Made available in DSpace on 2016-06-02T20:34:35Z (GMT). No. of bitstreams: 1 4393.pdf: 23560579 bytes, checksum: 0869152d96e7734e1dd6786fa23cb550 (MD5) Previous issue date: 2012-05-03 / Universidade Federal de Sao Carlos / The study of phytopathogenic fungi Alternaria alternata has improved the chemical of this micro-organism. The fungus was grown up in large scale incubated in rice medium for 26 days. After grown period was interrupted an extract of the microorganism was made and fractionated using several chromatographic techniques, obtaining 11 different classes of metabolites which are: alternariol monomethyl éter, alternariol, altertoxin, altenuen, altenusin, uridine, uracila, 5 -inosil, ergosterol, peróxido de ergosterol e triglycerides. Also, a study was made via LC-MS in order to identify some toxins produced by A. alternata like a pathogen of citrus, and it was possible to identify four of these. Other research via LC-MS was the study of co-cultivation of the fungus A. alternata, the causal agent of alternaria brown spot, and Guignardia citricarpa, the causal agent of citrus black spot. In this study was possible to observe the variation in concentration of alternariol and thenuazonic acid, mycotoxins produced by A. alternata when this fungus is in contact with G. citricarpa. The study identification of the volatiles produced by A. Alternaria in different ways contributed significantly to improve the knowledge of their chemistry. Once, the A. alternata is a pathogenic fungus of 'Murcott' a study was performed via scanning electron microscopy (ESEM). Thus it was possible to observe the penetration of this fungus by stomatal cavity of young leaves of citrus. In order to search for natural inhibitors to control the disease, were tested several classes of natural products against the A. alternata, and some alkaloids, a xxvi coumarin, a xanthyletin, and a flavonoid substances showed Higher inhibition of mycelial growth, spore germination and appressorium formation. With the aim to obtain information on natural inhibitors were tested metabolites produced by A. alternata opposite and G. citricarpa, the results indicated that alternariol monomethyl ether and alternariol were substances that had a potential fungistatic against the causal agent of citrus black spot. / O estudo do fungo fitopatógeno Alternaria alternata contribuiu com a química desse micro-organismo. O fungo foi cultivado em larga escala utilizando como meio de cultura arroz por um período de 26 dias. Após esse período o crescimento foi interrompido e foi feito um extrato do microorganismo o qual foi fracionado utilizando várias técnicas cromatográficas obtendo 11 metabólitos de distintas classes sendo eles: alternariol monometil éter, alternariol, altertoxina, altenueno, altenusina, uridina, uracila, 5 -inosil, ergosterol, peróxido de ergosterol e triglicerídeo. Além disso, foi feito um estudo via LC-MS buscando identificar algumas toxinas de citros produzidas por A. alternata, sendo que foi possível a identificação de quatro dessas. Outra pesquisa realizada via LC-MS foi o estudo de co-cultivo entre os fungos A. alternata, o agente causal da mancha marrom de alternaria, e Guignardia citricarpa, o agente causal da mancha preta de citros. Nesse estudo foi possível observar a variação de alternariol e ácido tenuazônico, micotoxinas produzidas por A. alternata, quando este fungo está em contato com outro fitopatógeno. O estudo de identificação dos voláteis produzidos por A. alternaria em diferentes meios contribuiu muito para um maior conhecimento de sua química. Sabendo que A. alternata é um fitopatógeno de tangor Murcott foi realizado um estudo via microscopia eletrônica de varredura (MEV) a qual foi possível observar a penetração desse fungo pela cavidade estomatal de folhas jovens desse citros. Visando a busca de inibidores naturais para o controle da doença, foram ensaiadas diversas classes de produtos naturais frente a A. alternata, sendo que alcalóides e uma cumarina, a xantiletina, foram as substâncias que apresentaram uma maior inibição no crescimento micelial, germinação de esporos e formação de apressórios. Pensando em inibidores naturais, foram ensaiados metabólitos produzidos pelo A. alternata frente ao G. citricarpa, e os resultados obtidos revelaram que alternariol monometil éter e alternariol foram as substâncias que apresentaram um potencial fungistático frente ao agente causal da mancha preta de citros.

Citrus is the main fruit crop in the world and Spain is the 6th producer and the major exporter for the fresh fruit market. Seedlessness is one of the most important fruit quality traits for this market since consumers do not accept seedy fruits. Recovery of triploid hybrids has become an important breeding strategy to develop new seedless citrus varieties and several of them have been already released from citrus breeding programs worldwide. Despite the undisputable importance of polyploidy in plant species, their genetics are much less well known than those of their diploid counterparts. Citrus triploid hybrids can be routinely recovered from sexual polyploidization (2x ¿ 2x) or interploid crosses (2x ¿ 4x and 4x ¿ 2x). In 2x ¿ 2x sexual crosses, spontaneous triploid hybrids arise from the union of an unreduced (2n) megagametophyte with haploid pollen. In the case of interploid sexual crosses (2x ¿ 4x and 4x ¿ 2x), triploid hybrids result from the fecundation of a diploid gamete arising from the tetraploid parent and a haploid gamete arising from the diploid parent. The genetic and phenotypic structures of triploid populations greatly depend on the parental heterozygosity restitution (HR) in the diploid gamete at each locus, which is mainly affected by the triploid recovery strategy. In 2x ¿ 2x crosses, HR depends on the underlying mechanism leading to the unreduced gamete formation, which are genetically equivalent to First Division Restitution (FDR) or Second Division Restitution (SDR) mechanisms. Moreover, under each restitution mechanism, HR also depends on the locus-centromere genetic distance. In the case of interploid crosses, parental heterozygosity restitution from tetraploid parents depends on the double reduction frequency. In citrus, the unreduced gamete formation mechanism is still controversial; FDR has been the mechanism proposed for sweet orange, whereas SDR has been proposed for clementine. On the other hand, inferring the allelic configurations of genetic markers is a main challenge in polyploidy crops to infer genotypic and gametic structures with the objective to analyze meiosis and inheritance mechanisms. According to this scientific context, the objectives of the thesis where: (i) to develop a new approach for allele dosis assignation when using co-dominant markers, (ii) to implement and apply methods for the analysis of 2n gametes origin and locate centromeres, and (iii) to take advantage of this knowledge to locate a major gene of resistance to Alternaria Brown Spot (ABS) which is a major constraint for triploid mandarin breeding. For microsatellite (SSR) markers, we have demonstrated that triploid progeny genotyping can be successfully performed using the microsatellite allele-counting peak ratio (MAC-PR) method. However, SSR analysis remains relatively costly and time consuming compared with actual SNP genotyping methods. Moreover, with the increasing availability of EST databases and whole genome sequences, SNPs have become the most abundant and powerful polymorphic markers that can be selected along the entire genome. In this thesis, a new method based on competitive allele-specific PCR has been developed to assign SNP allele dosage in an accurate, simple, and cost effective way. Combining the MAC-PR and the new developed SNP genotyping methods offers the possibility to utilize a broad range of molecular markers in genotyping triploid genotypes. Both methods have been used in further works included in this thesis. SDR has been demonstrated as the mechanism underlying unreduced gamete production in `Fortune¿ mandarin by genotyping triploid progenies with SSR markers. In addition, a new method to locate the centromere, based on the best fit between observed heterozygosity restitution within a linkage group and theoretical functions under either partial or no chiasma interference hypotheses has been developed and successfully applied. To expand the knowledge of the mechanism underlying unreduced gamete formation to other citrus genotypes besides clementines and `Fortune¿ mandarin, a maximum likelihood method based on parental heterozygosity restitution of centromeric loci was developed and successfully applied in sixteen mandarin cultivars. The new method developed in the study allows inferring the restitution mechanism both at population level and even at individual level. Maternal origin of 2n gametes was confirmed for all triploid hybrids and SDR was proposed as the restitution mechanism for all analyzed progenies. The information acquired from the mode of heterozygosity restitution in citrus was useful to determine the genetic and phenotypic structures of new triploid populations arising from different breeding strategies. We studied these structures for the resistance to Alternaria brown spot (ABS), a serious fungal disease producing necrotic lesions on fruits and young leaves in susceptible citrus genotypes. In the present work, different approaches were combined taking advantage of the particular genetic structures of 2n gametes resulting from SDR to map a genome region linked to ABS resistance in triploid citrus progeny. The monolocus dominant inheritance of the susceptibility, proposed on the basis of diploid population studies, was corroborated in triploid progeny. A 3.3 Mb genomic region linked to ABS resistance was located near the centromere on chromosome III, which includes clusters of resistance genes. SSR and SNP markers were developed for an efficient early selection of ABS resistant hybrids and they are currently used in our breeding program to perform marker assisted selection. The knowledge obtained in this thesis on the mechanism of sexual polyploidization and inheritance of concrete traits in citrus will allow implementing much more efficient triploid breeding programs on the basis of current and future needs. Indeed, applied outcomes of this PhD are already routinely used in the IVIA triploid breeding program. / Cuenca Ibáñez, J. (2013). Mechanisms of sexual polyploidization and inheritance in triploid citrus populations [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/34671 / TESIS / Premios Extraordinarios de tesis doctorales

Exposure to spores and hyphae of Alternaria alternata, an airborne ubiquitous fungus, is clinically associated with allergic airway disorders including allergic rhinitis, asthma, and chronic rhinosinusitis. Dendritic cells are known as the type of antigen presenting cells most often associated with allergic inflammation. In this study, we used mouse bone marrow-derived dendritic cells (BMDCs) as a model to study the ability of A. alternata spores and different components of the spore cell wall to stimulate innate immune responses. We found that BMDCs were highly sensitive to A. alternata spores, chitin and the major allergen Alt a 1. Following stimulation with these molecules, the expression of MHC II and other co-stimulators, like CD40, CD86, and OX40L, were highly up regulated. In order to determine how different cell wall components affect the T cells, we conducted co-culture experiments of BMDCs and lymphocytes in this study. Both spores and Alt a1 did not induce IL-4 in mixed lymphocytes reactions. Interestingly, we found that Alt a 1 induced the switching of the CD4+ T cell population to the Th17 type, with a major increase in IL-17A secretion. This study reveals that A. alternata components may balance the innate immune responses between Th2 and Th17 pathways, and/or contributes to the development and exacerbation of more serve neutrophilic forms of asthma. / Master of Science

Made available in DSpace on 2016-06-02T19:30:12Z (GMT). No. of bitstreams: 1 6721.pdf: 2764879 bytes, checksum: ca82bcb193d5ae0d2e21032afd6ed1dd (MD5) Previous issue date: 2015-03-30 / Financiadora de Estudos e Projetos / The Brazilian savanna holds the richest flora among the tropical savannas, being one of the hot spots for conservation of the biodiversity in the world. However, the chemical potential of its plant species is still underused while deforestation and loss of biodiversity are main issues. In the Brazilian savanna, plants are exposed to environmental pressures (competition, pathogens, drought, injurious UV radiation, etc) that may stimulate the production of secondary metabolites as a defense. These compounds can generally be explored due to their biological properties. The aim of this study was to assess the fungicidal and antioxidant properties of plants from the Brazilian savanna. First a screening was conducted with ethanolic leaf extracts of 28 plant species. There was no activity against Alternaria alternata, a filamentous fungi that causes one of the most severe diseases in tangerines (Alternaria brown spot disease). However, the same extracts presented a high antioxidant potential associated with the phenolic content. Other investigations were made using infusion extractions focusing on the Myrtaceae family, one of the most abundant families of plants in the Brazilian savanna. Most species also presented high antioxidant activity and phenolic content. At last, the extraction of piceid, a compound with high antioxidant activity, was optimized from roots of Psidium laruotteanum. A response surface methodology was applied to assess the influence of solvent, pH, amplitude, cycle, temperature and solvent-solid ratio using an ultrasound extraction. A high amount of piceid was extracted and Psidium laruotteanum root was found to be the most concentrated source of this compound reported up to date. These results point the plant species from the Brazilian savanna as an important source of bioactive compounds, emphasizing the need to preserve and study its biodiversity. / O cerrado possui a flora mais rica entre as savanas tropicais, sendo um dos pontos quentes para conservação da biodiversidade no mundo. No entanto, o potencial químico de suas espécies vegetais é ainda subutilizado, enquanto o desmatamento e a perda de biodiversidade são questões de grande relevância. No cerrado, as plantas estão expostas a pressões ambientais (competição, patógenos, seca, radiação UV nociva, etc) que podem estimular a produção de metabólitos secundários como defesa. Estes compostos geralmente podem ser explorados devido suas propriedades biológicas. O objetivo deste estudo foi avaliar as propriedades fungicidas e antioxidante de plantas do cerrado. Primeiro uma triagem foi realizada com extratos etanólicos de folhas de 28 espécies vegetais. Não houve nenhuma atividade contra Alternaria alternata, um fungo filamentoso que causa uma das doenças mais severas em tangerinas (mancha marrom de Alternaria). No entanto, os mesmos extratos apresentaram um potencial antioxidante elevado associado com o conteúdo fenólico. Outras investigações foram feitas usando extrações por infusão enfocando a família Myrtaceae, uma das famílias mais abundantes de plantas no cerrado. A maioria das espécies apresentou alta atividade antioxidante e alto conteúdo fenólico. Por fim, a extração de piceido, um composto com alta atividade antioxidante, foi otimizada de raízes de Psidium laruotteanum. Uma metodologia de superfície de resposta foi aplicada para avaliar a influência do solvente, pH, amplitude, ciclo, temperatura e razão solvente-sólido usando uma extração de ultrasonido. Uma alta quantidade de piceido foi extraída e a raiz de Psidium laruotteanum se mostrou como sendo a fonte mais concentrada deste composto relatada até hoje. Estes resultados apontam as espécies de plantas do cerrado como uma importante fonte de compostos bioativos, enfatizando a necessidade de preservar e estudar sua biodiversidade.

The transition from the Neoproterozoic to the early Cambrian world was a critical time in the co-evolution of life and the environment, when dramatic changes in the environment, including global glaciations and rising atmospheric oxygen levels precipitated the conditions in which variety of complex life emerged, diversified, and proliferated. However, questions persist regarding the timing and duration of those environmental events, and thus the extent of their direct effect on the evolution of life. These changing environmental conditions also produced unique taphonomic conditions that preserved a wide variety of organisms, and also produced unusual early diagenetic features that can be difficult to distinguish from fossilized organisms. In order to better resolve the link between life and the environment during the Neoproterozoic to Cambrian transition I present three chapters. The first chapter of this dissertation examines the carbonate clasts from a glacial diamictite deposited at the end of the Cryogenian Period in South China. By determining the source locality of those carbonate clasts using stable carbon and oxygen isotope compositions, we can better understand the extent of the Marinoan Glaciation and the intensity of its impact on the Earth's surface. In the second chapter of this dissertation, I investigate the changing chemical conditions of ocean waters following both the Marinoan glaciation and potentially one of the most intense disruptions to the carbon cycle in Earth History, the Ediacaran Shuram carbon isotope anomaly. I use Hg concentrations and stable isotope compositions to determine changes in sediment sourcing along with changes in marine redox, such as the development of photic zone euxinia. In the third chapter, I investigate the identity of Brooksella alternata, a purportedly cnidarian fossil that was later suggested to be a hexactinellid sponge fossil. My morphological and compositional analysis reveals B. alternata to be a concretion and thus a pseudofossil. / Doctor of Philosophy / The Earth experienced major changes between 635 and ~514 million years ago; the last global scale glaciation in the history of Earth ended, early animals arose, primary producers such as algae grew larger and more complex, and the first animals that were mobile and with hard skeletal parts evolved and diversified tremendously. Concomitantly, the environment on the Earth changed dramatically: the carbon cycle experienced one of the greatest disruptions in all of Earth history and the oceans and atmosphere gradually became more oxygen-rich, though areas with low levels of dissolved molecular oxygen and high concentrations of dissolved iron or hydrogen sulfide persisted, which may have impeded the diversification of complex animals. The exact timing and intensity of these changes are not fully resolved, and by investigating this time interval in the geologic record, we can better understand how the world changed and how life at the time responded. These changing environments also produced unique conditions which led to fossils being preserved in unique ways, though these conditions also produced non-biological structures that superficially resemble fossils. The first chapter of this dissertation investigates the source of clasts made of carbonate rock from a glacial diamictite (the poorly sorted mixture of the fine-grained and coarse-grained deposits left behind as glaciers recede) in South China deposited at the end of the last global scale glaciation about 635 million years ago. By determining the source, we can evaluate how far glacial activity transported sediments. I found that the previously suggested source of these clasts does not match the geochemical fingerprint of the carbonate clasts. I propose that these carbonate clasts may have come from a source that has since become "extinct" because of glacial erosion, or they may have come from another continent (for example, India). These scenarios highlight the magnitude of the global glaciation. In the second chapter, I investigated the mercury concentrations and stable isotope compositions from organic rich shales deposited ~550 million years ago. Mercury is an emerging tool for evaluating the level of dissolved molecular oxygen in ancient oceans. In this study, I found evidence of locally complex marine oxygen levels, including evidence for photic zone euxinia, meaning waters where oxygen was absent and hydrogen sulfide was present. Such conditions are toxic to animals and may have delayed the diversification of complex animal life. In the final chapter, I analyzed the ~503 million-year-old fossil Brooksella alternata. It was first described more than 100 years ago as a jelly-fish fossil. More recently it was suggested to be a sponge fossil (though it has been ascribed many other identities). Based on morphological analysis of a large collection, I determined that Brooksella alternata is not a fossil but rather a concretion with unusual shape.

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