Este trabalho utiliza a Análise de Fluxos Metabólicos para estudar o aumento da eficiência da linhagem Burkholderia sacchari (LFM101) na produção de PHB. Foram avaliadas as eficiências de conversão de açúcares em PHA de LFM101. Esta também foi cultivada em batelada alimentada em reator, apresentando o máximo teórico durante um estado pseudo-estacionário sob oferta de glicose. Estes dados, submetidos ao software Metatool, resultaram em mapa metabólico contendo os fluxos das reações centrais ede PHA ocorrido no experimento. Através do cultivo de LFM101 e C. necator sob oferta de glicose marcada com 13C, determinou-se que estas utilizam as mesmas vias para produção de PHA, não justificando a baixa eficiência de LFM101. Em um projeto paralelo, estudou-se a eficiência da produção de PHB utilizando melaço de cana, glicerol cru e acetato, em produtores de hidrogênio e PHA, onde verificou-se não apenas o aumento de PHA em mutantes nifh- de R. capsulatus mas a interação dos parâmetros luz e nitrogênio a partir das metodologias DOE e RSM. / This work applies Metabolic Flux Analysis to discuss the eficiency of Burkholderia sacchari (LFM101) in PHA production from sugars . Conversion yields of LFM101 and Cupriavidus necator from carbohydrates to PHA were assessed. LFM101 was also grown in reactor fed-batch cultivations, and presented the theoretical maximum in a pseudo-steady stage while grown on glucose. These data were submitted to Metatool and resulted in a metabolic network containing the experimental fluxes of central and PHA metabolism. Cultivation of LFM101 and C. necator under 13C- labeled glucose showed that both species use the same metabolic pathways for the biodegradable polymer synthesis. On a parallel project, the efficiency of biopolymer production from molasses, raw glycerol and acetate in strains producing hydrogen and PHA was tested. Results showed that there was not only an increase in PHA production by R. capsulatus nifH- mutants but also the interaction of light and nitrogen effects were studied by DOE and RSM.

L’établissement d’un marché intérieur procède, sur le plan juridique d’un double mécanisme : un mécanisme d’intégration négative et un mécanisme d’intégration positive. L'intégration négative désigne les interdictions adressées aux États et à leurs collectivités, d'entraver la circulation interétatique de marchandises, personnes, services et capitaux. La Constitution américaine et le traité sur le fonctionnement de l’Union européenne contiennent des dispositions permettant cette intégration des marchés étatiques. La Cour Suprême et la Cour de justice de l'Union européenne ont, dans ce cadre, joué un rôle majeur en précisant la portée qu'il convenait de donner à ces dispositions. Elles ont, à cet égard, développé un cadre d'analyse similaire consistant à identifier les mesures susceptibles de restreindre les libertés de circulation et à examiner si de telles mesures pouvaient être justifiées par un intérêt légitime des États. La marge de manœuvre laissée aux États diverge, néanmoins, sensiblement dans la mesure où les objectifs assignés à l’intégration négative ne présentent qu’une similarité partielle. La jurisprudence américaine est, en effet, essentiellement fondée sur la doctrine de l'anti-protectionnisme tandis que la jurisprudence communautaire a posé un principe plus dynamique de décloisonnement des marchés. Cette divergence est révélatrice d'une conception différente des finalités de chaque Union et du rôle du juge. Le juge américain veille avant tout au respect de la répartition des compétences, telle que prévue par la Constitution; le juge communautaire s’inscrit dans une logique fonctionnelle de réalisation d’un marché intérieur. / The establishment of an internal market results, in legal terms, from a dual mechanism: a mechanism of negative integration and a mechanism of positive integration. Negative integration refers to the prohibition addressed to the States and their local authorities, ta impede interstate movement of goods, persons, service and capital. The U.S. Constitution and the Treaty on the Functioning of the European Union contain provision allowing the integration of state markets. The Supreme Court and the Court of Justice of the European Union have played, in this context, a major role in specifying the meaning ta be given ta these provisions. They have in this regard, developed a similar framework to identify measures that are likely to restrict freedom of movement and ta examine whether such measures could be justified by a legitimate interest of the States. They way left to the States differs, however, substantially since the goals of negative integration are only partially identical. American case law is, indeed, essentially based on the anti-protectionist doctrine while F.U case law is based on a more dynamic principle of market liberalization. This difference is indicative of different approaches regarding the purposes of each Union and the roIe of the judge. The U .S. judge ensures, above all, that the division of competences, as provided by the Constitution, is enforced whereas the role of the ECJ is essentially to guarantee market integration.

In recent years, DNA nanotechnology has emerged as one of the most powerful strategies for bottom-up construction of nanomaterials. Due to the high programmability of DNA molecules, their self-assembly can be rationally designed. Engineered 3D DNA crystals, as critical products from the design of DNA self-assembly, have been proposed as the structural scaffolds for organizing nano-objects into three-dimensional, macroscopic devices. However, for such applications, many obstacles need to be overcome, including the crystal stability, the characterization methodology, the revision of crystal designs as well as the modulation of crystallization kinetics. My PhD research focuses on solving these problems for engineered 3D DNA crystals to pave the way for their downstream applications.<br>In this thesis, I started by enhancing the stability of engineered 3D DNA crystals. I developed a highly efficient post-assembly modification approach to stabilize DNA crystals. Enzymatic ligation was performed inside the crystal lattice, which was designed to covalently link the sticky ends at the crystal contacts. After ligation, the crystal became a covalently bonded 3D network of DNA motifs. I investigated the stability of ligated DNA crystals under a wide range of solution conditions. Experimental data revealed that ligated DNA crystals had significantly increased stability. With these highly stabilized DNA crystals, we then demonstrated their applications in biocatalysis and protein encapsulation as examples.<br>I also established electron microscope imaging characterization methods for engineered 3D DNA crystals. For crystals from large-size DNA motifs, they are difficult to study by X-ray crystallography because of their limited diffraction resolutions to no better than 10 Å. Therefore, a direct imaging method by TEM was set up. DNA crystals were either crushed or controlled to grow into microcrystals for TEM imaging. To validate the imaging results, we compared the TEM images with predicted models of the crystal lattice. With the advance in crystal characterization, DNA crystals of varying pore size between 5~20 nm were designed, assembled, and validated by TEM imaging.<br>The post-assembly ligation was further developed to prepare a series of new materials derived from engineered 3D DNA crystals, which were inaccessible otherwise. With the directional and spatial control of ligation in DNA crystal, I prepared new DNA-based materials including DNA microtubes, complex-architecture crystals, and an unprecedented reversibly expandable, self-healing DNA crystal. The integration of weak and strong interactions in crystals enabled a lot of new opportunities for DNA crystal engineering.<br>In the final chapter, I investigated the effect of 5’-phosphorylation on DNA crystallization kinetics. I found that phosphorylation significantly enhanced the crystallization kinetics, possibly by strengthening the sticky-ended cohesion. Therefore, DNA crystals can be obtained at much lower ionic strength after phosphorylation. I also applied the result to controling the morphology of DNA crystals by tuning the crystallization kinetics along different crystallographic axes. Together with previously methods to slow down DNA crystallization, the ability to tune DNA crystallization kinetics in both ways is essential for DNA crystal engineering.

Nas últimas décadas, a incidência de infecções hospitalares causadas por bactérias Gram-negativas multirresistentes vem crescendo de maneira vertiginosa em todo o mundo, de modo que a Organização Mundial de Saúde (OMS) recentemente reconheceu essas infecções como uma preocupação mundial devido ao seu impacto negativo sobre as taxas de mortalidade intra-hospitalar e dos custos da assistência à saúde, afetando tanto os países desenvolvidos quanto os em desenvolvimento. Atualmente considera-se que o uso racional de antimicrobianos, a higienização das mãos e o isolamento de contato são as principais medidas disponíveis para contenção desse avanço. Porém, elas são apenas parcialmente efetivas e de implementação trabalhosa e onerosa. Assim, considera-se necessário o desenvolvimento de formas mais simples e eficientes para lidar com esse problema. No presente estudo, nos propusemos a avaliar o impacto da administração de um produto simbiótico a pacientes colonizados e/ou infectados por bactérias Gram-negativas multirresistentes sobre a incidência subsequente de infecções hospitalares relacionadas ao trato respiratório e urinário. Trata-se de um ensaio clínico randomizado, duplamente cego, controlado com placebo, cuja intervenção consistiu na administração oral ou enteral diária de 1010 unidades de Lactobacillus bulgaricus e 1010 unidades de Lactobacillus rhamnosus associados a fruto-oligosacarídeos durante 7 dias, a pacientes internados em um hospital terciário, com colonização prévia por bactérias Gram-negativas multirresistentes, demonstrada por meio de cultura seletiva de swab retal. O desfecho primário do estudo foi a incidência de infecção hospitalar posterior à intervenção, que, na análise do tipo intenção de tratar foi 18/48 (37,50%) no grupo experimental e 12/53 (22,64%) no grupo controle (odds ratio ajustado=1,95, IC95%=0,69-5,50, p=0,21). Os desfechos secundários principais, também de acordo com a análise intenção de tratar, foram: o tempo de internação hospitalar; sendo a mediana de 17 dias no grupo controle e 31 dias no grupo experimental (p= 0,07), taxas de óbito; com valores de 3,77% no grupo placebo e 8,33% no grupo simbiótico (odds ratio ajustado = 1,34, IC95%= 0,454,00, p= 0,61) e ocorrência de eventos adversos; 7,55% no grupo que utilizou placebo e 6,25% no grupo sob intervenção (p= 1,00). Os dados obtidos pelo estudo nos levam à conclusão de que o simbiótico estudado demonstrou-se inefetivo na prevenção de infecções hospitalares do trato respiratório e urinário em pacientes colonizados e/ou infectados por bactérias Gram-negativas multirresistentes. / In recent decades the incidence of multidrug resistant Gram-negative nosocomial infections has been dramatically raising in the whole world. The World Health Organization (WHO) recently recognized nosocomial infections as a global concern due to its negative impact on patients, health care workers and health care institutions, affecting developed countries as well as developing ones. They negatively impact in-hospital mortality and healthcare related costs. Antibiotic stewardship, hand hygiene promotion and contact precautions are the main available measures to control such multidrug resistant Gram-negative organisms in hospitals. However, they are only partially effective as well as difficult to be implemented and expensive. Therefore, simpler and more effective actions are thought to be helpful and urgent. In the main study, we propose to analyze the impact of the administration of a symbiotic product on patients colonized and/or infected by Gram-negative multidrug resistant bacteria upon the subsequent incidence of respiratory and urinary tract nosocomial infections. A randomized, double- blinded, placebo controlled, clinical trial was proposed in order to provide oral or enteral daily administration of 1010 units of Lactobacillus bulgaricus and 1010 units of L. rhamnosus associated with fructo-oligosacharide (FOS) during 7 days, to previously colonized patients with multi-resistant Gram-negative bacteria, identified through selective culture of rectal swab, hospitalized in a tertiary-care hospital. The primary outcome was the incidence of nosocomial infections after the intervention, which in the intention to treat analysis was 18/48 (37,50%) in the experimental group versus 12/53 (22,64%) in the control group (adjusted odds ratio= 1,95, IC95%= 0,69-5,50, p=0,21). Secondary outcomes, according to intention to treat analysis, were hospital length of stay: median of 17 days in the control group and 31 days in the symbiotic group (p= 0,07), mortality rates: 3,77% in the placebo group versus 8,33% in the experimental group (adjusted odds ratio = 1,34, IC95%= 0,45 4,00, p= 0,61) and adverse effects: 7,55% in the control group and 6,25% in the intervention group (p= 1,00). The results of this study leads to the conclusion that the studied symbiotic proved to be ineffective to prevent nosocomial respiratory and urinary tract infections in patients colonized and/or infected by Gram-negative multi-resistant bacteria.

Objective: Recent research has supported the construction of a model of depression and anxiety in older adults ( > 60years), in which the decline in working memory with age is implicated in increasing propensity to engage in repetitive negative thinking, thereby predisposing older adults to anxiety and depression. The study detailed here intended to extend these research findings by exploring the efficacy and acceptability of a working memory intervention in older adults. Methods: The study employed a randomised multiple-baseline single case research design, in which six older adults participated in baseline assessment of mood, repetitive negative thinking and working memory. Participants then tracked their repetitive negative thinking daily throughout a baseline phase, the length of which was defined by the randomisation procedure. Following phase change, participants continued to rate their repetitive thinking daily, as well as engaging in a daily working memory training intervention. On completion of the intervention phase, outcome measures of mood, repetitive negative thinking and working memory were repeated. Full visual and statistical analysis of all data was undertaken to support exploration of the findings. Results: Results of the study indicated that that the working memory training program was unlikely to have elicited a significant impact upon participants’ working memory. However, two participants demonstrated reliable improvement in both repetitive thinking and mood. Analysis of relevant variables to predict the selective impact of the intervention was not fruitful, but may indicate that improvements in working memory underscore the improvements in thinking and mood, lending support to the proposed model. There appeared to be a small, non-significant decrease in daily repetitive negative thinking across five of the six participants. Conclusion: Further research is needed to identify factors that may predict response to working memory training within older adult populations. The research supports the on-going investigation of innovative working memory interventions within an older adult population, although results are not sufficiently robust to indicate wider adoption of these models within health services or as routine treatments for this population.

A presente tese consiste em percurso pelas concepções de manejo das transferências negativas nas produções de Freud, Ferenczi e Winnicott, explicitando os diferentes sentidos e enquadres da resistência e da destrutividade na teoria da clínica de cada um desses autores, e também ensaiando pontos de contato entre eles. Como fio condutor, ou como visada ética, a valorização de uma dimensão de positividade da destruição na clínica psicanalítica na medida em que, promotora de diferenças e limites, está implicada nos caminhos das transferências em análise. Ao longo do percurso, o estudo de figuras de submissão na clínica masoquismo em Freud, identificação ao agressor em Ferenczi e falso self em Winnicott nos permite mapear outras vias para o manejo da agressividade, especialmente em situações nas quais a resistência e ódio não aparecem no trabalho analítico ou, se aparecem, não são sentidos como próprios. Em comum entre Freud, Ferenczi e Winnicott, a crítica ao excesso interpretativo e às análises que ocorrem em nível estritamente intelectual. Em Freud, além da interpretação, princípio de abstinência, associação livre/atenção flutuante e luta para superação das resistências, forjados ao longo da primeira tópica, demonstramos que as noções de elaboração, construção, bem como a menção à lenta demolição do superego hostil, permitem o esboço de outras possibilidades de manejo que se desdobram nas teorias de Ferenczi e Winnicott. Em Ferenczi, abordamos o percurso clínico que culmina na elasticidade da técnica analítica e em sua discussão sobre a implicação e limites do analista no manejo do ódio traumático e clivado que reincide na clínica. Em Winnicott, o holding, a regressão à dependência, o brincar, o uso das falhas do analista e a destruição/sobrevivência do analista permitem abordar as diferentes facetas de sua teoria da agressividade no desenvolvimento emocional e diferentes posições do analista no manejo da transferência. Como recurso para explicitar os limites e possibilidades de manejo, a construção do texto é perpassada por extratos de casos clínicos. Em Freud, os casos Dora, Homem dos Ratos, Homem dos Lobos, Jovem Homossexual e AB são abordados; em Ferenczi, algumas considerações sobre a análise de Elisabeth Severn e S.I. são evocados; em Winnicott, são analisados os casos B. e Margaret Little. Por fim, nosso percurso permite afirmar que a possibilidade da destrutividade exercer sua função diferencial depende da disponibilidade do analista de se oferecer como suporte (o que inclui também oposição, resistência e limite) e de se permitir ser destruído e sobreviver à destruição ao longo do trabalho analítico / The present thesis consists of path through the management of negative transfers in Freud\'s, Ferenczi\'s and Winnicott\'s productions by making explicit different framing and meaning of resistance and destructiveness in treatment\'s theory of each of these authors, but also essaying points of contact among them. As a guidewire, or as ethic bias, the valorization of a dimension of positiveness of the destruction in psychoanalytic treatment insofar as, promoter of differences and limits, it is implied in the ways of the transfers in analysis. Along the course, the study of figures of submission in treatment masochism in Freud, identification with the aggressor in Ferenczi and false self in Winnicott allows one to map other ways for the management of aggressiveness, especially in situations in which resistance and hate do not appear in analytic work or, if they do, they are not felt as their own. Common to Freud, Ferenczi and Winnicott, the criticism to the interpretative excess and to analysis that occur in the strictly intellectual level. In Freud, besides interpretation, principle of abstinence, free association / floating attention and fight to overcome the resistances, forged along the first topic, we demonstrate that the notions of working through, construction, as well as the mention to the slow demolition of the hostile super-ego, permit outline other possibilities of management that unfold in Ferenczi\'s and Winnicott\'s theories. In Ferenczi, we approach the clinical course that culminates in the elasticity of the analytical technique and in its discussion about the implications and limits of the analyst in cleaved and traumatic hate management that relapses in treatment. In Winnicott, the holding, the regression to dependence, the play, the use of the analyst\'s fails and destruction/survival permit to approach the different facets of his theory of aggressiveness in emotional development and different positions of the analyst in transfer management. As a resource to make explicit the limits and possibilities of management, the construction of the text is permeated by extracts of clinical cases. In Freud, Dora, Rat Man, Wolf Man, Young Female Homosexual, and AB cases are addressed; in Ferenczi, some considerations about the analysis of Elisabeth Severn and S.I. are evoked; in Winnicott, B. and Margaret Little cases are analyzed. In conclusion, our course permits to affirm that the possibility of destructiveness to exert its differential function depends on the availability of the analyst to offer as a holding himself (which includes also opposition, resistance and limit) and to permit himself to be destroyed and himself to be destroyed and to survive to the destruction along the analytical work

Les cancers du sein triple-négatif (TNBC) (RE-, RP-, HER2) représentent 15% des cas de cancer du sein. Les patientes atteintes de TNBC sont traitées uniquement par chimiothérapie. A l’heure actuelle, il n’existe aucune thérapie ciblée efficace. Malgré une chimiosensibilité initiale, les rechutes sont fréquentes. Ainsi de nouveaux traitements sont nécessaires pour soigner ces patientes. Dans le cancer du sein, l’aspartyl protéase cathepsine D (cath-D), un marqueur de mauvais pronostic, est surexprimée par les cellules cancéreuses et est hyper-secrétée dans le microenvironnement tumoral. La cath-D stimule la prolifération des cellules cancéreuses, la croissance invasive des fibroblastes, la croissance tumorale, l’angiogenèse tumorale et la formation des métastases. Différentes études ont mis en exergue le rôle oncogénique de la cath-D extracellulaire dans le cancer du sein, suggérant qu’elle serait une cible thérapeutique d’intérêt. Afin de neutraliser sélectivement la forme sécrétée de la cath-D, le laboratoire a généré des anticorps humains IgG1 dirigés contre la cath-D par un crible de phage display (International patent N° PCT/EP2016/061454).Les principaux objectifs de ma thèse ont été i) de valider la cath-D comme une cible extracellulaire d’intérêt pour les patientes atteintes de TNBC, ii) d’évaluer les effets thérapeutiques et iii) de caractériser les mécanismes d’action des anticorps humains anti-cath-D.Nous avons montré que des niveaux élevés d'ARNm de CTSD sont corrélés à une survie sans récidive plus courte. Par analyse protéomique et étude immunohistochimique anti-cath-D réalisée sur Tissue Micro-Array, nous avons observé que la cath-D extracellulaire est détectée dans le microenvironnement tumoral des TNBC contrairement au tissu mammaire normal. Nos résultats mettent ainsi en exergue que la cath-D serait un biomarqueur tumoral extracellulaire, suggérant que les patientes atteintes de TBNC pourraient bénéficier d’une thérapie par des anticorps anti-cath-D. Par des analyses de SPECT-CT (Single Photon Emission Computed Tomography) et de biodistribution, nous avons validé que les anticorps humains anti-cath-D, F1 et E2, s’accumulent dans les xénogreffes de la lignée TNBC MDA-MB-231 chez la souris athymique. Les anticorps F1 et E2 inhibent la croissance tumorale des xénogreffes MDA-MB-231 et améliorent la survie des souris athymiques. Notre meilleur anticorps anti-cath-D, F1, inhibe également la croissance tumorale de deux lignées de TNBC PDX (patient-tumor derived xenografts). Au niveau mécanistique, l’anticorps F1 module le microenvironnement immunitaire dans les tumeurs issues des xénogreffes MDA-MB-231. L’ensemble de nos résultats suggèrent qu’une immunothérapie avec des anticorps humains anti-cath-D pourrait être une nouvelle approche thérapeutique pour les patientes atteintes de TNBC. / Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancer cases, and chemotherapy is the only available treatment. Thus identification of new therapeutic targets is required to improve TNBC outcome. In breast cancer, the aspartic protease cathepsin D (cath-D) is a marker of poor prognosis associated with metastatic risk. This protease is overexpressed by breast cancer cells and is abnormally hypersecreted into the tumor microenvironment. Cath-D affects both cancer and stromal cells in the breast tumor microenvironment by increasing the proliferation of breast cancer cells, fibroblast invasive outgrowth, tumor growth and angiogenesis, and metastasis formation. Many studies indicated that extracellular cath-D displays oncogenic activities, suggesting it could represent a novel therapeutic target in TNBC. In order to block its oncogenic actions, the laboratory generated human IgG1 antibodies against extracellular cath-D by phage display (International patent N° PCT/EP2016/061454). The aims of my PhD project was i) to validate the potential value of cath-D as a tumor-specific extracellular target in TNBC, ii) to evaluate the therapeutic activity, and iii) to characterize the mechanisms of action of these anti-cath-D human antibodies.We showed that elevated CTSD mRNA levels correlated with shorter recurrence-free survival. Using proteomics analysis and anti-cath-D immunohistochemistry performed on Tissue Micro-Array, we observed that extracellular cath-D was detected in the tumor microenvironment of TNBC, but not in matched normal breast stroma samples. Our results thus indicate that cath-D is a tumor cell-associated extracellular biomarker and strongly suggest that it could be a good candidate for antibody-based therapy in TNBC. We found that anti-cath-D human antibodies, F1 and E2, accumulated in TNBC MDA-MB-231 tumor xenografts in athymic mice by SPECT-CT (Single Photon Emission Computed Tomography) and biodistribution analysis. F1 and E2 antibodies inhibited tumor growth of MDA-MB-231 tumor xenografts and improved mice survival without apparent toxicity. F1, the best antibody candidate, inhibited tumor growth of two TNBC patient-derived xenografts (PDX). Mechanistically, F1 treatment modulates immune tumor microenvironment in the MDA-MB-231 tumor cell xenograft model. Together, our results indicate that antibody-based targeting of cath-D may have therapeutic efficacy for TNBC treatment.

No presente trabalho tentamos reconstruir os principais lineamentos da teoria estética e da teoria da sociedade de Theodor Adorno. Com tal propósito, partimos de uma revisão critica da interpretação canônica efetuada tanto por Jürgen Habermas como por Axel Honneth do potencial explicativo contido em obras clássicas da primeira geração da Escola de Frankfurt. O objetivo central deste confronto procura demonstrar que o conceito de cultura de Adorno y Horkheimer tem um potencial explicativo e critico que nem Habermas, nem Honneth souberam destacar. Para reexaminar esse potencial teórico subterrâneo resulta imprescindível referir-se as duas grandes obras da maturidade de Adorno, a Teoria estética e a Dialética negativa. Realizamos-nos esse trabalho procurando explicitar um conceito de cultura alternativo ao de Habermas e Honneth, para tentar logo extrair conclusões referidas à lógica interna das ciências sociais. / The object of this thesis is to reconstruct the basic lines of Adorno\'s aesthetic and social theory. We discuss Jürgen Habermas and Axel Honneths canonic interpretation about the theoretic potential of the classic works of the first generation of the Frankfurt School. The central aim of this confrontation is to demonstrate that the concept of culture developed by Adorno and Horkheimer has a theoretic potential that had not been perceived by Habermas and Honneth. In order to reevaluate this potential it is necessary to discuss the last Adornos works, which means, to discuss once again the Aesthetic Theory and the Negative Dialectics. We pretend to find, finally, a different concept of culture in order to use it in the critical reexamination of the internal logic of social sciences.

Introdução: Bactérias intestinais influenciam a resposta imune e conhecendo-se as ações imunomoduladoras da vitamina D passou-se a investigar sua relação com a microbiota. O status adequado desta vitamina está associado a uma composição mais saudável da microbiota, enquanto sua deficiência pode acarretar disbiose intestinal, endotoxemia, inflamação e resistência à insulina. O conhecimento de interações do status de vitamina D com a microbiota pode melhorar a compreensão da gênese de doenças crônicas mediadas pela inflamação. Objetivo: Examinou-se a associação da ingestão e concentração de vitamina D com a composição da microbiota fecal, marcadores inflamatórios e perfil bioquímico de participantes do Nutritionists Health Study. Métodos: Nesta análise transversal, 150 adultos jovens foram estratificados em tercis de consumo e de concentração de 25(OH)D e comparados quanto ao perfil clínico e inflamatório. A associação de 25(OH)D com a microbiota (sequenciamento do 16S rRNA, região V4, Illumina® MiSeq) foi testada por regressão linear múltipla. Resultados: A ingestão de vitamina D se associou aos níveis séricos (p < 0,05). Não foram observadas diferenças significantes de variáveis clínicas e inflamatórias entre os tercis de ingestão, exceto tendência de aumento do LPS com a redução da 25(OH)D (p-trend < 0,05). Prevotella foi mais abundante (log2FC 1,67; p < 0,01), e Haemophilus and Veillonella menos abundantes (log2FC -2,92 e -1,46; p < 0,01, respectivamente) no subgrupo com maior ingestão de vitamina D (referência) comparado aos outros grupos (primeiro e segundo tercis). PCR (r = -0,170; p = 0,039), selectina-E (r = -0,220; p = 0,007) e abundância de Coprococcus (r = -0,215; p = 0,008) e de Bifdobacterium (r = -0,269; p = 0,001) foram inversamente correlacionados com 25(OH)D. Após ajustes por idade, sexo, estação do ano e IMC, a 25(OH)D manteve associação inversa com Coprococcus ( = -9,414; p = 0,045) e Bifdobacterium ( = -1,881; p = 0,051), mas a significância desapareceu com a adição de marcadores inflamatórios aos modelos. Conclusão: Associações de ingestão e concentração de vitamina D com abundância de certos gêneros da microbiota sugerem que sua ação imunomoduladora poderia influenciar a composição bacteriana. Abundância relativamente maior de gram-negativos (Haemophilus e Veillonella) pode ter sido facilitada pela baixa ingestão e/ou concentração da vitamina. Menor proporção de bactérias benéficas (Coprococcus e Bifidobacterium) poderia estimular a resposta imune e inflamação. Concluímos que a participação da vitamina D na manutenção da homeostase imune deve ocorrer em parte pelas interações com a microbiota intestinal, embora o delineamento transversal impeça assegurar relações tipo causa-efeito. / Introduction: Gut bacteria influence the immune response and due the immunomodulatory actions of vitamin D, it has been investigated its relationship with the microbiota. Adequate status of this vitamin is associated with adequate composition of the microbiota, while its deficiency can cause gut dysbiosis, endotoxemia, inflammation and insulin resistance. The knowledge of interactions of vitamin D status with the microbiota may improve the understanding of the genesis of inflammation-mediated chronic diseases. Objective: We examined the association of vitamin D intake and concentration with the composition of fecal microbiota, inflammatory markers and biochemical profile of participants from the Nutritionists\' Health Study. Methods: In this cross-sectional analysis, 150 healthy young adults were stratified into tertiles of intake and concentrations of vitamin D and their clinical and inflammatory profiles were compared. The association between 25(OH)D and fecal microbiota (16S rRNA sequencing, V4 region, Illumina® MiSeq) was tested by multiple linear regression.) Results: Vitamin D intake was associated with its concentration (p<0.05). There were no significant differences in clinical and inflammatory variables across tertiles of intake, except for a trend of LPS increases with reduction of 25(OH)D (p-trend <0.05). Prevotella was more abundant (log2FC 1.67; p <0.01), and Haemophilus and Veillonella less abundant (log2FC -2,92 e -1.46; p <0.01, respectively) in subset with the highest vitamin D intake (reference) than that observed in the other subset (first plus second tertiles). CRP (r=-0.170, p=0.039), E-selectin (r=-0.220, p=0.007) and abundances of Coprococcus (r=-0.215, p=0.008) and Bifdobacterium (r=-0.269, p=0.001) were inversely correlated with 25(OH)D. After adjusting for age, sex, season and BMI, the 25(OH)D maintained inversely associated with Coprococcus (=-9.414, p=0.045) and Bifdobacterium (=-1.881, p=0.051), but significance disappeared following the addition of inflammatory markers in the regression models. Conclusion: Association of vitamin D intake and concentration with abundance of certain genera of microbiota suggests that its immunomodulatory action could influence the bacterial composition. Relatively higher abundance of gram-negative (Haemophilus and Veillonella) may have been facilitated by the low intake and/or concentration of the vitamin. Lower proportion of beneficial bacteria (Coprococcus and Bifidobacterium) could stimulate the immune response and inflammation. We conclude that the role of vitamin D in maintaining immune homeostasis should occur in part by interactions with the gut microbiota, although the cross-sectional design does not allow ensuring cause-effect relationships.

Uma suposição muito comum na análise de modelos de regressão é a de respostas independentes. No entanto, quando trabalhamos com dados longitudinais ou agrupados essa suposição pode não fazer sentido. Para resolver esse problema existem diversas metodologias, e talvez a mais conhecida, no contexto não Gaussiano, é a metodologia de Equações de Estimação Generalizadas (EEGs), que possui similaridades com os Modelos Lineares Generalizados (MLGs). Essas similaridades envolvem a classificação do modelo em torno de distribuições da família exponencial e da especificação de uma função de variância. A única diferença é que nessa função também é inserida uma matriz trabalho que inclui a parametrização da estrutura de correlação dentro das unidades experimentais. O principal objetivo desta dissertação é estudar como esses modelos se comportam em uma situação específica, de dados de contagem com sobredispersão. Quando trabalhamos com MLGs esse problema é resolvido através do ajuste de um modelo com resposta binomial negativa (BN), e a ideia é a mesma para os modelos envolvendo EEGs. Essa dissertação visa rever as teorias existentes em EEGs no geral e para o caso específico quando a resposta marginal é BN, e além disso mostrar como essa metodologia se aplica na prática, com três exemplos diferentes de dados correlacionados com respostas de contagem. / An assumption that is common in the analysis of regression models is that of independent responses. However, when working with longitudinal or grouped data this assumption may not have sense. To solve this problem there are several methods, but perhaps the best known, in the non Gaussian context, is the one based on Generalized Estimating Equations (GEE), which has similarities with Generalized Linear Models (GLM). Such similarities involve the classification of the model around the exponential family and the specification of a variance function. The only diference is that in this function is also inserted a working correlation matrix concerning the correlations within the experimental units. The main objective of this dissertation is to study how these models behave in a specific situation, which is the one on count data with overdispersion. When we work with GLM this kind of problem is solved by setting a model with a negative binomial response (NB), and the idea is the same for the GEE methodology. This dissertation aims to review in general the GEE methodology and for the specific case when the responses follow marginal negative binomial distributions. In addition, we show how this methodology is applied in practice, with three examples of correlated data with count responses.