Interplay of Virulence Factors in Complement Resistance of Streptococcus pneumoniae

Streptococcus pneumoniae (pneumococcus) is a normal inhabitant of the human nasopharynx. Symptoms occur in only a small proportion of those who become carriers, but the ubiquity of the organism in the human population results in a large burden of disease. S. pneumoniae is the leading bacterial cause of pneumonia, sepsis, and meningitis worldwide, causing the death of a million children each year. Middle-ear infection is the most common clinical manifestation of mucosal pneumococcal infections. In invasive disease, S. pneumoniae gains access to the bloodstream and spreads to normally sterile parts of the body. The progression from asymptomatic colonization to disease depends on factors characteristic of specific pneumococcal strains as well as the status of host defenses. The polysaccharide capsule surrounding the bacterium is considered to be the most important factor affecting the virulence of pneumococci. It protects pneumococci from phagocytosis and also may determine its affinity to the respiratory epithelium. S. pneumoniae as a species comprises more than 90 different capsular serotypes, but not all of them are equally prevalent in human diseases. Invasive serotypes are rarely isolated from healthy carriers, but relatively often cause invasive disease. Serotypes that are carried asymptomatically for a long time behave like opportunistic pathogens, causing disease in patients who have impaired immune defenses. The complement system is a collection of blood and cell surface proteins that act as a major primary defense against invading microbes. Phagocytic cells with receptors for complement proteins can engulf and destroy pneumococcal cells opsonized with these proteins. S. pneumoniae has evolved a number of ways to subvert mechanisms of innate immunity, and this is likely to contribute to its pathogenicity. The capsular serotype, proteins essential for virulence, as well the genotype, may all influence the ability of pneumococcus to resist complement and its potential to cause disease. Immunization with conjugate vaccines produces opsonic antibodies, which enhance complement deposition and clearance of the bacteria. The pneumococcal vaccine included in the Finnish national immunization program in 2010 contains the most common serotypes causing invasive disease. Clinical data suggest that protection from middle-ear infection and possibly also from invasive disease depends largely on the capsular serotype, for reasons hitherto unknown. The general aim of this thesis is to assess the relative roles of the pneumococcal capsule and virulence proteins in complement evasion and subsequent opsonophagocytic killing. The main question is whether differences between serotypes to resist complement explain the different abilities of serotypes to cause disease. The importance of particular virulence factors to the complement resistance of a strain may vary depending on its genotype. Prior studies have evaluated the effect of the capsule and virulence proteins on complement resistance of S. pneumoniae by comparing only a few strains. In this thesis, the role of pneumococcal virulence factors in the complement resistance of the bacterium was studied in several genotypically different strains. The ability of pneumococci to inhibit deposition of the complement protein C3 on the bacterial surface was found to depend on the capsular serotype as well as on other features of the bacteria. The results suggest that pneumococcal histidine triad (Pht) proteins may play a role in complement inhibition, but their contribution depends on the bacterial genotype. The capsular serotype was found to influence complement resistance more than the bacterial genotype. A higher concentration of anticapsular antibodies was required for the opsonophagocytic killing of serotypes resistant to C3 deposition. The invasive serotypes were more resistant to C3 deposition than the opportunistic serotypes, suggesting that the former are better adapted to resist immune mechanisms controlling the development of invasive disease. The different susceptibilities of serotypes to complement deposition, opsonophagocytosis, and resultant antibody-mediated protection should be taken into account when guidelines for serological correlates for vaccine efficacy evaluations are made. The results of this thesis suggest that antibodies in higher quantity or quality are needed for efficient protection against the invasive serotypes.

Mother-Infant Antibodies to Pneumococcal Polysaccharides and Proteins : Transfer and Persistence of Maternal Antibodies and Development of Vaccine-Induced and Naturally Acquired Antibodies in Infants

Symptomless nasopharyngeal carriage of Streptococcus pneumoniae (pneumococcus) is very common in young children. Occasionally the carriage proceeds into mild mucosal diseases, such as sinusitis or acute otitis media, or into serious life-threatening diseases, such as pneumonia, sepsis or meningitis. Each year, up to one million children less than five years of age worldwide die of invasive pneumococcal diseases (IPD). Especially in the low-income countries IPD is a leading health problem in infants; 75% of all IPD cases occur before one year of age. This stresses the need of increased protection against pneumococcus in infancy. Anti-pneumococcal antibodies form an important component in the defence against pneumococcal infection. Maternal immunisation and early infant immunisation are two possible ways by which potentially protective antibody concentrations against pneumococci could be achieved in early infancy. The aim of this thesis is to increase the knowledge of antibody mediated protection against pneumococcal disease in infants and young children. We investigated the transfer of maternal anti-pneumococcal antibodies from Filipino mothers to their infants, the persistence of the transferred antibodies in the infants, the immunogenicity of the 23-valent pneumococcal polysaccharide vaccine (PPV) in infants and the response of the children to a second dose of PPV at three years of age. We also investigated the development of antibodies to pneumococcal protein antigens in relation to culture-confirmed pneumococcal carriage in infants. Serum samples were collected from the mothers, the umbilical cords and from the infants at young age as well as at three years of age. The samples were used to determine the antibody concentrations to pneumococcal serotypes 1, 5, 6B, 14, 18C and 19F, as well as to the pneumococcal proteins PspA, PsaA, Ply, PspC, PhtD, PhtDC and LytC by the enzyme immunoassay. The findings of the present study confirm previously obtained results and add to the global knowledge of responses to PPV in young children. Immunising pregnant women with PPV provides the infants with increased concentrations of pneumococcal polysaccharide antibodies. Of the six serotypes examined, serotypes 1 and 5 were immunogenic already in infants. At three years of age, the children responded well to the second dose of PPV suggesting that maternal and early infant immunisations might not induce hyporesponsiveness to polysaccharide antigens after subsequent immunisations. The anti-protein antibody findings provide useful information for the development of pneumococcal protein vaccines. All six proteins studied were immunogenic in infancy and the development of anti-protein antibodies started early in life in relation to pneumococcal carriage.

Mediated music makers : Constructing author images in popular music

The subject of the thesis is the mediated construction of author images in popular music. In the study, the construction of images is treated as a process in which artists, the media and the members of the audience participate. The notions of presented, mediated and compiled author images are used in explaining the mediation process and the various authorial roles of the agents involved. In order to explore the issue more closely, I analyse the author images of a group of popular music artists representing the genres of rock, pop and electronic dance music. The analysed material consists mostly of written media texts through which the artists authorial roles and creative responsibilities are discussed. Theoretically speaking, the starting points for the examination lie in cultural studies and discourse analysis. Even though author images may be conceived as intertextual constructions, the artist is usually presented as a recognizable figure whose purpose is to give the music its public face. This study does not, then, deal with musical authors as such, but rather with their public images and mediated constructions. Because of the author-based functioning of popular music culture and the idea of the artist s individual creative power, the collective and social processes involved in the making of popular music are often superseded by the belief in a single, originating authorship. In addition to the collective practices of music making, the roles of the media and the marketing machinery complicate attempts to clarify the sharing of authorial contributions. As the case studies demonstrate, the differences between the examined author images are connected with a number of themes ranging from issues of auteurism and stardom to the use of masked imagery and the blending of authorial voices. Also the emergence of new music technologies has affected not only the ways in which music is made, but also how the artist s authorial status and artistic identity is understood. In the study at hand, the author images of auteurs, stars, DJs and sampling artists are discussed alongside such varied topics as collective authorship, evaluative hierarchies, visual promotion and generic conventions. Taken altogether, the examined case studies shed light on the functioning of popular music culture and the ways in which musical authorship is (re)defined.

Personality and social psychological studies on computer-mediated communication in school settings

The present study examined how personality and social psychological factors affect third and fourth graders' computer-mediated communication. Personality was analysed in terms of the following strategies: optimism, pessimism and defensive pessimism. Students worked either individually or in dyads which were paired homogeneously or heterogeneously according to the strategies. Moreover, the present study compared horizontal and vertical interaction. The study also examined the role that popularity plays, and students were divided into groups based on their popularity level. The results show that an optimistic strategy is useful. Optimism was found to be related to the active production and processing of ideas. Although previous research has identified drawbacks to pessimism in achievement settings, this study shows that the pessimistic strategy is not as debilitating a strategy as is usually assumed. Pessimistic students were able to process their ideas. However, defensive pessimists were somewhat cautious in introducing or changing ideas. Heterogeneous dyads were not beneficial configurations with respect to producing, introducing, or changing ideas. Moreover, many differences were found to exist between the horizontal and vertical interaction; specifically, the students expressed more opinions and feelings when teachers took no part in the discussions. Strong emotions were observed especially in the horizontal interaction. Further, group working skills were found to be more important for boys than for girls, while rejected students were not at a disadvantage compared to popular ones. Schools can encourage emotional and social learning. The present study shows that students can use computers to express their feelings. In addition, students who are unpopular in non-computer contexts or students who use pessimism can benefit from computers. Participation in computer discussions can give unpopular children a chance to develop confidence when relating to peers.

Distribution and adhesion-promoting activity of alpha4 and alpha5 chain laminins in human endothelia and carcinomas

Basement membranes are specialized sheets of extracellular matrix found in contact with epithelia, endothelia, and certain isolated cells. They support tissue architecture and regulate cell behaviour. Laminins are among the main constituents of basement membranes. Due to differences between laminin isoforms, laminins confer structural and functional diversity to basement membranes. The first aim of this study was to gain insights into the potential functions of the then least characterized laminins, alpha4 chain laminins, by evaluating their distribution in human tissues. We thus created a monoclonal antibody specific for laminin alpha4 chain. By immunohistochemistry, alpha4 chain laminins were primarily localized to basement membranes of blood vessel endothelia, skeletal, heart, and smooth muscle cells, nerves, and adipocytes. In addition, alpha4 chain laminins were found in the region of certain epithelial basement membranes in the epidermis, salivary gland, pancreas, esophagus, stomach, intestine, and kidney. Because of the consistent presence of alpha4 chain laminins in endothelial basement membranes of blood vessels, we evaluated the potential roles of endothelial laminins in blood vessels, lymphatic vessels, and carcinomas. Human endothelial cells produced alpha4 and alpha5 chain laminins. In quantitative and morphological adhesion assays, human endothelial cells barely adhered to alpha4 chain-containing laminin-411. The weak interaction of endothelial cells with laminin-411 appeared to be mediated by alpha6beta1 integrin. The alpha5 chain-containing laminin-511 promoted endothelial cell adhesion better than laminin-411, but it did not promote the formation of cell-extracellular matrix adhesion complexes. The adhesion of endothelial cells to laminin-511 appeared to be mediated by Lutheran glycoprotein together with beta1 and alphavbeta3 integrins. The results suggest that these laminins may induce a migratory phenotype in endothelial cells. In lymphatic capillaries, endothelial basement membranes showed immunoreactivity for laminin alpha4, beta1, beta2, and gamma1 chains, type IV and XVIII collagens, and nidogen-1. Considering the assumed inability of alpha4 chain laminins to polymerize and to promote basement membrane assembly, the findings may in part explain the incomplete basement membrane formation in these vessels. Lymphatic capillaries of ovarian carcinomas showed immunoreactivity also for laminin alpha5 chain and its receptor Lutheran glycoprotein, emphasizing a difference between normal and ovarian carcinoma lymphatic capillaries. In renal cell carcinomas, immunoreactivity for laminin alpha4 chain was found in stroma and basement membranes of blood vessels. In most tumours, immunoreactivity for laminin alpha4 chain was also observed in the basement membrane region of tumour cell islets. Renal carcinoma cells produced alpha4 chain laminins. Laminin-411 did not promote adhesion of renal carcinoma cells, but inhibited their adhesion to fibronectin. Renal carcinoma cells migrated more on laminin-411 than on fibronectin. The results suggest that alpha4 chain laminins have a counteradhesive function, and may thus have a role in detachment and invasion of renal carcinoma cells.

The Mediated Immediacy : João Batista Libanio and the Question of Latin American Liberation Theology

This study is a systematic analysis of mediated immediacy in the production of the Brazilian professor of theology João Batista Libanio. He stresses both ethical mediation and the immediate character of the faith. Libanio has sought an answer to the problem of science and faith. He makes use of the neo-scholastic distinction between matter and form. According to St. Thomas Aquinas, God cannot be known as a scientific object, but it is possible to predicate a formal theological content of other subject matter with the help of revelation. This viewpoint was emphasized in neo-Thomism and supported by the liberation theologians. For them, the material starting point was social science. It becomes a theologizable or revealable (revelabile) reality. This social science has its roots in Latin American Marxism which was influenced by the school of Louis Althusser and considered Marxism a science of history . The synthesis of Thomism and Marxism is a challenge Libanio faced, especially in his Teologia da libertação from 1987. He emphasized the need for a genuinely spiritual and ethical discernment, and was particularly critical of the ethical implications of class struggle. Libanio s thinking has a strong hermeneutic flavor. It is more important to understand than to explain. He does not deny the need for social scientific data, but that they cannot be the exclusive starting point of theology. There are different readings of the world, both scientific and theological. A holistic understanding of the nature of religious experience is needed. Libanio follows the interpretation given by H. C. de Lima Vaz, according to whom the Hegelian dialectic is a rational circulation between the totality and its parts. He also recalls Oscar Cullmann s idea of God s Kingdom that is already and not yet . In other words, there is a continuous mediation of grace into the natural world. This dialectic is reflected in ethics. Faith must be verified in good works. Libanio uses the Thomist fides caritate formata principle and the modern orthopraxis thinking represented by Edward Schillebeeckx. One needs both the ortho of good faith and the praxis of the right action. The mediation of praxis is the mediation of human and divine love. Libanio s theology has strong roots in the Jesuit spirituality that places the emphasis on contemplation in action.

Characterization of small GTPase Cdc42 from the ectomycorrhizal fungus Suillus bovinus and Agrobacterium tumefaciens-mediated transformation of fungi

Ectomycorrhizal formation between the host tree, Pinus sylvestris and fungal symbiont, Suillus bovinus was investigated at the molecular level by isolating genes regulating the organization of the actin cytoskeleton in the fungal partner S. bovinus. An Agrobacterium tumefaciens mediated transformation (ATMT) system was developed for the ectomycorrhizal fungi in order to assign specific functions to the cloned molecules. The developed ATMT system was also used to transform a plant pathogenic fungus, Helminthosporium turcicum, to hygromycin B resistance. Small GTPases Cdc42 and Rac1, the regulators of actin cytoskeleton in eukaryotes were isolated from S. bovinus. Sbcdc42 and Sbrac1, are both expressed in vegetative and in the symbiotic hyphae of S. bovinus . Using IIF microscopy, Cdc42 and actin were co-localized at the tips of vegetative hyphae and were visualized in association with the plasma membrane in swollen cells typical to the symbiotic hyphae. These results suggest that the small GTPases Cdc42 may play a significant role in the polarized growth of S. bovinus hyphae and regulate fungal morphogenesis during ectomycorrhiza formation through reorganization of the actin cytoskeleton. The functional equality of Cdc42 was tested in yeast complementation experiments using a Saccharomyces cerevisiae temperature sensitive mutant, cdc42-1ts. The genomic clone of CDC42 was isolated from S. bovinus genomic DNA via specific primers for Cdc42. The analogous S. cerevisiae cdc42 mutations, dominant active G12V and dominant negative D118A, were generated in the Sbcdc42 gene by in-vitro mutagenesis. The ectomycorrhizal fungi, S. bovinus, P. involutus and H. cylindroporum were transformed using ATMT and phleomycin as a selectable marker. PCR screeing suggested that the T-DNA was inserted in all the three fungal genomes but the fate of integration could not be proved by Southern blot analysis. An alternative Agrobacterium strain, AGL-1 and selection marker, hygromycin was used to transform our model fungus S. bovinus. PCR and Southern analysis suggested an improved efficiency of transformation. All the transformed fungal colonies selected for hygromycin gave positives in PCR and the Southerns showed multiple or single copy T-DNA integrations into the S. bovinus genome. Using the same Agrobacterium strain and the selectable marker, a maize pathogen, H. turcicum was also subjected to ATMT. The H. turcicum transformation data suggested the single copy T-DNA integrations into the genome of the screened transformants that further confirms wider applicability of the ATMT. The plasmids carrying the wild-type (pHGCDC42) and the mutated Sbcdc42 alleles (pHGGV; pHGDA) under Agaricus bisporus gpd promoter were constructed in an A. tumefaciens vector. ATMT was used to transform S. bovinus with the plasmids carrying the wild-type and mutated Sbcdc42 alleles. The isolation of Sbcdc42 and Sbrac1 genes and some other functionally related genes from ectomycorrhizal fungus, S. bovinus will form the basis of future work to resolve the signalling pathway leading to ectomycorrhizal symbiosis. The development of ATMT system will be a valuable tool in analysing the exact function of signalling pathway components in ectomycorrhizal symbiosis or in plant pathogenic interactions. The transformation frequency and broad applicability along with the simplicity of T-DNA integration make Agrobacterium a valuable, new and a powerfull tool for targeted and insertional mutagenesis in these plant associated fungi. The developed ATMT systems should therefore make it possible to generate large number of transformants with tagged genes which could then be screened for their specific roles in symbiosis and pathogenecity, respectively.

GABAa Receptor Mediated Signalling in the Brain: Inhibition, Shunting and Excitation

Within central nervous system, the simple division of chemical synaptic transmission to depolarizing excitation mediated by glutamate and hyperpolarizing inhibition mediated by γ-amino butyric acid (GABA), is evidently an oversimplification. The GABAa receptor (GABAaR) mediated responses can be of opposite sign within a single resting cell, due to the compartmentalized distribution of cation chloride cotransporters (CCCs). The K+/Cl- cotransporter 2 (KCC2), member of the CCC family, promotes K+ fuelled Cl- extrusion and sets the reversal potential of GABA evoked anion currents typically slightly below the resting membrane potential. The interesting ionic plasticity property of GABAergic signalling emerges from the short-term and long-term alterations in the intraneuronal concentrations of GABAaR permeable anions (Cl- and HCO3-). The short-term effects arise rapidly (in the time scale of hundreds of milliseconds) and are due to the GABAaR activation dependent shifts in anion gradients, whereas the changes in expression, distribution and kinetic regulation of CCCs are underlying the long-term effects, which may take minutes or even hours to develop. In this Thesis, the differences in the reversal potential of GABAaR mediated responses between dopaminergic and GABAergic cell types, located in the substantia nigra, were shown to be attributable to the differences in the chloride extrusion mechanisms. The stronger inhibitory effect of GABA on GABAergic neurons was due to the cell type specific expression of KCC2 whereas the KCC2 was absent from dopaminergic neurons, leading to a less prominent inhibition brought by GABAaR activation. The levels of KCC2 protein exhibited activity dependent alterations in hippocampal pyramidal neurons. Intense neuronal activity, leading to a massive release of brain derived neurotrophic factor (BDNF) in vivo, or applications of tyrosine receptor kinase B (TrkB) agonists BDNF or neurotrophin-4 in vitro, were shown to down-regulate KCC2 protein levels which led to a reduction in the efficacy of Cl- extrusion. The GABAergic transmission is interestingly involved in an increase of extracellular K+ concentration. A substantial increase in interstitial K+ tends to depolarize the cell membrane. The effects that varying ion gradients had on the generation of biphasic GABAaR mediated responses were addressed, with particular emphasis on the novel idea that the K+/Cl- extrusion via KCC2 is accelerated in response to a rapid accumulation of intracellular Cl-. The KCC2 inhibitor furosemide produced a large reduction in the GABAaR dependent extracellular K+ transients. Thus, paradoxically, both the inefficient KCC2 activity (via increased intracellular Cl-) and efficient KCC2 activity (via increased extracellular K+) may promote excitation.

Diffusive and ship-mediated spread of dinoflagellates in the Baltic Sea with Prorocentrum minimum as a special case

In this thesis the role played by expansive and introduced species in the phytoplankton ecology of the Baltic Sea was investigated. The aims were threefold. First, the studies investigated the resting stages of dinoflagellates, which were transported into the Baltic Sea via shipping and were able to germinate under the ambient, nutrient-rich, brackish water conditions. The studies also estimated which factors favoured the occurrence and spread of P. minimum in the Baltic Sea and discussed the identification of this morphologically variable species. In addition, the classification of phytoplankton species recently observed in the Baltic Sea was discussed. Incubation of sediments from four Finnish ports and 10 ships ballast tanks revealed that the sediments act as sources of living dinoflagellates and other phytoplankton. Dinoflagellates germinated from all ports detected and from 90% of ballast tanks. The concentrations of cells germinating from ballast tank sediments were mostly low compared with the acceptable cell concentrations set by the International Maritime Organization s (IMO s) International Convention for the Control and Management of Ships Ballast Water and Sediments. However, the IMO allows such high concentrations of small cells in the discharged ballast water that the total number of cells in large ballast water tanks can be very high. Prorocentrum minimum occurred in the Baltic Sea annually but with no obvious trend in the 10-year timespan from 1993 to 2002. The species occurred under wide ranges of temperatures and salinities and the abundance of the species was positively related especially to the presence of organic nitrogen and phosphorus. This indicated that the species was favoured by increased organic nutrient loading and runoff from land and rivers. The cell shape of P. minimum varied from triangular to oval-round, but morphological fine details indicated that only one morphospecies was present. P. minimum also is, according to present knowledge, the only potentially harmful phytoplankton species that has recently expanded widely into new areas of the Baltic Sea.