La réponse antivirale innée constitue la première réaction d’une cellule à une infection virale. Il s’agit d’une réponse rapide, transitoire, non-spécifique, ubiquitaire qui a été conservée sous différentes formes au cours de l’évolution. La synthèse d’interféron β (IFNβ) joue un rôle essentiel dans l’établissement de la réponse antivirale innée chez les vertébrés. L’expression du gène Ifnb1 codant pour l’IFNβ est finement régulée au niveau transcriptionnel, ce qui permet de passer de la répression de son expression en absence d’infection à son activation après infection par un virus. Bien qu’une forte et rapide expression d’IFNβ soit bénéfique lors d’une infection virale, une expression anormale d’IFNβ peut entrainer des troubles physiologiques importants (réactions auto-immunes, dérégulations inflammatoires). En s’intéressant plus particulièrement à la séquence promotrice du gène Ifnb1, codant pour l’IFNβ, nous avons identifié deux sites de liaison potentiels pour les facteurs de la famille des TCFs (T-Cell Factor). En présence de β-caténine nucléaire, les complexes TCF/β-caténine se forment au niveau des sites de liaison aux TCFs et recrutent des complexes co-activateurs de la transcription. Dans une première partie de ce travail, nous avons étudié le rôle des complexes TCF/β-caténine dans la régulation de l’expression d’Ifnb1 aussi bien en absence que suite à une infection virale. Nous avons ensuite montré que ce mécanisme était fonctionnel car il permettait aux cellules traitées par un inhibiteur de GSK3 (qui induit une accumulation de β-caténine) de mieux se protéger contre l’effet cytopathique induit par le VSV. Lors de l’étude des fonctions biologiques ciblées par le Virus de la Fièvre de la Vallée du Rift (VFVR) auquel j’ai participé, il est apparu que cet arbovirus hautement pathogène ciblait via sa protéine non-structurale NSs, les promoteurs de gènes codant pour des nombreux acteurs de la voie Wnt/β-caténine et de l’adhésion cellulaire. Dans une deuxième partie de ce travail, j’ai analysé l’effet d’une infection par la souche pathogène (+NSs) ou non-pathogène (-NSs) du VFVR sur le taux de β-caténine et la présence de celle-ci au niveau des jonctions adhérentes. Les résultats obtenus montrent que l’infection par la souche pathogène de VFVR entraine une diminution du taux global de β-caténine ex et in vivo ainsi que la redistribution de celle-ci en dehors des jonctions adhérentes, couplée à une très forte désorganisation du cytosquelette d’actine de la cellule infectée. Cette perturbation du réseau d’actine est corrélée à la dérégulation de l’expression de certains gènes codant pour des protéines affectant la morphologie cellulaire. / No abstract available

While antibiotics have saved the lives of millions of people since the discovery of the first β-lactam, penicillin, their continued effectiveness is being increasingly threatened by resistant bacteria. Bacterial resistance to β-lactams is mainly achieved through the production of serine-β-lactamases (SBLs) and metallo-β-lactamases (MBLs). Although both types of β-lactamases are commonly isolated in clinical settings, MBLs represent the greatest threat to public health since they are resistant to SBL inhibitors and most β-lactams. However, aspergillomarasmine A (AMA), a fungal natural product synthesized by Aspergillus versicolor, was shown to be a rapid and potent inhibitor against two clinically relevant MBLs: NDM-1 and VIM-2. In bacteria possessing these enzymes, AMA could rescue the activity of meropenem, a broad-spectrum β-lactam that is usually reserved for the treatment of the most severe bacterial infections. However, many questions remain revolving around AMA's inhibitory potency and spectrum. Therefore, the activity of AMA in combination with six β-lactams from three subclasses (carbapenem, penam, cephem) was explored against 19 MBLs from three subclasses (B1, B2, B3). After determining that AMA activity was linked to MBL zinc affinity and that AMA was more potent when paired with a carbapenem, the efficacy of an AMA/meropenem combination was evaluated with and without avibactam, a potent SBL inhibitor. This study used ten Escherichia coli and ten Klebsiella pneumoniae laboratory strains as well as 30 clinical strains producing at least one MBL and one SBL. Once establishing that the AMA/avibactam/meropenem combination was effective against carbapenemase-producing Enterobacterales, new Acinetobacter and Pseudomonas shuttle vectors were created. With these shuttle vectors, it was determined that the AMA/avibactam/meropenem combination was effective against some of the bacteria topping the World Health Organization’s priority pathogen list. / Thesis / Doctor of Philosophy (PhD) / Bacteria are all around us. While some bacteria can promote human health, others can cause serious infections. These infections are typically treated with antibiotics. β-Lactam antibiotics, such as penicillins and cephalosporins, are especially important to medicine. Unfortunately, an increasing number of bacteria employ enzymes, known as β-lactamases, which negate the effects of β-lactam antibiotics. Previous studies demonstrated that a natural product, known as aspergillomarasmine A (AMA), could inhibit some β-lactamase enzymes. Consequently, the inhibitory power of AMA was further explored against a larger number of β-lactamase enzymes and in combination with different β-lactam antibiotics. After discovering that AMA had more inhibitory power when combined with a β-lactam antibiotic known as meropenem, the efficacy of the AMA/meropenem pairing was evaluated against resistant bacteria in the presence and absence of avibactam, another β-lactamase inhibitor. The AMA/avibactam/meropenem combination was shown to be effective against some of the world’s most antibiotic-resistant bacteria.

L’utilisation fréquente de biomédicaments, composés principalement de protéines recombinantes, nécessite de développer les outils qui permettent de stabiliser les protéines. La lyophilisation est une technique couramment utilisée, dans domaine pharmaceutique, afin de convertir une formulation de protéine de l’état liquide à l’état sec, assurant ainsi une meilleure stabilité à la protéine. Une première partie de cette thèse a consisté à détailler, à l’échelle moléculaire, les mécanismes de bio-préservation des disaccharides pendant une procédure de lyophilisation, et comment une faible quantité de glycérol pouvait exacerber les propriétés bioprotectrices des disaccharides. L’analyse in-situ par imagerie Raman des trois étapes d’un cycle de lyophilisation a révélé que l’efficacité bioprotectrice résultait de la combinaison de propriétés physiques liées à la capacité du disaccharide de former un verre (valeur de Tg) et à celle de former un réseau de liaisons hydrogène rigide, caractérisé par des temps de vie des liaisons plus longs. Une seconde partie a été consacrée à l’étude d’un certain type de dérivé de β-cyclodextrines, qui sont des molécules permettant à la fois la libération contrôlée de molécules et d’inhiber des phénomènes d’agrégation. Les analyses Raman de la dénaturation chaude du lysozyme en présence de la HPβCD ont révélé des phénomènes originaux, en particulier un effet déstabilisateur ou stabilisateur suivant une vitesse de chauffe plus ou moins rapide de la solution. Cet effet cinétique a été relié à la capacité de la cyclodextrine à complexer les résidus de la protéine plus ou moins favorisée suivant la vitesse de chauffage. / The frequent use of biopharmaceuticals, mainly composed of recombinant proteins, requires the development of tools for stabilizing proteins. Freeze-drying is a commonly used technique in the pharmaceutical field to convert a liquid protein formulation into the dry state, thus providing better protein stability. A first part of this thesis focused in deciphering, on the molecular scale, the mechanisms of bio-preservation of disaccharides during a freeze-drying cycle, and in understanding how a small amount of glycerol could enhance the bio-protective properties of disaccharides. In situ Raman imaging analyzes, performed during the three steps of freeze-drying cycle revealed that the bio-protective efficiency resulted from the combination of physical properties related to the capacity of the disaccharide to be vitrified (Tg value) with that to form a rigid hydrogen-bond network, characterized by longer lifetime of the H-bonds. A second part has been devoted to the study of β-cyclodextrin derivatives, which can be used, both as drug delivery system and as inhibitor of protein aggregation. Raman analyzes performed during lysozyme thermal denaturation in presence of HPβCD, have revealed original results such as a destabilizing or stabilizing effect depending on the heating rate of the solution. This kinetic effect was related to the capability of cyclodextrins to include the protein residues inside their cage, probably favored by a slow heating.

La micro et la nanotechnologie a créé un bouleversement dans beaucoup de domaine tel que l’industrie ou la recherche. Pour la recherche, les enjeux économiques (quantité) et écologiques (déchets, risques chimiques) vont directement dans le sens de cette miniaturisation pour l’obtention de procédé sûr, propre et moins couteux. Cette thèse présente la mise en place d’un nouveau procédé de conception de BioMEMS assistée par plasma froid. L’objectif est le développement d’un microdispositif à partir d’un matériau non toxique, le Tetramethyldisiloxane (TMDSO), grâce à une technologie de dépôt de couche mince assisté par plasma, et intégrant une enzyme, pour la réalisation de réaction catalytique. Pour cela, un protocole d’immobilisation et d’intégration de l’enzyme, la β-galactosidase, a été développé afin de vérifier la capacité du TMDSO à retenir les enzymes et conserver sa fonction biologique. Ensuite, une évaluation de l’activité catalytique de l’enzyme immobilisée a été entreprise par la réalisation de réaction à l’échelle millifluidique, validant l’immobilisation ainsi que la biocompatibilité du ppTMDSO. Ensuite, un microréacteur à enzyme immobilisée a été réalisé, afin d’évaluer l’influence du passage à l’échelle microfluidique et de comprendre les phénomènes liés à la diffusion et la réaction des espèces au sein du dispositif. Enfin, la conception d’un microcanal en ppTMDSO et intégrant l’enzyme, a été réalisée afin de d’étudier la faisabilité d’une méthodologie « bio-integrante » pour la création d’un BioMEMS. L’utilisation d’une méthodologie bio-integrante peut être considérée comme une alternative prometteuse pour le développement de nouveaux outils de recherches. / The micro and nanotechnology has created an upheaval in many field such as industry or research. For research, economic issues (quantity) and ecological (waste, chemical hazards) go straight in the direction of this miniaturization process for obtaining safe, clean and less expensive. This thesis presents the development of a new BioMEMS design process assisted by cold plasma. The objective is to develop a micro-device from a non-toxic material, tetramethyldisiloxane (TMDSO), through a plasma enhanced thin film deposition technology, and incorporating an enzyme, for carrying out catalytic reaction. For this, an immobilizer protocol and integration of the enzyme, β-galactosidase, was developed to verify TMDSO's ability to retain enzymes and retain its biological function. Then, an evaluation of the catalytic activity of the immobilized enzyme was carried out by carrying out the reaction millifluidic scale, validating the asset and the biocompatibility of ppTMDSO. Then, an immobilized enzyme microreactor was conducted to assess the influence of the transition to the microfluidic scale and understand the phenomena related to the diffusion and reaction of the species within the device. Finally, the design of a microchannel ppTMDSO and incorporating the enzyme, was conducted to study the feasibility of a "bio-integral 'methodology for establishing a BioMEMS. The use of a bio-integral method may be regarded as a promising alternative for the development of new research tools.

Osteosarcoma is a mesenchymal tumour of bone common among children and young adults. Prognosis is poor if metastases are present at diagnosis. The transforming growth factor beta (TGF-β) signaling pathway plays a complex dual role in cancer. We hypothesized that alterations in the TGF-β signaling pathway are important in the tumorigenesis of osteosarcoma cell lines. Smad phosphorylation and nuclear localization, Smad4 expression, and TAZ expression were determined in the HOS osteosarcoma cell line and tumorigenic derivatives. Basal TGF-β activity and TAZ expression correlated with a tumorigenic phenotype in the KHOS cell lines as measured by Anchorage Independent Growth (AIG). In comparison, exogenous TGF-β suppressed AIG and acted as a tumour suppressor, while Smad4-deficient KHOS cells were resistant to the inhibitory TGF-β effect. In conclusion, basal TGF-β signaling and TAZ correlate with increased tumorigenic potential in osteosarcoma cell lines, whereas exogenous TGF-β acted as a tumour suppressor in a Smad4-dependent manner.

Osteosarcoma is a mesenchymal tumour of bone common among children and young adults. Prognosis is poor if metastases are present at diagnosis. The transforming growth factor beta (TGF-β) signaling pathway plays a complex dual role in cancer. We hypothesized that alterations in the TGF-β signaling pathway are important in the tumorigenesis of osteosarcoma cell lines. Smad phosphorylation and nuclear localization, Smad4 expression, and TAZ expression were determined in the HOS osteosarcoma cell line and tumorigenic derivatives. Basal TGF-β activity and TAZ expression correlated with a tumorigenic phenotype in the KHOS cell lines as measured by Anchorage Independent Growth (AIG). In comparison, exogenous TGF-β suppressed AIG and acted as a tumour suppressor, while Smad4-deficient KHOS cells were resistant to the inhibitory TGF-β effect. In conclusion, basal TGF-β signaling and TAZ correlate with increased tumorigenic potential in osteosarcoma cell lines, whereas exogenous TGF-β acted as a tumour suppressor in a Smad4-dependent manner.

Dissertação (mestrado)—Universidade de Brasília, Instituto de Química, Programa de Pós-Graduação em Tecnologias Química e Biológica, 2017. / Submitted by Fernanda Percia França (fernandafranca@bce.unb.br) on 2017-05-09T16:08:47Z No. of bitstreams: 1 2017_ErisleneSilvadeAlmeida.pdf: 14240509 bytes, checksum: 7a098ad412cfc0d9534c12ac3f92b1a4 (MD5) / Approved for entry into archive by Raquel Viana (raquelviana@bce.unb.br) on 2017-05-09T21:37:58Z (GMT) No. of bitstreams: 1 2017_ErisleneSilvadeAlmeida.pdf: 14240509 bytes, checksum: 7a098ad412cfc0d9534c12ac3f92b1a4 (MD5) / Made available in DSpace on 2017-05-09T21:37:58Z (GMT). No. of bitstreams: 1 2017_ErisleneSilvadeAlmeida.pdf: 14240509 bytes, checksum: 7a098ad412cfc0d9534c12ac3f92b1a4 (MD5) Previous issue date: 2017-05-09 / Antes de sua utilização, o óleo de palma deve ser refinado para remover compostos indesejáveis, como ácidos graxos livres, produtos de oxidação e pigmentos (carotenos), possibilitando a sua utilização em diversos produtos. A remoção dos pigmentos é realizada através da adsorção, na etapa do refino chamada branqueamento. Esse trabalho visa estudar o comportamento da adsorção de carotenos do óleo de palma híbrido utilizando dois tipos de adsorventes comerciais em condições similares ao processo industrial de branqueamento. Para a realização deste estudo, foi realizada a caracterização de dois adsorventes comerciais (um ácido e um neutro), e a determinação das cinéticas e isotermas de adsorção de carotenos na superfície destes. Os estudos foram realizados com óleo de palma híbrido com concentração inicial de 1757 mg/kg de carotenos totais. As análises de caracterização mostraram que o adsorvente ácido apresenta maior área superficial e maior volume de microporos quando comparado ao adsorvente neutro. Os dois materiais possuem raio de poro de 17 Å, e podem ser classificados como microporosos. Os dados cinéticos para ambos adsorventes foram bem descritos pelo modelo de pseudo-segunda-ordem. O modelo de difusão intra-partícula sugere que existe mais de um fenômeno limitante nesse processo adsortivo, sendo que para o ácido o processo em três etapas e para o neutro em duas. As isotermas mostraram que adsorvente ácido apresenta uma maior capacidade de adsorção, pois em todas as temperaturas trabalhadas a quantidade de caroteno adsorvido pelo adsorvente ácido foi maior. A 90 °C, por exemplo, o ácido adsorveu mais de 90 % de carotenos contra 50 % adsorvidos pelo neutro. A isoterma obtida com o adsorvente neutro apresentou comportamento desfavorável nas condições estudadas. / Prior to its use, palm oil should be refined to remove undesirable compounds such as free fatty acids, oxidation products and pigments (carotenes), making it possible to use them in several products. The removal of the pigments is carried out through adsorption, in the refining step called bleaching. This work aims to study the behavior of carotene adsorption of hybrid palm oil using two types of commercial adsorbents under conditions similar to the industrial bleaching process. For the accomplishment of this study, the characterization of two commercial adsorbents (acid activated and neutral), and the determination of the kinetics and isotherms of carotene adsorption onto the surface of these adsorbents. The studies were carried out with hybrid palm oil with initial concentration of 1757 mg / kg of total carotenes. The characterization analyzes showed that the acid adsorbent had a higher surface area and a higher volume of micropores when compared to the neutral one. The two materials have a pore radius of 17 Å and can be classified as microporous. The kinetic data for both adsorbents were well described by the pseudo-second-order model. The intra-particle diffusion model suggests that there is more than one limiting phenomenon in this adsorption process, being three and two steps, respectively, for the process carried out with acid and neutral adsorbents, respectively. The isotherms showed that acid adsorbent presents a greater capacity of adsorption, because in all the worked temperatures the amount of carotene adsorbed by the acid adsorbent was greater. At 90 ° C, for example, the acid adsorbed more than 90% of carotenes against 50% adsorbed by the neutral. The isotherm obtained with the neutral adsorbent showed unfavorable behavior under the studied conditions.

Porphyrins with extended π-electronic networks are promising candidates for a wide range of applications from medicine to nanotechnology owing to their unique optical and electronic properties. This dissertation is focused on synthesis, characterization and application of β-functionalized π-extended porphyrins. This dissertation is comprised of seven chapters. Chapter 1 focuses on the importance and objective of this work. Chapter 2 gives brief introduction to porphyrins and π-extended porphyrins. In chapter 3, a class of β-functionalized linear push-pull zinc dibenzoporphyrins YH1-YH3 were designed, synthesized, and utilized as light harvesters for DSSCs. In chapter 4, in order to further enhance the photovoltaic performance of β-functionalized benzoporphyrin dyes based DSSCs, a new class of push-pull dibenzoporphyrins YH4-YH7 bearing the phenylethynyl bridge was designed, synthesized and utilized as light harvesters for DSSCs. In chapter 5, in order to solve the photodegradation problem associated with YH7, a new series of push-pull dibenzoporphyrins YH8-YH10 bearing different diarylamino push groups was designed and synthesized. This class of push-pull porphyrins shows improved photostability and enhanced DSSC performance. In chapter 6, a new pentacene-fused diporphyrin with high stability and solubility was prepared and characterized. Chapter 7 includes the summary of this dissertation and describes possible future work.

The Wnt/β-catenin pathway is a fundamental regulator of embryonic development and adult tissue homeostasis. The key effector, β-catenin, is a multifunctional protein that occupies dual roles in signalling and intercellular adherens junctions. β-catenin primarily signals though the TCF/LEF transcription factors; however, many transcription factors, in addition to TCF/LEFs, interact with β-catenin, and the function of these interactions is poorly understood. To investigate novel β-catenin regulated signalling mechanisms with certainty, we developed TCF/LEF quadruple knockout (QKO) mESCs. In vitro differentiation of QKO cells reveals a neural differentiation bias, which is attenuated by overexpression of stabilized β-catenin. Our data indicate the presence of a TCF-independent β-catenin regulated neural differentiation blockade in mESCs. In addition to directly challenging the central dogma of canonical Wnt signalling, this finding has the potential to unveil new therapeutic targets for the treatment of many β-catenin-associated diseases, including forms of brain cancer that may arise from the oncogenic stimulation of neural stem cells. Furthermore, we describe an attempt to identify genome-wide TCF-independent β-catenin binding sites in QKO cells by ChIP-seq. Optimization trials provide proof of concept that the fold enrichment method of interpreting ChIP-qPCR results can be highly misleading when compared to the more comprehensive % input method of analysis. This conclusion has important implications for all fields of scientific research in which ChIP-seq methodology is employed. / Thesis / Master of Science (MSc)

Glycosylation O-linked β-N-acetylglucosamine (O-GlcNAc) is post-translational modification of proteins, regulated by β-N-acetylglucosaminyltransferase (OGT) and β-N-acetylglucosaminidase (OGA). This intracellular glycosylation differs from the other glycosylation types - it is dynamically regulated, similarly to phosphorylation, β-N-acetylglucosamine serves as a nutrient and stress sensor in cell. Chronically dysregulated O-linked glycosylation by GlcNAc is associated with pathology of various diseases, such as diabetes mellitus type II, oncological and neurodegenerative diseases. Expression of enzymes OGT and OGA is very sensitive for homeostasis of GlcNAc, which is the product of hexosamine biosynthetic pathway. Changes in expressions of these ezymes could be used as a potencial blood marker, e.g. in early stage of diabetes. The aim of this master thesis was to study changes in expression of genes encoding ezymes OGT and OGA in cohort of obese patients in comparison with healthy controls and also to compare the state before and after change of lifestyle (loosing weight). Analysed cohort comprised of 34 samples of isolated lymphocytes from peripheral blood from obese adolescent patients and 80 samples of adults patients. RNA was isolated by TriReagent, quantification of the expression of mRNA was...