με την clusterin του ανθρώπου, αντίστοιχα. Η μοριακή δομή των συναγομένων πρωτεϊνών αντιστοιχεί στην α΄ αλυσίδα της clusterin και ομοιάζει με εκείνη των θηλαστικών. Γονιδιωματικοί κλώνοι απομονώθηκαν και για τις δύο ισομορφές και μελετήθηκε η οργάνωση εξωνίων-εσωνίων των αντίστοιχων γονιδίων. Η οργάνωση των γονιδίων clusterin-1 και clusterin-2, με τα μέχρι στιγμής δεδομένα, δείχνει αντιστοιχία εξωνίων - εσωνίων με το γονίδιο της clusterin του ανθρώπου, στην περιοχή ομολογίας τους. Ως προς το μέγεθος, τα αντίστοιχα εξώνια φαίνονται συντηρημένα, ενώ τα εσώνια ποικίλουν, εμφανίζοντας μικρότερο μέγεθος συγκριτικά με εκείνα της clusterin του ανθρώπου. Ανάλυση κατά Southern έδειξε ότι η clusterin απαντάται σε δύο τουλάχιστον αντίγραφα στο γονιδίωμα της πέστροφας. Τα γονίδια clusterin-1 και clusterin-2 εμφανίζουν διαφορική έκφραση στο επίπεδο του mRNA σε διάφορους ιστούς πέστροφας, όπως διαπιστώθηκε με RT-PCR. Παρατηρήθηκαν υψηλά επίπεδα mRNA του γονιδίου TCLU-1 στο ήπαρ και στο σπλήνα, ενώ η έκφραση του γονιδίου TCLU-2 παρατηρήθηκε αυξημένη στην καρδιά και στο έντερο, χαμηλή στο ήπαρ και στο σπλήνα δεν ανιχνεύτηκε. Τέλος, η φυλογενετική ανάλυση των πρωτεϊνών της clusterin από διάφορους οργανισμούς εμφάνισε την TCLU-1 να ομαδοποιείται με την TCLU-2, ενώ η όλη υποομάδα προηγείται φυλογενετικά των ορθόλογων πρωτεϊνών του pufferfish και του zebrafish και αυτές των ορθόλογων των άλλων οργανισμών που αναλύθηκαν. Συμπερασματικά, το γονίδιο της clusterin ανιχνεύεται στην ιριδίζουσα πέστροφα, σε δύο τουλάχιστον ισομορφές και παρουσιάζει ομολογία με την clusterin του ανθρώπου, τόσο στο επίπεδο οργάνωσης του γονιδίου, όσο και στο επίπεδο της αμινοξικής αλληλουχίας και της προκύπτουσας δομής. Η παρουσία ισομορφών από εναλλακτικό μάτισμα που ισχύει στην clusterin του άνθρωπου δεν έγινε δυνατόν να πιστοποιηθεί. / The complement system consists of a group of plasma proteins and cell receptors that play a critical role in host defense, by interacting with components of both innate and adaptive immunity. Α group of plasma and cell membrane regulatory proteins protects the host cells from the autologous complement activation. The conformation of the lytic complex MAC, which results to the cytolysis, is regulated by the regulatory proteins: CD59, clusterin και vitronectin. Up to date, the regulatory proteins of MAC complex are not well characterized in low vertebrates. Bony fish represent an attractive model for studies on complement system, as they are, the only organisms that reveal a completely developed and extended system, via duplication and functional differentiation of many genes of the complement system. In order to elucidate the presence and the evolution of the clusterin regulatory molecule, which impedes the binding of C5b-7 complex to the membrane of the target cell, inhibiting cytolysis, we report here the cloning and characterization of the clusterin gene in rainbow trout (Oncorhynchus mykiss irideus). Two isoforms of clusterin gene, clusterin-1 (TCLU-1) and clusterin-2 (TCLU-2) were isolated from a trout liver cDNA library, by PCR using specific oligonoucleotides. The deduced amino-acid sequences of the isoforms TCLU-1 and TCLU-2 show 89% identity to each other, as well as 40 and 38% identity to human clusterin, respectively. The domain structure of the deduced proteins corresponds to the α΄ chain of clusterin and resembles to that of mammalian. Genomic clones were isolated for the two isoforms and intron-exon organization of corresponding genes was clarified. The organization of the clusterin genes TCLU-1 and TCLU-2, as the partial data have shown, is in line with the intron-exon organization of the human clusterin gene, at their homology region. Regarding the length, the corresponding exons seem to be conserved, but the introns in trout clusterin genes are shorter than human clusterin. Southern blot analysis has shown that clusterin can be found as two copies at least, in the trout genome. Also, the genes TCLU-1 and TCLU-2 reveal differential expression, at mRNA level, among several trout tissues, as it was found out by RT-PCR analysis. High levels of TCLU-1 mRNA are detected in liver and spleen. On the other hand, TCLU-2 is expressed mainly in heart and intestine, while is not detected any expression in spleen. Finally, the phylogenetic analysis of the clusterin proteins from various organisms, showed grouping of TCLU-1 and TCLU-2, while this subgroup precedes evolutionary the clusterin orthologs of pufferfish, zebrafish and the other examined organisms, afterwards. Conclusively, the clusterin gene in rainbow trout is detected as two isoforms and shows homology with human clusterin, not only at the level of gene organization, but also at the level of amino-acid sequence and domain architecture. The alternative splicing of human clusterin was not able to be certified in trout.