Produção de lipases de Aspergillus niger e Aspergillus fumigatus através de fermentação em estado sólido, avaliação da especificidade do substrato e seu uso em reações de esterificação e alcoólise: evaluation of substrate specificity and use in esterification and alcoholysis reactions

Filamentous fungi were cultured under solid state fermentation of soybean residues to produce lipases. Enzymes produced by Aspergillus niger esterified oleic and butyric acids in the presence of ethanol, while enzymes produced by Aspergillus fumigatus demonstrated no esterification activity toward lauric acid. In case of A. niger, direct lyophilization of fermented bran led to higher esterification activity. The esterification of oleic acid by enzymes of A. fumigatus was neither influenced by pH adjustment nor by the extraction process. Conversions to ethyl esters were higher after pH adjustment with lyophilized liquid extract of A. niger.

Identification and characterization of a Hydrangea ringspot virus isolate infecting hydrangea in São Paulo State

Hydrangea plants showing leaves with chlorotic and necrotic rings from Arujá Municipality, São Paulo State, were analyzed for the identification of the viral species. Elongated filamentous particles of 490 nm were visualized under transmission electron microscope. Oligonucleotides for Hydrangea ringspot virus (HdRSV), a potexvirus commonly found in Europe and in the United States, were tested using total RNA from hydrangea plants, amplifying two fragments, one around 550 and another one of 250 nucleotides. Nucleotide identity with HdRSV (accession number AJ 707100.1) was 96% and 88% for the longest and shortest fragment, respectively, indicating the presence of this virus. To evaluate its dissemination in the matrices of hydrangea used in the commercial production, 17 samples were collected in the region of Arujá, and eight were infected by HdRSV. For the analyzed viral replicase portion, the isolates from the varieties 'Azul LZR', 'Rosita', 'Renat Blue' and 'Vermelho Comum' did not differ in their amino acid sequences from isolates with sequences deposited in the GenBank (accession numbers AY 707100 and NC_006943). The isolates from 'Azul Rendado' and "Rosa Japonesa' showed few differences but were related to the remaining isolates. An antiserum was obtained for HdRSV and can be efficiently used to detect such virus in hydrangea and Primula malacoides, another ornamental plant also infected by HdRSV.

Cyanobacteria from the Baltic Sea and Finnish lakes as an energy source and modulators of bioenergetic pathways

Cyanobacteria are a diverse group of oxygenic photosynthetic bacteria that inhabit in a wide range of environments. They are versatile and multifaceted organisms with great possibilities for different biotechnological applications. For example, cyanobacteria produce molecular hydrogen (H2), which is one of the most important alternatives for clean and sustainable energy. Apart from being beneficial, cyanobacteria also possess harmful characteristics and may become a source of threat to human health and other living organisms, as they are able to form surface blooms that are producing a variety of toxic or bioactive compounds. The University of Helsinki Culture Collection (UHCC) maintains around 1,000 cyanobacterial strains representing a large number of genera and species isolated from the Baltic Sea and Finnish lakes. The culture collection covers different life forms such as unicellular and filamentous, N2-fixing and non-N2-fixing strains, and planktonic and benthic cyanobacteria. In this thesis, the UHCC has been screened to identify potential strains for sustainable biohydrogen production and also for strains that produce compounds modifying the bioenergetic pathways of other cyanobacteria or terrestrial plants. Among the 400 cyanobacterial strains screened so far, ten were identified as high H2-producing strains. The enzyme systems involved in H2 metabolism of cyanobacteria were analyzed using the Southern hybridization approach. This revealed the presence of the enzyme nitrogenase in all strains tested, while none of them are likely to have contained alternative nitrogenases. All the strains tested, except for two Calothrix strains, XSPORK 36C and XSPORK 11A, were suggested to contain both uptake and bidirectional hydrogenases. Moreover, 55 methanol extracts of various cyanobacterial strains were screened to identify potent bioactive compounds affecting the photosynthetic apparatus of the model cyanobacterium, Synechocystis PCC 6803. The extract from Nostoc XPORK 14A was the only one that modified the photosynthetic machinery and dark respiration. The compound responsible for this effect was identified, purified, and named M22. M22 demonstrated a dual-action mechanism: production of reactive oxygen species (ROS) under illumination and an unknown mechanism that also prevailed in the dark. During summer, the Baltic Sea is occupied by toxic blooms of Nodularia spumigena (hereafter referred to as N. spumigena), which produces a hepatotoxin called nodularin. Long-term exposure of the terrestrial plant spinach to nodularin was studied. Such treatment resulted in inhibition of growth and chlorosis of the leaves. Moreover, the activity and amount of mitochondrial electron transfer complexes increased in the leaves exposed to nodularin-containing extract, indicating upregulation of respiratory reactions, whereas no marked changes were detected in the structure or function of the photosynthetic machinery. Nodularin-exposed plants suffered from oxidative stress, evidenced by oxidative modifications of various proteins. Plants initiated strategies to combat the stress by increasing the levels of alpha-tocopherol, mitochondrial alternative oxidase (AOX), and mitochondrial ascorbate peroxidase (mAPX).

Outbreaks of rhinofacial and rhinopharyngeal zygomycosis in sheep in Paraíba, northeastern Brazil

Two outbreaks of zigomycosis with rhinofacial and two other with rhinopharyngeal lesions involving fungi with filamentous coaenocytic hyphae characteristic of entomoph-thoramycetous fungi are reported in the state of Paraíba, northeastern Brazil. One outbreak of rhinofacial zygomycosis occurred during the rainy season affecting 5 sheep. Another outbreak of the clinical form affected one out of 40 sheep during the dry season. Common clinical signs of the rhinofacial infection were bilateral serosanguineous nasal discharge with swelling of nostrils, upper lip, and the skin of the face. At necropsy the nasal mucosa showed dark brownish ulcerated areas which extended from the mucocutaneous region to 10cm inside the nasal vestibule. The mucosa of the hard palate was also ulcerated. The cutting surface of nostrils and palate showed a brownish or red spongeous tissue of friable consistency. One outbreak of rhinopharyngitis took place on an irrigated coconut farm; 7 out of 60 adult sheep were affected. Another outbreak affected a sheep in a flock of 80 during the dry season. Clinical signs as noisy respiration and dyspnoea due to mechanical blockage of the nasal cavities, swelling of the nostrils, and serosanguineous nasal discharge were observed. Six out of 8 sheep in this group showed exophthalmia, keratitis and unilateral corneal ulceration of the eye. The sheep either died of their infection or were euthanized after a clinical course of 7-30 days. At necropsy there was a dense yellow exudate in the nasopharyngeal area affecting the ethmoidal region, turbinate bones, paranasal sinuses, hard and soft palates, orbital cavity, pharynges, regional muscles and lymph nodes. Histopathologically both forms of the disease showed multifocal granulomas with an eosinophilic necrotic reaction (Splendore-Hoeppli phenomenon) containing ribbon-type coenocytic hyphae with 7-30mum in diameter similar to hyphae of zygomycetous fungi, possibly Conidiobolus spp. Outbreaks of both forms of mycotic rhinitis are common in northeastern Brazil and in other regions of the country.

Genes associated with pathogenicity of avian Escherichia coli (APEC) isolated from respiratory cases of poultry

The virulence mechanisms of avian pathogenic Escherichia coli (APEC) have been continually studied and are believed to be multi-factorial. Certain properties are primarily associated with virulent samples and have been identified in avian isolates. In this study a total of 61 E. coli, isolates from chicken flocks with respiratory symptomatology, were probed by Polimerase Chain Reation (PCR) for the presence of genes responsible for the adhesion capacity, P fimbria (papC) e F11 fimbria (felA), colicin production (cvaC), aerobactin presence (iutA), serum resistance (iss), temperature-sensitive hemagglutinin (tsh), and presence of K1 and K5 capsular antigens (kpsII). The iss gene was detected in 73,8%, tsh in 55,7%, iutA in 45,9%, felA in 39,3%, papC in 24,3%, cvaC in 23% and kpsII in18%.

Serogroups and virulence genes of Escherichia coli isolated from psittacine birds

Escherichia coli isolates from 24 sick psittacine birds were serogrouped and investigated for the presence of genes encoding the following virulence factors: attaching and effacing (eae), enteropathogenic E. coli EAF plasmid (EAF), pili associated with pyelonephritis (pap), S fimbriae (sfa), afimbrial adhesin (afa), capsule K1 (neu), curli (crl, csgA), temperature-sensitive hemagglutinin (tsh), enteroaggregative heat-stable enterotoxin-1 (astA), heat-stable enterotoxin -1 heat labile (LT) and heat stable (STa and STb) enterotoxins, Shiga-like toxins (stx1 and stx2), cytotoxic necrotizing factor 1 (cnf1), haemolysin (hly), aerobactin production (iuc) and serum resistance (iss). The results showed that the isolates belonged to 12 serogroups: O7; O15; O21; O23; O54; O64; O76; O84; O88; O128; O152 and O166. The virulence genes found were: crl in all isolates, pap in 10 isolates, iss in seven isolates, csgA in five isolates, iuc and tsh in three isolates and eae in two isolates. The combination of virulence genes revealed 11 different genotypic patterns. All strains were negative for genes encoding for EAF, EAEC, K1, sfa, afa, hly, cnf, LT, STa, STb, stx1 and stx2. Our findings showed that some E. coli isolated from psittacine birds present the same virulence factors as avian pathogenic E. coli (APEC), uropathogenic E. coli (UPEC) and Enteropathogenic E. coli (EPEC) pathotypes.

Platelet activation: ultrastructure and morphometry in platelet-rich plasma of horses

This study was conducted to investigate the activation ability of the platelet-rich plasma (PRP) by pharmacological agents, as well as to verify the need or not of this activation for therapeutic use. The PRP was obtained from four healthy crossbred geldings aged 13 to 16 years (15±1years), and was processed for observation and quantification of the platelet morphology by using the transmission electron microscopy. All PRP samples were activated with 10% calcium chloride (CaCl2) solution, pure bovine thrombin or associated with CaCl2. The control (pure PRP) was not pharmacologically activated. In the pure PRP samples, 49% of the platelets were classified as state of activation uncertain, 41% as resting, 9% as fully activated and 1% as irreversibly damaged. Treatment with 10% CaCl2 provided a distribution of 54% platelets in state of activation uncertain, 24% as fully activated, 20% as resting, and 2% as irreversibly damaged. The platelet morphology of the bovine thrombin treated samples did not fit into classification adopted, as showing irregular shape with emission of large filamentous pseudopods, appearance of ruptured and whole granules in the remaining cytoplasm and extracellular environment. There was effect of the treatment on the platelet morphology (P=0.03). The 10% CaCl2 is an adequate platelet-activating agent. However, in cases the use of PRP under its liquid form is necessary, the use of pure PRP is recommended, since besides presenting an adequate percentage of fully activated platelets it also has significant amount of the resting type, which can be activated by substances found in the injured tissue.

Methods of genetic diversity creation and functional display for directed evolution experiments

Protein engineering aims to improve the properties of enzymes and affinity reagents by genetic changes. Typical engineered properties are affinity, specificity, stability, expression, and solubility. Because proteins are complex biomolecules, the effects of specific genetic changes are seldom predictable. Consequently, a popular strategy in protein engineering is to create a library of genetic variants of the target molecule, and render the population in a selection process to sort the variants by the desired property. This technique, called directed evolution, is a central tool for trimming protein-based products used in a wide range of applications from laundry detergents to anti-cancer drugs. New methods are continuously needed to generate larger gene repertoires and compatible selection platforms to shorten the development timeline for new biochemicals. In the first study of this thesis, primer extension mutagenesis was revisited to establish higher quality gene variant libraries in Escherichia coli cells. In the second study, recombination was explored as a method to expand the number of screenable enzyme variants. A selection platform was developed to improve antigen binding fragment (Fab) display on filamentous phages in the third article and, in the fourth study, novel design concepts were tested by two differentially randomized recombinant antibody libraries. Finally, in the last study, the performance of the same antibody repertoire was compared in phage display selections as a genetic fusion to different phage capsid proteins and in different antibody formats, Fab vs. single chain variable fragment (ScFv), in order to find out the most suitable display platform for the library at hand. As a result of the studies, a novel gene library construction method, termed selective rolling circle amplification (sRCA), was developed. The method increases mutagenesis frequency close to 100% in the final library and the number of transformants over 100-fold compared to traditional primer extension mutagenesis. In the second study, Cre/loxP recombination was found to be an appropriate tool to resolve the DNA concatemer resulting from error-prone RCA (epRCA) mutagenesis into monomeric circular DNA units for higher efficiency transformation into E. coli. Library selections against antigens of various size in the fourth study demonstrated that diversity placed closer to the antigen binding site of antibodies supports generation of antibodies against haptens and peptides, whereas diversity at more peripheral locations is better suited for targeting proteins. The conclusion from a comparison of the display formats was that truncated capsid protein three (p3Δ) of filamentous phage was superior to the full-length p3 and protein nine (p9) in obtaining a high number of uniquely specific clones. Especially for digoxigenin, a difficult hapten target, the antibody repertoire as ScFv-p3Δ provided the clones with the highest affinity for binding. This thesis on the construction, design, and selection of gene variant libraries contributes to the practical know-how in directed evolution and contains useful information for scientists in the field to support their undertakings.

Light and storage on the germination of spores of Dicksonia sellowiana (Presl.) Hook., Dicksoniaceae

Spores of Dicksonia sellowiana are positively photoblastic and reach the maximum percentage of germination at 23 ± 2°C in white light after seven days of imbibition. The pre-induction phase for spores induced by white or red light for 24 hours was 72 hours. Gametophytes grown in white light were plane and bidimensional, while those grown under red light were filamentous. The higher the number of hours of light applied per day during 10 days, the higher the percentage of germination. Germination was higher for long white light treatments applied on a daily basis. The effect of different light intensities on germination was also investigated here. The lower percentages of germination were observed for spores kept under 43% and 2% of full sunlight, while those kept under 26, 19 and 4% presented higher percentages. Spores presented circa 82% of germination after 731 days of storage under refrigeration at aproximately 10°C.

Analysis of viral and cellular parameters which affect the fusion process of influenza viruses

In the present investigation we studied the fusogenic process developed by influenza A, B and C viruses on cell surfaces and different factors associated with virus and cell membrane structures. The biological activity of purified virus strains was evaluated in hemagglutination, sialidase and fusion assays. Hemolysis by influenza A, B and C viruses ranging from 77.4 to 97.2%, from 20.0 to 65.0%, from 0.2 to 93.7% and from 9.0 to 76.1% was observed when human, chicken, rabbit and monkey erythrocytes, respectively, were tested at pH 5.5. At this pH, low hemolysis indexes for influenza A, B and C viruses were observed if horse erythrocytes were used as target cells for the fusion process, which could be explained by an inefficient receptor binding activity of influenza on N-glycolyl sialic acids. Differences in hemagglutinin receptor binding activity due to its specificity to N-acetyl or N-glycolyl cell surface oligosaccharides, density of these cellular receptors and level of negative charges on the cell surface may possibly explain these results, showing influence on the sialidase activity and the fusogenic process. Comparative analysis showed a lack of dependence between the sialidase and fusion activities developed by influenza B viruses. Influenza A viruses at low sialidase titers (<2) also exhibited clearly low hemolysis at pH 5.5 (15.8%), while influenza B viruses with similarly low sialidase titers showed highly variable hemolysis indexes (0.2 to 78.0%). These results support the idea that different virus and cell-associated factors such as those presented above have a significant effect on the multifactorial fusion process

Expression of extracellular matrix components and their receptors in the central nervous system during experimental Toxoplasma gondii and Trypanosoma cruzi infection

Alterations in extracellular matrix (ECM) expression in the central nervous system (CNS) usually associated with inflammatory lesions have been described in several pathological situations including neuroblastoma and demyelinating diseases. The participation of fibronectin (FN) and its receptor, the VLA-4 molecule, in the migration of inflammatory cells into the CNS has been proposed. In Trypanosoma cruzi infection encephalitis occurs during the acute phase, whereas in Toxoplasma infection encephalitis is a chronic persisting process. In immunocompromised individuals such as AIDS patients, T. cruzi or T. gondii infection can lead to severe CNS damage. At the moment, there are no data available regarding the molecules involved in the entrance of inflammatory cells into the CNS during parasitic encephalitis. Herein, we characterized the expression of the ECM components FN and laminin (LN) and their receptors in the CNS of T. gondii- and T. cruzi-infected mice. An increased expression of FN and LN was detected in the meninges, leptomeninges, choroid plexus and basal lamina of blood vessels. A fine FN network was observed involving T. gondii-free and T. gondii-containing inflammatory infiltrates. Moreover, perivascular spaces presenting a FN-containing filamentous network filled with a4+ and a5+ cells were observed. Although an increased expression of LN was detected in the basal lamina of blood vessels, the CNS inflammatory cells were a6-negative. Taken together, our results suggest that FN and its receptors VLA-4 and VLA-5 might be involved in the entrance, migration and retention of inflammatory cells into the CNS during parasitic infections.

Regulatory roles of microtubule-associated proteins in neuronal morphogenesis. Involvement of the extracellular matrix

As a result of recent investigations, the cytoskeleton can be viewed as a cytoplasmic system of interconnected filaments with three major integrative levels: self-assembling macromolecules, filamentous polymers, e.g., microtubules, intermediate filaments and actin filaments, and supramolecular structures formed by bundles of these filaments or networks resulting from cross-bridges between these major cytoskeletal polymers. The organization of this biological structure appears to be sensitive to fine spatially and temporally dependent regulatory signals. In differentiating neurons, regulation of cytoskeleton organization is particularly relevant, and the microtubule-associated protein (MAP) tau appears to play roles in the extension of large neuritic processes and axons as well as in the stabilization of microtubular polymers along these processes. Within this context, tau is directly involved in defining neuronal polarity as well as in the generation of neuronal growth cones. There is increasing evidence that elements of the extracellular matrix contribute to the control of cytoskeleton organization in differentiating neurons, and that these regulations could be mediated by changes in MAP activity. In this brief review, we discuss the possible roles of tau in mediating the effects of extracellular matrix components on the internal cytoskeletal arrays and its organization in growing neurons.

Extended hydrogen photoproduction by nitrogen-fixing cyanobacteria

Cyanobacteria are the only prokaryotic organisms performing oxygenic photosynthesis. They comprise a diverse and versatile group of organisms in aquatic and terrestrial environments. Increasing genomic and proteomic data launches wide possibilities for their employment in various biotechnical applications. For example, cyanobacteria can use solar energy to produce H2. There are three different enzymes that are directly involved in cyanobacterial H2 metabolism: nitrogenase (nif) which produces hydrogen as a byproduct in nitrogen fixation; bidirectional hydrogenase (hox) which functions both in uptake and in production of H2; and uptake hydrogenase (hup) which recycles the H2 produced by nitrogenase back for the utilization of the cell. Cyanobacterial strains from University of Helsinki Cyanobacteria Collection (UHCC), isolated from the Baltic Sea and Finnish lakes were screened for efficient H2 producers. Screening about 400 strains revealed several promising candidates producing similar amounts of H2 (during light) as the ΔhupL mutant of Anabaena PCC 7120, which is specifically engineered to produce higher amounts of H2 by the interruption of uptake hydrogenase. The optimal environmental conditions for H2 photoproduction were significantly different between various cyanobacterial strains. All suitable strains revealed during screening were N2-fixing, filamentous and heterocystous. The top ten H2 producers were characterized for the presence and activity of the enzymes involved in H2 metabolism. They all possess the genes encoding the conventional nitrogenase (nifHDK1). However, the high H2 photoproduction rates of these strains were shown not to be directly associated with the maximum capacities of highly active nitrogenase or bidirectional hydrogenase. Most of the good producers possessed a highly active uptake hydrogenase, which has been considered as an obstacle for efficient H2 production. Among the newly revealed best H2 producing strains, Calothrix 336/3 was chosen for further, detailed characterization. Comparative analysis of the structure of the nif and hup operons encoding the nitrogenase and uptake hydrogenase enzymes respectively showed minor differences between Calothrix 336/3 and other N2-fixing model cyanobacteria. Calothrix 336/3 is a filamentous, N2-fixing cyanobacterium with ellipsoidal, terminal heterocysts. A common feature of Calothrix 336/3 is that the cells readily adhere to substrates. To make use of this feature, and to additionally improve H2 photoproduction capacity of the Calothrix 336/3 strain, an immobilization technique was applied. The effects of immobilization within thin alginate films were evaluated by examining the photoproduction of H2 of immobilized Calothrix 336/3 in comparison to model strains, the Anabaena PCC 7120 and its ΔhupL mutant. In order to achieve optimal H2 photoproduction, cells were kept under nitrogen starved conditions (Ar atmosphere) to ensure the selective function of nitrogenase in reducing protons to H2. For extended H2 photoproduction, cells require CO2 for maintenance of photosynthetic activity and recovery cycles to fix N2. Application of regular H2 production and recovery cycles, Ar or air atmospheres respectively, resulted in prolongation of H2 photoproduction in both Calothrix 336/3 and the ΔhupL mutant of Anabaena PCC 7120. However, recovery cycles, consisting of air supplemented with CO2, induced a strong C/N unbalance in the ΔhupL mutant leading to a decrease in photosynthetic activity, although total H2 yield was still higher compared to the wild-type strain. My findings provide information about the diversity of cyanobacterial H2 capacities and mechanisms and provide knowledge of the possibilities of further enhancing cyanobacterial H2 production.

Isolation of bovine plasma albumin by liquid chromatography and its polymerization for use in immunohematology

The aim of the method described here is to remove hemoglobin, the major contaminant in the bovine plasma obtained from slaughterhouses, by adding a mixture of 19% cold ethanol and 0.6% chloroform, followed by fibrinogen and globulin precipitation by the Cohn method and nonspecific hemagglutinin by thermocoagulation. The experimental volume of bovine plasma was 2,000 ml per batch. Final purification was performed by liquid chromatography using the ion-exchange gel DEAE-Sepharose FF. The bovine albumin thus obtained presented > or = 99% purity, a yield of 25.0 ± 1.2 g/l plasma and >71.5% recovery. N-acetyl-DL-tryptophan (0.04 mmol/g protein) and sodium caprylate (0.04 mmol/g protein) were used as stabilizers and the final concentration of albumin was adjusted to 22.0% (w/v), pH 7.2 to 7.3. Viral inactivation was performed by pasteurization for 10 h at 60°C. The bovine albumin for the hemagglutination tests used in immunohematology was submitted to chemical treatment with 0.06% (w/v) glutaraldehyde and 0.1% (w/v) formaldehyde at 37°C for 12 h to obtain polymerization. A change in molecular distribution was observed after this treatment, with average contents of 56.0% monomers, 23.6% dimers, 12.2% trimers and 8.2% polymers. The tests performed demonstrated that this polymerized albumin enhances the agglutination of Rho(D)-positive red cells by anti-Rho(D) serum, permitting and improving visualization of the results.

Oxygen Photoreduction in Cyanobacteria

Cyanobacteria are well-known for their role in the global production of O2 via photosynthetic water oxidation. However, with the use of light energy, cyanobacteria can also reduce O2. In my thesis work, I have investigated the impact of O2 photoreduction on protection of the photosynthetic apparatus as well as the N2-fixing machinery. Photosynthetic light reactions produce intermediate radicals and reduced electron carriers, which can easily react with O2 to generate various reactive oxygen species. To avoid prolonged reduction of photosynthetic components, cyanobacteria use “electron valves” that dissipate excess electrons from the photosynthetic electron transfer chain in a harmless way. In Synechocystis sp. PCC 6803, flavodiiron proteins Flv1 and Flv3 comprise a powerful electron sink redirecting electrons from the acceptor side of Photosystem I to O2 and reducing it directly to water. In this work, I demonstrate that upon Ci-depletion Flv1/3 can dissipate up to 60% of the electrons delivered from Photosystem II. O2 photoreduction by Flv1/3 was shown to be vital for cyanobacteria in natural aquatic environments and deletion of Flv1/3 was lethal for both Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 under fluctuating light conditions. The lethal phenotype observed in the absence of Flv1/3 results from oxidative damage to Photosystem I, which appeared to be a primary target of reactive oxygen species produced upon sudden increases in light intensity. Importantly, cyanobacteria also possess other O2 photoreduction pathways which can protect the photosynthetic apparatus. This study demonstrates that respiratory terminal oxidases are also capable of initiating O2 photoreduction in mutant cells lacking the Flv1/3 proteins and grown under fluctuating light. Photoreduction of O2 by Rubisco was also shown in Ci-depleted cells of the mutants lacking Flv1/3, and thus provided the first evidence for active photorespiratory gas-exchange in cyanobacteria. Nevertheless, and despite the existence of other O2 photoreduction pathways, the Flv1/3 route appears to be the most robust and rapid system of photoprotection. Several groups of cyanobacteria are capable of N2 fixation. Filamentous heterocystous N2- fixing species, such as Anabaena sp. PCC 7120, are able to differentiate specialised cells called heterocysts for this purpose. In contrast to vegetative cells which perform oxygenic photosynthesis, heterocysts maintain a microoxic environment for the proper function of the nitrogenase enzyme, which is extremely sensitive to O2. The genome of Anabaena sp. PCC 7120 harbors two copies of genes encoding Flv1 and Flv3 proteins, designated as “A” and “B” forms. In this thesis work, I demonstrate that Flv1A and Flv3A are expressed only in the vegetative cells of filaments, whilst Flv1B and Flv3B are localized exclusively in heterocysts. I further revealed that the Flv3B protein is most responsible for the photoreduction of O2 in heterocysts, and that this reaction plays an important role in protection of the N2-fixing machinery and thus, the provision of filaments with fixed nitrogen. The function of the Flv1B protein remains to be elucidated; however the involvement of this protein in electron transfer reactions is feasible. Evidence provided in this thesis indicates the presence of a great diversity of O2 photoreduction reactions in cyanobacterial cells. These reactions appear to be crucial for the photoprotection of both photosynthesis and N2 fixation processes in an oxygenic environment.