Orientador: Fausto Foresti / Resumo: Cerca de 15% dos organismos eucariotos possuem elementos genômicos adicionais dispensáveis chamados cromossomos B. Na maioria dos casos a composição e os efeitos da presença destes cromossomos é desconhecida. Estes elementos estão presentes em diversas espécies de peixes do gênero Astyanax. Assim, os objetivos do presente trabalho foram caracterizar a composição do cromossomo B de A. paranae, bem como os efeitos da presença deste cromossomo nesta espécie e sua relação com cromossomos B de espécies próximas. Foram identificados sete genes codificadores de proteínas no cromossomo B de A. paranae, dos quais quatro mostraram expressão diferencial nos ovários de fêmeas 0B e 1B. Além disto, quatro destes genes estão presentes nos cromossomos B de A. fasciatus e de A. bockmanni. Desta forma, nós concluímos que o cromossomo B de A. paranae interfere na expressão dos genes que carrega em seu próprio benefício e possui uma origem comum com cromossomos B encontrados em outras espécies de Astyanax. Além deste objetivo principal do trabalho, também foi realizada uma análise do DNA mitocondrial de A. paranae e, por extensão, dos vertebrados em geral. Esta análise revelou inicialmente que o conteúdo de bases do DNA mitocondrial dos vertebrados tende a um padrão comum. Adicionalmente, foi possível distinguir grupos entre as classes de vertebrados com conteúdos semelhantes na composição de bases do DNA mitocondrial, indicando que a evolução do DNA destas organelas poderia estar relacionada às... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: About 15% of eukaryotic organisms have additional and dispensable genomic elements called B chromosomes. In most cases the composition and effects of the presence of these chromosomes is unknown. These elements are present in several species of fish of the genus Astyanax. Thus, the objectives of the present work were to characterize the composition of the B chromosome of A. paranae, as well as the effects of the presence of this chromosome in this species and its relation with B chromosomes of nearby species. Seven protein-coding genes were identified on the B chromosome of A. paranae, of which four showed differential expression in the ovaries of females 0B and 1B. In addition, five of these genes are present on the B chromosomes of A. fasciatus and A. bockmanni. Thus, we conclude that the B chromosome of A. paranae interferes with the expression of the genes it carries for its own benefit and has a common origin with B chromosomes found in other species of Astyanax. In addition to this main objective of the work, an analysis of the mitochondrial DNA of A. paranae and, by extension, of the vertebrates in general was also performed. This analysis initially revealed that the base content of vertebrate mitochondrial DNA tends to a common pattern, and it was possible to distinguish groups among vertebrate classes with similar contents in mitochondrial DNA bases, indicating that the DNA evolution of these organelles could be related to the biological characteristics of each group... (Complete abstract click electronic access below) / Doutor

Submitted by DUÍLIO MAZZONI ZERBINATO DE ANDRADE SILVA null (duzerbinato@yahoo.com.br) on 2018-03-13T18:57:43Z No. of bitstreams: 1 Tese_Duilio_2018.pdf: 3388923 bytes, checksum: d3bd5397e2e92ef08a15e7b3a7fe9cc8 (MD5) / Approved for entry into archive by ROSANGELA APARECIDA LOBO null (rosangelalobo@btu.unesp.br) on 2018-03-16T19:40:48Z (GMT) No. of bitstreams: 1 silva_dmza_dr_bot.pdf: 3388923 bytes, checksum: d3bd5397e2e92ef08a15e7b3a7fe9cc8 (MD5) / Made available in DSpace on 2018-03-16T19:40:48Z (GMT). No. of bitstreams: 1 silva_dmza_dr_bot.pdf: 3388923 bytes, checksum: d3bd5397e2e92ef08a15e7b3a7fe9cc8 (MD5) Previous issue date: 2018-02-26 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Cerca de 15% dos organismos eucariotos possuem elementos genômicos adicionais dispensáveis chamados cromossomos B. Na maioria dos casos a composição e os efeitos da presença destes cromossomos é desconhecida. Estes elementos estão presentes em diversas espécies de peixes do gênero Astyanax. Assim, os objetivos do presente trabalho foram caracterizar a composição do cromossomo B de A. paranae, bem como os efeitos da presença deste cromossomo nesta espécie e sua relação com cromossomos B de espécies próximas. Foram identificados sete genes codificadores de proteínas no cromossomo B de A. paranae, dos quais quatro mostraram expressão diferencial nos ovários de fêmeas 0B e 1B. Além disto, quatro destes genes estão presentes nos cromossomos B de A. fasciatus e de A. bockmanni. Desta forma, nós concluímos que o cromossomo B de A. paranae interfere na expressão dos genes que carrega em seu próprio benefício e possui uma origem comum com cromossomos B encontrados em outras espécies de Astyanax. Além deste objetivo principal do trabalho, também foi realizada uma análise do DNA mitocondrial de A. paranae e, por extensão, dos vertebrados em geral. Esta análise revelou inicialmente que o conteúdo de bases do DNA mitocondrial dos vertebrados tende a um padrão comum. Adicionalmente, foi possível distinguir grupos entre as classes de vertebrados com conteúdos semelhantes na composição de bases do DNA mitocondrial, indicando que a evolução do DNA destas organelas poderia estar relacionada às características biológicas de cada grupo, como modo de respiração, ocupação do meio terrestre ou aquático, modo de vida em água doce ou salgada e taxa metabólica dos organismos. / About 15% of eukaryotic organisms have additional and dispensable genomic elements called B chromosomes. In most cases the composition and effects of the presence of these chromosomes is unknown. These elements are present in several species of fish of the genus Astyanax. Thus, the objectives of the present work were to characterize the composition of the B chromosome of A. paranae, as well as the effects of the presence of this chromosome in this species and its relation with B chromosomes of nearby species. Seven protein-coding genes were identified on the B chromosome of A. paranae, of which four showed differential expression in the ovaries of females 0B and 1B. In addition, five of these genes are present on the B chromosomes of A. fasciatus and A. bockmanni. Thus, we conclude that the B chromosome of A. paranae interferes with the expression of the genes it carries for its own benefit and has a common origin with B chromosomes found in other species of Astyanax. In addition to this main objective of the work, an analysis of the mitochondrial DNA of A. paranae and, by extension, of the vertebrates in general was also performed. This analysis initially revealed that the base content of vertebrate mitochondrial DNA tends to a common pattern, and it was possible to distinguish groups among vertebrate classes with similar contents in mitochondrial DNA bases, indicating that the DNA evolution of these organelles could be related to the biological characteristics of each group, such as the type of respiration, occupation of the terrestrial or aquatic environment, freshwater or salt water livelihood and metabolic rates of organisms. / 2013/24367-0

Quantitative microbial risk assessment (QMRA) is being increasingly used to support decision-making for food safety issues. Decision-makers need to know whether these QMRA results can be trusted, especially when urgent and important decisions have to be made. This can be achieved by setting up a quality assurance (QA) framework for QMRA. A Belgian risk assessment project (the METZOON project) aiming to assess the risk of human salmonellosis due to the consumption of fresh minced pork meat was used as a case study to develop and implement QA methods for the evaluation of the quality of input data, expert opinion, model assumptions, and the quality of the QMRA model (the METZOON model). The first part of this thesis consists of a literature review of available QA methods of interest in QMRA (chapter 2). In the next experimental part, different QA methods were applied to the METZOON model. A structured expert elicitation study (chapter 4) was set up to fill in missing parameters for the METZOON model. Judgements of experts were used to derive subjective probability density functions (PDFs) to quantify the uncertainty on the model input parameters. The elicitation was based on Cookes classical model (Cooke, 1991) which aims to achieve a rational consensus about the elicitation protocol and allowed comparing different weighting schemes for the aggregation of the experts PDFs. Unique to this method was the fact that the performance of experts as probability assessors was measured by the experts ability to correctly and precisely provide estimates for a set of seed variables (=variables from the experts area of expertise for which the true values were known to the analyst). The weighting scheme using the experts performance on a set of calibration variables was chosen to obtain the combined uncertainty distributions of lacking parameters for the METZOON model. A novel method for the assessment of data quality, known as the NUSAP (Numeral Unit Spread Assessment Pedigree) system (chapter 5) was tested to screen the quality of the METZOON input parameters. First, an inventory with the essential characteristics of parameters including the source of information, the sampling methodology and distributional characteristics was established. Subsequently the quality of these parameters was evaluated and scored by experts using objective criteria (proxy, empirical basis, methodological rigour and validation). The NUSAP method allowed to debate on the quality of the parameters within the members of the risk assessment team using a structured format. The quality evaluation was supported by graphical representations which facilitated decisions on the inclusion or exclusion of inputs into the model. It is well known that assumptions and subjective choices can have a large impact on the output of a risk assessment. To assess the value-ladenness (degree of subjectivity) of assumptions in the METZOON model a structured approach based on the protocol by Kloprogge et al. (2005) was chosen (chapter 6). The key assumptions for the METZOON model were first identified and then evaluated by experts in a workshop using four criteria: the influence of situational limitations, the plausibility, the choice space and the agreement among peers. The quality of the assumptions was graphically represented (using kite diagrams, pedigree charts and diagnostic diagrams) and allowed to identify assumptions characterised by high degree of subjectivity and high expected influence on the model results, which can be considered as weak links in the model. The quality assessment of the assumptions was taken into account to modify parts of the METZOON model, and allows to increase the transparency in the QMRA process. In a last application of a QA method, a quality audit checklist (Paisley, 2007) was used to critically review and score the quality of the METZOON model and to identify its strengths and weaknesses (chapter 7). A high total score (87%) was obtained by reviewing the METZOON model with the Paisley-checklist. A higher score would have been obtained if the model was subjected to external peer review, if a sensitivity analysis, validation of the model with recent data, updating/replacing expert judgement data with empirical data was carried out. It would also be advisable to repeat the NUSAP/Pedigree on the input data and assumptions of the final model. The checklist can be used in its current form to evaluate QMRA models and to support model improvements from the early phases of development up to the finalised model for internal as well as for external peer review of QMRAs. The applied QA methods were found useful to improve the transparency in the QMRA process and to open the debate about the relevance (fitness for purpose) of a QMRA. A pragmatic approach by combining several QA methods is recommendable, as the application of one QA method often facilitates the application of another method. Many QA methods (NUSAP, structured expert judgement, checklists) are however not yet or insufficiently described in QMRA related guidelines (at EFSA and WHO level). Another limiting factor is the time and resources which need to be taken into account as well. To understand the degree of quality required from a QMRA a clear communication with the risk managers is required. It is therefore necessary to strengthen the training in QA methods and in the communication of its results. Understanding the usefulness of these QA methods could improve among the risk analysis actors when they will be tested in large number of QMRAs.