T lymphocytes regulate the initiation, duration, and magnitude of adaptive immune responses and function as effector cells in cell mediated immunity. To become immunologically competent they must generate functional antigen receptors. This process takes place in the thymus and requires somatic recombination of T cell receptor (TCR) genes. It is mediated by the endonucleases recombination activating gene-1 (RAG1) and RAG2. Although the thymus regresses at puberty, T cells are present throughout life implying that other tissues must provide the proper milieu for T cell development. This thesis describes extrathymic T cell maturation in man. RAG1, RAG2, and the preTα-chain (pTα), which is exclusively utilized in developing T cells, were used as markers for TCR gene rearrangement. Two new exons (1A and 1B) encoding sequences in the 5’ untranslated region (5’UTR) of mRNA were discovered in the human RAG1 gene. The previously described 5’UTR exon (renamed 1C) was located between the new exons and exon 2, the latter containing the entire coding sequence. We found that small intestinal lymphocytes of the T cell lineage expressed the new exons in three different splice forms. RAG1 mRNA containing the 1C exon was not expressed in small intestinal lymphocytes. In contrast, splice forms containing the 1A exon were not expressed in thymocytes. RAG1 and pTα mRNA expressing lymphocytes were seen both within the epithelium and in lamina propria. Thymocyte-like CD2+CD7+CD3-, CD4+CD8+, CD1a+, and IL7-R+ lymphocytes were identified in the small intestinal mucosa. CD2+CD7+CD3- cells had the highest expression levels of mRNA for RAG1 and pTα, suggesting that the small intestinal mucosa is indeed a site for T cell maturation. Small intestinal T lymphocytes were also shown to kill via the Fas/FasL pathway in a TCR/CD3 independent manner and via the perforin/granzyme pathway in a TCR/CD3 dependent manner. The Fas/FasL-mediated cytotoxicity may reflect an ongoing selection process of extrathymically maturated T cells. The nasopharyngeal tonsil is the major inductive site for immune reactions against inhaled antigens. Previous demonstration of RAG1 expression in tonsillar B cells was interpreted as antigen driven receptor revision. The present study confirms the expression of RAG1 in B cells. We also found that RAG1, RAG2, and pTa mRNAs were expressed in lymphocytes of the T cell lineage. A small population of cells with the immature phenotype CD2+CD7+CD3- was demonstrated. This population had the highest expression levels of mRNA for RAG1, RAG2, pTα and terminal deoxynucleotidyl transferase. All four splice-forms of RAG1 mRNA were expressed. RAG1 and pTα mRNA expressing cells were mainly located in the proximity of the surface epithelium and in the outer rim of the follicles. These results suggest that the nasopharyngeal tonsil is a site where extrathymic T cell development and antigen driven TCR revision are occurring in parallel. Celiac disease (CD) is a small intestinal enteropathy characterized by permanent intolerance to gluten. Gluten reactive intestinal T cells are central in the pathogenesis and CD can be regarded as a failure to maintain tolerance to this food antigen. Expression of the RAG1 1A/2 splice form was significantly decreased in small intestinal T cell subsets of CD patients suggesting that impaired TCR gene rearrangement could contribute to failure of maintain tolerance in CD. Together, these findings show that both small intestinal and nasopharyngeal tonsillar lymphocytes of T cell lineage have the molecular machinery for antigen receptor rearrangement and that thymocyte-like lymphocytes are present in both tissues. Thus these organs are likely sites of T lymphocyte ontogeny as well as for secondary T cell receptor rearrangement in man.

In this study we tested whether the prevalence or synaptic distribution of NMDA receptor subunits would be altered in the brain of the MeCP2-null mouse model of Rett syndrome. Detergent resistant membranes (DRMs) and post-synaptic densities (PSDs) were isolated from the synaptic membranes treated with TritonX-100, and resolved by sucrose density gradient centrifugation. Immunoblot analysis of the resulting density gradient fractions revealed that the relative distribution of the different NMDA receptor subunits between the DRM fractions, soluble fractions, and insoluble postsynaptic density fractions was preserved in the MeCP2-null brain. However, analysis of the overall NMDA receptor subunit prevalence within these fractions revealed a significant decrease in the expression of the NR1 and NR2A subunits, but not the NR2B subunit, in the MeCP2-null brain. The preservation of distribution of NMDAR subunits to the synaptic membranes, together with the decrease in NR1 and NR2A prevalence, suggest an imbalance in equilibrium between the mature and the immature synapses in a mouse model of Rett syndrome.

In this study we tested whether the prevalence or synaptic distribution of NMDA receptor subunits would be altered in the brain of the MeCP2-null mouse model of Rett syndrome. Detergent resistant membranes (DRMs) and post-synaptic densities (PSDs) were isolated from the synaptic membranes treated with TritonX-100, and resolved by sucrose density gradient centrifugation. Immunoblot analysis of the resulting density gradient fractions revealed that the relative distribution of the different NMDA receptor subunits between the DRM fractions, soluble fractions, and insoluble postsynaptic density fractions was preserved in the MeCP2-null brain. However, analysis of the overall NMDA receptor subunit prevalence within these fractions revealed a significant decrease in the expression of the NR1 and NR2A subunits, but not the NR2B subunit, in the MeCP2-null brain. The preservation of distribution of NMDAR subunits to the synaptic membranes, together with the decrease in NR1 and NR2A prevalence, suggest an imbalance in equilibrium between the mature and the immature synapses in a mouse model of Rett syndrome.

Solute diffusion is critical to maintenance of cellular function and matrix integrity in articular cartilage. Nutrient deficiency due to transport limitations is thought to be one of the causes of the pathological degeneration of the cartilage tissue. Thus, a study of diffusion within cartilage will lead to a better understanding of the causes of cartilage degeneration. To accurately estimate diffusion coefficients in cartilage and other hydrated medium, we developed a finite-element based method, the Direct Diffusion Simulation Parameter Estimation method (DDSPE), to be used for quantitative determination of solute diffusivities from Fluorescence Recovery After Photobleaching data. Analyses of simulated and experimental FRAP data demonstrated that this method was more accurate than existing analytical methods, including having a low sensitivity to variations in the spot radius. Subsequently, the roles of extracellular matrix (ECM) composition and tissue orientation in solute diffusion within immature bovine cartilage were explored. Diffusivities were measured through the cartilage depth and in two different orientations (radial and transverse). Diffusivities were then correlated with ECM components. Matrix water content was found to be the best predictor of solute diffusion rates in immature cartilage. Although no specific experiments were done to measure the effect of structure, our results suggested that matrix structure did indeed modulate transport. Diffusional anisotropy, defined as the ratio of the diffusivities in both orientations, was observed to be significant in all the immature cartilage zones. As a consequence, the differences in solute diffusion between immature and mature bovine cartilage were investigated. Diffusion rates and diffusional anisotropy decreased in the mature cartilage superficial zone. The decrease in diffusivities observed in mature cartilage suggests that there may be a reduction in nutrient and growth factor supply to the cells. Nevertheless, healthy adult cartilage can still maintain its normal function even with a reduction in solute diffusion rates as nutrient diffusion distances are shorter in mature cartilage. However, any disruption in the mechanical or biological environment could cause an imbalance in tissue homeostasis, which when combined with decreased diffusivities, could trigger matrix degeneration. Thus, decreased diffusivity may be a necessary but not a sufficient prerequisite of matrix degeneration.

The date for the formation of the Benton Uplift, Ouachita orogeny, is bracketed by Carboniferous synorogenic sediments deposited to the north and Late Pennsylvanian to Early Permian isotopic dates from the weakly metamorphosed rocks within the uplift. We address the largely unknown structural history between these two constraints by presenting an improved 3-dimensional kinematic model using better constrained retrodeformable sections. These new sections are based on all surface and subsurface data, new zircon fission track dates and thermal maturation data including new ‘crystallinity’ data to constrain the maximum burial depth. Concordant zircon fission track ages range from 307 ± 18.8 Ma to 333.4 ± 38.9 Ma or from the Late Devonian to Early Permian. Maximum ‘crystallinity’ of both illite and chlorite indicate these exposed rocks experienced a temperature of ~300°C across the eastern Benton Uplift. This temperature is consistent with reconstructed burial depths using cumulative stratigraphic thickness without having to call on structural thickening. Comparing coarse and fine clay fractions, computed temperature for the fine clay fraction is less by ~100°C than that of the coarse clay fraction. This difference is the same for all formations studied. This uniform difference in temperature may indicate cooling of the orogen as it deformed or more than one thermal event.

The central role played by the Endoplasmic Reticulum (ER) in the correct folding and assembly of secretary and membrane proteins cannot be overstated. As the first compartment in the secretary pathway, it is responsible for the synthesis, modification and targeting of proteins to their proper destinations within the secretary pathway and the extracellular space. Protein folding in this specialized compartment is dynamic and involves a host of molecular chaperones and folding catalysts. Once inside the ER lumen, proteins fold into their native conformation and undergo a multitude of post-translational modifications, including N-linked glycosylation and disulfide oxidation. The proper conformational maturation of nascent proteins that traverse the secretary pathway is both aided and monitored by a complex process termed ER quality control. A variety of quality control mechanisms that rely on the chaperone systems operate in the ER. These act in close concert with the molecular machinery involved in degradation of non-native proteins to maintain homeostasis. The common goal of these mechanisms is to prevent expression and secretion of misfolded proteins. As a general rule, only those proteins that have successfully completed their folding and passed a stringent selection process are allowed to exit the ER on their way to their final destinations. The importance of the normal functioning of the ER is underlined by the fact that disruption in protein folding, resulting in ER stress, has now been identified as the biochemical basis of many ER storage diseases including Diabetes mellitus, Endocrinopathies and Hemophilia A. Processing events occurring inside the ER lumen are known to influence the efficiency of protein secretion. Vastly different rates of exocytose observed among secretary proteins have been found to correlate with the rate of exit from the ER. One such example is the interesting secretion property exhibited by Retinol Binding Protein (RBP) The principal carrier of retinol (Vitamin A) in plasma. RBP is a single domain protein consisting of three intramolecular disulfide bonds and helps transport retinol from the liver stores to the various target tissues in the body. Availability of its ligand, retinol, while not affecting its synthesis, is known to be the major factor in regulating RBP secretion from the liver. In the absence of retinol, apo-RBP has been shown to be retained in the ER by a hitherto unclear mechanism. Like most other secretary proteins, RBP is co-translationally targeted to the ER lumen, where it undergoes disulfide oxidation as the only modification. It has been shown to form a complex with another secretary protein, Transthyretin (TTR) in the ER and this complex formation is thought to prevent premature glomerular filtration of the otherwise small RBP with its bound retinol. Despite attaining a mature conformation, apo-RBP is not secreted and awaits conversion to its ligand-bound, holo form in order to exit the ER. It is widely believed that ligand binding may relieve this retention of RBP from the ER quality control machinery. However the precise mechanisms that mediate and regulate RBP folding, ligand binding, TTR assembly and secretion are not clearly understood. Though the folding and secretion properties of RBP have been described in HepG2 cells, its interactions with the ER resident chaperones have not been addressed. Apart from being an important cell biological question, the study of RBP assumes a lot of significance with its recent emergence as a key player in the pathogenesis of type 2 diabetes mellitus. It has been proposed that lowering of serum RBP levels could be a new strategy for treating type 2 diabetes mellitus. The present study was undertaken with the intention of analyzing the oxidative folding of RBP in the ER more closely. A systematic approach aimed at understanding the early events associated with folding and maturation of RBP, with particular emphasis on the role of ER-resident chaperones and the quality control machinery, is likely to provide interesting insights into the mechanisms involved in its ligand dependent secretion. Reconstitution of RBP biogenesis in a cell free system. The folding of RBP in cells is extremely quick with rapid oxidation kinetics. This makes it difficult to systematically analyze the early folding events in cultured cells. It was necessary to make use of a simplified system that would faithfully recapitulate the folding process in the ER. Therefore, a cell free translation system consisting of rabbit reticulocyte lysate and canine pancreatic microcosms as a source of ER-derived membranes was developed. This system affords the advantage of easy manipulation while still preserving the overall environment that prevails in the ER of intact cells. Extensive biochemical and functional characterization of the isolated microcosms was carried out and in vitro translation and microsomal translocation of RBP was established. Though initially confined to studies on membrane insertion and core glycosylate, the cell free system supplemented with microcosms has subsequently been used to analyze folding and assembly of a number of secretary and membrane proteins. A similar strategy has been adopted in the present study of RBP folding and maturation. Oxidative folding of RBP in isolated microcosms: Delineation of its disulfide oxidation pathway Using glutathione (GSSG) as the oxidant, co- and posttranslational disulfide oxidation of RBP was carried out in isolated microcosms. The ability to manipulate the redox status of this cell free system has helped to considerably slow down the oxidative folding of RBP so that a more careful analysis of the folding process could be performed. RBP was found to undergo oxidative folding with a t1/2 of 30 minutes and folding proceeded through at least one disulfide-bonded intermediate. Non-reducing SDS PAGE was used to resolve the folding intermediates. The pattern of oxidation was in good agreement with that reported earlier in HepG2 cells. No significant effect of retinol was observed on either the folding kinetics or the pattern of disulfide oxidation of RBP in isolated microsomes.A DTT sensitivity assay, used to probe the conformational maturity of folding RBP, revealed that RBP was capable of maturing into a DTT-resistant conformation in isolated microsomes. With the aid of disulfide mutants, the probable disulfide oxidation pathway of RBP in the ER has been determined. Single and double disulfide mutants of RBP were generated by site-directed mutagenesis and their posttranslational oxidation patterns were analyzed and compared with that of the wild type protein. Based on the results obtained, it was clear that the folding intermediate was made up of one of the two big disulfide loops and that the presence of both these loops was essential for RBP to fold into a fully oxidized, compact form. It has not been possible to determine the contribution of the third, smallest disulfide loop to the oxidative folding of RBP. Molecular events associated with the early oxidative folding of RBP To gain insights into the possible role of ER chaperones in the oxidative folding of RBP, the oligomeric state of folding RBP was analyzed by velocity sedimentation and chemical crosslinking assays. Velocity sedimentation analysis revealed that the reduced form of RBP was present in a large complex of size >100 S20,W. Upon disulfide oxidation, it readily dissociated from the complex and assumed a monomeric state. This was evident even during co-translational oxidation which suggested that RBP transiently associated with the large complex during its oxidative folding. Dynamic nature of this complex indicated that this could be a folding complex containing the chaperone machinery of the ER. These results were also supported by crosslinking analysis performed in unbroken microsomes using the homo-bifunctional crosslinker, DSP. The early folding forms of RBP could be crosslinked to a large complex while upon disulfide oxidation, RBP matured to its monomeric form and was no longer crosslinkable. Sedimentation and crosslinking analyses of the RBP disulfide mutants revealed that while the double disulfide mutant remained irreversibly associated with the large complex, the single mutants were released upon acquiring one of the two big disulfide loops. This suggested that despite the lack of one of the two major disulfides, these mutants were considered ‘folded’ by the quality control machinery in the ER while the double mutant probably resembled a molten globule state and was therefore considered ‘unfolded’ and irreversibly retained. Results from crosslinking analysis in microsomes not engaged in active translation suggested that chaperones of the ER were organized in a complex constitutively thereby lending support to the concept of ER-matrix, a large network of luminal proteins consisting of ER chaperones and accessory factors. Given this scenario, it is not unlikely that newly synthesized protein substrates transiently associate with this large pre-existing complex of chaperones and dissociate during late stages of their maturation. Conclusion In all, this study provides significant insights into some of the early events associated with the oxidative folding of RBP in the ER. The delineation of the disulfide oxidation pathway of RBP has been possible. The results obtained from this study suggest that RBP probably dissociates from the quality control quite early during its folding process and this step in its maturation might not be influenced by retinol. The stimulus for its ligant dependent secretion is likely to operate at a later stage of its sojourn in the ER, possibly consequent to positive cues from accessory binding factors such as TTR. Lastly, Perservation of the ER microenvironment in isolated microsomes, as evidenced from this study, augurs well for the use of this system to analyze mechanisms underlying folding, maturation, secretion and/or retention of secretory proteins.

El presente trabajo de tesis consistió en evaluar el efecto de la adición de Cu durante la maduración in vitro (MIV) de ovocitos bovinos y sus consecuencias en el desarrollo embrionario preimplantacional. Para tal fin, se establecieron los siguientes objetivos: 1) evaluar la asociación entre las concentraciones de Cu en licor folicular y en plasma; 2) estudiar el efecto del Cu sobre la MIV; 3) evaluar el efecto del Cu durante la MIV sobre la capacidad de desarrollo embrionario y 4) determinar el efecto del Cu en la MIV con ovocitos desnudos, con células del cúmulus en cocultivo o de COC completos. Para ello se utilizó como modelo experimental la Producción in vitro (PIV) de embriones bovino. La PIV se realizó con ovocitos obtenidos de ovarios bovinos de frigorífico. Los resultados mostraron que: 1) la concentración de Cu en el licor folicular está determinado por el estatus de Cu en el plasma; 2) el agregado de Cu al medio de MIV incrementó la concentración intracelular de GSH-GSSG en el ovocito y las CC, 3) disminuyó la tasa de apoptosis y el daño en el ADN en dichas células. Además, 4) el agregado de Cu incrementó la tasa de blastocistos y mejoró la calidad de los embriones; y 5) la tasa de blastocistos aumentó independientemente de la presencia de CC durante la MIV. En consecuencia, este estudio sugiere que el Cu actuaría como un factor importante durante la maduración del ovocito bovino favoreciendo el desarrollo embrionario temprano. Además, las evidencias obtenidas sugieren la posible existencia de transportadores de Cu en el complejo ovocito-cúmulus de bovino. / This study evaluated the effect of adding copper (Cu) during bovine in vitro maturation (IVM) on preimplantational embryo development. For this purpose, we established the following objectives: 1) Study the relationship between Cu concentrations in follicular fluids and plasma; 2) Study the effect of different Cu concentrations on IVM of bovine oocytes; 3) Study the effect of different Cu concentrations during IVM on subsequent embryo development to the blastocyst stage; and 5) Study the role of cumulus cells (CC) as metabolic bridge between the external medium and the oocyte. For this end, bovine in vitro embryo production (IVP) was used as experimental model, including IVM, in vitro fertilization and in vitro culture of embryos. The IVP was performed using bovine oocytes obtainer from slaughterhouse. The results showed that: 1) Copper concentrations in follicular fluid depend on Cu plasma concentrations, 2) The addition of Cu to IVM medium increased intracellular GSH-GSSG content in oocytes and CC; 3) The rates of apoptosis and DNA damage, in both oocytes and CC, were reduced when Cu was added to IVM medium; 4) The addition of Cu in IVM medium improved the blastocyst rate and embryo quality; and 5) The percentage of blastocysts was enhanced regardless of the presence of CC during IVM. Consequently, this study indicates that Cu acts as an important factor during bovine oocyte maturation promoting early embryo development. Besides, these results suggest that Cu transporters might exist in bovine cumulus-oocyte complex.

Pre-harvest sexual maturation in farmed Atlantic salmon, Salmo salar, remains a key biological bottleneck compromising biomass and financial output, production predictability, environmental respect, stock welfare and the overall sustainability of the on-growing industry. The management practices currently in place are not optimized and events of high maturation rate are still sporadically observed. From an ecological perspective, the escape of reproductively competent, domesticated Atlantic salmon constitutes a threat to the integrity of wild stocks. The forecasted expansion of the Scottish salmon industry compels the need for a comprehensive and more reliable control of sexual maturation. The general aim of this research project was to optimize the current management strategy (windows of light-manipulation and quality grading) and test alternative practices (lighting-technologies, selective harvest and triploidization) in the control of pre-harvest sexual maturation within the Atlantic salmon on-growing industry. In that end, a number of trials were performed using stock reared in sea-cages on a full commercial-scale or in tanks on an experimental scale. The results of this project are organized around three experimental chapters dealing consecutively with body-size dimorphism, grading and harvest quality; light manipulations and triploidy. In each chapter, two original manuscripts either published or in review are included. In addition to these experimental results, a literature review chapter composed of two review papers on the photoperiodic synchronization and developmental regulation of maturation in salmonids and on morphological skin colour changes in teleosts (published) are presented. In the first experimental chapters, we aimed at investigating the possibility of detecting and selectively harvesting a high proportion of sexually recruited fish before flesh quality deterioration. Results clearly showed that body-size dimorphisms between maturity cohorts at the end of the anabolic window of reproduction (June/July) are strong and standard predictors of maturation among related populations with the same freshwater history. Dimorphism can therefore be modelled to easily and accurately estimate maturation rate in a number of discrete rearing-units. If required, a high proportion of sexually recruited fish can be selectively harvested as superior quality product while leaving the immature fish for further on-growing. This provides an alternative to visual grilse grading that is not feasible in large-scale aquaculture systems, prevents downgrading and increases production predictability as compared to emergency harvests. Furthermore, our results showed immature males grow faster than immature females which should be further investigated to fully determine gender specific performances and nutritional requirements. Weight-grading performed earlier in the cycle affects the sex-ratio within individual pens and in turn apparent performance. This work also revealed that Atlantic salmon can exhibit significant variations in skin colouration resembling the onset of nuptial display but that are not related to sexual recruitment and do not correlate with reduced flesh quality. This originates from a lack of purine (silver) pigments which was also identified, to a larger extent, as characteristic of the nuptial display. This suggests a degree of desmoltification in these histologically immature fish. The instrumental colouration of the altered phenotype was shown to be improved towards a more silver-like appearance by direct ice-contact. This knowledge could facilitate post-harvest quality grading towards the most appropriate market channel and increase product acceptance and attractiveness. The second experimental chapter investigated the possibility of improving photoperiodic manipulation used to suppress early maturation, currently applied for 6-months during the second winter at sea using wide-spectrum, high-intensity lighting systems. Our results showed that the window of continuous artificial-light (LL) exposure can be reduced to 4-months following its onset in early January without compromising its efficiency in suppressing pre-harvest maturation. In addition, alternative lighting technologies were also highly potent at suppressing sexual maturation. The mean-irradiance (intensity) generated within a commercial sea-cage was inversely proportional to the suppression of nocturnal plasma melatonin (light perception hormone) and negatively correlates with the maturation rate within the commercial sea-pen. Threshold levels of light-intensity required to achieve optimal (total) suppression of sexual maturation are suggested. Alternative, narrow band-width lighting-technologies (cold cathode and light-emitting diodes) present an array of technical, practical, economic and welfare benefits comparing to the system currently in use. Clear improvements of the photoperiod-manipulation strategy were demonstrated and these would reduce economic and environmental costs but also potential impacts on animal welfare. The third experimental chapter showed the strong potential of sterile-triploid Atlantic salmon stocks both in freshwater and seawater. Triploid out-of-season smolts were produced for the first time using a classical accelerated "square-wave" photoperiod. Triploidization affected the smoltification pattern but had no detrimental effects on freshwater and early seawater performances under both a S0+ and S1 regime. This illustrates the need to adapt the timing of seawater transfer for successfully producing triploid Atlantic salmon post-smolts. Following one year of seawater rearing, the prevalence of external deformities was higher in triploids but remained within acceptable levels. Importantly, the incidence of vertebral deformities and ocular cataract was higher in triploids possibly due to their specific requirements. It is suggested that tailoring the diet to the nutritional requirements of triploids holds strong potential for remediation. This must be addressed if the use of sterile-triploid stock is to become a commercial reality. The present research project provides means to optimize the maturation management strategy within the Atlantic salmon on-growing industry through light-manipulation, maturation detection and selective harvest, and quality grading. Proposed improvements have the potential to increase biomass and financial output, production predictability, environmental respect and animal welfare and will allow standardization of the overall control of pre-harvest sexual maturation. Their implementation provides a comprehensive strategy likely to favour a sustainable expansion of the Atlantic salmon industry. From a longer term perspective, the rearing of sterile-triploid stocks is promising and should be actively investigated to isolate domesticated strains from their wild conspecifics. This would also eliminate the need for on-growers to deploy a maturation management strategy that that might still affect stock welfare and remains, despite the strong improvements demonstrated, not 100% reliable, costly, technical and protracted.

Το Μυελοδυσπλαστικό Σύνδρομο (ΜΔΣ) αποτελεί νόσημα με διαταραχή σε επίπεδο αρχέγονου αιμοποιητικού κυττάρου (stem cell) που χαρακτηρίζεται από μη αποδοτική αιμοποίηση και κυτταροπενίες του περιφερικού αίματος που περιλαμβάνουν μία ή περισσότερες αιμοποιητικές σειρές. Διάφορες ανοσολογικές διαταραχές των ασθενών με ΜΔΣ, όπως, αυξημένη ευαισθησία σε βακτηριακές λοιμώξεις, αυτοάνοσα φαινόμενα και υψηλή συχνότητα κακοηθειών του λεμφικού ιστού, υποδεικνύουν αδυναμία των ασθενών με ΜΔΣ για ανοσολογική απάντηση, οι αιτίες των οποίων παραμένουν άγνωστες μέχρι σήμερα. Τα Δενδριτικά Κύτταρα (ΔΚ) είναι κύτταρα του ανοσολογικού μηχανισμού που προέρχονται από το μυελό των οστών. Ως αντιγονοπαρουσιαστικά κύτταρα (APC), είναι εξειδικευμένα για τη πρόσληψη, επεξεργασία, μεταφορά και παρουσίαση του αντιγόνου στα Τ λεμφοκύτταρα. Στη παρούσα μελέτη πραγματοποιήθηκε ανάλυση διαφορετικών ποσοτικών και λειτουργικών παραμέτρων των ΔΚ από ασθενείς με Μυελοδυσπλαστικό Σύνδρομο, in vivo ή in vitro. Αρχικά διερευνήθηκε ο αριθμός, ο φαινότυπος, η ικανότητα ενδοκύττωσης και η αλλογενής διεγερτική δυνατότητα των ΔΚ, προερχόμενων από μονοκύτταρα του περιφερικού αίματος (ΜοΔΚ) ασθενών με ΜΔΣ και υγιών μαρτύρων, σε διαφορετικά στάδια διαφοροποίησης. Τα μονοκύτταρα των ασθενών με ΜΔΣ χαρακτηρίστηκαν από μειωμένη ικανότητα διαφοροποίησης σε ΔΚ, λόγω του μειωμένου αριθμού των διαφοροποιημένων κυττάρων και τη χαμηλή έκφραση του CD1a αντιγόνου επιφανείας. Τα ΜοΔΚ των ΜΔΣ ασθενών παρουσίασαν χαμηλή έκφραση του υποδοχέα της μανόζης και μειωμένη ικανότητα ενδοκύττωσης. ΜοΔΚ των ΜΔΣ ασθενών επέδειξαν μειωμένη απάντηση ύστερα από διέγερση με TNF-α, καθώς η έκφραση των CD83, CD80 και CD54 αντιγόνων και η αλλοδιεγερτική ικανότητα ήταν μειωμένη, ενώ η επίδραση με LPS είχε ως αποτέλεσμα να εμφανίσουν φαινοτυπικά χαρακτηριστικά και ικανότητα διέγερσης των Τ-κυττάρων, όμοια με τα ΜοΔΚ των φυσιολογικών μαρτύρων. Σε δύο από τους ασθενείς με σύνδρομο 5q-, σχεδόν όλα τα μονοκύτταρα και τα ΜοΔΚ περιείχαν τη χρωμοσωμική διαταραχή, υποδηλώνοντας την προέλευσή τους από τον παθολογικό κλώνο. Στη συνέχεια διερευνήθηκε το δυναμικό πολλαπλασιασμού και διαφοροποίησης των CD34+ προγονικών κυττάρων του μυελού ασθενών με ΜΔΣ σε δενδριτικά κύτταρα (CD34-ΔΚ) σε υγρή καλλιέργεια παρουσία κυτοκινών. Παράλληλα, έγινε ανάλυση των κυκλοφορούντων ΔΚ περιφερικού αίματος στους ίδιους ασθενείς. Τα CD34+ προγονικά κύτταρα παρουσίασαν χαμηλή δυνατότητα ανάπτυξης ΔΚ in vitro, καθώς ο αριθμός των παραγόμενων ΔΚ ανά CD34+ κύτταρο ήταν χαμηλότερος συγκριτικά με τα δείγματα των υγιών μαρτύρων. Παρά την αυξημένη απόπτωση των προγονικών κυττάρων του μυελού των ΜΔΣ ασθενών, η επιβίωση και ο πολλαπλασιασμός των CD34+ κυττάρων στην καλλιέργεια, δεν συσχετίστηκε με την απόπτωση και αποτελεί αξιοσημείωτη παρατήρηση. Φαινοτυπικά, τα CD34-ΔΚ των ΜΔΣ ασθενών δεν διέφεραν από τα ΔΚ που παρήχθησαν από τα CD34+ κύτταρα του μυελού των φυσιολογικών μαρτύρων καθώς επέδειξαν όμοια έκφραση των CD83, CD80, CD40, HLA-DR και CD54 αντιγόνων. Κυτταροεπιλεγμένα CD1a+ κύτταρα ασθενών είχαν όμοια διεγερτική ικανότητα αλλογενών Τ κυττάρων με τα CD34-ΔΚ των φυσιολογικών ατόμων. Το ποσοστό των κυκλοφορούντων μυελοειδών- και πλασματοκυτταροειδών- ΔΚ στους ασθενείς με ΜΔΣ ήταν σημαντικά μειωμένο συγκριτικά με τους υγιείς μάρτυρες. Στους ασθενείς με 5q έλλειψη, τόσο τα CD34-ΔΚ, όσο και τα ΔΚ του αίματος, είχαν τη χρωμοσωμική ανωμαλία. Τα παραπάνω αποτελέσματα υποδηλώνουν ότι η διαδικασία παραγωγής δενδριτικών κυττάρων από το μυελό (‘δενδριτοποίηση’) των ασθενών με ΜΔΣ, είναι μέρος της κλωνικής διαταραχής με αποτέλεσμα την μη αποδοτική παραγωγή ΔΚ από τα προγονικά κύτταρα του μυελού και το χαμηλό ποσοστό των κυκλοφορούντων πρόδρομων ΔΚ. Όλες οι ΔΚ υποομάδες προέρχονται από τον παθολογικό κλώνο και χαρακτηρίζονται από ποσοτικές και ποιοτικές ανωμαλίες. Το σύνολο αυτών των διαταραχών που παρατηρήθηκαν στα ΔΚ πολύ πιθανόν να συμβάλει στη διαταραγμένη ανοσολογική απάντηση έναντι παθογόνων οργανισμών, στην επιβίωση και στην επικράτηση του παθολογικού κλώνου, όπως επίσης και στην εμφάνιση αυτοάνοσων φαινομένων, που παρατηρούνται στους ασθενείς με ΜΔΣ. / Myelodysplastic syndrome (MDS) is a stem cell disorder characterized by ineffective hematopoiesis and blood cytopenias involving one or several myeloid lineages. Various immune disturbances in MDS such as increased susceptibility to bacterial infections, autoimmune phenomena and high incidence of lymphoid malignancies reveal an underlying defect of the immune response in MDS patients, the reasons for which still remain unclear. Dendritic cells (DCs) are bone marrow derived cells. As the most potent antigen presenting cells (APC), they are specialized for the uptake, processing, transport and presentation of Ag to T cells. In the present study different quantitative and functional parameters of DCs in patients with MDS were analyzed either in vivo or in vitro. The number, phenotype, endocytic ability, and allostimulatory capacity of DCs derived from peripheral blood monocytes (MoDCs) were investigated in patients with MDS and healthy controls at different stages of differentiation using the maturation stimuli-TNF-á and LPS. Monocytes in MDS showed low potential to differentiate into DCs, as determined by low cell yield and CD1a expression. MDS-MoDCs exhibited low expression of Mannose receptor and reduced endocytic capacity. When stimulated with TNF-á, MoDCs obtained from MDS patients showed a diminished response with low CD83, CD80 and CD54 expression and allostimulatory capacity, whereas in the presence of LPS MDS-MoDCs acquired phenotypic characteristics and ability to stimulate T-cells similar to MoDCs derived from controls. In two patients with 5q- syndrome the vast majority of both monocytes and MoDCs were positive for the 5q deletion, suggesting that they originate from the malignant clone. Second, we investigated the potential of bone marrow CD34+ progenitors in patients with MDS to proliferate and differentiate into DCs in a liquid cytokine supplemented culture system and also analyzed the status of blood DC subsets in those patients. CD34+ progenitors had low potential to generate DCs in vitro, as the number of DCs obtained from one CD34+ cell was significantly lower compared to controls. Interestingly, although the increased apoptotic level of bone marrow progenitors in MDS, the survival and proliferation of CD34+ cells in culture was not correlated to the degree of apoptosis. Phenotypically the MDS CD34-DCs did not differ from DCs obtained from normal BM CD34+ cells, exhibiting similar expression of CD83, CD80, CD40, HLA-DR, and CD54. FACsorted CD1a+ cells from MDS patients were as efficient stimulators of allogeneic T cells as normal CD34-DCs. The percentage of both circulating DC subsets, MDCs and PDCs in MDS patients was extremely diminished compared to controls. In cases with the 5q deletion both CD34-DCs and blood DCs harbor the cytogenetic abnormality. The results indicate that “dendritopoiesis” in MDS is affected by the transformation process resulting in ineffective production of DCs from bone marrow progenitors with low circulating blood precursors. All DC subsets were derived from the malignant clone and exhibited quantitative and qualitative abnormalities. This constellation of DCs defects probably contribute to the defective immune response against pathogens, escape and expansion of the malignant clone, as well as autoimmune phenomena, observed in MDS patients.

In times with elevated CO2 levels and global warming there is a need of finding alternatives to carbon based energy carriers. One such environmental friendly solution could be H2 produced by living organisms. Cyanobacteria are good candidates since they can produce H2 from sunlight and water through the combination of photosynthesis and H2 producing enzymes i.e. nitrogenases and/or [NiFe]-hydrogenases. This thesis investigates the maturation and transcriptional regulation of [NiFe]-hydrogenases in cyanobacteria, with a special focus on hydrogenase specific proteases. The core of all hydrogenases consists of the small and large subunit. The large subunit in which the catalytic site is located goes through an extenstive maturation process which ends with a proteolytic cleavage performed by a hydrogenase specific protease (HupW/HoxW). This thesis shows that within the maturation process of hydrogenases, the proteolytic cleavage is probably the only step that is specific with respect to different types of hydrogenases i.e. one type of protease cleaves only one type of hydrogenase. Further in-silico analysis revealed that these proteases and the hydrogenases might have co-evolved since ancient time and that the specificity observed could be the result of a conserved amino acid sequence which differs between the two types of proteases (HupW/HoxW). A number of different transcription factors were revealed and shown to interact with the promoter regions of several of the genes encoding maturation proteins. The results indicate that the hydrogenase specific proteases are regulated on a transcriptional level in a similar manner as the hydrogenases they cleave. This thesis contributes with knowledge concerning transcriptional regulation and protein regulation of hydrogenases which will be useful for designing genetically engineered cyanobacteria with an improved and adjustable H2 production. / Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 722