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.

Thesis (MScMed (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2007. / The expansion of the Mycobacterium tuberculosis PE and PPE gene families seems to be linked to that of the immunologically-important ESAT-6 (esx) gene clusters secretion system, as the ancestral members of these families are found only within the ESAT-6 gene cluster regions. These ancestral members are also the only copies in the earlier mycobacteria like M. smegmatis. The later duplications of the PE and PPE families belonging to the PGRS and MPTR subgroups, have been implicated in virulence and are only found within the genomes of the pathogenic mycobacteria closely related to the M. tuberculosis complex. The aim of this study was to compare the subcellular localization of the later duplications of the PE and PPE gene families belonging to the PGRS and MPTR subgroups with that of the ancestral PE and PPE proteins found in M. smegmatis and to investigate whether the ESX secretion apparatus is involved in the trafficking of these proteins. The PE (Rv3872) and PPE (Rv3873) genes from M. smegmatis were PCR amplified with a C-terminal HA tag using M. smegmatis genomic DNA as template. Two PPE-MPTR genes, Rv0442c and Rv0878c, and one PE_PGRS gene, Rv2615c, were also PCR amplified with a C-terminal HA tag using M. tuberculosis genomic DNA as template. All genes were cloned into the mycobacterial expression vector p19Kpro. Expression and localization was investigated using SDS-PAGE and Western blotting. The PE and PPE genes expressed in M. smegmatis were found to be present within the cell wall, membrane, and cytosol fractions, but not in the culture filtrate, indicating no secretion. The PPE-MPTR and PE_PGRS genes expressed in M. smegmatis, were also found to be present within the cell wall, membrane and cytosol fractions, but not in the culture filtrate, indicating that they are also not secreted. We hypothesize that their secretion is dependent on ESAT-6 gene cluster region 5, which is absent from the genome of M. smegmatis. Ancestral PE and PPE proteins are secreted efficiently in M. tuberculosis. The ESAT-6 gene cluster Region 3 and Region 4 of M. smegmatis were knocked out, and these knockout mutants could be used in future studies to investigate if the ESAT-6 gene cluster region 1 is involved in the secretion of the ancestral and recent PE and PPE proteins.

Proteomic techniques were used to evaluate the protein profile of the earthworm, (Lumbricus terrestris), following a bacterial challenge. One control group received no injection; a second control group received injections of phosphate buffer solution (PBS). The experimental group received injections of PBS containing (Aeromonas hydrophila). After incubation for 12 hours at 20°C, coelomic fluid was collected from each group for analysis by 2-D electrophoresis. There were significant differences in spot appearance and density between control and experimental groups. Sixteen spots showed a two-fold increase in density and 63 showed at least a two-fold decrease in density between samples from control and bacteria-challenged earthworms, respectively, suggesting up- and down-modulation of proteins potentially involved in the earthworm's response to bacterial challenge.

Chez les vertébrés gnathostomes, le système immunitaire repose en grande partie sur les lymphocytes T qui se développent dans un organe conservé évolutivement : le thymus. Chez les mammifères, cet organe constitue une cible privilégiée pour les œstrogènes. La question soulevée ici est donc de savoir si c’est également le cas chez les poissons téléostéens. Dans ce but, la distribution des différents sous-types de récepteurs aux œstrogènes a d’abord été étudiée dans le contexte d’une description de l’anatomie fonctionnelle du microenvironnement thymique. Par la suite, l’expression de gènes relatifs à la fonction thymique et aux différents sous-types de lymphocyte T a été analysée dans le thymus, le rein-antérieur et la rate de bars exposés au 17ß-œstradiol. De plus, la capacité de flambée oxydative a été évaluée sur des leucocytes du rein-antérieur et de rate à la suite d’expositions in vivo et in vitro. Finalement, la variation du nombre de thymocytes a été examinée sur des bars capturés durant trois ans. La thèse fournit de nouvelles connaissances concernant l’évolution des fonctions immunomodulatrices des œstrogènes sur la différenciation des cellules T. En effet, en plus d’une organisation morpho-fonctionnelle fortement conservée, la distribution des sous-types de récepteurs aux œstrogènes ainsi que les effets œstrogéniques apparaissent conservés au cours de l’évolution. Nos résultats suggèrent que, chez le bar comme chez les mammifères, les œstrogènes (1) stimulent une voie alternative de maturation des lymphocytes T ayant des propriétés similaires aux cellules immunitaires innées, (2) augmentent la tolérance immunitaire et (3) régulent la plasticité du thymus. / Jawed vertebrates have developed an efficient adaptive immune system partly based on T lymphocytes. They develop in an evolutionarily conserved organ, the thymus. In mammals, endogenous oestrogens are well known to regulate thymus function and plasticity. The question is, therefore, whether this is also the case in lower vertebrates, such as teleosts. To achieve these aims, firstly the distribution of oestrogen receptor subtypes was investigated on the background of a detailed description of the functional anatomy of the thymic microenvironment. Secondly, thymic function- and T cell-related gene expression was analysed in the thymus, the head-kidney and the spleen of sea bass exposed to 17ß-oestradiol. Moreover, the oxidative burst capacity in the two latter organs was evaluated in vivo and in vitro in leucocytes of the head-kidney and spleen following exposure to oestrogen. Eventually, age- and size-dependent variations in thymocyte number were examined in sea bass caught at various time points over three years. The thesis provides new insights into the evolution of the immunomodulatory function of oestrogen with respect to the thymic and peripheral T cell differentiation in vertebrates. As a matter of fact, in addition to a highly conserved morpho-functional organisation, the distribution of oestrogen receptor subtypes as well as the oestrogenic effects appear to be evolutionarily conserved. Our results suggest that in sea bass, similar to mammals, oestrogen (1) stimulates a thymic alternative pathway of T cell maturation with innate-like properties, (2) enhances immune tolerance by promoting Treg differentiation, and (3) actively regulate thymic plasticity.