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Prediction of 3D structure and ligand-binding properties : ABC transporters and flavodiiron proteins from Synechocystis sp. strain PCC 6803

Cyanobacteria are unicellular, non-nitrogen-fixing prokaryotes, which perform photosynthesis similarly as higher plants. The cyanobacterium Synechocystis sp. strain PCC 6803 is used as a model organism in photosynthesis research. My research described herein aims at understanding the function of the photosynthetic machinery and how it responds to changes in the environment. Detailed knowledge of the regulation of photosynthesis in cyanobacteria can be utilized for biotechnological purposes, for example in the harnessing of solar energy for biofuel production. In photosynthesis, iron participates in electron transfer. Here, we focused on iron transport in Synechocystis sp. strain PCC 6803 and particularly on the environmental regulation of the genes encoding the FutA2BC ferric iron transporter, which belongs to the ABC transporter family. A homology model built for the ATP-binding subunit FutC indicates that it has a functional ATPbinding site as well as conserved interactions with the channel-forming subunit FutB in the transporter complex. Polyamines are important for the cell proliferation, differentiation and apoptosis in prokaryotic and eukaryotic cells. In plants, polyamines have special roles in stress response and in plant survival. The polyamine metabolism in cyanobacteria in response to environmental stress is of interest in research on stress tolerance of higher plants. In this thesis, the potd gene encoding an polyamine transporter subunit from Synechocystis sp. strain PCC 6803 was characterized for the first time. A homology model built for PotD protein indicated that it has capability of binding polyamines, with the preference for spermidine. Furthermore, in order to investigate the structural features of the substrate specificity, polyamines were docked into the binding site. Spermidine was positioned very similarly in Synechocystis PotD as in the template structure and had most favorable interactions of the docked polyamines. Based on the homology model, experimental work was conducted, which confirmed the binding preference. Flavodiiron proteins (Flv) are enzymes, which protect the cell against toxicity of oxygen and/or nitric oxide by reduction. In this thesis, we present a novel type of photoprotection mechanism in cyanobacteria by the heterodimer of Flv2/Flv4. The constructed homology model of Flv2/Flv4 suggests a functional heterodimer capable of rapid electron transfer. The unknown protein sll0218, encoded by the flv2-flv4 operon, is assumed to facilitate the interaction of the Flv2/Flv4 heterodimer and energy transfer between the phycobilisome and PSII. Flv2/Flv4 provides an alternative electron transfer pathway and functions as an electron sink in PSII electron transfer.

The seasonal and ovarian status effects on in vitro production of domestic cat embryos between Equator and Tropic of Capricorn

From the Tropic of Capricorn to Equator, the seasonality of domestic cat is known to be absent, i.e., these animals are considered non-seasonal breeders at these regions. We hypothesized that this particularity might have some influence on in vitro embryo production. The aim of this experiment was to determine the percentage of cleavage and morulae and blastocyst formation produced from oocytes recovered from queen ovaries of three distinct status - follicular, luteal or inactive - during two different reproductive seasons experienced by cats in southeast of Brazil (22°53'09" S and 48°26'42" W) - non breeding season (NBS), comprehending January to March; and breeding season (BS), August to October. Thirty queens were neutered. Ovaries were classified according to their status and were sliced in PBS for cumulus oocyte complex (COC) releasing. Grade I COC were washed three times in H-MEM supplemented with BSA, glutamine, sodium pyruvate, cysteine, streptomycin and penicillin. Oocytes were incubated in groups of 20-30 in 400µL of DMEM supplemented with FSH, LH, estradiol, IGF-I and basic fibroblast growth factor under mineral oil for 30 or 36 hours at 38°C in humidified environment of 5% de O2, 5% CO2 and 90% N2. COC were fertilized in Ham's F-10 medium supplemented with BSA, cysteine, pyruvate and streptomycin/penicillin (culture medium) with fresh semen selected through swim up technique. Eighteen hours later, the presumptive zygotes were denuded, the percentage of cleavage was determined and every 10 zygotes were transferred to 100mL drops of culture medium for culture during three days. After 72 hours of culture the percentage of morulae formation was evaluated and these structures were transferred to drops of the same culture medium. At the eighth day of culture blastocyst formation was analyzed. During NBS, from a total of 272 (inactive), 162 (luteal) and 134 (follicular) fertilized oocytes, the percentage of cleaved zygotes, morulae and blastocysts derived from inactive ovaries were 24.63, 16.54 and 8.09 respectively; for those derived from luteal ovaries, the percentage was 21.6, 12.96 and 8.64, and for those from follicular ovaries, they were 24.62, 16.41 and 8.21. Considering BS, from a total of 102 (inactive), 198 (luteal) and 86 (follicular) fertilized oocytes, the relative frequency (%) of cleaved zygotes, morulae and blastocysts derived from inactive ovaries were 64.7, 41.17 and 23.53 respectively; for those derived from luteal ovaries, the percentage was 64.14, 40.41 and 23.73, and for those from follicular ovaries, they were 63.95, 39.54 and 24.41. The results of this experiment demonstrate that no statistically significant difference (P<0.05) was verified in the frequency of cleaved embryos and morulae and blastocyst formation when comparing the three ovarian conditions in the same season. However the breeding season presented better results considering cleavage and morulae and blastocyst formation.

Stop bugging me! : diversity in appearance of warning coloration

In nature, many animals use body coloration to communicate with each other. For example, colorations can be used as signals between individuals of the same species, but also to recognise individuals of other species, and if they may comprise a threat or not. Many animals use protective coloration to avoid predation. The two most common strategies of protective coloration are camouflage and aposematism. Camouflaged animals have coloration that minimises detection, usually by matching colours or structures in the background. Aposematic animals, on the other hand, signal to predators that they are defended. The defence can be physical structures, such as spikes and hairs, or chemical compounds that make the animal distasteful or even deadly toxic. In order for the warning signal to be effective, the predator has to recognise it as such. Studies have shown that birds for example, that are important visual predators on insects, learn to recognise and avoid unpalatable prey faster if they contrast the background or have large internal contrasts. Typical examples of aposematic species have conspicuous colours like yellow, orange or red, often in combination with black. My thesis focuses on the appearance and function of aposematic colour patterns. Even though researchers have studied aposematism for over a century, there is still a lot we do not know about the phenomenon. For example, as it is crucial that the predators recognise a warning signal, aposematic colorations should assumingly evolve homogeneously and be selected for maximal conspicuousness. Instead, there is an extensive variation of colours and patterns among warning colorations, and it is not uncommon to find typical cryptic colours, such as green and brown in aposematic colour patterns. One hypothesis to this variation is that an aposematic coloration does not have to be maximally signalling in order to be effective, instead it is sufficient to have distinct features that can be easily distinguished from edible prey. To be maximally conspicuous is one way to achieve this, but not the only way. Another hypothesis is that aposematic prey that do not exhibit maximal conspicuousness can exploit both camouflage and aposematism in a distance-dependent fashion, by being signalling when seen close up but camouflaged at a distance. Many prey animals also make use of both strategies by shifting colour at different ecological conditions such as seasonal variations, fluctuations in food resources or between life stages. Yet another explanation for the variation may be that prey animals are usually exposed to several predator species that vary in visual perception and tolerance towards various toxins. The aim with this thesis is, by studying their functions, to understand why aposematic warning signals vary in appearance, specifically in the level of conspicuousness, and if warning coloration can be combined with camouflage. In paper I, I investigated if the colour pattern of the aposematic larva of the Apollo butterfly (Parnassius apollo) can switch function with viewing distance, and be signalling at close range but camouflaged at a distance, by comparing detection time between different colour variants and distances. The results show that the natural coloration has a dual distance-dependent function. Moreover, the study shows that an aposematic coloration does not have to be selected for maximal conspicuousness. A prey animal can optimise its coloration primarily by avoiding detection, but also by investing in a secondary defence, which presence can be signalled if detected. In paper II, I studied how easily detected the coloration of the firebug (Pyrrhocoris apterus), a typical aposematic species, is at different distances against different natural backgrounds, by comparing detection time between different colour variants. Here, I found no distance-dependent switch in function. Instead, the results show that the coloration of the firebug is selected for maximal conspicuousness. One explanation for this is that the firebug is more mobile than the butterfly larva in study I, and movement is often incompatible with efficient camouflage. In paper III, I investigated if a seasonal related colour change in the chemically defended striated shieldbug (Graphosoma lineatum) is an adaptation to optimise a protective coloration by shifting from camouflage to aposematism between two seasons. The results confirm the hypothesis that the coloration expressed in the late summer has a camouflage function, blending in with the background. Further, I investigated if the internal pattern as such increased the effectiveness of the camouflage. Again, the results are in accordance with the hypothesis, as the patterned coloration was more difficult to detect than colorations lacking an internal pattern. This study shows how an aposematic species can optimise its defence by shifting from camouflage to aposematism, but in a different fashion than studied in paper I. The aim with study IV was to study the selection on aposematic signals by identifying characteristics that are common for colorations of aposematic species, and that distinguish them from colorations of other species. I compared contrast, pattern element size and colour proportion between a group of defended species and a group of undefended species. In contrast to my prediction, the results show no significant differences between the two groups in any of the analyses. One explanation for the non-significant results could be that there are no universal characteristics common for aposematic species. Instead, the selection pressures acting on defended species vary, and therefore affect their appearance differently. Another explanation is that all defended species may not have been selected for a conspicuous aposematic warning coloration. Taken together, my thesis shows that having a conspicuous warning coloration is not the only way to be aposematic. Also, aposematism and camouflage is not two mutually exclusive opposites, as there are prey species that exploit both strategies. It is also important to understand that prey animals are exposed to various selection pressures and trade-offs that affect their appearance, and determines what an optimal coloration is for each species or environment. In conclusion, I hold that the variation among warning colorations is larger and coloration properties that have been considered as archetypically aposematic may not be as widespread and representative as previously assumed.

Prediction of transients and control reactions in a transonic wind tunnel

This work describes a lumped parameter mathematical model for the prediction of transients in an aerodynamic circuit of a transonic wind tunnel. Control actions to properly handle those perturbations are also assessed. The tunnel circuit technology is up to date and incorporates a novel feature: high-enthalpy air injection to extend the tunnel’s Reynolds number capability. The model solves the equations of continuity, energy and momentum and defines density, internal energy and mass flow as the basic parameters in the aerodynamic study as well as Mach number, stagnation pressure and stagnation temperature, all referred to test section conditions, as the main control variables. The tunnel circuit response to control actions and the stability of the flow are numerically investigated. Initially, for validation purposes, the code was applied to the AWT ("Altitude Wind Tunnel" of NASA-Lewis). In the sequel, the Brazilian transonic wind tunnel was investigated, with all the main control systems modeled, including injection.

Seasonal variation in migratory body condition, corticosterone secretion, oxidative stress and biotransformation activity in barn swallows (Hirundo rustica L.)

I studied the associations between migration-related physiological regulation (corticosterone) and body condition of barn swallows (Hirundo rustica L.). An additional purpose was to determine whether oxidative stress and biotransformation activity vary seasonally. Since physiological regulation, biotransformation activity and the stress involved may be important factors for body condition during migration; they may have direct effects on migration success. This in turn may influence other important life history stages, such as breeding and moult. In the thesis I used barn swallow data of the Finnish Ringing Centre (1997–2009), consisting of all juveniles ringed in the nests and recaptured from night roosts later the same autumn. Before the autumn migration in Finland I also captured, ringed and sampled barn swallows from night roosts in 2003, 2006, 2007 and 2011. Samples preceding spring migration in South Africa were collected in 2007. Juvenile barn swallows started to migrate southward in mid-August (first broods). Second broods started their migration at a younger age and almost a month later than first broods (mid-September). Barn swallows increased body mass and accumulated fat for the autumn migration. In the course of the autumn they seemed to be able to prevent the loss of energy already accumulated, since the proportional overnight mass loss, fat loss and faecal production decreased. Surprisingly, corticosterone, the major energy-regulating hormone in birds, seemed not to be involved in the fuelling process. Previous studies with warblers, sparrows and shorebirds had shown that during migration, the baseline levels of corticosterone were elevated in order to facilitate fuelling. It is possible that for Finnish barn swallows the most important fuelling place is in southern Europe, since northern and eastern populations migrate via the Balkan Peninsula. However, the adrenocortical stress response of Finnish barn swallows in good body condition was lower than that of those in poor body condition. Birds clearly suppressed the response, probably to prevent the catabolic effects of excessive corticosterone levels; birds cannot afford to lose muscle mass before migration. South African barn swallows had high levels of baseline corticosterone, but this may have been associated with the high oxidative damage and biotransformation activity of those birds. Barn swallows in spring and summer had low biotransformation activity and intermediate oxidative stress, which was probably related to breeding. Autumn birds had low biotransformation activity and oxidative stress but high redox enzyme activities in some migration-related enzymes.

Linear prediction in optical spectroscopy

A linear prediction procedure is one of the approved numerical methods of signal processing. In the field of optical spectroscopy it is used mainly for extrapolation known parts of an optical signal in order to obtain a longer one or deduce missing signal samples. The first is needed particularly when narrowing spectral lines for the purpose of spectral information extraction. In the present paper the coherent anti-Stokes Raman scattering (CARS) spectra were under investigation. The spectra were significantly distorted by the presence of nonlinear nonresonant background. In addition, line shapes were far from Gaussian/Lorentz profiles. To overcome these disadvantages the maximum entropy method (MEM) for phase spectrum retrieval was used. The obtained broad MEM spectra were further underwent the linear prediction analysis in order to be narrowed.

Prediction of market switching and delisting events from OMX First North Nordic multilateral stock exchange

This thesis studies the predictability of market switching and delisting events from OMX First North Nordic multilateral stock exchange by using financial statement information and market information from 2007 to 2012. This study was conducted by using a three stage process. In first stage relevant theoretical framework and initial variable pool were constructed. Then, explanatory analysis of the initial variable pool was done in order to further limit and identify relevant variables. The explanatory analysis was conducted by using self-organizing map methodology. In the third stage, the predictive modeling was carried out with random forests and support vector machine methodologies. It was found that the explanatory analysis was able to identify relevant variables. The results indicate that the market switching and delisting events can be predicted in some extent. The empirical results also support the usability of financial statement and market information in the prediction of market switching and delisting events.

Estrutura e diversidade do componente arbóreo da floresta na Estação Ecológica dos Caetetus, Gália, SP

O estudo fitossociológico, se realizado em diferentes estratos verticais da floresta, fornece dados de grande valia para a compreensão da dinâmica da comunidade. Efetuou-se levantamento das espécies arbóreas presentes em diferentes estratos, em área de 6.000 m² de floresta madura, na Estação Ecológica dos Caetetus, Gália, SP. Foram utilizadas 60 parcelas de 10 x 10 m, para indivíduos com diâmetro do caule a 1,30 m acima do nível do solo (DAP) a partir de 5 cm (estrato superior); 10 x 2 m, para indivíduos com DAP entre 1 e 5 cm (estrato intermediário) e 60 sub-parcelas de 2 x 2 m, para indivíduos menores que 1 cm de DAP (estrato inferior). Verificou-se que a diversidade, a composição florística e a densidade relativa das espécies variam entre estratos. No estrato superior, com 1.080 indivíduos.ha-1 e área basal de 31,2 m².ha-1, encontraram-se 62 espécies (28 famílias), tendo sido as mais importantes, em ordem decrescente de IVI: Metrodorea nigra, Savia dictyocarpa, Ocotea indecora, Aspidosperma polyneuron e Trichilia catigua. O estrato intermediário apresentou-se com densidade de 3.525 indivíduos.ha-1, pertencentes a 30 espécies (17 famílias), sendo as mais importantes em ordem decrescente de IVI: Metrodorea nigra, Actinostemon concolor, Trichilia catigua, Aspidosperma polyneuron e Trichilia clausenii. No estrato inferior a densidade foi de 28.875 indivíduos.ha-1, pertencentes a 37 espécies (19 famílias), sendo as mais abundantes, em ordem decrescente de densidade relativa: Metrodorea nigra, Actinostemon concolor, Trichilia catigua, Aspidosperma polyneuron e Eugenia blastantha. Constatou-se que há espécies do estrato superior da floresta que não foram observadas em regeneração, especialmente heliófitas de estádios sucessionais iniciais (p. ex. Croton floribundus). Outras ocorreram com densidade relativa decrescente nos estratos inferiores (p. ex. Savia dictyocarpa e Ocotea indecora). Para outras espécies, as sementes germinaram, mas não se desenvolveram à sombra, estando ausentes no estrato intermediário (p. ex. Cariniana estrellensis). Observou-se, ainda, um grupo de espécies, de estádios sucessionais mais avançados, que apresentaram a mesma densidade relativa em todos os estratos (p. ex. Metrodorea nigra e Aspidosperma polyneuron).

Composição, estrutura e relações florísticas do componente arbóreo de uma floresta estacional no Rio Grande do Sul, Brasil

A composição específica de florestas estacionais no Rio Grande do Sul é fortemente influenciada por dois contingentes florísticos diferentes, um coincidente com as florestas atlânticas do leste e outro com as florestas paranaense-uruguaias do oeste. Um levantamento fitossociológico de uma floresta central sul-rio-grandense foi realizado para detectar a estrutura comunitária do componente arbóreo e a participação local dos diferentes contingentes florísticos. Todas as árvores com DAP > ou = 5 cm foram registradas em uma área de 1 ha, subdividida em 100 unidades amostrais de 10 x 10 m. As espécies amostradas foram classificadas como amplas ou restritas, segundo suas distribuições geográficas, considerando as afinidades florísticas leste ou oeste. A densidade total por hectare foi de 1855 indivíduos, pertencentes a 23 famílias, 46 gêneros e 55 espécies. As famílias com maior riqueza específica foram Fabaceae e Myrtaceae. As espécies mais importantes foram Gymnanthes concolor Spreng., Euterpe edulis Mart., Sorocea bonplandii (Baill.) Burger, Lanj. & Boer, Pachystroma longifolium (Nees) I.M. Johnst. e Trichilia claussenii C.DC., acumulando 55,2% do total do valor de importância. A diversidade específica (H') foi estimada em 2,244 (nats). As espécies do oeste formam um contingente mais diversificado, geralmente ocorrendo como árvores do dossel ou emergentes. As espécies do leste constituem um contingente bem menos diversificado, porém com uma alta participação quantitativa como árvores de porte médio do sub-bosque. Aspectos gerais da composição e da estrutura sugerem a importância de árvores do dossel de florestas estacionais interiores como formadoras de ambientes favoráveis para diversas árvores do sub-bosque de florestas pluviais costeiras.

Fenologia de Rubiaceae do sub-bosque em floresta Atlântica no sudeste do Brasil

A comparação da fenologia de diferentes espécies de uma família, ocorrendo no mesmo hábitat, pode auxiliar na compreensão dos fatores reguladores das fenofases. Os padrões fenológicos vegetativos e reprodutivos de dez espécies de Rubiaceae foram estudados durante 14 meses no sub-bosque de floresta Atlântica, Núcleo Picinguaba, Ubatuba, SP. A produção e queda de folhas ocorreram simultaneamente nas espécies estudadas e ao longo de todo o período de estudo, proporcionando ao sub-bosque uma aparência sempre verde. A floração foi pouco sazonal, com aumento no número de espécies durante o período de maior precipitação e temperatura, porém não apresentando correlações significativas com os fatores climáticos. Este resultado contrasta com o padrão de floração sazonal, correlacionado ao fotoperíodo e temperatura, observado nas espécies de Rubiaceae de dossel na mesma área. A produção de frutos ocorreu ao longo de todo período de estudo, com aumento no número de espécies com frutos maduros no final da estação chuvosa, resultando em disponibilidade constante desses frutos para frugívoros. Os padrões fenológicos reprodutivos diferiram entre as espécies, sugerindo que fatores distintos devem estar regulando a ocorrência das fenofases nestas espécies de Rubiaceae.

Phenology, litterfall, growth, and root biomass in a tidal floodplain forest in the Amazon estuary

The dynamics of forests subject to inundation appears to be strongly influenced by the frequency and intensity of natural disturbances such as flooding. In a late successional tidal floodplain forest near the Amazon port of Belém, Brazil, we tested this prediction by measuring seasonal patterns of phenology and litterfall in relation to two key variables: rainfall and tide levels. In addition, we estimated the root biomass and the annual growth of the forest community by measuring stem increments over time. Our results showed high correlations between phenological events (flowering and fruiting) and rainfall and tide levels, while correlations between litterfall and these variations were generally weaker. Contrary to our prediction, root biomass to 1 m depth showed no significant differences along the topographic gradient, and the root biomass at all topographic levels was low to intermediate compared with other neotropical forests. Both litterfall and total stem increment were high compared to other tropical forest, indicating the high productivity of this ecosystem.

Fenologia de espécies do dossel e do sub-bosque de duas Florestas de Restinga na Ilha do Mel, sul do Brasil

Devido à ampla distribuição geográfica, a Floresta Atlântica e os ecossistemas associados estão sujeitos a condicionantes ambientais que variam de acordo com a latitude e que devem influenciar nos processos dinâmicos das comunidades. Para avaliar se a fenologia de florestas da região atlântica pode ser determinada por tais variações, plantas do dossel e do subbosque (total 55 espécies) de duas Florestas de Restinga (Floresta não inundável e Floresta inundável) foram acompanhadas por dois anos na Ilha do Mel, localizada em região meridional da distribuição da Floresta Atlântica e com pouca diferença climática entre o período superúmido (setembro a maio) e úmido (junho a agosto). Apesar das diferenças florísticas e estruturais, as duas florestas apresentaram padrões semelhantes, com pico de queda de folhas (outubro a dezembro), brotação (dezembro a janeiro), floração (dezembro a janeiro) e frutificação (março a abril) ocorrendo sucessivamente ao longo da estação superúmida, o que esteve correlacionado principalmente com as variações do comprimento do dia e da temperatura. Dossel e sub-bosque apresentaram padrões fenológicos distintos, sendo que no primeiro houve maior sincronia interespecífica. Os resultados mostraram que mesmo localizadas na situação marginal de distribuição do clima tropical, as Florestas de Restinga da Ilha do Mel apresentam semelhanças fenológicas com outros ecossistemas da região atlântica, o que deve refletir a similaridade florística entre estas áreas.

Structure and dynamics of a woody plant community of a tropical semi-deciduous seasonal forest in the "Estação Ecológica do Panga", municipality of Uberlândia, Minas Gerais, Brazil

Studies of plant community dynamics are essential in understanding the demographic patterns of species since changes in demographic rates can affect the floristic composition and future structure. The purpose of the present study was to analyze the changes in the community structure and floristic composition of woody plants in a tropical semi-deciduous forest in Uberlândia in central Brazil, during a 10-years period. The data were collected in 1989 and in 2000 in 50 quadrats (10 m x 10 m) where all trees with a minimum circumference at breast height of 10 cm were sampled. In 1989, 93 species and 1103 individuals were registered. Over a period of 10 years, seven new species were added to the community, although eight disappeared. The main change that occurred during this period in the floristic composition was the replacement of savannah species occurring in forest gaps by those from the forest understory.

Sementes recalcitrantes: intolerantes a baixas temperaturas? Embriões recalcitrantes de Inga vera Willd. subsp. affinis (DC.) T. D. Penn. toleram temperatura sub-zero

Sementes recalcitrantes são intolerantes à dessecação e a baixas temperaturas, que são as principais formas de conservação de sementes e, portanto, são de difícil armazenamento. Inga vera subsp. affinis produz sementes denominadas recalcitrantes, uma vez que não toleram redução do teor de água para valores inferiores a 35%. Neste trabalho é demonstrada a tolerância desses embriões a temperatura de -2 ºC. Embriões de I. vera de diferentes estádios de maturação foram armazenados por até 45 dias nas temperaturas de 8 ºC a -18 ºC, com ou sem secagens prévias até -4,0 e -6,0 MPa. Os resultados permitiram observar que, embora nenhum tratamento tenha proporcionado resistância à temperatura de -18 ºC, a secagem dos embriões maduros a -4 MPa proporcionou maior tolerância à redução de temperatura até níveis de congelamento da água (-2 ºC).