L’évolution de la consommation électrique est un point clé pour les choix à venir, tant pour les moyens de production d’électricité, que pour le dimensionnement du réseau à toutes ses échelles. Aujourd’hui, ce sont majoritairement des modèles statistiques basés sur les consommations passées et des tendances démographiques ou économétriques qui permettent de prédire cette consommation. Dans le contexte de la transition énergétique, des changements importants sont en cours et à venir, et la consommation future ne sera certainement pas une continuation des tendances passées. Modéliser ces changements nécessite une modélisation fine de type bottom-up de chaque contributeur de la consommation électrique. Ce type de modèle présente des challenges de modélisation, car il nécessite un grand nombre de paramètres d’entrée qui peuvent difficilement être renseignés de façon réaliste à grande échelle. En même temps, les données et informations de tout type n’ont jamais été autant disponibles. Cela représente à la fois un atout pour la modélisation, mais aussi une difficulté importante notamment à cause de l’hétérogénéité des données. Dans ce contexte, cette thèse présente une démarche de construction d’un simulateur de consommation électrique bottom-up capable de simuler différentes alternatives énergétiques à l’échelle de la France. Un travail de recensement, de classification et d’association des bases de données pour expliquer la consommation électrique a d’abord été mené. Ensuite, le modèle de consommation électrique a été présenté ; il a été validé et calibré sur une grande quantité de mesures de consommation électrique des départs HTA fournie par Enedis. Ce modèle a enfin pu être utilisé pour simuler différentes alternatives énergétiques afin d’aider au dimensionnement du réseau de distribution. / Future trend of electricity demand is a key point for sizing both the electricity network and the power plants. In order to forecast future electricity demand, current models mostly use statistical approaches based on past demand measurements and on demographic and economic trends. Because of current context of energy transition which comes along with important changes, future electricity demand is not expected to be similar to past trends. Modeling these changes requires a bottom-up modeling of each contributor to electricity demand. This kind of model is challenging because of the large number of input data required. At the same time, data and information are more and more available. Such availability can be considered both as an asset for modeling and as an important issue because of data heterogeneity. In this context, this dissertation offers an approach to build a bottom-up load curve simulator which enables to simulate prospective scenarii at the scale of France country. Firstly, an assessment, classification, and matching of the large databases explaining the electricity demand have been performed. Then, the electricity demand model has been presented. It has been validated and calibrated on Enedis’ large volumes of electricity demand measurements of medium voltage feeders. Finally, this model has been used to simulate several prospective scenarii in order to improve the electricity distribution network sizing.

Wetlands are globally important biogeochemical hotspots, and their roles as either significant sources of greenhouse gas or carbon sinks are strongly controlled by environmental drivers such as carbon, nitrogen, and phosphorus availability, which can stimulate emission of three greenhouse gases: nitrous oxide (N2O), carbon dioxide (CO2) and methane (CH4). Mounting evidence suggests that aquatic invertebrate activity can enhance wetland greenhouse gas flux, through bioturbation and bioirrigation of surrounding sediments. However, waterbirds reduce the density of aquatic invertebrates, which could potentially reduce greenhouse gas flux. Alternatively, the nutrient contents of waterbird guano may stimulate microbial activity responsible for these gas fluxes. The research project FLAMMINGGOS (Functional Links in Avian, Microbial, Macrophyte, and INvertebrate Greenhouse Gas Output Stimulation) aims to test the relative strength of top-down and bottom-up effects of predatory waterbirds on wetland greenhouse gas fluxes. This study is a part of the FLAMMINGGOS project and studied the bottom-up stimuli effects of methane flux, through addition of waterbird guano to sediment cores sampled from three sites in Doñana, Spain. Guano did not enhance methane flux; on the contrary, the overall flux rates decreased after guano addition for one site. There was also a positive correlation between methane flux and the presence of benthic invertebrates. This suggests that the top-down gas-stimuli effects of waterbirds may be stronger than the bottom-up; benthivorous waterbirds, such as flamingos, may lessen the emissions of CH4 through predation on benthic invertebrates. Further research is however required to fully understand these interactions and gas flux stimuli effects. / FLAMMINGGOS

Esta pesquisa analisa os processos de urbanismo bottom-up a partir do contexto social em que emergiram e de sua atuação em meios digitais. Como contexto são analisados os movimentos sociais contemporâneos, a consolidação da sociedade em rede por meio da popularização das TICs e a emergência de novas culturas organizacionais. A análise histórica desse universo é sempre vinculada a seu impacto no processo de produção das cidades, até a apresentação da situação contemporânea. Sobre a atuação nos meios digitais a pesquisa investiga por meio da mineração de dados e análise de redes, a atuação no Facebook, a partir de um estudo de caso. As investigações revelam as dinâmicas dos principais atores, como o número e frequência de eventos, o engajamento com o público, os diálogos no espaço virtual e as ações de transformação urbana. Como resultado, a pesquisa não pretende apresentar conclusões precisas e definições de conceitos, mas apontamentos que permitem uma melhor compreensão e planejamento do urbanismo contemporâneo. / This research analyzes the processes of bottom-up urbanism from the social context in which they emerged and from their presence in digital media. As context, are analyzed the contemporary social movements, the consolidation of the network society through the popularization of ICTs and the emergence of new organizational cultures. The historical analysis of this universe is always linked to its impact on the production process of cities. About the presence in the digital media the research investigates through the data mining and analysis of networks, the action in Facebook, from a case study. The investigations reveal the dynamics of the main actors, such as the number and frequency of events, engagement with the public, dialogues in virtual space and actions of urban transformation. As a result, the research does not intend to present precise conclusions and defi nitions of concepts, but notes that allow a better understanding and planning of contemporary urbanism.

Inúmeros estudos experimentais contribuíram para o desenvolvimento da teoria das redes tróficas lacustres, revelando o importante papel dos peixes, ignorados no campo da limnologia durante décadas. A maioria dos estudos foi desenvolvida na Europa e Estados Unidos e a generalidade da teoria ainda não foi amplamente testada em ambientes subtropicais e tropicais. Apesar das controversas, as hipóteses de cascata trófica e de bottom-up : top-down são os modelos conceituais mais utilizados. Neste contexto, este trabalho baseia-se na hipótese de que as relações tróficas em cascata, relacionadas aos efeitos bottom-up e top-down, podem ser encontradas no Reservatório de Itaipu, inserido em região subtropical. Com base em dados disponibilizados pela Itaipu Binacional, para o período de 1999 a 2004. Análises de Covariância (ANCOVAs) foram realizadas para determinar as relações das variáveis dependentes clorofila-a, cianobactéria e transparência da água sobre as demais variáveis limnológicas. Análises de correlações também foram realizadas para verificar a existência de algum tipo de efeito na cascata trófica. Os resultados obtidos caracterizam o reservatório como mesotrófico, com situações de oligotrofia na zona lacustre. Efeito negativo foi verificado para transparência da água em relação aos sólidos suspensos totais e turbidez. A clorofila apresentou relação positiva com nitrogênio total, mas não para fósforo total. Os peixes onívoros, detritívoros e insetívoros exerceram efeito negativo (controle) na densidade das cianobactérias e na concentração de clorofila. A estimativa da produção de peixes no reservatório foi melhor relacionada com as cianobactérias do reservatório, gerando um modelo estatístico através desta análise. Os efeitos top-down e bottom-up foram confirmados, sendo que as forças top-down foram encontradas apenas no primeiro nível trófico e as demais apresentaram efeito bottom-up. / Many experimental studies contributed for the development of the theory of the lacustrine food web, disclosing the important paper of the fish, for decades ignored by the limnology. These studies, in its majority, had been developed in Europe and North America and the generality of the hypotheses supporting the theory was not tested yet in subtropical and tropical environments. Although controversial, the trophic cascade and bottom-up: top-down hypotheses are the two main conceptual models presently used. This work is based on the hypothesis of the trophic cascade relations, particularly related to bottom-up and top-down effect can be detected in Itaipu Reservoir, a subtropical system. Using the made data available by Itaipu Binacional, from 1999 to 2004. Analyses of Covariance (ANCOVAs) were accomplished to determine the relationships of the dependent variable on the chlorophyll-a, cyanobacterias and transparency of the water (independent variables) and between the other limnologycal variables. Analyses of correlations were also accomplished to verify the existence of some effect type in the trophic cascade. The results show that the reservoir presented mesotrophic, and oligotrophic and characteristics in the lacustrine area. A negative effect was verified between the water transparency in relation to suspended solids and turbid. The chlorophyll presented positive relationship with total nitrogen, but didn\'t present any relationship with the total phosphorus. The omnivorous, and detritivorous and insectivorous fishes presented a negative effect (control) in the cyanobacteria concentrations and chlorophyll. The estimate of the production of fish in the reservoir was better related with the concentrations of cyanobacterias of the reservoir, generating a statistical model through this analysis. The top-down and bottom-up effect had been confirmed, the forces top-down had been found only in the first trophic level, and the others presented bottom-up effect.

Com este estudo procurou se identificar os resultados produzidos pela aprendizagem do tipo Bottom-Up (BU) em organizações, tendo-se em vista que as variáveis Cultura Organizacional de Aprendizagem (COA), Mecanismos de Coordenação e Integração (MCI) e Liderança (LID) podem interferir na relação entre o método de aprendizagem e os resultados obtidos pela organização. Para se compreender o fenômeno, após uma pesquisa bibliográfica, foi desenvolvido um estudo multicaso pela metodologia proposta por Yin (2009). Foram pesquisadas quatro organizações atuantes em segmentos distintos, nomeadas neste estudo como \"Empresa X\" (setor farmacêutico), \"Empresa Y\" (setor alimentício/franquia), \"Empresa Z\" (indústria e comércio de calçados) e \"Empresa W\" (setor farmacêutico). Nestas empresas foram inquiridos o Diretor, o Vendedor e os responsáveis pelo setor da inovação, produção, comercial, financeiro e RH. Os respondentes foram entrevistados e preencheram um questionário, além de ter existido coleta prévia de dados secundários para maior compreensão dos fatos relacionados às empresas. Como resultado, constatou-se que nos casos analisados, aparentemente existe uma combinação entre o fator MCI e os constructos LID e COA, que possibilita a existência da aprendizagem do tipo BU. Os resultados provenientes desta combinação tendem a se manifestar em termos financeiros, competitivos e mercadológicos. A combinação do fator e os constructos não necessariamente conduzem à inovatividade da organização. / This study sought to identify the results produced by the Bottom-Up learning (BU) in organizations having in mind that the variables Organizational Learning Culture (OLC), Coordination and Integration Mechanisms (CIM) and Leadership (LEAD) can interfere with the relationship between the learning method and the results obtained by the organization. To understand the phenomenon, after a bibliographic research, it was developed a multi case study by the methodology proposed by Yin (2009). They were surveyed four organizations active in different segments, named in this study as \"Empresa X\" (pharmaceutical industry), \"Empresa Y\" (franchiser in the food sector), \"Empresa Z\" (footwear industry and retail trade) e \"Empresa W\" (pharmaceutical industry). In these companies were surveyed the diretor, the salesman and and those responsible for industry innovation, production, commercial, financial and HR. Respondents were interviewed and filled out a questionnaire, and have existed prior collection of secondary data for greater understanding of the facts related to business. As a result, it was found that in the cases analyzed, there is apparently a combination of CIM factor and constructs LEAD and OLC, which allows the existence of BU learning. The results from this combination tend to express in financial terms, competitive and related to the market. The combination of the factor and the constructs do not necessarily lead to the innovativeness of the organization.

O presente estudo teve por objetivo investigar a dinâmica temporal da percepção de tamanho de objetos inseridos em gradientes de textura, provenientes de desenho gerado computacionalmente (gradiente de perspectiva) e de fotografia com elementos naturais (gradiente fotografado). Além disso, verificou-se a ocorrência e padrão dos movimentos oculares nestas condições. Para isso, foram realizados 2 experimentos. No primeiro experimento, dois círculos negros eram apresentados no meridiano vertical e a tarefa dos 96 participantes foi indicar em qual parte do campo visual, superior ou inferior, foi apresentado o maior círculo. Usando o método das escadas duplas, estes círculos eram apresentados brevemente (50, 100, 150 ou 200 ms) em três condições de fundos de tela (sem textura, gradiente de perspectiva ou gradiente fotografado). Foram calculados a inclinação da função psicométrica e o ponto de igualdade subjetivo (PIS) como medidas da sensibilidade discriminativa e da distorção perceptiva, respectivamente. Os resultados mostraram que os participantes discriminaram melhor o tamanho dos estímulos à medida que o tempo de exposição aumentava, independentemente do gradiente de textura. A análise do PIS revelou que há uma forte distorção do tamanho aos 100 ms para o gradiente de perspectiva e aos 150 ms para o gradiente fotografado. No segundo experimento, 24 participantes realizaram uma tarefa semelhante ao do primeiro experimento e utilizou-se um eye tracker para registrar o movimento ocular. Os resultados mostraram que, em apresentações de 150 e 200 ms, os movimentos oculares ocorreram em menos de 10% das tentativas em todos os fundos de tela. Isto indica que a redução das distorções de tamanho após os 100 ms observada no primeiro experimento não é explicada completamente pela alocação da atenção. Tomados em conjunto, estes resultados sugerem que o processamento e integração das informações de tamanho e profundidade são mediados principalmente por mecanismos bottom-up. Ademais, em uma condição de menor restrição temporal (1500 ms), a presença de informações de profundidade afetou a dimensão espacial das fixações e sacadas, mas não a dimensão temporal. Os participantes olharam preferencialmente para o estímulo apresentado no campo visual superior, o que indica que a atenção foi alocada às posições de maior profundidade. Os resultados também indicaram que os estímulos maiores capturaram tanto a atenção transitória quanto a atenção sustentada. Assim, este estudo permitiu um maior conhecimento sobre os mecanismos perceptivos e atentivos envolvidos no processamento das informações de tamanho e de profundidade. / This study aimed to investigate the temporal dynamics of object size perception under texture gradients from line-drawing (perspective gradient) and photograph with natural elements (photographed gradient). Additionally, it was analyzed the occurrence and the pattern of eye movements in these conditions. For this, we carried out 2 experiments. In the first experiment, two black circles were displayed in the vertical meridian and 96 participants reported whether the bigger one was presented at the lower or upper visual field. By using double staircase psychophysics method, these circles were briefly presented (50, 100, 150 or 200 ms) in three background conditions (no texture, perspective gradient or photographed gradient). The slope of the psychometric function and the points of subjectivity equality (PSE) were calculated and used as discrimination sensitivity and size distortion measures, respectively. The results showed a greater sensitivity to size discrimination as exposure time increased, regardless the texture gradient. The analysis of PSE indicated greater size distortions in perspective gradient at 100 ms and in photographed gradient at 150 ms. In the second experiment, 24 participants performed a task similar to that in Experiment 1 while an eye tracker recorded their eye-movements. The results showed that, at 150 and 200 ms, eye movements occurred in less than 10% of trials in all backgrounds. It indicates that the size distortion reduction after 100 ms observed in the first experiment cannot be fully explained by attention allocation. Taken together, these results suggest that the process and integration of size and depth information are mainly mediated by bottom-up mechanism. Moreover, in a less time constrain condition (1500 ms), depth cues affected the spatial measures of fixations and saccades, but not the temporal ones. The participants looked more frequently at the stimulus on the upper visual field, indicating that attention has been allocated to positions of greater depth. The results also indicated that large stimulus capture both transient and sustained attention. Thus, this study provides a better understanding of perceptual and attentive mechanisms involved in size and depth process.

O desenvolvimento sustentável no final do século XX surgiu para expressar preocupações com graves problemas que causam riscos a vida no planeta. O uso do planeta de forma sustentável exigirá diferentes estratégias de sustentabilidade. Neste sentido têm-se as estratégias que abordam o nível mais elevado do ecossistema referidas como Top-down e as estratégias que abordam componentes locais ou regionais referidas como bottom-up. A escola traz essa compreensão nos conceitos e fenômenos ecológicos. No entanto, a compreensão dessas abordagens com apoio interdisciplinar faz com que o aluno entenda os fenômenos ecológicos de forma mais crítica. Assim, para um aprendizado motivador, o professor pode utilizar como recurso didático além dos espaços formais de educação também espaços não formais de educação, ou seja, aulas de campo em ambientes naturais. Com o intuito de promover no ensino de ecologia a alfabetização ecológica dos alunos no Ensino Médio, este trabalho elaborou uma proposta didática com apoio interdisciplinar (Biologia, Língua Portuguesa e Geografia) e com ênfase nas estratégias Top-down e bottom-up abordadas tanto em espaços formais quanto não formais de educação. O método de ensino em espaço formal de educação apesar de estar estruturado didaticamente para abranger a ecologia não se apresentou motivadora ao aluno e muitos dos conceitos e fenômenos ecológicos não foram assimilados por eles. Em contrapartida, a integração interdisciplinar em associação com as estratégias de Top-down e bottom-up em espaços não formais de educação foi motivadora para o aluno melhorando o seu desempenho com relação à assimilação dos conceitos e fenômenos biológicos promovendo a alfabetização ecológica dos alunos. Espera-se com esse trabalho ter contribuído para o ensino e aprendizagem do tema sustentabilidade através da promoção da alfabetização ecológica dos alunos de Biologia no Ensino Médio em escolas públicas com a formação de cidadãos mais conscientes e críticos em assuntos ambientais. / Sustainable development in the late 20th century has emerged to express concerns about serious life-threatening problems on the planet. The use of the planet in a sustainable way will require different sustainability strategies. In this sense we have the strategies that approach the highest level of the ecosystem referred to as \"top-down\" and the strategies that approach local or regional components referred to as \"bottom-up\". The school brings this understanding into concepts and ecological phenomena. However, understanding these approaches with interdisciplinary support makes the student understand ecological phenomena more critically. Thus, for a motivating learning, the teacher can use as a didactic resource beyond the formal spaces of education also non-formal spaces of education, that is, field lessons in natural environments. In order to promote ecological literacy among students in high school, this work elaborated a didactic proposal with interdisciplinary support (Biology, Portuguese Language and Geography) and with emphasis on top-down and bottom-up strategies addressed in both formal and non-formal education settings. The method of teaching in formal educational space despite being structured to cover ecology did not present itself as motivating to the student and many concepts and ecological phenomena were not assimilated by them. In contrast, interdisciplinary integration in association with top-down and bottom-up strategies in non-formal education spaces was motivating for the student to improve his performance in relation to the assimilation of concepts and biological phenomena by promoting literacy of the students. It is hoped that this work contributed to the teaching and learning of the sustainability theme through the promotion of the ecological literacy of Biology students in High School in public schools with the formation of citizens more conscious and critical in environmental issues.

Le secteur résidentiel en Algérie consomme 25 % de l'énergie finale et émet en moyenne 21 % des gaz à effet de serre. Or, il existe un important gisement d'économie d'énergie dans ce secteur. Ce constat a conduit à rechercher la manière de rendre le secteur du logement plus efficient en termes de consommation d'énergie et d'émission de CO2. Cette thèse vise à développer différentes méthodes d'analyse quantitative dédiées à l'amélioration performantielle du secteur résidentiel à l'horizon 2050 . Il s'agit, en clair, d'infléchir les consommations d'énergie et de stabiliser les émissions de CO2. La comparaison entre la France et l'Algérie doit mettre de mettre en lumière les différences, tant dans les dynamiques des structures de parcs que dans celles induites par les différentiels climatiques.Ce travail s'inscrit dans une démarche prospective. Il s'appuie sur les principes de l'exercice prospectif développés par Jacques Arcade et Michel Godet, et il s'inspire pleinement de nombreux travaux de recherche (ETHEL, CLIP…). Il a pour ambition de développer un modèle de prospective énergétique bottom-up, capable de mesurer différentes trajectoires de l'évolution des consommations d'énergie dans le secteur résidentiel et les émissions qui en découleraient à l'horizon 2050.Ce modèle s'articule à partir des données du scénario central de la démographie et du parc de logements de l'Office national des statistiques algérien, ainsi que les hypothèses argumentées relatives aux évolutions démographiques des ménages. La poursuite des tendances actuelles, en termes de consommations d'énergie et d'émissions à l'horizon 2050, permet de tracer un scénario de référence sur lequel la thèse s'appuie pour identifier les actions à mettre en œuvre afin d'y apporter d'éventuelles solutions. En se basant en effet sur un ensemble de leviers technologiques, notre modèle peut établir des scénarios volontaristes dans lesquels l'on suppose plusieurs trajectoires de l'évolution du couple consommations/émissions.Outre l'intérêt scientifique de ce travail, le développement de ce modèle peut constituer un outil d'aide à la mise en oeuvre d'une politique énergétique portant sur l'amélioration de l'efficacité énergétique dans le secteur du logement ; les scénarios réalisés peuvent en outre constituer une feuille de route pour de nouvelles stratégies énergétiques. / The residential sector in Algeria consumes 25 % of the final energy and emits about 21% of greenhouse gas emissions. However, there is a significant potential of energy saving in this sector. This leads to looking for ways to make the sector of housing more efficient in terms of energy consumption and CO2 emissions. This thesis aims to develop different methods of quantitative analysis dedicated to improving the performance of the résidential sector in 2050. In other words, the objective is to reduce energy consumption and stabilize CO2 emissions. The comparison between France and Algeria should help to clarify the differences in both dynamic structures housing stock as well as in those induced by climate differentials.This research work fits into a prospective approach. It is based on the principles of prospective exercise developed by Jacques Arcade, Michel Godet, and it is influenced by numerous research works (ETHEL, CLIP). It has the ambition to develop a model of bottom-up energy prospective capable of measuring various trajectories of the evolution of energy consumption in the residential sector and resulting emissions by 2050.This model is based on the data of the central scenario of demography and the housing stock of the Algerian National office of Statistics, and on the well-argued assumptions concerning the démographic evolution of households. The continuation of current trends in terms of energy consumption at the approach of 2050 allows us to draw up a reference scenario on which the thesis is based to identify the actions to be implemented to identify possible solutions. Indeed, by basing itself on a set of technological levers, our model can establish voluntaris scenarios in which several trajectories of the evolution of the consumption/emissions pair can be envisaged.Besides the scientific interest of this work, the developement of this model can establish a help tool for the implementation of an energy policy concerning the improvement of the energy efficiency in the sector of housing ; moreover, the scenarios undertaken could constitute a road map for new energy strategies.

Grâce aux récents progrès en terme d'instrumentation analytique, la protéomique, en tant que science qui étudie le protéome d'un organisme ou d'un milieu, a connu un véritable tournant et a permis d'étendre le champ des connaissances sur le fonctionnement du vivant dans son ensemble (structure, fonction, métabolisme, dynamisme). Néanmoins, l'étude des protéomes représente un challenge pour de nombreux biologistes, chimistes et biochimistes, en raison notamment de la complexité des échantillons étudiés. De nombreux protocoles analytiques ont d'ores et déjà été développés. Cependant, dans leur ensemble, ces stratégies sont relativement longues et multi-étapes et le plus souvent ciblées sur une protéine donnée.Dans ce contexte, ce travail de thèse a pour objectif de simplifier les protocoles expérimentaux existants afin de limiter les pertes en protéines et ainsi amplifier leurs signaux au cours d'une analyse « Bottom-up » par LC-HRMS (analyse « hors gel »). Chaque étape du processus a été décortiquée et optimisée. Dans un premier temps, les paramètres UPLC-HRMS/MS ont été optimisés afin d'améliorer la détection et la quantification des protéines présentes à des concentrations très variables dans les milieux étudiés. Ensuite, une approche de purification simplifiée qui repose sur une seule et unique étape de lavage et solubilisation des protéines a été mise au point. La démonstration de son efficacité « chimique » et « biologique » a ensuite été réalisée via une étude mécanistique au cours de laquelle les changements de conformation des protéines ont été étudiés à chacune des étapes de purification proposées. Enfin, certains paramètres influençant l'extraction des protéines à partir de ces mêmes matrices ont été étudiés afin de proposer à terme un protocole d'extraction à la carte compatible avec une analyse directe par LC-HRMS/MS. / Recent advances in proteomics have been spurred by the rapid development of hybrid and/or high-resolution mass analysers (HRMS/MS). These powerful instrumentations have led to significant improvements in « Bottom-up » approach and have enabled to deepen our knowledge on the functionality of biological systems (structure, function, metabolism, dynamic, etc).Despite their high sensibilities, the potential of such instruments could be significantly lessened by an imperfect sample pre-treatment. In this context, current sample pretreatments follow multi-steps experimental workflows, which alternatively lead to low recoveries of proteins. In this line, this study aims at developing a simple and versatile strategy in order to reduce protein losses and enhance their detection in gel-free LC-MS analysis. First, an analytical method based on liquid chromatography and tandem mass spectrometry was developed to detect and quantify complex peptides mixture. Secondly, a universal, simple and fast purification approach was designed with the aim to purify protein extracts in only one-step. For this purpose, the molecular reactivity, dynamics and conformational changes of proteins at each development step were comprehensively investigated with a set of spectroscopic techniques, in order to select the best strategy. Finally, different factors influencing extraction of proteins were investigated with the goal in the long term to propose an on-demand extraction protocol for direct analysis of proteins by LC-HRMS/MS.

Different approaches for the estimation of the uncertainty related to measurement results are found in the literature and in published guidelines. In the present work three approaches were used to estimate the uncertainty of the determination of aflatoxins (AB1, AB2, AG1, AG2) and deoxynivalenol in maize by liquid chromatography coupled to mass spectrometry tandem (LC-MS/MS): the Bottom up approach, adapted from the Guide to the Expression of Uncertainty in Measurement (ISO GUM); the Monte Carlo Method (MCM), which propagates distributions assigned to the input quantities through a numerical simulation; and the Kragten approach wich calculates standard deviations and confidence intervals with a universally applicable spreadsheet technique. A measurement equation was developed for mycotoxins analysis and a cause-and-effect diagram was draft to assist in the identification of the sources of uncertainty associated with the method. Detailed analysis of contributions of the various uncertainty sources was carried out. Measurement uncertainty was determined by the addition of the variances of the individual steps of the test procedure, according to each approach employed. The Bottom up, MCM and Kragten approaches produced very similar estimates to the combined uncertainty and the coefficient of variation (CV) between them was smaller than 1.0%. The main contribution to overall uncertainty is the intermediate precision with contributions over 90,0% for each mycotoxin. The results obtained with this research conclude that the three approaches are adequate for estimating the uncertainty in mycotoxin assays with LC-MS/MS technique. Among them, Bottom up is the most appropriate approach, since it requires that the analyst performs a detailed investigation about dominant components of the measurement uncertainty, allowing for better understanding and improvement of the measurement process. The Monte Carlo and Kragten methods are indicated the data generated by the Bottom up approach. / A literatura cita diferentes abordagens para estimar a incerteza de medição em ensaios quantitativos. No presente trabalho três abordagens foram utilizadas para estimar a incerteza da determinação de aflatoxinas (AB1, AB2, AG1, AG2) e desoxinivalenol em milho por cromatografia líquida acoplada à espectrometria de massa tandem (LC-MS/MS): a abordagem Bottom up, adaptada do Guia para a Expressão da Incerteza de Medição (ISO GUM); o método de Monte Carlo (MMC), que propaga distribuições atribuídas às grandezas de entrada através de uma simulação numérica; e a abordagem de Kragten que calcula desvios padrão e intervalos de confiança com uma técnica planilha universalmente aplicável. Um modelo matemático foi desenvolvido para os ensaios de micotoxinas e um diagrama de causa-efeito foi proposto para auxiliar na identificação das fontes de incerteza associadas ao método. A análise detalhada das contribuições das várias fontes de incerteza foi realizada. A incerteza de medição foi determinada pela adição das variações dos passos individuais do procedimento de ensaio, de acordo com cada abordagem. Os métodos Bottom up, MMC e Kragten geraram estimativas similares para a incerteza combinada, com um coeficiente de variação (CV) menor que 1,0% entre elas. O principal componente de incerteza é a precisão intermediária, com contribuições acima de 90,0% para cada micotoxina. Como resultado da pesquisa, conclui-se que as três abordagens são adequadas para estimar a incerteza nos ensaios para quantificação de micotoxinas por LC-MS/MS. Dentre elas, a abordagem Bottom up é a mais apropriada, pois requer que o analista avalie o método detalhadamente para identificar os principais componentes de incerteza, possibilitando a implementação de melhorias no sistema de medição. Os métodos de Monte Carlo e Kragten são indicados como uma ferramenta de confirmação dos resultados obtidos pela abordagem Bottom up.