Mycobacterium tuberculosis (Mtb) causes human tuberculosis, and more people die of it than of any other pathogen in the world. Immunodominant antigens elicit the large majority of T cells during an infection, making them logical vaccine candidates. Yet, it is still unknown whether these immunodominant antigen-specific T cells recognize Mtb-infected cells. Two immunodominant antigens, TB10.4 and Ag85b, have been incorporated into vaccine strategies. Surprisingly, mice vaccinated with TB10.4 generate TB10.4-specific memory CD8+ T cells but do not lead to additional protection compared to unvaccinated mice during TB. Ag85b-specific CD4+ T cells are also generated during vaccination, but the literature on whether these cells recognize Mtb-infected cells is also inconsistent. We demonstrate that TB10.4-specific CD8+ T cells do not recognize Mtb-infected cells. However, under the same conditions, Ag85b-specific CD4+ T cells recognize Mtb-infected macrophages and inhibit bacterial growth. In contrast, polyclonal CD4+ and CD8+ T cells from the lungs of infected mice can specifically recognize Mtb-infected macrophages, suggesting macrophages present antigens other than the immunodominant TB10.4. The antigen location may also be critical for presentation to CD8+ T cells, and live Mtb may inhibit antigen presentation of TB10.4. Finally, we propose that TB10.4 is a decoy antigen as it elicits a robust CD8+ T cell response that poorly recognizes Mtb-infected macrophages, allowing Mtb to evade host immunity.

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<p>Virulence in <em>Staphylococcus aureus </em>is mediated by the <em>accessory gene regulator </em>(agr) quorum sensing system. This regulatory system is activated by a secreted thiolactone peptide termed autoinducing peptide (AIP) and its receptor histidine kinase, AgrC. Interaction of extracellular AIP with a cognate AgrC receptor generates an intracellular signal that is transduced by conformational changes and phosphorylation events in a two-component sensor histidine kinase system. At the heart of the <em>agr</em> quorum-sensing cascade lies the two-component histidine kinase, AgrC, and the response regulator protein, AgrA. Interaction of AgrC and AgrA, and the resulting phosphotransfer event results in expression from the divergent promoters P2 and P3, inducing expression of the master quorum-sensing regulator RNAIII and upregulating the <em>agr</em> operon respectively. Signal transduction systems function as intracellular information-processing pathways that link sensation of external stimuli to specific adaptive processes. In <em>S. aureus</em> , these include the up-regulation of virulence factors and hemolysis production, biofilm formation, and colonization-based regulation of surface proteins and adhesion factors. As such, the interactions of these systems have become key targets in the design of small inhibitor compounds.</p> <p>Through the creation of a protein truncation series, we proposed the development of a small protein for the inhibition of key protein-protein interactions involved in <em>S. aureus</em> <em>agr</em> two-component signaling. Herein, we demonstrate the efficacy of these protein truncations as dominant negative inhibitors of AgrC:AgrA interactions, likely acting as a dominant phosphoacceptor in place of endogenous AgrA. We provide evidence of this function through <em>in vitro </em>hemolysis assays and phosphate-detection based gel electrophoresis.</p> / Master of Science (MSc)

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Doctor of Philosophy / Department of Biology / Rollie J. Clem / Apoptosis is an extremely conserved process among metazoans. This dissertation will describe apoptotic regulation in two orders of insects, Lepidoptera and Diptera. In the lepidopteran host Trichoplusia ni, we describe phenotypes of infection with the baculovirus AcMNPV lacking the caspase inhibitor gene P35. In the lepidopteran host Spodoptera frugiperda, infection with this mutant virus results in apoptosis, which dramatically hinders spread of the virus in the host. In T. ni, however, infection with this mutant virus is similar to wild-type with normal spread, but the end result of liquefaction does not occur. Experiments indicated that infection of T. ni cells with the P35 mutant virus (P35Δ) resulted in caspase activation, and the P35Δ virus lacked the ability to inhibit these active caspases. With the P35Δ virus a slower entry phenotype was observed, but when the P35Δ virus was grown in the presence of a caspase inhibitor the entry phenotype was rescued. This indicated that caspases have detrimental effects on budded virions, and illustrated that P35 is needed to make robust virions. With regards to Diptera, apoptosis-regulatory genes were annotated in the yellow fever mosquito, Aedes aegypti. The genes annotated included multiple caspases and caspase regulators. Phylogenetic relationships were determined among the caspases from Anopheles gambiae, Ae. aegypti and Drosophila melanogaster, expression patterns were determined for all the annotated genes in Ae. aegypti, and one of the genes, an IAP antagonist named IMP, was functionally characterized. Further characterization of the phylogenetic relationships of caspases from fifteen dipteran species was performed by obtaining gene models for caspases of recently sequenced genomes for twelve Drosophila species and three mosquito species. Furthermore, several Drosophila and mosquito species were found to contain caspase genes with substitutions in critical active site residues. These genes were proposed to encode caspase-like decoy molecules. While these have been found in humans and nematodes, this is the first report for these molecules in insects. One of the caspase-like decoy molecules was found to increase the activity of its paralog caspase.

The goal of the following thesis research was to develop a systematic approach for the refinement of low-resolution protein models, as a part of the protein structure prediction procedure. Significant progress has been made in the field of protein structure prediction and the contemporary methods are able to assemble correct topology for a large fraction of protein domains. But such approximate models are often not detailed enough for some important applications, including studies of reaction mechanisms, functional annotation, drug design or virtual ligand screening. The development of a method that could bring those structures closer to the native is then of great importance. The minimal requirements for a potential that can refine protein structures is the existence of a correlation between the energy with native similarity and the scoring of the native structure as being lowest in energy. Extensive tests of the contemporary all-atom physics-based force fields were conducted to assess their applicability for refinement. The tests revealed flatness of such potentials and enabled the identification of the key problems in the current approaches. Guided by these results, the optimization of the AMBER (ff03) force field was performed that aimed at creating a funnel shape of the potential, with the native structure at the global minimum. Such shape should facilitate the conformational search during refinement and drive it towards the native conformation. Adjusting the relative weights of particular energy components, and adding an explicit hydrogen bond potential significantly improved the average correlation coefficient of the energy with native similarity (from 0.25 for the original ff03 potential to 0.65 for the optimized force field). The fraction of proteins for which the native structure had lowest energy increased from 0.22 to 0.90. The new, optimized potential was subsequently used to refine protein models of various native-similarity. The test employed 47 proteins and 100 decoy structures per protein. When the lowest energy structure from each trajectory was compared with the starting decoy, we observed structural improvement for 70% of the models on average. Such an unprecedented result of a systematic refinement is extremely promising in the context of high-resolution structure prediction.

Submitted by Guilherme Leão (guilherme89leao@gmail.com) on 2013-08-08T20:52:46Z No. of bitstreams: 1 dissertação guilherme_final_.pdf: 1058270 bytes, checksum: 4c6f6359e94a68739ede38306bf6ab14 (MD5) / Approved for entry into archive by ÁUREA CORRÊA DA FONSECA CORRÊA DA FONSECA (aurea.fonseca@fgv.br) on 2013-08-13T16:25:58Z (GMT) No. of bitstreams: 1 dissertação guilherme_final_.pdf: 1058270 bytes, checksum: 4c6f6359e94a68739ede38306bf6ab14 (MD5) / Approved for entry into archive by Marcia Bacha (marcia.bacha@fgv.br) on 2013-08-15T13:08:22Z (GMT) No. of bitstreams: 1 dissertação guilherme_final_.pdf: 1058270 bytes, checksum: 4c6f6359e94a68739ede38306bf6ab14 (MD5) / Made available in DSpace on 2013-08-15T13:08:59Z (GMT). No. of bitstreams: 1 dissertação guilherme_final_.pdf: 1058270 bytes, checksum: 4c6f6359e94a68739ede38306bf6ab14 (MD5) Previous issue date: 2012-08-29 / Several models and neoclassical economic theories are based on the principles of rationality of individuals. However, many studies have shown how these principles can be flawed in numerous contexts. This research aimed to contribute to this literature by developing an experiment conducted with undergraduate students , testing applications of rationality biases, known as Decoy Effect and Anchor Effect . We analyzed 142 questionnaires that apparently sought to profile the students by asking questions about their preferences for job opportunities by analyzing various aspects (remuneration, stability, workload and chances of professional growth ). Throughout the issues, however, decoys and anchors were inserted . As a result, we found that when looking for a job , most students predominantly valued the chances of professional growth and the remuneration offered by the job , while the workload was the attribute less weight ed in their decisions. While the rest of their responses were quite consistent with the outlined profile, the bias of rationality proved influential. The decoys used led to changes in responses 7 - 8%, while the use of anchors have been shown to influence the minimum amount by which the re spondents would accept a paid service, violating the principles of rationality. / Diversos modelos e teorias econômicas neoclássicas se baseiam nos princípios de racionalidade dos indivíduos. No entanto, muitos estudos vêm mostrando como tais princípios podem ser falhos em diversos contextos. Esta pesquisa buscou contribuir com tal literatura através do desenvolvimento de um experimento realizado com alunos de graduação no qual foram testadas aplicações dos vieses de racionalidade conhecidos como Efeito Chamariz e Efeito Âncora. Foram analisados 142 questionários que aparentemente visavam traçar o perfil dos estudantes através de perguntas sobre suas preferências quanto a oportunidades de emprego considerando múltiplos aspectos (remuneração, estabilidade, carga horária e chances de crescimento profissional). Ao longo das questões, no entanto, foram inseridas âncoras e chamarizes. Como resultados, observamos que, ao procurar um emprego, a maioria dos estudantes valoriza principalmente as chances de crescimento profissional e a remuneração oferecida pelo trabalho, ao passo que a carga horária é o atributo de menor peso em suas decisões. Embora o restante de suas respostas fosse bastante coerente com o perfil traçado, os vieses de racionalidade se mostraram influentes. Os chamarizes usados levaram a alterações nas respostas de 7 a 8%, enquanto que o uso de âncoras se mostraram capazes de influenciar o valor mínimo pelo qual os respondentes aceitariam prestar um serviço, violando os princípios de racionalidade.

De la confrontation problématique d’un projet de jeu vidéo aux conditions plurielles desa création, ce travail de thèse tente de mettre à jour les contraintes et les interrogationsqu’elles suscitent. Le jeu vidéo, contemporain de la société de l’information est un témoinde l’évolution complexe du paradigme postmoderne, né avec la cybernétique. De la genèsedu jeu vidéo au temps présent, les technologies, les façons de jouer et les productions serévèlent significatives de visions du monde opposées. La thèse montre que derrière l’imaged’un divertissement et d’une industrie consensuelle, une résistance peut s’exprimer d’unpoint de vue technique, artistique et politique. A travers l’histoire et le devenir du jeuvidéo peuvent se lire certains enjeux de société en lien avec de nouvelles formes de pouvoir.C’est dans un interstice contre culturel que se développe une oeuvre résistante, nourrie d’unexamen critique préalable. Une création originale se construit progressivement en contreculturedu modèle libéral dominant, dans un mouvement d’opposition à l’action irréfléchie,l’accélération et la dématérialisation... Du point de vue plastique, la thèse interroge le retourdes technologies de réalité virtuelle, et élabore deux nouvelles notions, le véhicule expérientielet le leurre suffisant. Profitant de ce nouveau champ d’investigation de création, la thèseinvite le joueur à laisser l’usage pour le faire. / From the problematic confrontation of a video game project with the multiple conditions ofits creation, the present thesis tries to highlight the constraints and the interrogations whichthey arouse. Contemporary to the information society, the video game is a witness of thecomplex evolution of the postmodern paradigm born from cybernetics. From the genesis ofthe video game to present time, technologies, the ways of playing and the productions revealthe significant oppositions in their visions of the world. The thesis shows that behind theimage of entertainment and of a consensual industry, a form of resistance can express/voiceitself from a technological, artistic and political point of view. Through the history of videogame and its future, one can read some of society’s stakes/challenges linked to new formsof power. It is within a counter cultural interstice, and fed by a prior critical exam, thata resistant work/piece is developed. An original creation is progressively built in reflexivecounter cultural opposition to the mainstream dominant liberal world and its accelerationand dematerialization... From a plastic point of view, the thesis questions the return of thevirtual reality technologies, and elaborates two new notions: the experiential vehicle and thesufficient decoy. Taking advantage of this new field of investigation of creation, the thesisinvites the player to let it be used so as to do so. Keywords: Video games. Counter culture.Experiential vehicle. Counter dematerialization. Sufficient decoy.