Submitted in fulfilment of the requirements of the degree of Master of Technology: Biotechnology, Durban University of Technology, 2014. / Cyanobacteria contain phycocyanin a light harvesting pigment found to have numerous biotechnological applications, such as: a natural colorant in food and cosmetics, fluorescent tags employed in clinical and immunological research and also in therapeutic processes. Successful phycocyanin production depends on growth characteristics, ability to accumulate high quantities of the pigment, and an effective downstream process. Therefore, the aim of this research was to optimize the extraction method and production by determining the optimal cultivation conditions for phycocyanin producing cyanobacterium. This cyanobacterium was isolated from a hypersaline water body in Kwa-Zulu Natal, and subsequently purified using traditional streak and spread plate techniques. Different cell disruption techniques and a range of buffers were evaluated for the extraction of phycocyanin. The buffer concentrations and pH was subsequently optimized. Results showed that maximum phycocyanin was extracted when cells were suspended in 50mM sodium phosphate buffer (pH-7.5) supplemented with 10 % lysozyme and then disrupted using the freeze–thaw method at -20 & 4°C. The UV-Vis absorption spectral scan of the crude extracted pigments showed a peak at 620 nm. This corresponds to phycocyanin production. Unwanted proteins were removed using a 25and 50% saturated ammonium sulphate precipitation, followed by dialysis. SDS-PAGE showed two subunits with molecular masses of 19 and 20 kDa. These masses corresponded to phycocyanin α and β subunits. Furthermore, a food grade purity ratio (A615/A280) of 1.20 was achieved. The effects of various abiotic factors (temperature, light and pH) on growth and phycocyanin production of the Cyanothece sp. was investigated. Temperature ranging from 20-45°C and pH (5-10) was evaluated for 2 weeks. Cultures were then subjected to four photoperiods (24:0, 18:06 12:12 and 8:16 h light: dark) three light intensities (25, 75 and 125 µmol photons per m2 per –s) at varying wavelengths i.e. blue, red and green and Grolux light. Ideal conditions were observed at 35°C, 125 µmol photons.m2.s-1 of Grolux light for a 16:8 light and dark photoperiod. It was observed that the highest biomass and phycocyanin production was found to be at 35°C, temperatures below or above resulted in a decrease in both growth and pigment synthesis. Phycocyanin concentration changed in response to light quality and intensity. A significantly higher (p<0.05) phycocyanin yield was found when the culture was exposed to 125 µmol photons.m2.s-1 of Grolux light compared with the other three light conditions. Using Design of experiments, a series of fractional factorial experiments were carried out to optimize media components for pigment production. The final optimized growth medium was determined from a central composite design using response surface plots together with a mathematical point-prediction tool and consisted of 2g/L NaNO3, 0.06g/L K2HPO4, 0.12 g/L MgSO4.7H2O, 0.033 g/L CaCl2.2H2O, 100g/L NaCl, 12mL minor nutrients and 0.5 trace metal. A 72 % increase in phycocyanin was observed. This research revealed that this particular Cyanothece sp. shows great potential as a reliable source of phycocyanin.

The goal of this research is to identify and characterize enzymes involved in bilin attachment to the phycobiliprotein allophycocyanin in the cyanobacterium Synechococcus sp. PCC 7002. Candidates for lyases responsible for attachment of phycocyanobilin to allophycocyanin are two cpeS-like genes termed cpcS and cpcU, and one cpeT-like gene termed cpcT. In vitro bilin attachment reactions were conducted in the presence of the recombinant substrate apo-allophycocyanin (HT-ApcAB). Size exclusion HPLC showed that CpcS and HT-CpcU form a 1:1 heterodimeric complex and that HT-ApcAB is present as a monomer (áâ). Absorbance and fluorescence spectroscopy illustrated that both CpcS and HT-CpcU were required to get holo-allophycocyanin with phycocyanobilin attached to the cysteine-81 residue. Absorbance of the product at 615 nm was consistent with holo-monomeric allophycocyanin. Experiments were performed with HT-ApcD ApcB and HT-ApcF ApcA, but size exclusion HPLC showed they were in aggregated form.

The goal of this research is to investigate the role of the slr1098 gene in the cyanobacterium Synechocystis sp. PCC 6803, a gene with similarity to cpcE which encodes a subunit of an enzyme involved in bilin attachment to phycocyanin. This protein is hypothesized to be involved in oligomerization of phycocyanin due to previous results showing the mutant made shorter phycocyanin rods. The recombinant Slr1098 protein was produced and purified from E. coli cells. Binding assays showed interaction between Slr1098 and both apo- and holo-phycocyanin, but not to apo-allophycocyanin. Slr1098 blocked bilin addition at Cys-82 on CpcB by the CpcS/CpcU bilin lyase. Size exclusion chromatography and sucrose density gradient analysis of complexes formed suggest that Slr1098 strongly interacts with all intermediate forms of phycocyanin and may be an important checkpoint in the biosynthesis and oligomerization of this protein, but that by itself, Slr1098 does not increase oligomerization of phycocyanin.

The biological significance of photosynthesis is indisputable as it is necessary for nearly all life on earth. Photosynthesis provides chemical energy for plants, algae, and bacteria, while heterotrophic organisms rely on these species as their ultimate food source. The initial step in photosynthesis requires the absorption of sunlight to create electronic excitations. Light-harvesting proteins play the functional role of capturing solar radiation and transferring the resulting excitation to the reaction centers where it is used to carry out the chemical reactions of photosynthesis. Despite the wide variety of light-harvesting protein structures and arrangements, most light-harvesting proteins are able to utilize the captured solar energy for charge separation with near perfect quantum efficiency. This thesis will focus on understanding the energy transfer dynamics and photophysics of a specific subset of light-harvesting antennae known as phycobiliproteins. These proteins are extracted from cryptophyte algae and are investigated using steady-state and ultrafast spectroscopic techniques.

The biological significance of photosynthesis is indisputable as it is necessary for nearly all life on earth. Photosynthesis provides chemical energy for plants, algae, and bacteria, while heterotrophic organisms rely on these species as their ultimate food source. The initial step in photosynthesis requires the absorption of sunlight to create electronic excitations. Light-harvesting proteins play the functional role of capturing solar radiation and transferring the resulting excitation to the reaction centers where it is used to carry out the chemical reactions of photosynthesis. Despite the wide variety of light-harvesting protein structures and arrangements, most light-harvesting proteins are able to utilize the captured solar energy for charge separation with near perfect quantum efficiency. This thesis will focus on understanding the energy transfer dynamics and photophysics of a specific subset of light-harvesting antennae known as phycobiliproteins. These proteins are extracted from cryptophyte algae and are investigated using steady-state and ultrafast spectroscopic techniques.

Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2017-03-14T18:27:56Z No. of bitstreams: 1 Rizzo, Roberta Ferreira [Dissertação, 2014].pdf: 3622671 bytes, checksum: 2e33786bbb464784e93274b48c446ebd (MD5) / Made available in DSpace on 2017-03-14T18:27:56Z (GMT). No. of bitstreams: 1 Rizzo, Roberta Ferreira [Dissertação, 2014].pdf: 3622671 bytes, checksum: 2e33786bbb464784e93274b48c446ebd (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Corantes são aditivos amplamente utilizados pelas indústrias de alimentos. O aumento de investimento em pesquisa sobre novas fontes de pigmentos naturais, como a partir de micro-organismo, para aplicação em alimentos vem sendo justificada pelo potencial toxicológico dos corantes artificiais e/ou pela baixa estabilidade dos corantes naturais. Cianobactérias são micro-organismos produtores de pigmentos como as ficobiliproteínas, e estudos relatam a ocorrência de ficocianina, aloficocianina e pequena fração de ficoeritrina na cianobactéria A. platensis. Há descrição de que o gênero Arthrospira realize adaptação cromática, fenômeno que leva a reestruturação dos ficobilissomas, induzindo ao acúmulo de ficobiliproteínas específicas na biomassa. O presente trabalho visou ao estudo do crescimento de A. platensis em diferentes qualidades de irradiância luminosa, em diferentes níveis, avaliando a produção de metabólitos de interesse. Os cultivos foram realizados em meio Zarrouk, a 31 ± 2ºC. A cianobactéria foi cultivada nos níveis de irradiância, 50, 100 e 150 mol fotons.m-2.s-1, fornecida por lâmpadas branca ou verde. Foi acompanhada a produção de biomassa, proteína e ficobiliproteínas e foi avaliada a estabilidade de cor dos extratos obtidos. As condições sob luz branca apresentaram maior produção de biomassa que os cultivos sob luz verde. A condição de 150 μmol fotons.m-2.s-1 sob luz branca foi a que promoveu maior produção de biomassa com 2.115,24 mg/L. Não houve diferença estatisticamente significativa na produção de proteína total e ficobiliproteínas nas condições estudadas. Não foi evidenciado o fenômeno da adaptação cromática complementar na cepa de A. platensis estudada, cultivada sob iluminação de qualidade verde. O efeito combinado do pH, temperatura e tempo de tratamento térmico, demonstrou que o extrato obtido no crescimento sob luz verde é termoestável quando comparado ao extrato obtido no crescimento sob luz branca. O armazenamento por um período de 60 dias, em temperatura ambiente e de refrigeração, com extratos em diferentes valores de pH em diferentes irradiâncias ou ausência de luz, mostrou que o extrato obtido sob luz verde se mantém estável independente da temperatura de armazenamento, enquanto o extrato sob luz branca necessita ser mantido sob refrigeração para manutenção de sua coloração azul / Colorants are additives extensively used by food industry. The increase of investment in research of new sources of natural pigments, like by microorganisms, for use in food products has been justified by toxicological potential of artificial dyes and/or the low stability of natural dyes. Cyanobacteria are microorganisms pigments producers such as phycobiliproteins, and studies have reported the occurrence of phycocyanin, allophycocyanin and small fraction of phycoerythrin in cyanobacteria Arthrospira platensis. There is report of the genus Arthrospira perform chromatic adaptation, a phenomenon that leads to restructuring of phycobilisomes, resulting to accumulation of specific phycobiliproteins in biomass. The present work aimed to study the growth of A. platensis in different light qualities of irradiance, at different levels, evaluating the production of desired metabolites. Cultures were performed in Zarrouk medium, at 31 ± 2ºC. The cyanobacteria was cultivated in irradiance levels, 50, 100 and 150 μmol fotons.m-2.s-1, provided by white or green light. Biomass, protein and phycobiliproteins production was measured and the color stability of extracts was evaluated. The conditions under white light produced more biomass than the cultures under green light. The condition of 150 μmol fotons.m-2.s-1 under white light was the best condition for biomass production with 2115.24 mg/L. There was no statistically significant difference in total protein and phycobiliproteins in the studied conditions. There is no evidence the complementary chromatic adaptation phenomenon in the strain of A. platensis studied when cultivated under green light. The combined effect of pH, temperature and time of heat treatment showed that the extract obtained in growth under green light is thermostable when compared to extract obtained in growth under white light. The storage for a period of 60 days at room temperature and refrigerated, with extracts in different pH values, in different light intensities or absence of light, showed that the extract obtained under green light was stable independent of the storage temperature, while the extract in white light needs to be kept under refrigeration for maintaining its blue color

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2018-10-03T11:42:26Z No. of bitstreams: 2 Dissertação - Renata Klícia Mendes Caetano - 2018.pdf: 2847916 bytes, checksum: 2eaea78563b91986afd782253b20323a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-10-03T11:47:01Z (GMT) No. of bitstreams: 2 Dissertação - Renata Klícia Mendes Caetano - 2018.pdf: 2847916 bytes, checksum: 2eaea78563b91986afd782253b20323a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-10-03T11:47:01Z (GMT). No. of bitstreams: 2 Dissertação - Renata Klícia Mendes Caetano - 2018.pdf: 2847916 bytes, checksum: 2eaea78563b91986afd782253b20323a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-08-29 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / The growing interest in bioactive compounds from renewable natural sources has stimulated more and more studies aimed at increasing the supply of this market. An example is phycobiliproteins, dyes used in various areas, such as food, pharmacological and cosmetic. The microalgae arthrospira platensis (Spirulina platensis) is one of the organisms that stands out most in the production of these dyes, producing allophycocyanin and phycocyanin; these two dyes are the ones that today have more demand in the market. These proteins have their application determined from their degree of purity obtained with the absorbance ratio read at 620 nm of the extract used for the absorbance read at 280 nm. For use of these dyes, in the food area, a purity value greater than 0.7 should be obtained, and for use in the analytical area, the purity should be greater than 4.0. In view of the importance of these dyes and their degree of purity, in order to develop means of obtaining phycobiliproteins in a purity satisfactory for use in food industries, phase separation techniques were used in aqueous biphasic systems (ABS) to evaluate the action of pH, phosphate salt concentration and the concentration of low chlorine content cationic polymer and polyethylene glycol 4000 (PEG 4000) on the efficiency of the extraction and purification process of Spirulina crude extract and also in the pre-purified extract of phycocyanin obtained by centrifugation. For this, a first experiment was carried out using a factorial design 2 4 to evaluate the variables that most influenced the extraction and purification of phycocyanin, allophycocyanin and purity of the extract. Values of 2.6 mg.mL -1 were reached for phycocyanin concentration, 1.4 mg.mL -1 for the allophycocyanin concentration and 1.6 for the purity of the extract. The significance of the PEG 4000 and PBC polymers was observed for extraction and purification of phycobiliproteins. Then, a second design was carried out, a Rotational Central Composite Design (RCCD) 2 2 , fixing the phosphate salt, pH and varying the PEG and low chlorine content cationic polymer concentrations. At the end of the extraction, it was possible to find concentrations of 2.7 mg.mL -1 for Phycocyanin, 1.3 mg.mL -1 of allophycocyanin and 2.5 for the purity of the extract, evidencing that the techniques used are effective for the extraction process and purification of phycobiliproteins, in which the levels achieved allow their use and application in food industry. / O crescente interesse por compostos bioativos de fontes naturais renováveis tem estimulado cada vez mais estudos que visam aumentar a oferta deste mercado. Um exemplo são as ficobiliproteínas, corantes utilizados em diversas áreas, como alimentícia, farmacológica e cosmética. A microalga Arthrospira platensis (Spirulina platensis) é um dos organismos que mais se destaca na produção destes corantes, produzindo a Aloficocianina e, principalmente, a Ficocianina, sendo estes dois corantes os que, hoje, mais têm demanda no mercado. Essas proteínas têm sua aplicação determinada a partir de seu grau de pureza, obtido com a relação da absorbância lida a 620 nm do extrato utilizado pela absorbância lida de 280 nm. Para utilização destes corantes na área alimentícia, deve-se obter valor de pureza maior que 0,7 e para sua utilização na área analítica, a pureza deve ser maior que 4,0. Tendo em vista a importância destes corantes e seu grau de pureza, a fim de se desenvolver meios de obtenção das ficobiliproteínas em um grau de pureza satisfatório para utilização em indústriasalimentícias, foram utilizadas técnicas de separação por fases em sistemas aquosos bifásicos (SAB) para avaliar a ação do pH, da concentração de sal fosfato, e da concentração dos polímeros catiônicos com baixo teor de cloro e polietilenoglicol 4000 (PEG 4000) na eficiência do processo de extração e purificação do extrato bruto da Spirulina, e também, em extrato pré-purificado de ficocianina obtido por processo de centrifugação. Para tal, realizou-se um primeiro experimento utilizando um planejamento fatorial 2 4 para a avaliação das variáveis que mais influenciaram na extração e purificação da ficocianina, da aloficocianina e da pureza do extrato. Foram atingidos valores de 2,6 mg.mL -1 para concentração de ficocianina, valor de 1,4 mg.mL -1 para concentração de aloficocianina e 1,6 mg.mL -1 para a pureza do extrato. Foi observada a significância dos polímeros PEG 4000 e polímeros catiônicos com baixo teor de cloro para extração e purificação das ficobiliproteínas. Então foi realizado um segundo delineamento, sendo um DCCR - Composto Central Rotacional 2 2 , fixando as variáveis de sal fosfato e pH; e variando as concentrações de PEG 4000 e polímeros catiônicos com baixo teor de cloro. Ao final da extração, foi possível encontrar concentrações de 2,7 mg.mL -1 para Ficocianina, 1,3 mg.mL -1 de Aloficocianina e 2,5 para pureza do extrato, evidenciando que as técnicas utilizadas são eficazes para o processo de extração e purificação das ficobiliproteínas;em que os níveis alcançados permitem sua utilização e aplicação em indústrias do setor alimentício.

Additionally, cyclosporin A, an inhibitor of the mitochondrial permeability transition (MPT) pore, failed to prevent hIAPP-induced DeltaPsim collapse, cytochrome c and AIF release and caspase-3 activation, indicating that the MPT pore was not involved in hIAPP-induced apoptosis. On the other hand, potential crosstalk between the extrinsic and intrinsic apoptotic pathways was demonstrated by cleavage of Bid by caspase-8 in the apoptotic process triggered by hIAPP. / It is widely accepted that human islet amyloid polypeptide (hIAPP) aggregation plays an important role in the loss of insulin-producing pancreatic beta cells. Insulin secretion impairment and cell apoptosis can be due to mitochondrial dysfunction in pancreatic beta cells. hIAPP-induced cytotoxicity is mediated by the generation of reactive oxygen species (ROS). Phycocyanin (PC) is a natural compound from blue-green algae that is widely used as food supplement. Currently, little information is available about the effect of hIAPP on mitochondrial function of beta cells and protection of PC against hIAPP-induced cytotoxicity. In this thesis, I hypothesize that hIAPP may impair beta cell function with the involvement of mitochrondrial dysfunction, and this effects could be attenuated by PC. Therefore, the aim of this study was to investigate the role of mitochondria in hIAPP-induced apoptosis, the in vitro protective effects of PC and explore the underlying mechanisms. / It was found that hIAPP induced apoptosis in INS-1E cells with the disruption of mitochondrial function, as evidenced by ATP depletion, mitochondrial mass reduction, mitochondrial fragmentation and loss of mitochondrial membrane potential (DeltaPsim). Further molecular analysis showed that hIAPP induced changes in the expression of Bcl-2 family members, release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria into cytosol, activation of caspases and cleavage of poly (ADP-ribose) polymerase. Interestingly, the hIAPP-induced mitochondrial dysfunction in INS1-E cells was effectively restored by co-treatment with PC. / Our results showed that hIAPP inhibited the INS-1E cell growth in a dose-dependent manner. However, cytotoxicity of hIAPP was significantly attenuated by co-incubation of the cells with PC. hIAPP induced DNA fragmentation and chromatin condensation, which were key characteristics of cell apoptosis. These changes were inhibited by PC as examined by TUNEL assay and DAPI staining. Moreover, PC significantly prevented the hIAPP-induced overproduction of intracellular ROS and malonaldehyde (MDA), as well as changes of activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) enzymes. Furthermore, hIAPP triggered the activation of mitogen-activated protein kinases (MAPKs) such as c-Jun N-terminal kinase (JNK) and p38 kinase, and these effects were effectively suppressed by PC. / Taken together, I have demonstrated for the first time the involvement of mitochondrial dysfunction in hIAPP-induced INS-1E cell apoptosis, which was attenuated by PC through attenuating oxidative stress, modulating JNK and p38 pathways and reducing mitochondrial dysfunction. / Li, Xiaoling. / Adviser: Juliana Chung Ngor Chan. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 150-159). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.