The aim of this master thesis is to analyze the approaches of members of the North American Free Trade Agreement (NAFTA) to the Trans-Pacific Partnership (TPP). The work is divided into three chapters. The first chapter is devoted to the analysis of NAFTA, primarily to its creation and approaches of the individual members during negotiations, main provisions and objectives of this free trade area. The second chapter analyzes the main reasons that led the states to join NAFTA and their current positions within the zone. The last chapter is dedicated to TPP, its main provisions and position of NAFTA members in this agreement.

Memoria para optar al título de Periodista

La régulation de l’expression génétique est essentielle afin qu’un organisme puisse s’adapter aux changements environnementaux. Chez les bactéries, la régulation peut s’effectuer à plusieurs étapes de l’expression des gènes (transcription, stabilité de l’ARN, traduction, maturation et dégradation des protéines) et par des mécanismes impliquant différents types de molécules (ADN, ARN, protéines, métabolites ou ions inorganiques). Traditionnellement, les protéines se situaient au centre de ces mécanismes de régulation. On sait maintenant que certains ARN, dont les riborégulateurs, ont également un grand rôle à jouer dans ce processus. Un riborégulateur est un élément génétique retrouvé dans une région non-codante de certains ARN messagers (ARNm), qui peut lier directement un ligand spécifique afin de réguler l’expression de son transcrit. Chez Escherichia coli (E. coli), trois riborégulateurs lient la thiamine pyrophosphate (TPP). Un d’entre eux, le riborégulateur thiB, n’a toujours pas été étudié. On croit qu’il contrôlerait, au niveau de l’initiation de la traduction, l’expression de l’opéron thiBPQ encodant un transporteur ABC de la thiamine. De plus, un petit ARN nommé SroA a précédemment été identifié grâce à des techniques de séquençage d’ARN. SroA correspond à l’aptamère du riborégulateur thiB, mais aucun rôle ne lui a été attribué à ce jour. Le présent mémoire porte sur la caractérisation du riborégulateur thiB d’E. coli. Dans un premier temps, nous avons démontré que le riborégulateur est fonctionnel. Le mécanisme permettant la régulation génétique en cis est cependant plus complexe que seulement la régulation prédite au niveau traductionnel. Dans un second temps, nous avons utilisé deux approches différentes à l’échelle transcriptomique afin de vérifier si SroA régule l’expression de certains ARNm en trans. Plusieurs cibles potentielles découlent de cette étude. Une caractérisation préliminaire de certaines d’entre elles est présentée ici et devra être poursuivie par des travaux subséquents. Les résultats présentés ici suggèrent que le riborégulateur thiB régulerait l’expression de l’opéron thiBPQ (en cis) et d’autres gènes en trans.

Mitochondria are organelles present in eukaryotic cells that play a key role in regulating cells’ metabolic processes as well as cell death. The main function of mitochondria is to produce ATP, by oxidizing nutrients in a process called oxidative phosphorylation (OXPHOS). Besides this, mitochondria also play a critical role in calcium homeostasis, cell signaling, and apoptosis. Mitochondrial dysfunction is implicated in a plethora of diseases including neurodegenerative diseases, metabolic disorders as well as ageing and cancer. The triphenylphosphonium (TPP+) moiety has been used as a carrier to direct a wide variety of therapeutic and diagnostic cargo to mitochondria, in an effort to study and treat mitochondrial dysfunction. Studies in recent years show that TPP+ is not an inert carrier as previously thought. Many TPP+ conjugates have been shown to exert a negative effect on mitochondrial and cellular bioenergetics by decreasing the efficiency of OXPHOS. This phenomenon is called ‘mitochondrial uncoupling’. While mitochondrial uncoupling is undesirable for the TPP+ moiety to function as a carrier of cargo to mitochondria, controlled uncoupling has therapeutic applications in treatment of obesity and cancer. The extent of mitochondrial accumulation as well as potency of mitochondrial uncoupling caused by the TPP+ moiety increases with increasing length of the linker functionality in TPP+ conjugates. Most of the studies to date have focused on altering the linker length of the TPP+-linker-cargo conjugate to optimize the balance between safety and efficacy. However, very little is known about how structural modification of the TPP+ moiety itself affects mitochondrial uncoupling potency. Therefore, there is a need to understand the structure activity relationship (SAR) between modification of TPP+ structure and the effect of these structural changes on mitochondrial uncoupling and uptake. Towards this end, the first goal of this study was to understand the effect of modulating electron density on the phosphorus atom of TPP+ on the potency of uncoupling OXPHOS. Modifications to the TPP+ moiety included substitution of electron withdrawing and donating groups on the phenyl rings of TPP+, and replacing phenyl rings with bulkier napthyl rings. Modified TPP+ moieties were conjugated to five different linkers, which varied in length and lipophilicity, and the effect of these conjugates on mitochondrial bioenergetics was studied. The second goal of the study was to evaluate if the propensity of TPP+ to uncouple mitochondrial respiration can be modulated, independently of mitochondrial uptake. For this purpose, the uptake of modified TPP+-linker conjugates into isolated mitochondria and the uptake of fluorescently labeled modified TPP+-linker conjugates into mitochondria within whole cells was investigated. The ability of modified TPP+ to protect cells from oxidative stress by successfully delivering an anti-oxidant cargo to mitochondria within cells was also assessed. The results of these studies establish the first SAR for modulating TPP+ structure to either eliminate, optimize, or maximize uncoupling of mitochondrial OXPHOS, and led to identification of lead molecules for potential applications in the fields of mitochondrial delivery, anti-obesity therapy and anti-cancer therapy.

Šio darbo tikslas buvo ištirti antimikrobinių medžiagų ir daugiavaisčio atsparumo siurblių slopiklių poveikį bakterijų sąveikai su TPP+ jonais. Šiam tikslui pasiekti reikėjo ištirti antimikrobinių peptidų įtaką bakterinių apvalkalėlių laidumui, įvertinti M. smegmatis bakterijų apvalkalėlio sąveiką su TPP+ jonais ir jų pasiskirstymą veikiant šias polikatijoniniais junginiais bei ištirti daugiavaisčio atsparumo siurblių slopiklių įtaką TPP+ jonų pasiskirstymui abipus M. smegmatis bakterijų apvalkalėlio. Šio darbo metu buvo parodyta, kad peptidų pVEC ir cys-pVEC sąveika su gramneigiamųjų E. coli bakterijų apvalkalėliu beveik nesiskiria: peptidai mažai laidina E. coli bakterijų išorinę membraną, bet depoliarizuoja jų plazminę membraną. Peptidas pVEC depoliarizuoja ir gramteigiamųjų B. subtilis bei mikobakterijų M. smegmatis plazminę membraną. Be to, buvo parodyta, kad TPP+ jonų srautų tyrimai gali padėti įvertinti mikobakterijų energetinę būseną. Polimiksinas B – antibiotikas prieš gramneigiamąsias bakterijas – nelaidina mikobakterijų M. smegmatis apvalkalėlio TPP+ jonams, bet depoliarizuoja jų plazminę membraną. Daugiavaisčio atsparumo siurblių slopiklis chlorpromazinas veikia mikobakterijų M. smegmatis plazminę membraną depoliarizuojančiai ir sumažina ampicilino koncentracijas, reikalingas mikobakterijų augimo slopinimui. Nauji potencialūs DVA siurblių slopikliai neturi įtakos M. smegmatis ląstelių jautrumui ampicilinui, bet keletas junginių sąveikauja su TPP+ išmetančiais... [toliau žr. visą tekstą] / The aim of this work was to estimate the effect of membrane permeabilizing compounds and multidrug resistance pump inhibitors on interaction of bacterial envelope with TPP+ ions. To achieve the aim we worked on antimicrobial peptides and their interaction with bacterial envelopes, on estimation of interaction of M. smegmatis envelope with TPP+ ions and equilibration of these ions across mycobacterial envelope. We have found that antimicrobial peptides pVEC and cys-pVEC have very similar effect on the envelope of gram-negative E. coli cells. They have little permeabilizing effect on the outer membrane but effectively depolarize the plasma membrane of E. coli. The peptide pVEC has depolarizing effect on both B. subtilis and M. smegmatis plasma membrane. We described interaction of M. smegmatis envelope with TPP+ ions. Polymyxin B has no permeabilizing effect on the mycobacterial cell wall, but it depolarizes the plasma membrane of M. smegmatis. Multidrug resistance pump inhibitor chlorpromazine increases the efficacy of ampicillin against M. smegmatis and has a depolarizing effect on mycobacterial plasma membrane. New potential multidrug resistance pump inhibitors have no effect on M. smegmatis sensitivity to ampicillin, however some of the compounds interacted with TPP+ efflux pumps.

Indiana University-Purdue University Indianapolis (IUPUI) / Thiamin diphosphate (ThDP)-dependent enzymes catalyze a wide range of reactions including the oxidative and nonoxidative decarboxylation of 2-keto acids, carboligation reactions, the cleavage of C-C bonds, and the formation of C-S, C-N, and C-O bonds. Surprisingly, given this diversity, all ThDP-dependent enzyme catalyzed reactions proceed through essentially the same intermediate. This suggests that these enzymes share a common ancestry and have evolved to become the diverse group of enzymes seen today. Sequence alignments have revealed that all ThDP-dependent enzymes share two common ThDP binding domains, the PYR domain and the PP domain. In addition to these conserved domains, over time, other domains have been added creating further diversity in this superfamily. For instance, the TH3 domain, found in many ThDP-dependent enzymes, serves the function of binding additional cofactors such as FAD in enzymes like acetohydroxyacid synthase (AHAS) but in others, like pyruvate decarboxylase (PDC), it has lost this function completely. The work presented here focuses on ThDP-dependent decarboxylases. In this thesis, several evolutionary aspects of this group of enzymes will be examined including (i) the characterization of an evolutionary forerunner in the presence of a mechanism-based inhibitor, (ii) the characterization of the minor isozymes of pyruvate decarboxylase from Saccharomyces cerevisiae, and (iii) the development of a selection method to increase the efficiency of the site-saturation mutagenesis used to study ThDP-dependent enzyme evolution.

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Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-10-17T13:31:20Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) JANIRA DISSERTAÇÃO PPGIT UFPE 2016.pdf: 2767067 bytes, checksum: c6f9b4694fc912f845955ae3a11a3c37 (MD5) / Made available in DSpace on 2016-10-17T13:31:20Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) JANIRA DISSERTAÇÃO PPGIT UFPE 2016.pdf: 2767067 bytes, checksum: c6f9b4694fc912f845955ae3a11a3c37 (MD5) Previous issue date: 2016-03-15 / CNPq / O desenvolvimento de sistemas de administração de fármacos utilizando biopolímeros, como a goma de cajueiro e quitosana tem-se destacado pela formação de nanoestruturas por complexação polieletrolítica, realizada pela interação das diferentes cargas dos materiais e devido as suas promissoras propriedades, dentre elas: excelente biodegradabilidade, elevada biocompatibilidade, baixa toxicidade. Usados no desenvolvimento nanotecnológico de formulações para proteção de fármacos peptídicos, como a insulina, para sua administração por via oral, no qual faz-se necessário pela sua instabilidade em variações de pH. Este trabalho teve como objetivo a síntese de nanopartículas a partir de biopolímeros por complexação polieletrolítica para administração oral de insulina e avaliação analítica do fármaco e seu principal produto de degradação o A-21 em meio ácido. Nanopartículas de goma de cajueiro e quitosana foram sintetizadas por complexação polieletrolítica com e sem adição do tripolifosfato de sódio, como agente de reticulação. Foram caracterizadas quanto ao seu tamanho, carga superficial, índice de polidispersão e adicionalmente a Espectroscopia da Região do Infravermelho (FTIR), Microscopia Eletrônica de Varredura (MEV), Análise Termogravimétrica (TG). Para a determinação analítica da insulina e seu principal produto de degradação o A-21 foi realizado um planejamento experimental fatorial, a fim de analisar quais variantes iriam influenciar para obtenção de um menor tempo de análise das moléculas por HPLC. Vinte e sete experimentos foram realizados e destacou-se dentre os valores observados nos tempos de retenção de 5,18 min para insulina e 9,10 min para o A-21, na condição onde a temperatura da coluna foi de 35°C, fluxo do eluente de 1,0 mL/min-1 e na proporção da fase móvel de 52% KH2PO4, 31% acetonitrila e 17% metanol, uma análise final com cerca de 11 minutos. Em média os fatores que mais influenciaram no sistema foram o fluxo do eluente e a proporção da fase móvel, demonstrados através da estimativa dos efeito, análise da variância e teste F. O modelo linear obtido demonstrou ter uma boa predição para o método, com pontos reais de distribuição aleatórios condizentes aos resultados obtidos experimentalmente, a variância total explicada de 91,14% e o modelo deixa de explicar 8,86% dos resíduos. A adequabilidade cromatográfica da combinação das variáveis independentes resultaram em valores próximos ou dentro dos limites estabelecidos. Os resultados das avaliações de concentração dos biopolímeros, proporção dos agentes de reticulação, ordens de adição, pH das soluções mostraram-se bastante significativos na influência dos tamanhos das partículas, potencial zeta e índice de polidispersão. Houve uma grande distinção das preparações com e sem TPP, pois foi evidenciada uma eficaz ação reticulante entre os polieletrólitos, as ligações cruzadas realizadas foram importantes para aperfeiçoamento dos parâmetros, demonstrado a redução dos tamanhos das nanopartículas, de modo homogêneo e com baixos índices de polidispersão, potenciais zeta na faixa neutra diferindo nos valores de acordo com a concentração adicionado. Através das caracterizações realizadas por FTIR, MEV e TG houve a demonstração da interação entre os materiais estudados. As características e propriedades apresentadas pelas nanoesferas formadas pela interação GC/TPP/QT indicam um promissor sistema de liberação de fármacos e proteção para moléculas instáveis ao ambiente gastrointestinal. / The development of drug delivery systems using biopolymers such as cashew gum and chitosan it has been highlighted by the formation of nanostructures by complexation polyelectrolytic held by the interaction of the different loads of materials and due to their promising properties, among them: excellent biodegradability , high biocompatibility, low toxicity. Used in nanotechnological development of formulations for protection peptide drugs such as insulin, for administration orally, in which it is necessary for its instability at pH variations. This study aimed to the synthesis of nanoparticles from biopolymers by complexation polyelectrolytic for oral administration of insulin and analytical evaluation of the drug and its main degradation product A-21 in acid. cashew gum and chitosan nanoparticles were synthesized by complexation polyelectrolytic with and without addition of sodium tripolyphosphate as crosslinking agent. They were characterized as to their size, surface charge, polydispersity and additionally Spectroscopy Infrared Region index (FTIR), Scanning Electron Microscopy (SEM), Thermogravimetric Analysis (TG). For the analytical determination of insulin and its main degradation product A-21 factorial experimental design was conducted to analyze variants which would influence to obtain a shorter analysis of molecules by HPLC. Twenty-seven experiments were conducted and it was highlighted among the values observed at 5.18 min retention time of 9.10 min for insulin and A-21 in the condition where the column temperature was 35 ° C, flow of the eluent of 1.0 ml / min-1 and the rate of mobile phase of 52% KH2PO4, 31% acetonitrile and 17% methanol, with a final analysis about 11 minutes. On average the factors that most influence the system were the eluent flow and the proportion of the mobile phase, demonstrated by estimating the effect of variance analysis and test F. The resulting linear model was shown to have a good prediction for the method, with points real random distribution consistent with the results obtained experimentally, the total explained variance of 91.14% and the model fails to explain 8.86% of the waste. Chromatographic suitability of the combination of the independent variables resulted in close to or within the established limits. The results of evaluations concentration of biopolymers, the proportion of cross-linking agents, addition orders, the solutions pH were quite significant in the influence of particle size, zeta potential and molecular weight distribution index. There was distinction of preparations with and without TPP, as was evidenced effective action crosslinking of polyelectrolytes, cross-connections made were important for improvement of the parameters shown to reduce the size of the nanoparticles, homogeneous way and with low levels of polydispersity, zeta potential in the neutral range in differing values according to the concentration added. Through the characterizations made by FTIR, SEM and TG was the demonstration of the interaction between the studied materials. The characteristics and properties presented by the nanospheres formed by the interaction GC / TPP / QT indicate a promising system of drug delivery and protection for unstable molecules to the gastrointestinal environment.

I januari 2018 kommer det nuvarande EU-direktivet PSD att ersättas av det nya betaltjänstdirektivet Payment Services Directive 2 (PSD2). Motiven bakom direktivets införande är bland andra att bidra till en förenkling för tredjepartsaktörer att inträda betaltjänstmarknaden samt öka valmöjligheten för konsumenten. Idag domineras betaltjänstmarknaden främst av banker, något som direktivet utformats för att motverka. Syftet med denna studie är att undersöka hur väl några av direktivets motiv stämmer överens med aktörernas uppfattning av direktivets påverkan samt även undersöka vilka möjligheter och utmaningar banker och tredjepartsaktörer ser att det nya direktivet innebär för dem och marknaden. Uppsatsen är genomförd som en fallstudie där semistrukturerade intervjuer med banker och tredjepartsaktörer varit den främsta datainsamlingsmetodiken. Fallstudien visade att aktörernas uppfattning av direktivet till stora delar stämmer bra överens med motiven för direktivet. Samtliga aktörer har en förhållandevis positiv bild av direktivet och ser en mängd olika möjligheter med direktivet. Samarbetet mellan banker och tredjepartsaktörer ses som en av de större möjligheterna. Utmaningarna som aktörerna står inför är varierande beroende på typ av aktör. Studien visade att det finns en del utmaningar som kan komma att begränsa antalet nya aktörer på marknaden. Samtliga möjligheter och utmaningar presenteras i fallstudiens sjätte avsnitt. / In January 2018, the current EU directive PSD will be replaced by the new Payment Services Directive 2 (PSD2). The motives behind the implementation of the directive are, inter alia, to help simplify third party providers entering the payment service market and increasing consumer choice. Today, the payment service market is uppermost dominated by banks, something that the directive is designed to counteract. The purpose of this study is to investigate how well some of the motives of the directive are in line with the actors' perception of the impact of the directive, as well as investigate what opportunities and challenges banks and third party providers see that the new directive implies for them and the market. The essay was conducted as a case study, where semistructured interviews with banks and third party players were the primary data collection methodology. The case study showed that the actors' perception of the directive largely complies with the motives for the directive. All actors have a relatively positive view of the directive and see a wide range of possibilities with the directive. Cooperation between banks and third parties is seen as one of the major opportunities. The challenges that the stakeholders face are varied depending on the type of actor they represent. The study showed that there are some challenges that may limit the number of new players in the market. All possibilities and challenges are presented in the sixth section of the case study.