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Accelerated global warming plus an increasing need for energy is an equation not easily solved, thus new forms of sustainable energy production are urgently requested. In this context hydrogen production based on a cyanobacterial system offers an environmentally friendly alternative for energy capture and conversion. Cyanobacteria can produce hydrogen gas from sun light and water through the combination of photosystems and hydrogenases, and are suitable to cultivate in large scale. In the present thesis the maturation process of [NiFe]-hydrogenases is investigated with special focus on transcription of the accessory genes encoding proteins needed for assembly of the large and possibly also for the small hydrogenase subunit. The cyanobacteria used are two N2-fixing, filamentous, heterocystous strains; Nostoc sp. strain PCC 7120 and Nostoc punctiforme PCC 73102. For a biotechnological exploration of hydrogen production tools for regulatory purposes are important. The transcription factor CalA (cyanobacterial AbrB like) (Alr0946 in the genome) in Nostoc sp. strain PCC 7120 was found to be involved in hydrogen metabolism by regulating the transcription of the maturation protein HypC. Further the bidirectional hydrogenase activity was down-regulated in the presence of elevated levels of CalA, a result important to take into account when optimizing cyanobacteria for hydrogen production. CalA regulates at least 25 proteins in Nostoc sp. strain PCC 7120 and one of the down-regulated proteins was superoxide dismutase, FeSOD. The characterization of FeSOD shows that it has a specific and important function in the oxidative stress tolerance of Nostoc sp. stain PCC 7120. Since CalA is involved in regulation of both the hydrogen metabolism as well as stress responses these findings indicate that Alr0946 is an important transcription factor in Nostoc sp. strain PCC 7120 active on a global level in the cell. This thesis adds more knowledge concerning maturation and regulation of cyanobacterial hydrogenases which might be useful for future large scale hydrogen.

Whooping cough (pertussis) is a highly contagious acute respiratory disease caused by the strict human pathogen Bordetella pertussis, a gram-negative coccobacillus. The worldwide mass-vaccination was started in 1940s and to date, a number of whole-cell (Pw) and acellular pertussis vaccine (Pa) formulations were developed. Yet the current vaccines are incapable of providing sustained, lifelong immunity and eliminating subclinical infections, which pose a threat especially for unimmunized infants as well as adolescents and adults. Thus, finding new protein candidates with high immune protective capacities is necessary to enhance the clinical efficacy of current acellular pertussis (Pa) vaccines. In this study, iron-superoxide dismutase (FeSOD) protein was investigated for its capacity of conferring protectivity as well as stimulating humoral and cellular responses against B. pertussis infection in a mouse model. For this purpose, sodB gene, which encodes iron-superoxide dismutase FeSOD protein, was amplified from the genomic DNA of the universal B. pertussis strain &lsquo / Tohama I&rsquo / and sequentially cloned to pGEM&reg / -T subcloning and pET-28a(+) expression vectors. Afterwards sodb/pET28a(+) construct was introduced to E. coli BL21(DE3) cells and the gene was overexpressed therein via IPTG induction. The expressed FeSOD protein was then purified by affinity chromatography and its previously reported immunogenicity was confirmed by Western blot. After filter-sterilization, the protein was adsorbed to alum [Al(OH)3] adjuvant and introduced to BALB/c twice at three weeks intervals intraperitoneally at a concentration of 20 &mu / g purified FeSOD protein/mouse. Another group of mice were immunized in tandem with heat-inactivated whole-cell suspension of B. pertussis. Ten days after the second immunization, mice were intranasally challenged with the local &lsquo / Saadet&rsquo / strain of B. pertussis. Next the lungs of groups of mice were excised, homogenized and plated as serial dilutions on days 5, 8 and 14 post-challenge, and viable lung CFU counts were carried out. Whole cell immunization conferred complete bacterial clearance following B. pertussis intranasal infection while FeSOD immunization failed to attain such protection. In addition to the protectivity assay, ELISA was performed to assess the humoral (i.e. IgG) immune response triggered upon FeSOD- and whole-cell immunizations and a statistically significant increase in anti-FeSOD IgG production was observed in FeSOD-immunized group. Finally, cellular immune response was tested via cytokine (IFN-&gamma / ) assay, in which spleens of mice were excised, splenocytes were cultured and the level of IFN-&gamma / production upon FeSOD addition to the cultures was measured via ELISA. This test showed that whole-cell immunization triggered IFN-&gamma / production at significant levels while FeSOD-immunization did not / indicating the failure of alum-adsorbed FeSOD immunization in inducing cell-mediated immune response.