ICP27 is an HSV-1 immediate early protein required for the switch from early to late gene expression. ICP27 binds viral and cellular RNAs, and recently a yeast three-hybrid (Y3-H) analysis has identified an array of viral RNA sequences that interact with ICP27. Presented here are analyses of functional assays using a selection of the Y3-H identified RNA sequences inserted into the 5’ untranslated region (UTR) of a chloramphenicol acetyl transferase (CAT) reporter plasmid. A set of plasmids was transfected into baby hamster kidney (BHK) cells and CAT assays carried out to analyse the effects of the sequences on gene expression. Results indicated that expression was increased when ICP27-binding sequences were present even though no viral proteins were present. Comparison of sequences revealed that no common activation code or RNA structure was present that could be responsible for the increase in CAT gene expression. The levels of expression were further determined in the presence of wild type (wt), ICP27-null or ICP27 mutant HSV-1 infection to investigate whether ICP27 had any affect on CAT expression when ICP27-binding sequences were present. Interestingly, enhanced expression was observed during wt HSV-1-infection when ICP27-binding sequences were present, whereas little to no enhancement was observed during ICP27-null or mutant virus infections. However, a higher fold increase in CAT gene expression was observed during a HSV-1 infection when ICP27-binding sequences were not present. This indicated that an inhibitory effect on CAT expression observed during wt HSV-1 infection when ICP27-binding sequences were present was ICP27-depdnent. As a control, a noncoding, protein binding regulatory HPV RNA sequence was inserted into the CAT reporter plasmid, transfected into BHK cells and then infected with wt HSV-1, ICP27-null or ICP27 mutant viruses. Surprisingly, CAT expression was increased, albeit to only a limited extent, indicating that the previously observed increase in gene expression was not HSV-1 sequence specific. However, upon transfection of plasmids with the HPV control sequence inserted in the reverse orientation and a subsequent infection of cells no increase in CAT expression was observed. Analysis of the control sequence in the reverse orientation identified a shortage in G residues, which led to the construction of CAT reporter plasmids containing homopolymer sequences to inserted into the 5’UTR. A series of transfections and subsequent mock, wt HSV-1 or ICP27-null virus infections were carried out using this set of constructs. CAT assay analysis revealed an increase in CAT expression, to levels similar to those observed when the HSV-1 sequences were present, when poly(G) homopolymers were used as inserts during wt HSV-1 infection, whereas poly(A), (C) and (T) gave low levels of expression.

Ca2+ is an important messenger which mediates several physiological functions, including muscle contraction, fertilisation, heart regulation and gene transcription. One major way its cytosolic level is raised is via a G-protein coupled receptor (GPCR)- mediated release from intracellular stores. GPCR’s are the target of approximately 50% of all drugs in clinical use. Hence, understanding the underlying mechanisms of signalling in this pathway could lead to improved therapy in disease conditions associated with abnornmal Ca2+ signalling, and to the identification of new drug targets. To gain such insight, this thesis builds and analyses a detailed mathematical model of key processes leading to Ca2+ mobilisation. Ca2+ signalling is considered in the particular context of the M3 muscarinic receptor system. Guided by available data, the Ca2+ mobilisation model is assembled, first by analysing a base G-protein activation model, and subsequently extending it with downstream details. Computationally efficient designs of a global parameter sensitivity analysis method are used to identify the key controlling parameters with respect to the main features of the Ca2+ data. The underlying mechanism behind the experimentally observed, rapid, amplified Ca2+ response is shown to be a rapid rate of inositol trisphosphate (IP3) formation from Phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis. Using the same results, potential drug targets (apart fromthe GPCR) are identified, including the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and PIP2. Moreover, possible explanations for therapeutic failures were found when some parameters exerted a biphasic effect on the relative Ca2+ increase. The sensitivity analysis results are used to simplify the process of parameter estimation by a significant reduction of the parameter space of interest. An evolutionary algorithm is used to successfully fit the model to a significant portion of the Ca2+ data. Subsequent sensitivity analyses of the best-fitting parameter sets suggest that mechanistic modelling of kinase-mediated GPCR desensitisation, and SERCA dynamics may be required for a comprehensive representation of the data.

Proteins containing selenocysteine (selenoproteins) have been found to exist in organisms from all domains of life. Selenoproteins are important for many in vivo processes such as the removal of reactive oxygen containing species (ROS), redox disulfide shuffling reactions, and pro-hormone activation. Structurally and functionally analogous to cysteine, selenocysteine's lower pKa appears to be the defining chemical difference between these two amino acids. Using a single-molecule electrical recording technique, rate constants for the reaction of selenocysteine with small molecule disulfides were obtained over a pH range of 6 - 10. Analogous single molecule ~riments carried out ~ .. - using cysteine, revealed that, after correcting for the ratio of selenolate to selenol and thiolate to thiol based on the pKa of each amino acid, the nuc1eophilicity of selenocysteine was comparable to that of cysteine. The selenium atom of the selenylsulfide bond was found to be substantially more electrophilic than a sui fur atom of the analogous disulfide bond and the leaving group ability of the selenolate of selenocysteine compared to the thiolate of cysteine were found to be comparable. Another biologically relavant interaction that occurs in vivo is the reaction between selenocysteine and organoarsenic (Ill) molecules. It is known that arsenic (Ill) compounds are toxic to organisms, and that this toxicity stems from the ability to coordinate to the thiol and selenol groups of the cysteine and selenocysteine residues within proteins. The reaction of selenocysteine with an organoarsenic species was investigated at the single molecule level over the pH range 6.5 - 8.5. By carrying out an analogous reaction between cysteine and the organoarsenic (Ill) species, it was found that selenocysteine and cysteine exhibit similar reaction rates. The organoarsenic reagent could exist in a range of different protonation states in solution and it was concluded that the rate of reaction was governed by the equilibrium of the arsenic molecule, where only some of the forms were reactive towards the selenocysteine and cysteine groups.

No description available.

Heat shock proteins (Hsps) are increasingly being seen as having roles other than those of intracellular molecular chaperones, particularly with regard to their potential to act as cytokines, and to stimulate the innate immune system. Hsps have also been found to promote bone resorption and osteoclast formation in vitro, although the mechanism has not been previously identified. The overall aims of this thesis were to determine whether Hsps could stimulate bone resorption by affecting the RANKL/OPG pathway, and to address the hypothesis that Hsps can act as a danger signal to the innate immune system. In order for Hsps to affect either the RANKL/OPG system of bone resorption or act as danger signals they would need to be actively released from cells, ideally in a controlled manner following exposure to the source of stress. Hsp60 and Hsp70 were found to be released from a range of immune cells including the cell lines Jurkat and U937, and also PBMCs, T-cells and B-cells. This release was not due to cell damage. The release of Hsp60 and Hsp70 were downregulated by inhibitors of protein secretion, in particular Hsp70 release was reduced by compounds that inhibited lysosomal pathways and Hsp60 release by classical secretion inhibitors. Hsp60, Hsp70, GroEL and LPS all affected the RANKL/OPG system of bone regulation; OPG production and release was down-regulated in the MG63 and GCT osteoblast-like cell lines following treatment with Hsp60, Hsp70 and LPS, and RANKL expression was upregulated following treatment with Hsp60, Hsp70, GroEL and LPS. This effect on the RANKL/OPG system was found to translate into an effect on osteoclast formation when conditioned media from treated osteoblasts was added to osteoclast precursors in the presence of M-CSF. A range of different factors that affected Hsp release were identified; PHA activation of PBMCs was found to upregulate Hsp60 release from PBMCs. GroEL and LPS caused an upregulation in Hsp70 release from PBMCs and GCT osteoblast like cells, and Hsp70 was found to stimulate Hsp60 release from PBMCs and GCT cells. These responses of Hsp release were used to form a theory of a cascade-like danger signal that may occur when cells are exposed to bacterial infection and which would result in activation of antigen presenting cells via previously identified receptors for Hsps such as CD14/TLR4 or by unidentified pathways. The elevated release of Hsps in response to GroEL and LPS was also identified as a mechanism that could stimulate bone loss during infection or autoimmuniry by affecting the RANKL/OPG system. hi conclusion, Hsp60 and Hsp70 can be released from immune cells under normal conditions, and from both immune and osteoblast-like cells following stimulation with LPS and other Hsps. The observed release responses provide a mechanism through which Hsps can act as danger signals to the innate immune system, and also as promoters of bone resorption via the RANKL/OPG system.

O Coup¬TF, αποτελεί ορφανό μέλος της υπεροικογένειας των υποδοχέων των στεροειδών/θυρεοειδών ορμονών και κατέχει κυρίαρχο ρόλο στην ανάπτυξη των εμβρύων όλων των μεταζώων. Στην παρούσα Διατριβή μελετήθηκε η cis¬ ρυθμιστική περιοχή του γονιδίου του, με σκοπό την ένταξή του στο γονιδιακό ρυθμιστικό δίκτυο του εμβρύου του αχινού. Με πειράματα in situ υβριδοποίησης βρέθηκε ότι το γονίδιο PlCoup¬TF εκφράζεται στο στοματικό εξώδερμα του γαστριδίου και στη βλεφαριδωτή ζώνη στον πλουτέα, στο είδος Paracentrotus lividus. Από παλαιότερα πειράματα είχε βρεθεί ότι το τμήμα της ανοδικής περιοχής που εκτείνεται από το -232 ως το ¬532 (τμήμα a), είναι απαραίτητο και επαρκές για την έκφραση του γονιδίου αναφοράς (gfp) στη βλεφαριδωτή ζώνη του πλουτέα. Εντός της περιοχής a ανευρέθησαν τρία πιθανά ρυθμιστικά στοιχεία (¬ 453, ¬432 και ¬377) του γονιδίου PlCoup¬TF, τα οποία αναγνωρίζονται από πρωτεΐνες εμβρυικού πυρηνικού εκχυλίσματος. Στοχευμένες μεταλλάξεις των στοιχείων αυτών, οδήγησαν σε μείωση της έκφρασης του γονιδίου αναφοράς (στοιχείο ¬453) και στην εκτοπική έκφρασή του (στοιχεία ¬432 και ¬377). Περαιτέρω μελέτη των παραγόντων που αναγνωρίζουν τα στοιχεία αυτά, οδήγησε στο συμπέρασμα ότι ο μεταγραφικός παράγοντας PlElk αναγνωρίζει το στοιχείο ¬ 453 και ρυθμίζει θετικά το γονιδίο του PlCoup¬TF και ο μεταγραφικός παράγοντας PlOtx αναγνωρίζει το στοιχείο -377 και καταστέλλει την έκφραση του PlCoup¬TF στο αντιστοματικό εξώδερμα. Τα αποτελέσματα της παρούσης εργασίας οδήγησαν στην ένταξη του γονιδίου PlCoup¬TF και των δύο ρυθμιστών του στο γονιδιακό ρυθμιστικό δίκτυο που καθορίζει τη διαφοροποίηση της βλεφαριδωτής ζώνης εντός του εμβρυικού εξωδέρματος. / Coup­TF, an orphan member of the nuclear receptor super family, has a fundamental role in the development of metazoan embryos. The study of the gene's regulatory circuit in the sea urchin embryo will facilitate the placement of this transcription factor in the well­studied embryonic Gene Regulatory Network (GRN). The Paracentrotus lividus Coup­TF gene (PlCoup­TF) is expressed throughout embryonic development preferentially in the oral ectoderm of the gastrula and the ciliary band of the pluteus stage. Two overlapping λ genomic clones, containing three exons and upstream sequences of PlCoup­TF, were isolated from a genomic library. The transcription initiation site was determined and 5′ deletions and individual segments of a 1930 bp upstream region were placed ahead of a GFP reporter cassette and injected into fertilized P.lividus eggs. Module a (−532 to −232), was necessary and sufficient to confer ciliary band expression to the reporter. Comparison of P.lividus and Strongylocentrotus purpuratusupstream Coup­TF sequences, revealed considerable conservation, but none within module a. 5′ and internal deletions into module a, defined a smaller region that confers ciliary band specific expression. Putative regulatory cis­acting elements (RE1, RE2 and RE3) within module a, were specifically bound by proteins in sea urchin embryonic nuclear extracts. Site­specific mutagenesis of these elements resulted in loss of reporter activity (RE1) or ectopic expression (RE2, RE3). It is proposed that sea urchin transcription factors, which bind these three regulatory sites, are necessary for spatial and quantitative regulation of the PlCoup­TF gene at pluteus stage sea urchin embryos. Additional experiments led us to the conclusion that transcription factor PlElk binds to the ­453 regulatory element and positively regulates PlCoup­TF gene in the ciliary band. Furthermore, PlOtx binds to the ­377 regulatory element and negatively regulates PlCoup­TF gene in the aboral ectoderm. These findings lead to the hierarchical positioning of PlCoup­TF within the embryonic GRN

Η λεκτίνη ΜΒL συμμετέχει στην φυσική ανοσία, αφ΄ενός σαν ενεργοποιητής του συστήματος του συμπληρώματος και αφ΄ετέρου σαν οψωνίνη που προσδένεται σε συγκεκριμένες υδατανθρακικές δομές των μικροοργανισμών. Οι λεκτίνες τύπου C, είναι ασβέστιο εξαρτώμενες και φέρουν περιοχή δέσμευσης σε υδατάνθρακες (CRD). Προκειμενου να διασαφηνιστεί περαιτέρω η εξελικτική πορεία της λεκτινικής οδού του συμπληρώματος, απομονώθηκαν, κλωνοποιήθηκαν και χαρακτηρίστηκαν δύο ισομορφές της λεκτίνης ΜBL, οι ΜΒL1 και MBL2, στην ιριδίζουσα πέστροφα (Oncorhynchus mykiss). Οι συναγώμενες αμινοξικές αλληλουχίες των MBL1 και ΜΒL2 είναι 185 και 186 αμινοξέα, αντίστοιχα, παρουσιάζουν μεταξύ τους ταυτοσημία της τάξης του 83% ενώ εμφανίζουν το υψηλότερο σκορ ταυτοσημίας, 43% και 41% αντίστοιχα με τους τελεόστεους ιχθύες Αtlantic salmon και zebrafish. Το σκορ ταυτοσημίας των πρωτεϊνών της πέστροφας με τις αντίστοιχες πρωτεΐνες των πτηνών και των θηλαστικών κυμαίνεται μεταξύ 28 και 32%. Επιπλέον,οι περιοχές CRD των πρωτεϊνών αυτών χαρακτηρίζονται από την ύπαρξη του δομικού μοτίβου EPN που σχετίζεται με την εξειδίκευση της δέσμευσης ως προς την μαννόζη. Τα αποτελέσματα έδειξαν επίσης πως τα μόρια MBL1 και ΜBL2, εκφράζονται, σε επίπεδο mRNA, στο ήπαρ και στον σπλήνα της πέστροφας, αντίστοιχα. / Mannose-binding lectin(MBL) participates in the innate immune system as an activator of the complement system and as an opsonin after binding to certain carbohydrate structures on microorganisms. C-type lectins are all Ca2+ -dependent and they share a tightly folded carbohydrate recognition domain (CRD). In order to stydy the evolution of the complement lectin pathway, we report the isolation and characterization of two mannose-binding lectin isoforms, MBL1 and MBL2, in rainbow trout (Oncorhynchus mykiss). The deduced amino acid sequences of trout MBL1 and MBL2 are 185 and 186aa, respectively, presenting 83% identity to each other. These proteins exhibit the highest identity score 43 and 41% with the atlantic salmon and zebrafish counterparts. The identity with the bird and mammalian MBLs ranges from 28 to 32%. The trout MBL molecules contain in the CRD domain the EPN motif of mannose-binding C-type lectins, which is important for mannose specificity. Trout MBL1 and MBL2 are expressed exclusively in liver and spleen, respectively.

H νουκλεολίνη είναι μια πρωτεΐνη μοριακού βάρους 110 kDa και απαντάται στον πυρήνα, το κυτταρόπλασμα αλλά και στην επιφάνεια των κυττάρων, ενώ υπερεκφράζεται σε καρκινικά κύτταρα και σε ενεργά ενδοθηλιακά κύτταρα. Πρόσφατες έρευνες υποδεικνύουν την συμμετοχή της στις διαδικασίες ανάπτυξης καρκινικών όγκων και στην αγγειογένεση. Στην επιφάνεια του κυττάρου δρα ως υποδοχέας χαμηλής συγγένειας αυξητικών παραγόντων, μορίων κυτταρικής προσκόλλησης (ιντεγκρίνες και σελεκτίνες) λιποπρωτεϊνών και ορισμένων ιών (HIV-1 και coxsackie B). Παρουσιάζει πλήθος λειτουργιών, αλληλεπιδρώντας τόσο με πρωτεΐνες, όσο και με νουκλεϊνικά οξέα. Το HB-19 και το Ν6L είναι συνθετικά πεπτίδια που προσδένονται στην νουκλεολίνη της κυτταρικής επιφάνειας και καταστέλλουν τόσο την ανάπτυξη καρκίνου όσο και την αγγειογένεση. Στη συγκεκριμένη εργασία μελετήσαμε την βιολογική δράση των πεπτιδίων αυτών και βρέθηκε ότι αναστέλλουν τον in vitro πολλαπλασιασμό και την in vitro προσκόλληση των κυττάρων HUVEC. Τα πεπτίδια αυτά παρουσιάζουν in vivo ογκοκατασταλτική δράση και βρέθηκε να αναστέλλουν την αγγειογένεση στο σύστημα της χοριοαλλαντοϊδικής μεμβράνης εμβρύου όρνιθας. Επίσης αναστέλλουν τη μετανάστευση των κυττάρων HUVEC στο in vitro σύστημα μελέτης της μετανάστευσης Boyden chamber. Το πεπτίδιο HB-19 αναστέλλει την έκφραση διαφόρων αγγειογενετικών αυξητικών παραγόντων όπως ο FGF, VEGF και των υποδοχέων τους σε επίπεδο mRNA. Το HB-19, όπως και το Ν6L επηρεάζουν την έκφραση της μεταλλοπρωτεϊνάσης MMP-2 τόσο σε επίπεδο πρωτεΐνης όσο και σε επίπεδο έκφρασης γονιδίου. Επιπλέον, κανένα από τα δύο πεπτίδια δεν προκαλεί απόπτωση in vitro στα κύτταρα HUVEC. Τα πεπτίδια HB-19 και Ν6L δεσμεύονται στη νουκλεολίνη της επιφάνειας του κυττάρου και φαίνεται ότι για την άσκηση των βιολογικών τους δράσεων διαμεσολαβούν οι κινάσες SRC, ERK1/2, AKT και FAK καθώς αναστέλλουν την ενεργοποίησή τους σε κύτταρα HUVEC. Τέλος, μείωση της έκφρασης της νουκλεολίνης με χρήση siRNA επιβεβαίωσε τον ρόλο της νουκλεολίνης στην βιολογική δράση των πεπτιδίων HB-19 και N6L. Συνοψίζοντας τα παραπάνω αποτελέσματα, φαίνεται ότι τόσο το HB-19 όσο και το Ν6L εμφανίζουν βιολογικές δράσεις που τα καθιστούν υποψήφια μόρια για αντινεοπλασματική χρήση. / Nucleolin is a 110 kDa protein, located in the nucleus and the cytoplasm while it is over expressed on the surface of tumor and endothelial cells. Recent studies have demonstrated the involvement of nucleolin in tumor growth and angiogenesis. Thus, this cell surface molecule serves as a receptor for various ligands implicated in pathophysiological processes such as growth factors, cell adhesion molecules (integrins and selectins), laminin-1, lipoproteins and viruses (HIV and coxsackie B). HB-19 and N6L are synthetic peptides that bind cell surface expressed nucleolin and inhibit both tumor growth and angiogenesis. In the present work, we investigated the biological actions of peptides HB-19 and N6L. Our results show that both peptides inhibit the in vivo angiogenesis on the chicken embryo CAM assay and inhibit the in vitro adhesion, proliferation, migration and motility of HUVEC cells. HB-19 peptide inhibits the expression of growth factors FGF and VEGF mRNA as well as the expression of their receptors FGFR1 and FLT-1 respectively. We also found that both enzyme activity and expression of MMP-2 was inhibited by HB-19 and N6L. Furthermore, we found that HB-19 and N6L treatment shows no toxicity in HUVEC cells in vitro. The above biological actions seem to be regulated by SRC, ERK1/2, AKT and FAK kinases as we found that HB-19, as well as N6L, inhibit their activation in HUVEC cells. Finally, down regulation of nucleolin using siRNA confirmed the implication of nucleolin in the biological actions of these peptides. Taken all the above results into account, it is indicated that HB-19 and N6L could constitute an interesting tool for tumor therapy strategy targeting nucleolin.

La ténascine-X (TNX) est une glycoprotéine de la matrice extracellulaire. Son expression est fortement réprimée dans de nombreux cancers et l’invasion tumorale est accrue chez des souris TNX-/-. La TNX apparaît donc comme un répresseur potentiel du développement des tumeurs. L’objectif de notre travail est d’étudier cet effet présumé et d’en comprendre les mécanismes, en analysant in vitro le rôle de la TNX sur la croissance et la migration de cellules de fibrosarcome HT-1080 dans des modèles de culture bi- et surtout tridimensionnels, plus représentatifs de l’environnement cellulaire in vivo. Nos résultats montrent que la TNX inhibe la croissance des cellules tumorales, sans induire de mort apoptotique ou nécrotique. Des observations par microscopie confocale ont montré que la présence de TNX réduit l’étalement des cellules ainsi que leur efficacité de migration. Nous avons pu mettre en évidence que la TNX provoque un ralentissement de la migration des cellules tumorales ainsi qu’une diminution de la directionnalité de leurs trajectoires. L’observation de la protéolyse du collagène de type I par les cellules en migration montre qu’elle est inhibée en présence de TNX. Par ailleurs, la TNX réduit l’expression et l’activation des MMP 2, MMP-9, et MT1 MMP. Certaines voies de signalisation associées ont été étudiées : la TNX inhibe la phosphorylation de FAK sur sa tyrosine 397, ainsi que l’activation des GTPases RhoA et Rac, sans affecter celle de Cdc42. Par une régulation fine de ces molécules, qui sont impliquées dans le contrôle de la croissance et de la migration cellulaire, la TNX se caractérise comme un inhibiteur extracellulaire de l’invasion tumorale / Tenascin-X (TNX) is involved not only in the organisation of the extracellular matrix architecture but also in the regulation of cell behaviour. This matrix glycoprotein is down-regulated in many tumor types, while tumor invasion is promoted in TNX-deficient mice. In order to decipher the mechanisms by which TNX modulates tumor cell growth and migration, we compared the behaviour of HT1080 fibrosarcoma cells in conventionnal 2D culture model or embedded in 3D collagen gels, both containing or not recombinant TNX. Some experimentations have permit us to demonstrate that TNX inhibits tumor cells growth, without inducing apoptotic or necrotic cell death. Laser confocal microscopy observations demonstrated that the presence of TNX reduces cell spreading and migration efficency. Moreover, video time-lapse analysis showed that TNX reduces both velocity and directionnality of cell migration. This result is partly due to a decrease of pericellular proteolysis, as observed in situ using FITC-collagen-containing gels. Besides, we showed that TNX led to a decrease of MMP 2, MMP-9, MT1 MMP expression and activity. Then, we determined that both FAK phosphorylation on tyrosine 397 and activation of Rac1/2/3 and RhoA small GTPases were inhibited in TNX conditions. An inactivation of these small GTPases of the Rho family is known to deregulate cell cycle and highly decrease tumor cell spreading and migration efficiency in 3D environment. Taken together, these results indicate that TNX is an extracellular inhibitor of cell invasion, which acts by downregulating the main signalling pathways responsible for cell growth and motility in 3D-collagen gels

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