La Leucémie Lymphoïde Chronique (LLC) se caractérise par une hétérogénéité d’évolution avec des formes indolente et progressive. Cette dernière reste incurable avec les options thérapeutiques classiques. Mon objectif de thèse a consisté à définir phénotypiquement et fonctionnellement le potentiel régulateur des lymphocytes B de LLC induisant un contexte favorable à leur survie. Après purification des cellules B leucémiques de 30 patients LLC, nos résultats montrent que les sous-populations B sécrètent des cytokines immuno-régulatrices dont l’IL-10 et le TGFβ1. De façon intéressante, ces sous-populations lymphocytaires expriment également le facteur de transcription FOXP3, caractéristique des cellules T régulatrices. La signature phénotypique de ces sous-populations est spécifique au néoplasme avec des marqueurs de lymphocytes B mémoires activés. Nos approches fonctionnelles in vitro démontrent que ces sous-populations B modulent la prolifération et orientent la différenciation et les sécrétions des cellules T, contribuant à l’absence d’immuno-surveillance chez les patients. Enfin, une analyse statistique combinant les expressions de l’IL-10, du TGFβ1 et de FOXP3 dans ces sous-populations B permet de définir un indice polyfonctionnel qui corrèle avec deux facteurs clés du risque de progression de la LLC. L’ensemble de mes travaux de thèse a permis de caractériser de nouvelles sous-populations B impliquées dans la progression de la maladie et donne un rationnel à la survie des cellules leucémiques dans l’environnement ganglionnaire. / Chronic Lymphocytic Leukemia (CLL) is a clonal B cell malignancy of the elderly. This neoplasm is characterized by a heterogeneous clinical course from indolent chronic disease to progressive lymphadenopathy that remains incurable. The aim of my PhD was to undertake a comprehensive phenotypic and functional analysis of the B cell subpopulations responsible for their survival advantage. CLL B cell purification from 30 patients indicate the presence in various extents of leukemic B cell subpopulations producing immune-regulatory cytokines, notably IL-10 and TGFβ1. Remarkably, these CLL subpopulations express the FOXP3 transcription factor, a marker of regulatory T cells. These subpopulations present a phenotypic signature, distinguishable from regulatory B cells populations, with specific markers of activated memory B cells. Functional studies prove their regulatory capacities on T cell differentiation, proliferation and secretion, contributing to the T cell dysfunction observed in CLL. Finally, statistical analysis combining IL-10, TGFβ1 and FOXP3 expressions for these subpopulations allows generating a poly-functional index correlated with two major risk factors of CLL progression. Our data characterize novel CLL B cell subpopulations that are involved in disease progression and give a rational to CLL B cell survival in secondary lymphoid organs.

This study examines the influence of various facets of perceived risk on the attitudes toward really new services (RNS) and adoption intentions. Although there is considerable research examining perceived risk and consumer adoption of innovations, three aspects of the relationship have been neglected. First, much of the research on really new innovations is product-focused with little attention to services. Second, there is limited research examining perceived risk as a multidimensional construct. Third, consumer characteristics that affect the relationship between perceived risks and adoption intentions have not been included in most of the innovation studies. Thus, this study seeks to provide answers to the questions of “which types of perceived risk are more likely to affect adoption intentions of RNS?” and “which consumer characteristics affect the relationship between perceived risks and adoption intentions of RNS?”. The findings of the study show the influence of perceived risk in the service innovations area. We contribute to theory and practice by identifying the specific risks that cause consumers to resist adopting RNS as well as showing the explanatory power of Regulatory Focus Theory (RFT) to understand why consumers react differently when they encounter service innovations. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2017. / FAU Electronic Theses and Dissertations Collection

Thesis advisor: Michelle M. Meyer / As the factory responsible for producing proteins, ribosomes are of great importance. In bacteria, ribosomes are composed of three ribosomal RNAs (rRNA) of different sizes, and around 50 ribosomal proteins (r-protein). During ribosome biogenesis in bacteria, synthesis of rRNAs and r-proteins are both tightly regulated and coordinated to ensure robust growth. In particular, a group of cis-regulatory RNA elements located in the 5' untranslated regions or the intergenic regions in r-protein operons are responsible for the regulation of r-protein biosynthesis. Based on the fact that RNA-regulated r-protein biosynthesis is essential and universal in bacteria, such unique and varied regulatory RNAs could provide new targets for antibacterial purpose. In this thesis, we report and experimentally verify a novel r-protein L1 regulation model that contains dual L1-binding RNA motif, and for the first time, a S6:S18 dimer-binding RNA structure in the S6 operon. We also describe Escherichia coli-based and Schizosaccharomyces pombe-based reporter systems for in vivo characterization of RNA-protein interactions. So far, both in vivo systems failed to report RNA-protein interactions, and thus need further tuning. In addition, we performed phage-display to select for regulatory RNA-binding small peptides and examined their effects on bacteria viability. One selected peptide, N-TVNFKLY-C, caused defective growth when overexpressed in E. coli. Yet, further studies must be conducted to verify the possibility that bacteria were killed by direct RNA-peptide interaction that disrupted the native r-protein regulation. / Thesis (MS) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

As células T regulatórias (Tregs) são essenciais para a manutenção da tolerância periférica, prevenção de doenças autoimunes e limitantes nas doenças inflamatórias crônicas. Além disso, essas células exercem um papel fundamental no controle da rejeição de transplantes. Diferentes protocolos mostraram que é possível obter Tregs a partir de células T naive CD4+ in vitro. Para tal, é consenso que o TGF-? e a interleucina-2 (IL-2) são capazes de direcionar as células T naive CD4+ a se tornarem regulatórias após um estímulo antigênico (anti-CD3/CD28). Nosso grupo recentemente notou que, durante a imunomodulação de linfócitos T pelas células estromais mesenquimais (CTMs), estas eram capazes de produzir adenosina que, por sua vez, participa do processo de imunorregulação. Outros trabalhos indicam que as CTMs suprimem a proliferação dos linfócitos T pela geração de Tregs e que as CTMs induzem a geração de Tregs através da regulação negativa da via TCR e da via AKTmTOR. Evidências apontam que a adenosina pode atuar regulando negativamente a via mTOR. Portanto, acredita-se que a adenosina possa participar do processo de geração de Tregs através da modulação da via mTOR. Além disso, estudos recentes indicam que a ativação de receptores de adenosina, mais especificamente A2a, com agentes agonistas, leva ao aumento da produção de células Tregs, enquanto que a utilização de agentes antagonistas destes receptores leva à diminuição da diferenciação de Tregs. Porém, estes estudos mostram a geração de Tregs a partir de células T naive de camundongos. Visto a grande importância das Tregs no contexto imunológico, a produção eficiente de Tregs in vitro tem importância fundamental para o desenvolvimento de novos protocolos terapêuticos para o tratamento de doenças autoimunes e no combate à rejeição de transplantes. Assim, o objetivo central deste trabalho foi avaliar a participação de agonistas e antagonistas de receptores de adenosina na indução de células T regulatórias geradas in vitro (iTreg) pela ativação de células T CD4+ naive isoladas de sangue de cordão umbilical (SCU) humano. Para isso, células mononucleares foram isoladas de bolsas de SCU e as células T naive foram isoladas imunomagnéticamente. Essas células foram ativadas com beads ligadas a anticorpos anti-CD2/CD3/CD28 e cultivadas por cinco dias na presença de IL-2 e diferentes concentrações de drogas agonistas e antagonistas de receptores de adenosina. Em seguida, foram avaliados os principais marcadores de células T regulatorias por meio de citometria de fluxo e o meio de cultura foi coletado ao final da geração para quantificação de citocinas. Além disso, o RNA total foi extraído de todas as condições de cultivo para a análise da expressão de genes envolvidos na geração e desenvolvimento das Tregs, por PCR quantitativo. O potencial de supressão de células T efetoras também foi avaliado. / Regulatory T cells (Tregs) are essential for the maintenance of peripheral tolerance, prevention of autoimmune and limiting diseases in chronic inflammatory diseases. In addition, these cells play a key role in the control of transplant rejection. Different protocols have shown that it is possible to obtain Tregs from naive CD4+ T cells in vitro. To this end, there is consensus that TGF-? and interleukin-2 (IL-2) are capable of directing the naive CD4 + T cells to become regulatory following an antigenic stimulus (anti-CD3/CD28).. Our group recently noted that during the immunomodulation of T lymphocytes by mesenchymal stromal cells (MSCs), they were able to produce adenosine which in turn participates in the immunoregulation process. Other studies indicate that MSCs suppress the proliferation of T lymphocytes by generation of Tregs and that MSCs induce generation of Tregs by downregulation of the TCR pathway and the AKT-mTOR pathway. Evidence indicates that adenosine may act by downregulating the mTOR pathway. Therefore, it is believed that adenosine may participate in the generation of Tregs by modulating the mTOR pathway. In addition, recent studies indicate that activation of adenosine receptors, more specifically A2a, with agonist agents, leads to increased production of Treg cells, whereas the use of antagonistic agents of these receptors leads to a decrease in Treg differentiation.. However, these studies show the generation of Tregs from naive T cells of mice. In view of the great importance of Tregs in the immunological context, the efficient production of Tregs in vitro is of fundamental importance for the development of new therapeutic protocols for the treatment of autoimmune diseases and in the fight against transplant rejection. Thus, the central objective of this study was to evaluate the participation of adenosine receptor agonists and antagonists in induction of regulatory T cells generated in vitro (iTreg) by the activation of naive CD4+ T cells isolated from human umbilical cord blood (SCU). For this, mononuclear cells were isolated from SCU and naive T cells were immunomagnetic isolated. These cells were activated with beads bound to anti-CD2/CD3/CD28 antibodies and cultured for five days in the presence of IL-2 and different concentrations of agonist drugs and antagonists of adenosine receptors. Next, the major regulatory T-cell markers were assessed by flow cytometry and the culture medium was collected at the end of the generation for quantification of cytokines. In addition, total RNA was extracted from all culture conditions for the analysis of the expression of genes involved in the generation and development of Tregs by quantitative PCR. The potential for suppression of effector T cells was also evaluated.

In nature, biofilm is a complex structure resulted of multicellular bacterial communities that provide important nutritional functions and the acquisition of protective traits such as antibiotics resistance and horizontal gene transfer. The development from the planktonic, lonely bacteria, to the mature multilayered biofilm structure consists of three main phases: motility, attachment and biofilm maturation. At cellular level, the process is controlled by several genes such as flhD, fliA, rpoS, csgD, adrA, cpxR all acting as master regulators. Additionally, the global regulators CRP, IHF, Fis, and others in less frequency, have been related to biofilm formation, although blurry information has been provided. In this thesis we used synthetic, molecular and cellular biology approaches to understand the effect of CRP, IHF and Fis in the transcriptional regulatory network in the bacterium Escherichia coli. In the first chapter, we employed network analysis to reconstruct and analyze part of the entire regulatory network described to modulate the flagella-biofilm program. With this analysis we identified some critical interactions responsible for the planktonic-biofilm transition. Next, we selected the top ten effectors nodes of the network and cloned the promoter region of those genes in a reporter system. As extensively explained in chapter II, this system allowed us to validate as well as suggest new interactions in the network. Additionally, the measurement of the promoter activity during bacterial development show that CRP, IHF and Fis differentially modulate most of the surveyed genes suggesting that those Global Regulators participate to modulate gene expression in different phases of the planktonic-biofilm development. At chapter three, to get a better overview of the entire process, we performed motility, adherence/early biofilm and mature biofilm assays. We describe the intrinsic ability of E. coli to perform motility, adherence and mature biofilm at 37?C. In contrast, the absence of ihf, fis as well as Carbon Catabolite Repression (CCR), lead to altered phenotypes at both motility and biofilm development. At the end, we discussed how the changes of promoter activity of target genes, together with our network analysis, could explain part of the altered phenotypes observed. For instance, we observed changes at the main stress responders rpoS and rpoE that, in combination with alterations at specific genes such as fliA, can explain the enhanced motility in the E. coli ?ihf strain. Altogether, in this thesis, we provided evidence that CRP, IHF and Fis control the activity of the promoter regions of genes involved in the planktonic-biofilm development. / Na natureza, o biofilme é uma estrutura complexa resultante de comunidades bacterianas multicelulares que fornece importantes funções nutricionais e a aquisição de traços de proteção como resistência a antibióticos e transferência horizontal de genes. O desenvolvimento das bactérias planctônicas solitárias para uma estrutura de biofilme maduro consiste em três fases principais: motilidade, fixação e maturação do biofilme. Ao nível celular, o processo é controlado por vários genes tais como flhD, fliA, rpoS, csgD, adrA, cpxR, todos agindo como reguladores mestre. Além disso, os reguladores globais CRP, IHF, Fis e outros em menor freqüência, têm sido relacionados à formação de biofilme, embora tenham sido fornecidas informações nao conclusivas sobre esse processo. Nesta tese foram utilizadas abordagens de bioinformática, assim como de biologia molecular e celular para entender o efeito de CRP, IHF e Fis na rede reguladora da transição de motilidade para biofilme na bactéria Escherichia coli. No primeiro capítulo, utilizamos a análise de rede para reconstruir e analisar parte da rede regulatória descrita para modular o programa flagelo-biofilme. Com esta análise identificamos algumas interações críticas responsáveis pela transição planctônica-biofilme. Em seguida, selecionamos os dez principais nós efetores da rede e clonamos a região promotora desses genes em um sistema repórter. Conforme explicado amplamente no capítulo II, este sistema nos permitiu validar e sugerir novas interações na rede. Adicionalmente, a medição da atividade do promotor durante o desenvolvimento bacteriano mostra que a CRP, a IHF e a Fis modulam diferencialmente a maioria dos genes analisados sugerindo que estes Reguladores Globais participam para modular a expressão génica em diferentes fases do desenvolvimento de estado planctónico para biofilme. No capítulo três, para obter uma melhor visão geral de todo o processo, realizamos ensaios de motilidade, aderência / biofilme precoce e biofilmes maduros. Descrevemos a capacidade intrínseca de E. coli para realizar motilidade, adesão e biofilme maduro a 37 °C. Em contraste, a ausência de ihf, fis, bem como o fenômeno de Repressão de Catabolite de Carbono (CCR), levam a fenótipos alterados, tanto na motilidade como no desenvolvimento do biofilme. No final, discutimos como as mudanças da atividade do promotor de genes alvo, juntamente com a nossa análise de rede, poderia !xi explicar parte dos fenótipos alterados observados. Por exemplo, observamos mudanças nos principais respondedores de estresse rpoS e rpoE que, em combinação com alterações em genes específicos como fliA, podem explicar a motilidade aumentada na estirpe de E. coli ?ihf. Em conjunto, nesta tese, apresentamos evidências de que CRP, IHF e Fis controlam a atividade das regiões promotoras de genes envolvidos no desenvolvimento planctônico-biofilme.

At the core of gene regulatory networks are transcription factors (TFs) that recognize specific DNA sequences and target distinct gene sets. Characterizing the DNA binding specificity of all TFs is a prerequisite for understanding global gene regulatory logic, which in recent years has resulted in the development of high-throughput methods that probe TF specificity in vitro and are now routinely used to inform or interpret in vivo studies. Despite the broad success of such methods, several challenges remain, two of which are addressed in this thesis. Genomic DNA can harbor different epigenetic marks that have the potential to alter TF binding, the most prominent being CpG methylation. Given the vast number of modified CpGs in the human genome and an increasing body of literature suggesting a link between epigenetic changes and genome instability, or the onset of disease such as cancer, methods that can characterize the sensitivity of TFs to DNA methylation are needed to mechanistically interpret its impact on gene expression. We developed a high-throughput in vitro method (EpiSELEX-seq) that probes TF binding to unmodified and modified DNA sequences in competition, resulting in high-resolution maps of TF binding preferences. We found that methylation sensitivity can vary between TFs of the the same structural family and is dependent on the position of the 5mCpG within the TF binding site. The importance of our in vitro profiling of methylation sensitivity is demonstrated by the preference of human p53 tetramers for 5mCpGs within its binding site core. This previously unknown, stabilizing effect is also detectable in p53 ChIP-seq data when comparing methylated and unmethylated sites genome-wide. A second impediment to predicting TF binding is our limited understanding of i) how cooperative participation of a TF in different complexes can alter their binding preference, and ii) how the detailed shape of DNA aids in creating a substrate for adaptive multi-TF binding. To address these questions in detail, we studied the in vitro binding preferences of three D. melanogaster homeodomain TFs: Homothorax (Hth), Extradenticle(Exd) and one of the eight Hox proteins. In vivo, Hth occurs in two splice forms: with (HthFL) and without (HthHM) the DNA binding domain (DBD). HthHM-Exd itself is a Hox cofactor that has been shown to induce latent sequence specificity upon complex formation with Hox proteins. There are three possible complexes that can be formed, all potentially having specific target genes: HthHM-Exd-Hox, HthFL-Exd-Hox, and HthFL-Exd. We characterized the in vitro binding preferences of each of these by developing new computational approaches to analyze high-throughput SELEX-seq data. We found distinct orientation and spacing preference for HthFL-Exd-Hox, alternative recognition modes that depend on the affinity class a sequence falls into, and a strong preference for a narrow DNA minor grove near Exd's N-terminal DBD. Strikingly, this shape readout is crucial to stabilize the HthHM-Exd-Hox complex in the absence of a Hth DBD and can thus be used to distinguish HthHM from HthFL isoform binding. Mutating the amino acids responsible for the shape readout by Exd and reinserting the engineered protein into the fly genome allowed us to classify in vivo binding sites based on ChIP-seq signal comparison between “shape-mutant” and wild-type Exd. In summary, the research presented here has investigated TF binding preferences beyond sequence context by combining novel high-throughput experimental and computational methods. This interdisciplinary approach has enabled us to study binding preferences of TF complexes with respect to the epigenetic landscape of their cognate binding sites. Our novel mechanistic insights into DNA shape readout have provided a new avenue of exploiting guided protein engineering to probe how specific TFs interact with their co-factors in a cellular context, and how flanking genomic sequence helps determine which multi-TF complexes will form and which binding mode a complex adopts.

Esta dissertação compreende um estudo acerca da estrutura de governança regulatória do Brasil, a partir da análise dos setores de telecomunicação e eletricidade. Tal estudo é feito à luz do modelo teórico desenvolvido por Pablo Spiller e Mariano Tommasi que define os meios pelos quais as instituições de um país afetam a habilidade dos atores políticos de sustentar políticas ao longo do tempo e, conseqüentemente, de garantir a credibilidade regulatória do país. Guiada pelo modelo teórico em questão, a análise das características mais marcantes da dotação político-institucional do país, bem como dos processos políticos que deram origem às estruturas regulatórias dos dois setores permite identificar, entre outros aspectos, que o papel de destaque ocupado pelo Executivo no sistema politico brasileiro se confirma também nas arenas das políticas regulatórias dos setores regulados. A análise do desenho das estruturas de governança regulatória desses setores evidencia que, apesar da preocupação em instituir uma regulação de caráter técnico e imune a pressões políticas, nem sempre o desenho escolhido se adapta eficientemente às peculiaridades e características da dotação institucional do País, coibindo o comportamento oportunista dos atores políticos nos setores estudados. A inferência descritiva realizada permite a avaliação do modelo teórico em questão e evidencia a importância de que ao mesmo sejam incorporadas outras variáveis, como as preferências dos atores e os atores com poder de veto. Feito isso, o modelo poderá explicar e avaliar com mais precisão os diversos desenhos de estruturas de governança regulatória. / This thesis consists of a study of Brazils regulatory governance structure, through an analysis of the telecommunications and electricity sectors. The analytical efforts are guided by the theoretical model - developed by Pablo Spiller and Mariano Tommasi that defines the means through which a countrys institutions can affect the ability of politicians to engage in intra-temporal exchanges and, as a consequence, determines the countrys regulatory credibility. Guided by this theoretical model, the analysis of Brazils institutional endowment and of the processes that led to the creation of the regulatory institutions in each sector allows the conclusion that the broad role played by the Executive in the Brazilian political system was also reflected in the specific arenas where the regulatory bodies were shaped. The analysis of the design of the regulatory governance structures demonstrates that, despite initial concerns about the implementation of technical and nonpolitical regulation, these structures are yet to be completely and effectively adapted to the peculiarities and characteristics of Brazils institutional endowment. In addition, the descriptive inference highlights the importance of including the preferences of actors, and the influence of veto players, within the models variables, in order to refine its explanatory power.

A competition agency represents an independent regulatory institution, which takes the form of an administrative body. A competition authority enables the development of markets and displays to market operators and new players a dedication to the principles of free markets and fair competition. In other words, a competition authority should intervene in a timely manner to correct any anti-competitive behaviour and implement the necessary remedies; it should be equipped with an adequate knowledge of the market in order to make its decisions. Moreover, its involvement should be predictable, that is, it should have a positive influence on markets. Furthermore, a competition agency should continuously evaluate its role as public institution and law enforcer by following the economic and legal evolution of the jurisdiction in which it operates. Until recently, the debate has predominantly revolved around the substance of competition law. However, in recent years, the evaluation of the performance of competition agencies has been embraced by numerous countries, including developing ones. This is because most emerging countries around the world have progressively been opening their domestic markets to competition, which led to giving more power to competition agencies to monitor markets. As this perspective has not been explored in the context of Maghreb countries, which also represent developing economies, this research endeavours to do so. Therefore, the aim of this research is to analyse the extent to which the performance of competition agencies in Maghreb countries influences the enforcement of competition law.

Infection with human gastrointestinal (GI) parasites, such as Trichuris trichiura, affects more than billion people worldwide, causing significant morbidity and health problems especially in poverty-stricken developing countries. Despite extensive research, the mechanisms of induction and regulation of effector immune responses against these parasites are incompletely understood, which hinders the development of anti-parasite therapies. Infection with GI parasite is usually chronic suggesting that parasites are capable of modulating immune responses of their host to prevent expulsion. However, mechanisms by which parasites control host immunity to allow infection are still ill-defined. The aim of this PhD study was to characterise the role of different immunoregulatory mechanisms in immunity to GI parasite infection, with a focus on dendritic cells (DCs), regulatory T cells (Tregs) and the regulatory cytokine transforming growth factor β (TGF-β).Here we showed for the first time that loss of TGF-β-activating integrin alphavβ8 specifically on DCs resulted in protection from chronic infection with Trichuris muris, a mouse model of T. trichiura infection in man. Accelerated expulsion was immune-mediated and although increased levels of protective Th2 cytokines were observed very early during infection, elevated levels of non-protective Th1 cytokines were also detected. Partial depletion of CD4+ or FcεRI+ cells had no effect on the observed phenotype. Since deletion of alphavβ8 on DCs results in decreased numbers of Tregs in the gut, we tested whether depletion of Tregs using a mouse model that allows conditional ablation of Foxp3+ Tregs (DEREG mice) would alter infection development. Although transient Treg depletion at the beginning of infection had no major effect on expulsion kinetics, we observed a tendency for enhanced Th2 responses in DEREG mice. Moreover, even though DC-mediated TGF-β activation via alphavβ8 integrin was essential for T. muris infection development, transient depletion of DCs had no effect on the induction of Th2 responses or parasite expulsion. These data indicate a novel role for the TGF-β-activating integrin alphavβ8 and DCs in regulating effector immune responses during T. muris infection and may contribute to the development of new anti-parasite therapies.

The aim of the diploma thesis is to analyze the evolution of banking regulation from the turn of the 19th and 20th century with regard to the introduction of global standards of regulation and supervision. Great emphasis was placed on the status and functionality of the current regulation and also on its future shape as Basel III rules. In the theoretical part Basel I and Basel II projects are presented. The first of these is the concept from 1987 (Basel I) with the emphasis on greater stability and reliability of the international banking system. Basel I, however, represented a very simple and straightforward form of regulation, where the only monitored standard is bank's credit exposure. The amendment to Basel I and primarily Basel II, introduced a pillar regulation structure, which is still valid and provides national regulator a sufficient power to carry out its activities. The second, practical part of the thesis is devoted to current development and problems of banking regulation. This part introducing Basel III represents not only the key point of the gradual increase in the amount of regulatory capital until 2019, but also the introduction of uniform standards for measuring liquidity and strengthening the supervisory powers.