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Frugivoria de Ficus (Moraceae) por aves em paisagens com diferentes níveis de fragmentação florestal no Estado de São Paulo / Frugivory of Ficus (Moraceae) by birds in areas with different levels of forest fragmentation in São Paulo StateMariana Esther Lapate 27 November 2009 (has links)
Ficus (Moraceae) é o gênero mais importante para animais frugívoros nas florestas tropicais, considerado um recurso-chave durante períodos de escassez de frutos. Esses animais dispersam suas sementes e contribuem para a manutenção das populações de figueiras. A perda de hábitat e a fragmentação da paisagem podem afetar a sobrevivência de populações e a composição de comunidades e suas interações. Os objetivos deste trabalho foram (1) comparar a frugivoria e a dispersão de sementes por aves frugívoras entre espécies de Ficus encontradas em remanescentes de Floresta Estacional Semidecidual e (2) testar se a perda de hábitat influencia a frugivoria e dispersão de sementes dessas árvores. Estudamos três áreas de reservas florestais no Estado de São Paulo, sudeste do Brasil, com diferentes áreas e nível de fragmentação da paisagem: Parque Estadual do Morro do Diabo (33.845 ha), Estação Ecológica dos Caetetus (2.176 ha) e Estação Ecológica de Ribeirão Preto (180 ha). Ao longo de 443 h de observação árvore-focal, registramos todas as espécies de aves frugívoras se alimentando de figos e estimamos a taxa de remoção de figos e sementes. Como nossos dados não se adequaram a métodos estatísticos tradicionais, utilizamos a reamostragem por bootstrap para estimar os intervalos de confiança das taxas de remoção e dispersão de sementes e testar as diferenças entre as espécies de figueira e as áreas de estudo. Trinta e duas espécies de aves foram observadas consumindo figos de seis espécies de Ficus. As figueiras que apresentam figos verdes, de tamanho médio ou grande, foram consumidas por uma boa variedade de espécies, mas apenas uma das aves de cada assembléia foi responsável pela remoção da maior parte dos figos. Ficus luschnathiana, com figos pequenos e vermelhos quando maduros, foi consumida de forma mais equitativa pelos seus visitantes, e apresentou taxa de dispersão de sementes significativamente maior que as demais espécies. A fragmentação reduziu a remoção de figos e sementes de F. citrifolia. Ficus eximia não demonstrou nenhum padrão claro, uma vez que só teve um consumo expressivo em um microhábitat específico, não incluído na análise da fragmentação. No fragmento menor, aves de grande porte, consideradas bons dispersores de sementes, não foram encontradas e as aves menores e generalistas não compensaram a perda do serviço de dispersão de sementes. Essas espécies de Ficus, no entanto, devem depender mais de morcegos e macacos-prego que das aves no processo de dispersão, e devem inclusive ser beneficiadas pelo aumento na extensão de bordas. Mesmo que essa comunidade de figueiras no fragmento menor não estabeleça uma interação forte com a avifauna, ainda é relevante testar se a baixa densidade de indivíduos de F. luschnathiana é consequencia da ausência de bons dispersores de sementes. / Ficus (Moraceae) is the most important plant genus for tropical frugivores and is considered a keystone resource during periods of general fruit scarcity. Frugivorous animals disperse their seeds and contribute for the maintenance of fig trees populations. Habitat loss and landscape fragmentation can affect species survival as well as community composition and their interactions. The goals of this study were (1) to compare the frugivory and seed dispersal by frugivorous birds among Ficus species found in Semideciduous Seasonal Forest remnants and (2) to assess whether habitat loss influences frugivory and seed dispersal of these trees. We studied three natural reserves in São Paulo State, southeastern Brazil, which differ in area and landscape fragmentation: Morro do Diabo State Park (33.845 ha), Caetetus Ecological Station (2.176 ha) and Ribeirão Preto Ecological Station (180 ha). During 443 h focal tree observation, we registered all frugivorous birds eating figs and estimated their fruit removal and seed dispersal. As traditional statistical methods were not suitable for our data, we used bootstrap data resampling to estimate confidence limits of fruit removal and seed dispersal and test for differences between fig trees and forest fragments. A total of 32 bird species were observed consuming figs of six Ficus species. Fig trees bearing green-fruited and medium or large figs were consumed by a great diversity of birds, but showed a dominance of only one species over the others on the fruit removal. Ficus luschnathiana, whose figs are small and red when ripe, was more equally consumed by all visitors and had significantly more seeds dispersed per hour than the other species. Fragmentation reduced fruit removal and seed dispersal of F. citrifolia. Ficus eximia did not show a clear pattern, once it was only highly consumed by birds in a specific microhabitat not included on the fragmentation analysis. The effect upon other Ficus species remains to be tested. In the smallest forest remnant, large-bodied birds, considered high quality seed dispersers, cannot be found anymore and small and generalist birds are not able to compensate for this loss on seed dispersal service. These Ficus species, however, may depend more upon bats and capuchin monkeys than on birds for seed dispersal, and may even benefit from the increase in extension of forest edges. Even if this Ficus community as a whole does not seem to interact with birds, it remains relevant, though, to test whether F. luschnathiana\'s low density in the smallest fragment is due to the absence of good seed dispersers.
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Paleofauna e paleoambientes do Pleistoceno Superior no município de Jaguarari, norte da BahiaXavier, Márcia Cristina Teles 29 February 2012 (has links)
Conselho Nacional de Desenvolvimento Científico e Tecnológico / Numerous studies of paleo-environmental reconstruction (vegetation and climate) have been carried out in southern, southeastern, and central Brazil, based on the pollen deposited in lacustrine and peatbog sediments. Such studies are scarce in the Brazilian Northeast, however. Studies of this type permit the development of inferences on the ecological processes affecting local plant and animal communities. The present study aimed to understand the paleo-environmental dynamics of a tank-type fossil outcrop in the municipality of Jaguarari, part of the Senhor do Bonfim micro-region in central-western Bahia state, located within the Caatinga biome. Fossils of neopleistocene mammals were collected, together with samples of soil and plants for isotopic analyses (13C). Six mammalian taxa were identified: Eremotherium laurillardi, Notiomastodon platensis, Toxodontinae, Felidae, Panochthus greslebini, and Equus (Amerhippus) neogeus. The paleo-environmental interpretation of the ecological characteristics of these taxa indicated that this assemblage was found with a predominantly open environment, associated with areas of denser habitat. The isotopic analysis indicated that the vegetation of the area has been stable up to the present day, with no exchange of C3 to C4 at the study sites. The presence of charcoal fragments at different depths in the soil indicates the occurrence of wildfires throughout the Holocene, possibly as a consequence of a relatively much drier climate. The wildfires may have been anthropogenic, given that the evidence was found in association with vestiges of ancient human populations. The dynamics of the vegetation observed at the site to the depth excavated indicated a predominance of arboreal vegetation since approximately 15,000 BP, finally returning to a more humid climate similar to that of the present day. The vegetation of the study region may not have suffered major modifications of the vegetation during the drier periods of the mid-Holocene, with forest formations predominating. During excavations, the need for a patrimonial education program was perceived, in order to promote measures for the conservation of paleontological sites, together with the local population of Lajedo II village. The results of this initiative indicated that this approach is a useful tool for the generation of information on natural patrimony, basic concepts of conservation, the legal basis for this process and the possible sanctions that enforce it, as well as contributing to the development of new knowledge and perspectives in the local community. / Estudos de reconstrução paleoambiental (vegetação e clima) são mais abundantes nas regiões Sul, Sudeste e Central do Brasil utilizando pólen depositado em sedimentos lacustres e turfeiras. Por outro lado, na região Nordeste, trabalhos análogos são mais escassos. A partir deste tipo de estudo é possível inferir sobre os processos ecológicos associados às comunidades vegetais e animais. Neste trabalho buscou-se entender a dinâmica paleoambiental ocorrida em um afloramento fossilífero do tipo tanque localizado no município de Jaguarari, o qual faz parte da Microrregião de Senhor do Bonfim Centro-Oeste da Bahia, em domínio de Caatinga. Para tanto, coletou-se fósseis de mamíferos neopleistocênicos; solo e plantas para análises isotópicas (13C). A fauna identificada é composta por 6 taxa: Eremotherium laurillardi; Notiomastodon platensis; Toxodontinae; Felidae; Panochthus greslebini; Equus (Amerhippus) neogeus. Interpretações paleoambientais das características ecológicas dos taxa sugerem que este conjunto faunístico estava associado há um ambiente com predomínio de áreas abertas em associação com fisionomias mais fechadas. Os resultados isotópicos apontam para estabilidade vegetacional na região até o presente, sem troca de vegetação C3 para C4 nos locais estudados. A presença de fragmentos de carvão soterrados em várias profundidades do solo sugere a ocorrência de paleoincêndios durante todo o Holoceno, com a possível influência de um clima mais seco para a região. Estes incêndios podem ser de natureza antrópica, já que foram encontrados alguns vestígios de ocupação de populações antigas na região. A dinâmica da vegetação observada para Jaguarari, até a profundidade de solo estudada, sugere tendência a uma vegetação arbórea dominante, desde aproximadamente 15.000 anos AP. com retorno a um clima mais úmido e provavelmente similar ao atual. A região estudada, possivelmente, não sofreu modificação acentuada na vegetação no médio Holoceno, quando do estabelecimento de períodos mais secos, permanecendo com áreas de vegetação de floresta. Durante os trabalhos de escavação dos fósseis, sentiu-se a necessidade de desenvolver um trabalho de Educação Patrimonial junto à população do povoado de Lajedo II, para subsidiar ações de conservação dos sítios paleontológicos. Os resultados mostraram que esta ferramenta é um instrumento capaz de fornecer informações sobre patrimônio, noções de conservação, leis que normatizam estas práticas, as sanções legais previstas, como também, proporcionar a construção de novos conceitos e uma aprendizagem significativa na comunidade alvo.
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Uso e características dos fragmentos florestais urbanos da cidade de Manaus/AMGontijo, Jessica Cancelli Faria 26 August 2008 (has links)
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Previous issue date: 2008-08-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The main goal of this study is to relate FFUs spatial distribution and area with the characteristics as of avocation from the city. 56 FFU were identified ranging in size between three and 578.6 hectares. Regarding the property regime 26 FFU were private and 30 were public land. Preliminary results on the status of property (private = 1; public = 0) showed significant relationship between the status and FFU areas. Six categories of FFU occupation were identified in the urban areas surroundings the fragments. The FFU varied 17 to 90 years in agreement to the history of the city expansion and producing FFU from 2.25 to 14.88 km from city downtown and the distance of these fragments as far as the city centre variation 2.25 the one 14.88 Km.
The relation between human population and natural resources listed 32 uses in four fragments: UFAM, SUMAÚMA, SESI an MINDÚ, separated in customs and socials uses. The use curve of accumulation based in participatory diagnostic, interviews did not establishment and suggest there are another uses did not related, whereas that must add in around 40 customs. The most diversity were in SESI, as well as richness and dominance,
and suggests that did not relation between area and uses in fragments. / O estudo investigou a variação nos atributos de fragmentos florestais urbanos (FFU)
(representando 48% da área total da cidade) em relação ao seu grau de isolamento dentro da área urbana. Foram estudados 56 FFUs entre três e 578 ha (0.578 km²) distribuídos em seis zonas administrativas (norte, sul, leste, oeste, centro-sul, centro-oeste), sendo 26 FFU de
propriedade privada e 30 públicos, entre 10 e 90 anos de idade. A distância dos fragmentos em relação ao centro da cidade variou de 2,25 a 14,88 km. Resultados de regressão logística sugerem que quanto maior a distância do centro significativamente menos fragmentos são
de propriedade privada. A matriz do entorno dos fragmentos apresentou seis categorias sendo que em fragmentos menores do que 90 ha a categoria habitação foi mais abundante (48,9%) e em fragmentos maiores do que 90 ha foi a categoria instituição (42,8%). A relação entre os recursos naturais existentes nos FFU e as populações humanas foi estudada em quatro fragmentos: UFAM (578ha), SUMAÚMA (51ha), SESI (52ha) e MINDÚ (40ha). Foram listados 32 recursos utilizados sendo divididos em usos de subsistência e usos sociais. A curva de acumulação de uso de recursos baseada na coleta de
informação através de mapeamento participativo, entrevistas individuais com usuários e visitas de campo ainda não se estabilizou, sugerindo haver mais usos não relatados, considerando que devem incluir perto de 40 usos e recursos diferentes. A maior diversidade de usos de recursos foi no fragmento do SESI, assim como riqueza e dominância, e sugere que não haja relação entre a área do fragmento e a diversidade de usos, pois o fragmento da UFAM era o maior dentre os quatro fragmentos (578ha), mas apresentou a segunda maior diversidade. Os fragmentos do SUMAÚMA e SESI tiveram maior similaridade de usos, sendo 14 usos em comum, enquanto o MINDÚ se destacou dos demais na análise de
agrupamento.
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Fragmentos de Mata e PlantaÃÃo de cafÃ: valoraÃÃo dos bens e serviÃos do Ecossistemas / Fragments of forest and plantation of coffee: valuation of the goods and services of ecosystemPedro Silveira MÃximo 09 August 2006 (has links)
O pouco que resta do bioma da mata AtlÃntica no Estado de Minas Gerais està representado na forma de fragmentos, como à o caso de ViÃosa-MG, regiÃo considerada de extrema importÃncia ecolÃgica e que vem sendo intensivamente degradada nos Ãltimos anos, pela atividade antrÃpica. Esta paisagem fragmentada deve-se, principalmente a aÃÃes dos produtores de cafà que destroem suas Ãreas de PreservaÃÃo Permanente (APPâs) e Reservas Legais RLâs. Somente no municÃpio de ViÃosa-MG 4000 hectares foram plantados com cafÃ, causando assim, uma reduÃÃo drÃstica das matas nativas da regiÃo, bem como uma reduÃÃo significativa no que os economistas chamam de Bens e ServiÃos de Ecossistema - BSE. Assim, foram selecionados os cafeicultores do PRO-CAFà (OrganizaÃÃo local de cafeicultores), como pÃblico alvo, creditando a tal escolha o fato desse grupo caracterizar o cafeicultor de regiÃo montanhosa. Nesse sentido, essa pesquisa teve como objetivo determinar as principais variÃveis que afeta a percepÃÃo ambiental dos cafeicultores, e valorar monetariamente as matas, tendo em vista que isso pode ser uma estratÃgia Ãnica para preservaÃÃo ambiental. Para tal, foi elaborado um formulÃrio com 33 perguntas envolvendo informaÃÃes sobre caracterÃsticas sÃcio - econÃmicas, o uso da metodologia de valoraÃÃo de contingente (MVC), e do veÃculo de pagamento dos âJogos de Lancesâ que revelou a DisposiÃÃo a Aceitar uma CompensaÃÃo (DAC) na troca de um hectare de cafà por um hectare de mata. Os resultados demonstraram que os cafeicultores possuem uma boa impressÃo sobre a importÃncia dos fragmentos, sendo importante destacar que os entrevistados vincularam a existÃncia das APPâs e RLâs como responsÃvel direto pela provisÃo dos BSEâs. Contudo constatou-se que mesmo cientes dessa importÃncia nÃo tinham uma atitude conservacionista, associando a isso, a falta de uma educaÃÃo ambiental clara e a impunidade. A estimaÃÃo da DAC mostrou que caso o governo esteja disposto a aumentar a provisÃo de mata em 70 hectares, ele deveria despender 254.200 reais por ano, tratando apenas dos cafeicultores vinculados ao PRO-CAFà e 1.147.000 reais por ano caso o governo estivesse disposto a trabalhar com toda populaÃÃo de cafeicultores de ViÃosa, o que representa 314 hectares de mata / The little that still remains of the biome of the Atlantic forest, in the state of Minas Gerais, Brazil, is represented in the form of fragments, which is the case of the county of ViÃosa, a region considered to be of extreme ecological importance and that has been intensely degraded over the last years, due to anthropic activity. This fragmented scenery is due mainly to the actions of coffee planters that destroy its permanent preservation areas (PPAs) and legal reserves (LRs). Considering only the county of ViÃosa 4000 hectares has been planted with coffee, causing in this way, a drastic reduction of the native forest of the region, as well as a significant reduction of what economists call ecosystem goods and services - EGS. Thus, the coffee planters in the PRO-CAFà (organization of local coffee planters) were selected as target public, this choice being credited to the fact that this group characterizes the coffee planters of the mountainous region. In this respect this investigation had as a purpose to determine the main variables that affect the coffee planters environmental perception, and monetarily valorization of the forests, since this might be the a single strategy for environmental preservation. For this a formulary was developed with 33 questions involving information on socio economical characteristics, the use of the methodology of contingent valorization (MCV), and the vehicle of payment of the âoffer gameâ that reveled the willingness to accept a compensation (WAC) in exchange of a hectare of coffee for a hectare of forest. The results show that the coffee planters have a good understanding of the importance of the fragments, being important to stress that the interviewees linked the existence of the PPAs and LRs as the direct responsible for the provisions of the EGSs. However, it was found that even though aware of this importance a conservationist attitude was not perceived, associated to this the absence of a clear environmental education, as well as impunity. The DAC estimate shows that in the case that the government is willing to increase the provision of forest to 70 hectares, they should pay out 254,200 reais (around 116,000 dollars), dealing only with the coffee planters linked to the PRO-CAFà organization and 1,147,000 reais per year if the government should be willing to work with the entire coffee planters population in the county of ViÃosa, which represents 314 hectares of forests.
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Fragmentos e totalidades no conhecimento científico e ensino de ciências / Fragments and wholes in scientific knowledge and in science teachingJosé André Peres Angotti 25 April 1991 (has links)
Ciência e Tecnologia são considerados empreendimentos culturalmente referenciados, p ira além de históricos e sociais. A categoria dual Fragmentos e Totalidades é contemplada tanto na criação científica quanto no seu ensinar-aprender. Diante das prevalências do conhecimento fragmentado e do componente inercial que resiste ao novo na educação científica dos vários níveis de escolarização, é proposta a adoção de conceitos supradisciplinares e unificadores para ancorar o tratamento didático-pedagógico desse conhecimento. As possibilidades de conquistas de ganhos culturais pelos educandos na estruturação de unidades interligadas de saber são discutidas e avaliadas. / Science and Technology are determined by cultural relations, besides their constraints with historical and societal process. The dualistic category Fragments & Wholeness is considered in epistemological and pedagogical references. In opposition to the fragmentation process and the inercial component to the new knowledge that still remains in all the Science Education levels, the adoption of unifying concepts as anchors for the didatic treatment of this knowledge is proposed. The possibilities of cultural acquirements conquered by the students during the construction of knowledge structures in the perspective of linkages and unities are discussed and evaluated.
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Análise da Avifauna da RPPN Rio dos Pilões (Santa Isabel, SP), visando à conservação das espécies de um "Hotspot" da Mata Atlântica / Analysis of Avifauna from RPPN Rio dos Piloes (Santa Isabel, SP), aim at on the conservation from the species by one hotspot from Brazilian Atlantic ForestLuccas Guilherme Rodrigues Longo 13 July 2007 (has links)
A maior parte da biodiversidade global está concentrada nas florestas tropicais. Um dos principais fatores que ocasionam o desaparecimento dessa diversidade é a perda de hábitats pelas ações antrópicas, como o desmatamento e o crescimento urbano desordenado. Fragmentos florestais que possuem elevada biodiversidade, altas taxas de endemismo e fortes pressões antrópicas, são chamados de hotspots. A Floresta Atlântica é um dos hotspots brasileiros mais devastados. Por serem sensíveis as alterações do ambiente, as aves são consideradas importantes bioindicadores da qualidade dos ecossistemas. O presente estudo foi realizado na Reserva Particular do Patrimônio Natural (RPPN) Rio dos Pilões, em uma região antropizada e com remanescentes de Mata Atlântica (Floresta Ombrófila Densa Montana), localizada no município de Santa Isabel, no Estado de São Paulo. A RPPN pertence a um empreendimento imobiliário que visa à integração entre as atividades de proteção dos recursos naturais com as ações humanas. Com o intuito de conhecer e analisar a comunidade de aves da RPPN estudou-se três ambientes principais: um ambiente de campo antrópico (ACA), um ambiente antrópico inundado (AAI) e um ambiente florestal antropizado (AFA). O estudo foi realizado de maio de 2005 a maio de 2006, utilizandose o método de captura e recaptura com redes-neblina e o método de observações em trajetos irregulares, sendo o primeiro aplicado no AFA e o segundo no ACA e no AAI. Foram registradas 141 espécies de aves em um total de 1.824 horas de trabalhos. Estas espécies estão distribuídas em 20 ordens, 46 famílias, 125 gêneros, resultando em 2.243 indivíduos observados, 184 capturados e 17 recapturados. A curva acumulada de espécies não mostrou tendência à estabilização, sugerindo que o esforço de coleta despendido não tenha sido suficiente para amostrar toda a comunidade, indicando assim que novas espécies possam ser registradas. Os Não-Passeriformes somaram 56 espécies, com maior representatividade nas famílias Ardeidae e Trochilidae. Os Passeriformes foram os mais representativos, com 85 espécies, sendo Tyrannidae a de maior número. O ambiente que apresentou maior riqueza foi o AAI com 85 espécies, seguido pelo AFA com 72 e o ACA com 52 espécies. As espécies mais freqüentes (FR) foram Patagioenas picazuro no ACA, Thraupis sayaca no AAI e Chiroxiphia caudata no AFA. A análise da freqüência de ocorrência (FO) mostrou que a maioria das espécies teve FO abaixo de 25%, enquanto que poucas espécies apresentaram FO maior que 75%. As espécies com maiores FO foram Vanellus chilensis, Pitangus sulphuratus, T. sayaca e Basileuterus leucoblepharus. A comunidade foi agrupada em 13 guildas tróficas, sendo a insetívora a de maior predomínio em todos os ambientes. Das espécies registradas, 04 são migrantes e 03 estão ameaçadas de extinção. Embora a RPPN apresente um elevado grau de degradação, possui elevada riqueza e diversidade de espécies de aves, fator este que pode contribuir não somente para aumentar seu valor de conservação, como também auxiliar na implantação de projetos de restauração ecológica dos remanescentes. / The most of global biodiversity is intent in the tropical forests. One of the main factors that they cause the disappearance of this diversity is the loss of habitats for the anthropic actions, as the deforestation and the disordered urban growth. Forest fragments that possess high biodiversity, high taxes of endemism and forts anthropics pressures, are called hotspots. The Atlantic Forest is a Brazilian hotspots more deforested. By being sensible the alterations of the environment, the birds are considered the most important bioindicators of the quality of ecosystems. The present study River of the Piloes was carried through in the Particular Reserve of Patrimony Natural (RPPN), in an anthropic region and with remainders of Atlantic Forest, located in the city of Santa Isabel, State of Sao Paulo. The RPPN belongs to a real estate enterprise that aims at the integration enters the activities of protection of the natural resources with the actions human beings. Whit the intention to know and to analyze the community of birds of the RPPN one studied three main environments: environment of anthropic field (ACA), anthropic environment flooded (AAI) and the anthropic forest environment (AFA). The study has been at the May of 2006 to May of 2005, using the capture method with mist-nets and the method of irregular courses, being the first one applied in the AFA, and as in the ACA and the AAI. They had been registered 141 species of birds in a total of 1.824 working hours. These are distributed in 20 orders, 46 families, 125 genders, resulting in 2.243 observed individuals, 184 captured and 17 recaptured. The accumulated curve of species as soon as did not show to trend to the stabilization, suggesting that the expended effort of collection has not been enough to show to all the community, indicating new species can be registered. The Non- Passeriformes had added 56 species, with bigger representation in the families Ardeidae and Trochilidae. The Passeriformes had been most representative, with 85 species and in Tyrannidae are the bigger. The environment that presented greater wealth was the AAI with 85 species, AFA with the 72 and the ACA with 52 species. The most frequent species (FR) had been Patagioenas picazuro in ACA, Thraupis sayaca in AAI and Chiroxiphia caudata in AFA. The analysis of the occurrence frequency (FO) showed that the majority of the species had FO below of 25%, whereas few species had presented bigger FO that 75%. The bigger with FO had been Vanellus chilensis, Pitangus sulphuratus, T. sayaca and Basileuterus leucoblepharus. The community was grouped in 13 trophic guilds, being the insectivora of the bigger predominance in all environments. Of the registered species, 04 are migrants and 03 are threatened of extinguishing. Although the RPPN presents one high degree of degradation, it possess high wealth and diversity of species of birds, it is the factor that can contribute to not only increase its value of conservation, as well as auxiliary in the implantation of projects of ecological restoration of the remainders.
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Diversidade e riqueza funcional de assembleias de aves na Mata Atlântica / Functional richness and diversity of bird assemblages in the Atlantic ForestAlex Augusto de Abreu Bovo 02 February 2016 (has links)
A composição das assembleias de aves sofre mudanças de acordo com alterações na paisagem em que ocorrem. A transição de florestas para outros usos do solo pode modificar também o conjunto de funções e, consequentemente, impactar os processos ecológicos desempenhados pelas aves. Desse modo, a presente dissertação teve como objetivos principais: 1) identificar características da estrutura da paisagem que apresentem relação com a diversidade e riqueza funcional de assembleias de aves; e 2) identificar alterações morfológicas na assembleia de aves consumidoras de frutos e discutir seus possíveis impactos no processo de dispersão de sementes. Para isso, um levantamento bibliográfico foi realizado resultando em um banco de dados com 48 assembleias de fragmentos florestais da Mata Atlântica oriundas de 33 estudos. Para cada fragmento foi utilizado um buffer de 2000 m de raio para criar uma paisagem focal, o uso do solo foi classificado em \'floresta\' ou \'não-floresta\', e métricas foram calculadas. As assembleias de aves foram caracterizadas a partir de índices de composição de diversidade e riqueza funcional, usando diversas características morfológicas e de habitat. Regressões lineares e modelos mistos foram usadas para investigar a relação entre características da estrutura da paisagem e i) diversidade e riqueza funcional de aves florestais (obj. 1) e ii) riqueza de espécies, diversidade funcional e características morfológicas para aves consumidoras de frutos (obj. 2). Os resultados mostraram que a área do fragmento, juntamente com área nuclear foram as características mais importantes em relação à manutenção das funções na assembleia de aves florestais, demonstrando a importância de grandes áreas florestais. Para as alterações morfológicas, a diminuição na quantidade de cobertura florestal foi relacionada à diminuição de valores de massa corpórea, índice de asa e largura de bico. A redução desses valores pode implicar na dispersão de sementes menores, na redução de dispersão de espécies com sementes grandes e na redução da área abrangida pela chuva de sementes. / Bird assemblages composition suffer alteration according to landscape changes. The substitution of forest by others land uses can affect the group of functions, and consequently, the ecological process played by birds. This dissertation had two main goals: 1) to identify landscape metrics which has relation with functional diversity and richness of bird assemblages; and 2) to identify morphological changes in fruit-eater bird assemblage and discuss the potentials impacts on the seed dispersal process. To reach this, a database was created using literature data, with 48 bird assemblages of Atlantic Forest remnants from 33 studies. From the most central point of each fragment, was created a focal landscape of 2000 meters buffer, the land use was classified in \'forest\' or \'non-forest\' and metrics was calculated. The index of bird assemblages used were composition and functional diversity and richness, calculated using morphological and habitat traits. Linear regression and mixed models were used to investigate relationships between landscape metrics and i) functional diversity and richness of forest birds (obj. 1) and ii) functional diversity, species richness and morphological traits of fruit-eater birds (obj. 2). The results showed that fragment area and core area were the most important traits to preserve the functions in bird assemblage, demonstrating the importance of big blocks of forest. To morphological changes, the decreasing on forest over was related to decreases on values of: body mass, hand-wing index and bill width. The reduction of these values can promote the dispersal of smaller seeds, reduction on dispersal of plants with big seed and decreases of area cover by seed rain.
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Determinação de tamanhos de parcelas para otimização amostral em remanescentes de florestas nativas em Itatinga-SP / Determination of plot size for optimization of sampling in remnant natural forests in Itatinga - SPRenan Fischer Goffe 28 August 2015 (has links)
Devido a atual situação de degradação da vegetação remanescente do estado de São Paulo, existe com relativa urgência uma grande necessidade de estudos que apoiem práticas de monitoramento e conservação dos fragmentos florestais para melhor gerenciá-los. Parte destas demandas é referente ao campo de amostragem, um conjunto de técnicas específicas para pesquisas ecológicas, onde a unidade amostral (parcela) é um dos fatores determinantes de sua eficiência. O objetivo deste estudo foi definir e propor o tamanho ideal de parcelas para otimizar o inventário florestal de áreas remanescentes de Floresta Estacional Semidecidual (FES) e de Cerrado (CER), assim como também de Floresta Estacional Semidecidual em processo de regeneração (FESreg). O trabalho foi realizado na Estação Experimental de Ciências Florestais de Itatinga - SP (EECFI/ESALQ/USP), visando à otimização da amostragem para as variáveis diâmetro à altura do peito (DAP), altura total, densidade populacional, área basal, volume total, volume de fuste, volume de galho, biomassa e índices de Shannon, de Simpson e de Riqueza. O delineamento experimental foi realizado com o auxílio de uma grade amostral, na qual foram distribuídas aleatoriamente 15 parcelas de 1.000 m2 cada, sendo cinco repetições em cada uma das três fisionomias florestais estudadas, onde todos os indivíduos com circunferência à altura do peito (CAP) acima de 16 cm foram mensurados e identificados. Cada unidade amostral foi divida em 10 subparcelas, permitindo assim avaliar os tamanhos de 100 m2 (10X10 m), 200 m2 (10x20 m), 300 m2 (10x30 m), 400 m2 (10x40 m e 20x20 m), 500 m2 (10x50 m), 600 m2 (20x30 m), 800 m2 (20x40 m) e 1.000 m2 (20x50 m). Os tratamentos (tamanhos) foram analisados através de análise de variância, métodos da curvatura máxima e esforço amostral. Com base nos resultados, recomenda-se respectivamente para Cerrado, Floresta Estacional Semidecidual e Floresta Estacional Semidecidual em regeneração o uso de parcelas de 200 m2, 200 m2 e 600 m2 para estimar qualquer uma das variáveis dendrométricas ou parcelas de 200 m2 para estimativas apenas de diversidade. Esses resultados são bastante úteis por embasarem uma série de pesquisas ambientais, deste modo, aperfeiçoando operações de inventário florestal para que o processo de amostragem não tenha esforço amostral desnecessário sem prejuízo da precisão. / Because of advanced status of degradation of the natural forest of the state of São Paulo, there is a relatively urgent need for the conduction of studies to support monitoring and conservation practices of forest fragments for their better management. Of these needs refers to sampling, with the use specific techniques for ecological research, in which characteristics of the sampling unit is of outmost importance. The objective of this study was to define and propose optimal plot sizes for inventories remnant areas of Semideciduous Forest (FES) and Cerrado (CER), as well as of Semideciduous forest in regeneration process (FESreg). The study was conducted at Estação Experimental de Ciências Florestais de Itatinga - SP (EECFI/ESALQ/USP), aiming to optimize the sampling for the variables diameter at breast height (DBH), total height, density, basal area, wood volume, volume of stem, branch volume, biomass as well as Shannon, Simpson and Richness indexes. The experiment was carried out with the aid of a sampling grid, in which 15 sampling units of 1,000 m2 each were randomly determined, with five replicates in each type of forest five. In each unit, all trees with circumference (girth) at breast height (GBH) above 16 cm were measured and identified. Each unit was divided into 10 subplots, so as to allow the evaluation in areas of 100 m2 (10x10 m), 200 m2 (10x20 m), 300 m2 (10x30 m), 400 m2 (10x40 and 20x20 m), 500 m2 (10x50 m), 600 m2 (20x30 m), 800 m2 (20x40 m) and 1,000 m2 (20x50 m). Treatments (sizes) were analyzed by analysis of variance, maximum curvature methods and sampling effort. The results indicated that for areas of Cerrado, Semideciduous Forest and Semideciduous Forest in regeneration plot sizes of 200 m2, 200 m2 and 600 m2 respectively should be used to estimate any of the dendrometric variables or plot size of 200 m2 should be used for estimates of diversity only. These results are very useful for a number of environmental researches, improving forest inventory operations by preventing unnecessary sampling effort without jeopardizing accuracy.
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Avaliação de bactérias fototróficas em lagoas de estabilização: diversidade, purificação e identificação / Evaluation of phototropic bacteria in stabilization lagoons: diversity, purification and identificationNora Katia Saavedra del Aguila 01 June 2007 (has links)
As bactérias fototróficas freqüentemente apresentam florescimentos em lagoas de estabilização utilizadas no tratamento de esgoto sanitário, formando uma camada de cor púrpura na sua superfície. Portanto, o estudo das condições que propiciam tais florescimentos, a diversidade microbiana, o potencial de remoção da matéria orgânica e o estabelecimento das relações entre tais conhecimentos, permitem compreender o metabolismo do sistema. Nesse sentido, o objetivo deste trabalho foi avaliar a diversidade de bactérias (domínio Bacteria), bactérias fototróficas púrpuras e bactérias redutoras de sulfato (BRS) em lagoas de estabilização do Vale do Ribeira (Cajati, SP). Para tal, foram realizadas coletas sazonais (primavera, verão, outono e inverno) na sub-superfície, camada intermediária e interface água-sedimento, em dois horários (14:00 h e 02:00 h), nas lagoas anaeróbia e facultativa. Para analisar os diferentes grupos de microrganismos, utilizou-se a técnica de PCR/DGGE, com primers específicos. Nas análises de filogenia realizou-se o seqüenciamento parcial do gene RNAr 16S e da subunidade M do centro de reação fotossintético das bactérias fototróficas púrpuras. Análises físico-químicas, tais como sulfato, DQO, sólidos, nitrogênio e fósforo foram realizadas, além da determinação da concentração de oxigênio dissolvido, pH, temperatura e radiação solar fotossinteticamente ativa incidente. No outono observou-se maior diversidade de microrganismos do domínio Bacteria, bactérias fototróficas púrpuras e BRS, enquanto na primavera foi verificada a menor diversidade desses microrganismos para as duas lagoas. Na lagoa facultativa foi observada maior diversidade do domínio Bacteria e das BRS em relação à lagoa anaeróbia. Verificou-se maior diversidade de bactérias fototróficas púrpuras na lagoa anaeróbia, caracterizada por duas populações predominantes nas quatro estações e nas diferentes profundidades. A concentração de matéria orgânica (DQO) variou de 60,3 mg/L (inverno) a 298,0 mg/L (primavera) e a maior concentração de sulfato observada foi de 51,0 mg/L (inverno). Bacilo curvo Gram negativo, semelhante à bactéria fototrófica púrpura não sulfurosa, presente em amostra proveniente da sub-superfície da lagoa anaeróbia foi purificado e apresentou 92% de similaridade com Rhodopseudomonas palustris. Em ambas as lagoas foram identificadas bactérias semelhantes a Chromobacterium suttsuga (95%), Clostridium sp. (99%), Rhodobacter sphaeroides (99%), Rhodopseudomonas palustris (99%), Lampropedia hyalina (97%), Campylobacter fetus (99%), Desulfovibrio vulgaris (95%), Rhodospirillum rubrum (95%) e diferentes bactérias não cultivadas. / The phototrophic bacteria frequently blossom in the stabilization lagoons that are used in sanitary sewer treatment, forming a purple layer on its surface. Therefore, the study of the conditions that propitiate such blooms, the microbial diversity, the removal of the organic matter and the establishment of the relations between them permit to understand the metabolism of the system. The objective of this work was to evaluate the diversity of the bacteria (Bacteria domain), purple phototrophic bacteria and sulfate reducing bacteria (SRB) in stabilization lagoons of Vale do Ribeira (Cajati - SP). For this, it was made seasonal collects (spring, summer, autumn and winter) from the sub-surface, intermediate layer and interface water-sediment, at two times (14:00 h and 02:00 h) of the anaerobic and facultative lagoons. To analyze the different groups of microorganisms it was used the PCR/DGGE technique, with specific primers; for the phylogenic analysis it was realized the DNA partial sequencing of the 16S RNAr gene and of the subunit M of the photosynthetic center of reaction of the purple photosynthetic bacteria. It was determined: the concentration of dissolved oxygen, pH, temperature and photosynthetically active incident solar radiation, and the physical-chemistry analysis as: COD, solids, nitrogen and phosphorus. In the autumn it was observed greater diversity of microorganisms of the Bacteria domain, the group of the purples phototrophic bacteria and SRB, while in the spring it was verified minor diversity of these microorganisms in the two lagoons studied. In the facultative lagoon it was observed greater diversity of the Bacteria domain and of the SRB with respect to the anaerobic lagoon. It was verified greater diversity of the purple phototrophic bacteria in the anaerobic lagoon, of what in the facultative lagoon, which was characterized by the two predominant populations in the four seasons and in the different points of collect. The concentration of the organic matter (COD) varied from 60,3 mg/L (winter) to 298,0 mg/L (spring) and the greater concentration of sulfate observed was of 51,0 mg/L (winter). Arched bacillus Gram-negative similar to purple not sulfurous bacteria, from a sample of the sub-surface of the anaerobic lagoon was purified and presented 92% of similarity with Rhodopseudomonas palustris. In both lagoons it was identified bacteria similar to Chromobacterium suttsuga (95%), Clostridium sp. (99%), Rhodobacter sphaeroides (99%), Rhodopseudomonas palustris (99%), Lampropedia hyalina (97%), Campylobacter fetus (99%), Desulfovibrio vulgaris (95%), Rhodospirillum rubrum (95%).
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Insights into Substrate Specificity in Sortase Enzymes from Structural Studies on a Novel Class of Housekeeping Sortase (SrtE) Identifying Functionally Important Cis-Peptide Containing Segments in Proteins and their utility in Molecular Function AnnotationDas, Sreetama January 2016 (has links) (PDF)
Understanding protein function is fundamental to the fields of protein engineering and drug design. While most of the previous efforts in this direction have focused on the sequence-structure-function paradigm, recent studies have pointed to protein dynamics as being integral to its activity. The work in the current thesis follows this overall theme of obtaining insights into protein function from its structure and dynamics. It can be broadly divided into two sections. In the first section, the thesis candidate has tried to elucidate the residues modulating the substrate specificity of a particular family of enzymes, known as sortases, through structural and computational studies (including dynamics simulations) on a novel member in the family. This work has been carried out in collaboration with Dr. R.P. Roy, National Institute of Immunology, New Delhi (biochemical characterization was performed by Mr. Vijay Pawale at Dr. Roy‟s laboratory). In the second half of this thesis, the candidate has described a structure-based method involving the use of cis-peptide containing segments for the function annotation of proteins. The incorporation of dynamics information leads to an improvement of our annotation approach, which is also demonstrated. This part of the work has been carried out in collaboration with Dr. Debnath Pal, Department of Computational and Data Sciences, Indian Institute of Science. Following is a chapter-wise description of the overall layout of the thesis.
Section I: Insight into substrate specificity in sortase enzymes from structural studies on a novel housekeeping sortase of class E (SrtE)
Chapter 1| A brief account of sortases: This chapter provides a brief survey of the literature on sortases and the scope of the work presented in the thesis.
Many surface proteins in Gram-positive bacteria are incorporated into the cell wall through covalent ligation by a class of cysteine transpeptidases known as Sortase. These surface proteins contain a cell wall sorting signal (CWSS) which is recognized by sortase, enzymatically cleaved and subsequently joined covalently to the pentaglycine branch of lipid II (a peptidoglycan precursor) in general, which is finally incorporated into the peptidoglycan cell wall.
Six classes of sortases have been identified on the basis of their sequence. These sortases differ in the substrate motif that they recognize and the function performed. The class A sortase (SrtA) is expressed ubiquitously in Gram-positive bacteria. It is involved in the cell surface anchoring of a large number of functionally distinct proteins which contain an LPXTG recognition motif in their CWSS, and is referred to as the „house-keeping‟ sortase. Sortases of other types are not ubiquitous and are meant to perform specialized functions. Sortase B is involved in iron acquisition, sortase C in pilus formation and sortase D in sporulation. The substrate motifs recognized by these sortases are, in general, different from the recognition motif in SrtA substrates. Several Gram-positive bacteria with a high GC content in their genome have been suggested to use a sortase E (SrtE) instead of SrtA to perform the housekeeping activity. These sortase sequences share low identity with sortases of classes A-D. The substrates of SrtE have been proposed to contain an LAXTG recognition motif instead of LPXTG based on genomic analyses. Class F consists of sortases from several Actinobacteria. However, the biological function of these sortases is not well understood. To date, structures of sortases from classes A-D have been determined, all of which display an eight-stranded beta barrel fold (termed the sortase fold), a conserved catalytic triad of His-Cys-Arg and a TLXTC motif at the active site (C: catalytic Cysteine; X varies across the different classes of sortases). Sortase B and C are augmented by additional secondary structure features which are absent in sortase A. SrtA from Staphylococcus aureus is the most well studied among sortases of known structure.
Several of the surface proteins attached by sortases are responsible for bacterial virulence. SrtA deletion mutants have been found to exhibit reduced virulence without affecting cell viability. Moreover, the localization of sortase in the cell membrane and the absence of eukaryotic homologs have made sortase an attractive target for the development of novel therapeutics. In addition, the transpeptidase activity of sortase has found extensive applications in biotechnology. The prototype SrtA from Staphylococcus aureus is commonly used for these applications; however, its use is limited by its obligate Ca2+ ion-dependent activity and the stringent preference for an LPXTG motif. Hence, characterization of new sortases with altered substrate recognition profiles and rational modification of
known sortases has tremendous potential for biotechnological applications and
advancements.
While sortases of classes A-D have been studied extensively to date and their structures determined, no structural data is available for a class E sortase. The thesis candidate has solved the first high resolution crystal structure of a putative housekeeping Sortase E in Streptomyces avermitilis (SavSrtE), a bacterium with a GC rich genome. Biochemical experiments performed by our collaborator on this protein have demonstrated Ca2+ independent transpeptidase activity and a preference for LAXTG-containing peptides as its cognate substrate over the LPXTG motif that is recognized by sortase A. Moreover, the protein exhibits a preference for small uncharged residues in the position succeeding the penta-peptide motif. This thesis documents the results of crystal structure analyses, molecular docking studies and dynamics simulations to understand the structural basis for these experimental findings. Finally, sequence analyses were performed to detect possible residues which modulate substrate specificity. Based on these analyses, mutations were performed. The thesis also documents the crystal structure solution and analysis of an active site mutant (residue T196 at the position X in the TLXTC motif).
Chapter 2| Methods for the analyses of Sortase E from S. avermitilis (SavSrtE): This chapter provides a description of the procedures used to carry out the thesis work.
An N-terminus truncated construct (∆N50) of wild type SavSrtE and its mutant T196V were cloned, expressed and purified in the laboratory of our collaborator, Dr. R.P. Roy (NII, New Delhi), and provided to us for structure and sequence analyses. Initially, crystallization trials were carried out on the wild type protein using commercially available screening kits and the sitting drop vapor diffusion method. The condition which gave crystals was optimized further. Finally, diffraction quality crystals were obtained in a drop containing 1μL of protein (4 mg/mL in 10 mM Tris-HCl buffer pH 7.2, 100 mM NaCl and 2 mM beta-mercaptoethanol) mixed with 1μL solution of the crystallization condition containing 1.6 M ammonium sulfate, 0.1 M citric acid at pH 3.75 using the hanging drop vapor diffusion method. The crystals were cryo-protected in a 10% sucrose solution and diffraction data collected at the European Synchrotron Radiation Facility (BM-14, ESRF). The crystals diffracted to 1.65Å. The protein crystallized in the P3221 space group with unit cell parameters a = b = 85.84Å, c = 48.20Å, α = β = 90°, γ = 120°. Calculation of Matthews coefficient indicated the presence of one molecule in the asymmetric unit. T196V mutant protein yielded diffraction quality crystals in the same condition as the wild type protein. The crystals were cryo-protected using sucrose and diffraction data were collected at the BM-14 beamline.
The mutant crystals diffracted to 1.70Å. The protein crystallized in the P3221 space group with unit cell parameters a = b = 84.98Å, c = 48.00Å, α = β = 90°, γ = 120° and one molecule in the asymmetric unit. The quality of the datasets was assessed by SFCHECK and data were found to be of appropriate quality for structure solution.
SavSrtE has low sequence identity (25 – 34%) to other class A sortases of known structure. Hence the scaled data, sequence information and model coordinates (sortase A from Streptococcus agalactiae, PDB ID: 3rcc) were submitted to the MR (molecular replacement) phasing option in the EMBL-Hamburg AutoRickshaw pipeline. The model generated from the server was used as input to PHASER for MR. The MR solution was subjected to one cycle of rigid body refinement followed by several cycles of restrained refinement using REFMAC from the CCP4 suite, with alternate rounds of inspection and manual model building in COOT for model improvement. The convergence of the refinement procedure was checked from the reduction in R-factors. The most essential refinement statistics for the final models of the wild type protein and T196V mutant are tabulated below.
Table 1 Wild type (5GO5) T196V (5GO6)
Resolution 1.65 Å 1.70 Å
Rwork / Rfree (%) 16.11 / 19.05 17.31 / 20.82
R.M.S. bond lengths (Å) 0.012 0.019
R.M.S. bond angles (°) 1.53 1.89
Average B-factors (Å2)
Protein 19.1 32.5
Water 32.6 42.4
SO42- 58.7 60.8
Gly 36.0 -
Ramachandran map statistics
Most favoured region (%) 86.8 89.8
Additional allowed region (%) 13.2 10.2
Generously allowed region (%) 0.0 0.0
Outliers (%) 0.0 0.0
The genome of S. avermitilis was searched using the ScanProsite tool to identify putative substrates, details of which are also documented in this chapter. Additionally, the thesis candidate performed Mutual Information analysis on an alignment of 1569 sortase sequences from different classes to identify the residues possibly regulating substrate specificity. Based on this analysis, mutations were performed of which the T196V mutant has been studied in this thesis.
Finally, this chapter describes the protocol used to perform protein peptide docking and subsequent molecular dynamics simulations to understand how dynamics may influence substrate specificity.
Chapter 3| Analyses of SavSrtE sequence and structure: This chapter provides a description of the analyses on the wild type SavSrtE and the T196V mutant.
The overall fold of SavSrtE is very similar to that observed in the structures of other sortases, although the sequence similarity to other classes is low. Variations are observed in the loop regions (longer β1/β2 and β6/β7 loops). The active site is comprised by residues from the β2/H1 loop, β3/β4 loop, β4 strand, β6/β7 loop, β7 strand, β7/β8 loop and β8 strand. It also does not carry any cluster of electronegative residues close to the active site and therefore, is expected to have Ca2+ ion independent activity, which is observed in biochemical experiments (Dr. R.P. Roy‟s lab). Comparison with other housekeeping sortases showed that the β6/β7 loop in SavSrtE is in a closed conformation, indicating the presence of a preformed binding pocket for the LAXTG substrate binding, contrary to the prototype SrtA from Staphylococcus aureus which requires a Ca2+ ion to stabilize the closed conformation. Moreover, a small pocket is observed adjacent to the catalytic triad which contained electron density fitting a Gly molecule. This pocket is proposed to be the binding site for the second substrate that resolves the protein-peptide intermediate through a nucleophilic attack. Our docking simulations showed that a Gly of a triglycine moiety can be positioned in this pocket.
Biochemical experiments established that SavSrtE recognizes the substrate motif LAXTG instead of LPXTG which is preferred by class A sortases. It also prefers Gly based nucleophiles as the second substrate. Additionally, the protein is found to prefer neutral residues over charged residues in the position succeeding the Gly of the LAXTG motif.
Structure analyses showed the presence of a bulky Tyr residue (Y112) at the active site pocket which, according to molecular docking studies, hinders the productive binding of Pro-containing peptides (LPXTG) over Ala-containing ones (LAXTG). The OH group of Y112 is involved in a hydrogen bond with the backbone nitrogen of the second Ala in the ALANT peptide but not in the Pro-containing peptide. Y112 is held rigidly in place via interactions with neighbouring residues and a network of hydrogen-bonded water molecules in the crystal structure. A Tyr residue is found to be present in an equivalent position in several sortase sequences of Class E, and may be a general feature responsible for the specificity of sortase Es to putative LAXTG-containing substrates in their genomes. It may be mentioned that class D sortases, which contain a Phe residue at the equivalent position, recognize the LPXTA substrate motif. The side chain of this Phe displays different rotamers in the NMR structure of Bacillus anthracis SrtD, pointing to its flexibility, whereas Y112 in S. avermitilis SrtE is rigid. In addition, molecular dynamics simulations on the models of protein-peptide complex (obtained from docking) showed that the two peptides have similar backbone dynamics, unlike the case of S. aureus SrtA where the Ala-containing peptide does not maintain a kinked conformation similar to the Pro-containing cognate peptide. Hence the Tyr at the active site appears to be the main factor behind the discrimination of the two peptides.
Substrate sequences in the S. avermitilis genome contain small neutral residues in the position succeeding the Thr-Gly peptide bond in the substrate. This preference is also observed in biochemical assays. Docking calculations showed that the protein cannot accommodate large side chains in the site where this residue is positioned.
To detect the residues involved in altering the substrate specificity of SavSrtE, we performed a multiple sequence alignment using 1569 sortase sequences and carried out mutual information (MI) analysis on this data. Our analysis implicated several residue pairs lining the active site pocket in modulating substrate specificity. These included the aforementioned Tyr residue as well as the position X (T196 in SavSrtE) in the TLXTC motif at the active site. Mutations were performed at these positions and crystallization trials performed. We could successfully crystallize and solve the structure of the T196V mutant, which has been documented in this thesis.
The mutant protein has the same overall structure as the wild type. Moreover, the catalytic Cys residue was observed to be unmodified in this structure, compared to the wild type which was presumably altered by β-mercaptoethanol added during protein purification. The mutated residue (Val) was found to have a different side chain rotamer than T196. Moreover, the absence of any polar atom in the side chain of V196 disrupted the hydrogen-bonded network of water molecules observed at the active site in the wild type structure. Experiments on the mutant showed a reduction in activity, implying that T196 is important for substrate recognition. The altered side chain orientation of V196 is expected to be responsible for the reduction in activity, though a peptide-bound crystal structure would be necessary to clearly understand the mechanism. In this respect, future crystallization trials may be performed with modified peptides that bind covalently to the active site Cys residue, similar to the strategy employed for S. aureus SrtA and Bacillus anthracis SrtA.
Our structure and sequence analyses have pointed to some residue positions responsible for the modified substrate specificity. While only one mutant has been characterized, the other mutants also need to be studied (through biochemical asssays and structure analysis) to understand how they contribute to substrate recognition. In this context, double mutants may also be generated to understand the combined effect. For example, single mutations of E105 and E108 were found to reduce the activity of Staphylococcus aureus SrtA, while the double mutant resulted in Ca2+ ion independent activity. Additional structure and sequence analysis coupled with experiments are necessary to detect residues which may be mutated to enhance the activity of SavSrtE, similar to what has been performed for S. aureus SrtA.
To summarize, our studies show that the substrate specificity of SavSrtE is different from that of class A sortases, and provide an explanation for it using structure analyses and computation. This altered specificity profile, orthogonal to that of S. aureus SrtA, and Ca2+ ion independent activity make it a potential candidate for use in simultaneous conjugation of multiple peptide substrates to their target. Moreover, this structure may be used firstly as a model to design inhibitors for housekeeping srtEs from pathogenic organisms like Corynebacterium diphtheriae and Tropheryma whipplei. Secondly, most of the previous studies on inhibitor design for sortases documented small molecules or peptidomimetics binding to the pocket of the first substrate. Since distinct binding pockets have been observed
for the two substrates in SavSrtE, this information may be used to build inhibitors targeting the second pocket or spanning both the pockets.
Section II: Identifying functionally important cis-peptide containing segments in proteins and their utility in molecular function
annotation Chapter 4| Functionally important cis-peptide fragments in proteins: detection and relevance: This chapter describes the relevance of cis-peptides to protein function and a method to detect such functionally important cis-peptides in proteins.
Cis-peptide bonds are comparatively rare in proteins due to the steric strain associated with the 1,4-atomic clash in the peptide chain. Consequently, only about 0.03% of Xaa-Xnp (Xaa: any amino acid; Xnp: any amino acid other than Pro) peptide bonds occur in the cis conformation; the occurrence is somewhat higher (5%) for imino peptide-containing Xaa-Pro cases. Despite their low occurrence, cis-peptides have been found to be evolutionarily conserved, pointing to their important role in structure and function. Cis-Xnp peptide bonds exhibit a significant disposition towards ligand-binding sites and dimerization interfaces, whereas cis-Pro bonds have been found to occur in a rare „touch-turn‟ motif at functional sites. Cis-trans isomerization is expected to play a regulatory role in many cellular processes. Non-conservation of these peptides is implicated in the evolution of different function among similar protein folds. Hence, there has been a renewed interest in detecting cis-peptides from residue patterns and linking them to molecular function.
The importance of proteins as molecular 'workhorses' makes it imperative to understand how they function. However, a vast majority of the proteins catalogued in public sequence and structure databases do not have experimentally verified functional annotation. Experimental approaches are inadequate to manually curate these large numbers of un-annotated proteins. This necessitates the use of computational function prediction tools. The simplest prediction methods involve the assessment of similarity in sequence and three-dimensional structure with homologous proteins of known function. The presence of high overall similarity, however, does not predict function unambiguously since certain protein folds are associated with multiple functions while proteins with different folds may share functional traits. Often proteins with different global structure are found to have structural similarity at the local level of segments of residues that are responsible for the similarity in function. This has given rise to fragment-based (FB) function annotation methods. FB methods may involve locating functionally relevant surface patches or cavities formed by sequentially distant residues, or the presence of structurally conserved, contiguous residue fragments with proven relevance to function. The direct relevance of the cis-peptide bond to protein function suggests its use for the purpose of function annotation in a FB approach, yet no method exists to exploit it.
This chapter describes a method using geometric clustering and level-specific Gene Ontology (GO) molecular-function (MF) terms to identify, in a statistically significant manner, cis-peptide embedded fragments (henceforth referred to as cis-fragments) in a protein linked to its molecular function. Such fragments were associated with GO MF based propensity value ≥ 20 at p-value ≤ 0.05, indicating the statistical significance of our results. The relevance of the identified cis-fragments to protein function was further verified through a literature survey. The features of these fragments are discussed in this chapter. Some of these fragments do not overlap with known PROSITE patterns, depicting the utility of these fragments as sequence patterns. Moreover, the thesis candidate identified contiguous stretches of functionally important trans-peptide fragments and cis-fragments forming extended structure-based functional signatures.
Chapter 5| Use of functionally important cis-fragments in annotation: In this chapter, the candidate describes how a library of cis-peptide embedded fragments with proven association to molecular function can be useful for annotating proteins with known structure (and having cis-peptide) but unknown function.
The functionally important fragments detected in the previous chapter were searched for exact matches in sequence and cis-peptide in a test set of PDB entries of known function at different thresholds of sequence redundancy and p-value. Additionally, the match or mis-match in GO MF term between the functionally important fragment and the test protein was also evaluated. To assess the efficiency of our method in annotation, true positive rate (TPR) and false positive rate (FPR) were calculated at each threshold as follows:
TPR TP and FPR FP
TP FN FP TN
The following table explains how the numbers of cases with TP, FP, etc. were assigned.
Cases with match in Match in cis-peptide No match in cis-
sequence peptide
Match in GO MF TP FN
No match in GO MF FP TN
The cis-fragments alone were sufficient to identify other proteins with similar function. Over different thresholds, TPR >0.91 and FPR <0.23 were observed. Annotation recall benchmarks interpreted using receiver-operator-characteristic-plot returned >0.9 area-under-curve, corroborating the utility of the annotation method. Further, the applicability of our method in fragment-based function annotation is illustrated for cases where homology-based annotation transfer is not possible. The work presented here adds to the repertoire of function annotation approaches and also facilitates engineering, design and allied studies around the cis-peptide neighbourhood of proteins.
The results presented in chapters 4 and 5 have already been published (reprint enclosed) with the thesis candidate as the first author. Chapter 6| Molecular dynamics information improves cis-peptide based function annotation of proteins:
The preceding chapters have demonstrated the use of functionally relevant cis-peptide segments in a homology-independent, fragment match-based protein function annotation method. However, proteins are not static molecules; their dynamics is integral to their activity. Hence we have incorporated the dynamics (obtained using an in-house coarse-grained forcefield) of functionally important cis-peptide segments in our annotation method. This is the first study to include both static and dynamics information to improve the prediction of protein molecular function.
To ascertain the improvement upon incorporating dynamics, the ACV-based dynamics profiles (details in chapter) were compared in a dataset consisting of 102 pairs each of positive data (PDB entries with match in fragment sequence and cis-peptide) and negative data (PDB entries with match in fragment sequence but no match in cis-peptide). Our analyses depicted that using only cis-peptide information gave less false positives and a low FPR (0.11), which is desirable, but also a relatively low TPR (0.72). This is due to large FN (trans-peptide with matching GO MF), which can arise when the cis-fragment undergoes cis-trans isomerization to accomplish its function and coordinates have been obtained for the segment in the test data in the trans-state, or if there is an error in assignment of the omega angle during structure solution. On the other hand, using only dynamics information increases the numbers of both true and false positives and hence the TPR (0.95) and FPR (0.51). This is due to false-positive matches for cases where fragments with similar secondary structure show similar dynamics, but the proteins do not share a common function. Combining the predictions from the two methods reduces errors while detecting the true matches, thereby enhancing the utility of our method in function annotation (TPR: 0.95 and FPR: 0.07). Subsequently, we have combined static and dynamics information to annotate proteins of unknown function. A combined approach, therefore, opens up new avenues of improving existing automated function annotation methodologies.
The work described in this chapter has been submitted to a peer reviewed journal. Future prospects include the development of a web server to facilitate the application of our method by a wide research community. A possible improvement includes identification and comparison of the dynamics of additional sites close to the identified cis-fragment, in an automated manner, to improve the accuracy of our annotation.
Appendix 1 gives a description of the results of biochemical experiments performed in the laboratory of our collaborator Dr. R.P. Roy, NII, New Delhi. Appendix 2 contains additional data supplementary to chapter 4.
Appendix 3 provides additional data supplementary to chapter 5.
Appendix 4 provides additional data supplementary to chapter 6.
Appendix 5 contains reprints of publications.
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