My thesis involves studying the nature of chimera through history and how certain aspects of chimeras represent specific features of dualities in human nature. The research was reflected in a series of sketches, where one sketch was finalized into a fully realized 3D model. To aid in this goal, I created a system that will randomly generate chimera based on different characteristics. This system was created using Max 5, and was programmed to place images with alphas on top of each other to create unique chimeras. The variables within this system are derived from the research on chimeras depicted in art and mythology throughout history, and will be used as an inspirational tool to help generate unique combinations of chimeras that may not otherwise have been imagined.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 5 uppsatser.

Part I: Coral reefs are essential to coastal economies, protecting coastlines from storms, and harboring high biodiversity. However, reefs are declining due to local anthropogenic stressors and ocean warming. Sedimentation, a local stressor, aggravates the impacts of warming on corals and hinders their survival and growth. Therefore, it is important to investigate whether sedimentation and temperature have a synergistic effect on vulnerable coral species, especially during earlier stages of development. To quantify these effects, survival and growth of newly settled Acropora cervicornis corals were measured at two temperatures (29 and 31°C, representing current and predicted for 2050 Summer temperatures) and three sediment concentrations (30, 60 and 120 mg.cm-2, representing a range from natural sedimentation to dredging conditions). The intent of this study was to mix multiple genotypes to test temperature and sedimentation among genotypic unique individuals. However, only 20% of colonies spawned, and spawning was asynchronous by genotype. Therefore, individuals were produced from self-fertilization. The overall high mortality seen in this study suggests that self-fertilization in A. cervicornis does not produce viable juveniles. Although temperature did not have a significant effect on the survival of self-fertilized juveniles, sediment concentration did. The lowest sediment concentration led to the highest juvenile survival in both ambient and heated conditions. The growth of A. cervicornis selfed individuals was not significantly affected by temperature or sedimentation. These results suggest that reducing sedimentation in dredging and coastal construction areas around coral reefs facilitates the survival of Acropora cervicornis juveniles and may help to ensure their persistence in the future. Because self-fertilized larvae were used, these results need to be interpreted with caution, and this research needs to be repeated with outcrossed A. cervicornis. What is clear is that genotypic diversity is needed for A. cervicornis population growth and resilience. Part II: Chimeras occur when two or more genetically unique individuals of the same species fuse together. The presence of chimerism can aid in the survival and evolution of organisms. This study investigated whether the prevalence of chimerism differs between coral species of different reproduction modes and growth rates. To fulfill this goal, the surface area of egg and/or larvae of three coral species, Montastraea cavernosa, Acropora cervicornis, and Porites astreoides, were measured and compared with the respective surface areas of the newly settled juveniles. This comparison suggested that M. cavernosa displayed a greater tendency to form chimeras than A. cervicornis and P. astreoides. Observations during embryogenesis confirmed this prediction. Montastraea cavernosa is a slow grower and has the smallest eggs of all three study species. Chimerism during embryogenesis may increase this species’ tendency to start the sessile stage at a slightly bigger size and thus increases its competitive abilities for reef space. In contrast, A. cervicornis is a broadcast spawner, fast grower and has a relatively larger egg size, possibly explaining the reduced chimeric tendency during embryogenesis. It is possible that P. astreoides formed chimeras during embryogenesis within the polyp, but they did not form them in the swimming planulae stage. The lack of chimerism during P. astreoides planulae development may be attributed to its brooding reproductive mode, directly releasing large competent larvae that have large initial sizes at settlement. Therefore, the ability to form chimeras in an early developmental stage might provide an ecological advantage to M. cavernosa that contributes to its abundance in Broward County: the greater size at settlement caused by chimerism during embryogenesis may provide this species a competitive advantage for reef space.

Introdução e Objetivos. Fosfatases são enzimas promissoras para aplicação na degradação de organofosforados. Por exemplo, a enzima paraoxonase 1 (PON1), associada à lipoproteína de alta densidade (HDL), hidrolisa lactonas, ésteres aromáticos e compostos organofosforados (OP) neurotóxicos. \"Módulos de ligação a carboidrato\" (CBM) têm diversas aplicações biotecnológicas. Nosso objetivo é a obtenção de proteínas quiméricas contendo fosfatases ligadas a um módulo de ligação de celulose, o que possibilitaria a imobilização dessas enzimas em suportes de celulose. Resultados. Como prova de conceito, uma proteína quimérica contendo uma \"fosfatase ácida\" (appA) de E.coli e CBM familia 2 (CBM2) de uma celulase de Xanthomonas axonopodis pv citri foi montada e produzida em E. coli como uma proteína recombinante solúvel. appA-CBM2 purificada demonstrou ser totalmente funcional exibindo atividade de ligação à celulose microcristalina (Avicel PH101) e atividade de fosfatase sobre p-nitrofenil fosfato. A ligação à Avicel evidenciou um comportamento de saturação descrito por uma \"constante de ligação\" (Kb) de 26 mg e um \"máximo de ligação\" (Bmax) de 4,45 U/µg. Além disso, a ligação de appA-CBM2 em Avicel foi maior em pH 2,5 e diminuiu acima de pH 6,5, como observado anteriormente para CBM2. Finalmente, o efeito de concentração de p-nitrofenil fosfato na atividade catalítica de appA-CBM2 e appA foi idêntico, exibindo um Km de 2,8 mM. Portanto, esses dados mostram que o conceito de uma proteína que combina as propriedades da fosfatase e do domínio de ligação à celulose é possível e funcional. De forma similar, os segmentos de DNA que codificam para o CBM2 e para a PON1 de Homo sapiens, foram fusionados resultando em um segmento que codifica para uma proteína quimérica (PON1-CBM2). PON1 nativa e PON1-CBM2 foram produzidas na forma solúvel e ativa em E.coli cepa Arctic. Embora não tenha sido viável sua purificação, estas enzimas foram caracterizadas. PON1-CBM2 liga-se em Avicel PH101 com um comportamento de saturação, descrito por uma constante de ligação (Kb) de 27 mg, valor idêntico àquele observado para appA-CBM2, o que sugere que o domínio CBM2 é igualmente funcional nestas duas enzimas quiméricas. PON1-CBM2 também exibe atividade paraoxonásica com Km similar àquele observado para PON1 nativa (1,3 mM), sugerindo que o \"domínio\" PON1 encontra-se totalmente funcional na enzima quimérica. Conclusão. Uma estratégia para a construção e expressão heteróloga em E. coli de PON1 e das enzimas quiméricas appA-CBM2 e PON1-CBM2 foi desenvolvida. As enzimas quiméricas mostraram-se totalmente funcionais e conservaram as propriedades de seus \"domínios\" constituintes. / Introduction and Aims. Phosphatases are promising enzymes for application in the degradation of organophosphates, whereas carbohydrate binding module has significant and demonstrated biotechnological applications. The high-density lipoprotein-associated enzyme paraoxonase 1 (PON1) hydrolyzes lactones, aromatic esters, and neurotoxic organophosphorus (OP) compounds. Our aim is to obtain chimeric proteins containing a phosphatase domain linked to a carbohydrate binding module (CBM), which could be immobilized on a cellulose supports. Results. As a proof of concept, a chimeric protein combining an acid phosphatase (appA) from E.coli and a CBM family 2 (CBM2) from Xanthomonas axonopodis pv. citri was assembled and produced in E.coli as a recombinant soluble protein. Purified appA-CBM2 was fully functional, was bound to microcrystalline cellulose and exhibited phosphatase activity upon p-nitrophenyl phosphate. The binding to microcrystalline cellulose Avicel PH101 exhibited saturation with a binding constant (Kb) of 26 and a maximum binding (Bmax) of 4,45 U/µg. In addition, the binding was higher at pH 2.5 and decreased above pH 6.5, as previously observed for CBM2. Finally, effect of p-nitrophenyl phosphate concentration on appA-CBM2 and native appA activities were identical, exhibiting a Km of 2.8 mM. Taken together, these data show that the conceptual design of a protein combining the properties and biotechnological advantages of phosphatases and cellulose binding domains is possible and functional. Similarly, DNA segments coding for CBM2 and for PON1 from Homo sapiens combined resulting in a segment coding for a chimeric protein (PON1-CBM2). Native PON1 and PON1-CBM2 were produced as recombinant protein in E. coli Arctic. Although purification was not accomplished, these enzymes were characterized. PON1-CBM2 binds to microcrystalline cellulose, exhibiting a saturation behavior described by a Kb of 27 mg. PON1 and PON1- CBM2 have the same Km for paraoxon (1.3 mM), indicating that the phosphatase domain was fully functional. Conclusion. An effective strategy for heterologous expression of the native PON1 and chimeric appA-CBM2 and PON1-CBM2 in E. coli was attained. The chimeric enzymes were fully functional and maintained the properties of their original domains

L’émergence de bactéries multirésistantes aux antibiotiques est la conséquence de leur utilisation à mauvais escient au cours de ces dernières décennies. Ce phénomène constitue un problème de santé publique majeur, et face à cette urgence sanitaire, il est nécessaire de trouver rapidement de nouveaux agents antibactériens.Les colicines, au regard de leurs propriétés antimicrobiennes intrinsèques, constituent des candidats intéressants. Naturellement produites par E. coli dans le but de tuer des souches compétitrices de la même espèce ou d’espèces apparentées, elles exercent en général leur activité cytotoxique par le biais d’une activité ionophorique ou nucléasique. Parmi les nombreuses colicines connues à ce jour, la colicine M (ColM) est la seule à interférer avec la voie de biosynthèse du peptidoglycane, macromolécule essentielle et spécifique au monde bactérien. En effet, une fois dans le périplasme de E. coli, la ColM clive le lipide II, dernier précurseur de la voie de biosynthèse du peptidoglycane, conduisant de ce fait à la lyse bactérienne. Plusieurs homologues de la ColM ont été identifiés chez d’autres genres bactériens (Pseudomonas, Pectobacterium et Burkholderia) mais aucune cytotoxicité croisée n’a été mise en évidence à ce jour, d’où un spectre d’action restreint pour les membres de cette nouvelle famille d’enzymes antibactériennes.Ce travail traite de l’étude structurale et biochimique de la ColM et de certains de ses homologues. L’étude structurale de différents variants de la PaeM, homologue issu de P. aeruginosa, a permis d’identifier une molécule d’eau conservée au sein du site actif qui joue probablement un rôle central dans le mécanisme catalytique de cette famille d’enzyme. L’expression des homologues de la ColM issus de Pseudomonas et de Pectobacterium, directement dans le périplasme de E. coli, a permis de démontrer leur activité lytique, prouvant ainsi le grand potentiel de ces bactériocines en tant qu’alternatives aux antibiotiques. Enfin, la construction de plusieurs colicines chimères entre la ColM et ses homologues, capables de dégrader le lipide II in vitro et d’induire la lyse d’E. coli suite à leur expression périplasmique, ouvre la voie à de futurs espoirs thérapeutiques. / The misuse of antibiotics during the last decades led to the emergence of multidrug resistant pathogenic bacteria. This phenomenon constitutes a major public health issue. Given that urgency, the finding of new antibacterials in the short term is crucial.Colicins, due to their antimicrobials properties, constitute good candidates. They are protein toxins produced by E. coli to kill competitors belonging to the same or related species. In most cases, they exhibit their cytotoxic activity through an ionophoric or nucleasic activity. Among the twenty colicins known to date, colicin M (ColM) is the only one known to interfere with peptidoglycan biosynthesis. It develops its lethal activity in the E. coli periplasm, in three steps deeply linked to its structural organization in three domains. Once in the periplasm, ColM degrades the lipid II, i.e. the last precursor in the peptidoglycan biosynthesis pathway, in two products that cannot be reused, thereby leading to cell lysis. Several ColM homologues have been identified in other bacterial genera, such as Pseudomonas, Pectobacterium and Burkholderia, but no cross activity has been shown to date, explaining the narrow antibacterial spectrum displayed by the members of this new family of antibacterial enzymes.This work deals with the structural and biochemical study of ColM and some of its homologues. Structural studies on several variants of PaeM, the ColM homologue from P. aeruginosa, led to identify a conserved water molecule in the active site, probably playing a central role in the catalytic mechanism of this enzyme family. Moreover, expression of ColM homologues from Pseudomonas or Pectobacterium species directly in the E. coli periplasm showed that all these homologues were able to induce E. coli cell lysis, thus demonstrating the great potential of these bacteriocins as an alternative to antibiotics. Following these results, several chimera colicins were created between ColM and its homologues, which were shown to degrade lipid II in vitro and to induce E. coli cell lysis after their periplasmic expression, opening the way to future new therapeutic options.

The performances of two D-Wave 2 machines (476 and 496 qubits) and of a 1097-qubit D-Wave 2X were investigated. Each chip has a Chimera interaction graph G. Problem input consists of values for the fields hj and for the two-qubit interactions Ji,j of an Ising spin-glass problem formulated on G. Output is returned in terms of a spin configuration {sj}, with sj = +1 or -1. We generated random spanning trees (RSTs) uniformly distributed over all spanning trees of G. On the 476-qubit D-Wave 2, RSTs were generated on the full chip with Ji,j = -1 and hj = 0 and solved one thousand times. The distribution of solution energies and the average magnetization of each qubit were determined. On both the 476- and 1097-qubit machines, four identical spanning trees were generated on each quadrant of the chip. The statistical independence of the these regions was investigated.

No description available.

Flaviviruses are a genus of vector-transmitted viruses that are nearly globally distributed, and flavivirus infections can result in life threatening diseases. Many flaviviruses such as Dengue, West Nile, yellow fever and Zika viruses are globally distributed. Zika virus (ZIKV) is a single strand positive-sense RNA virus, and its disease has been linked to Guillain Barré Syndrome (i.e., a debilitating autoimmune disorder that affects the nerves) in adults and congenital birth defects including microcephaly (i.e., a neurodevelopmental disorder due to impaired neural cell proliferation) in newborns. Insect-specific flaviviruses (ISFVs) are understudied given their apathogenic characteristics to humans and animals. However, given their close genetic relationship to vertebrate infectious flaviviruses, ISFVs can serve as a delivery system (i.e., vector) for flavivirus antigenic proteins. Aripo virus (ARPV) is a recently discovered ISFV isolated in Trinidad. We developed a chimeric Zika vaccine, Aripo-Zika, by substituting the pre-membrane and envelope genes of ZIKV into the ARPV genome. Here, we explored (i) the efficacy of Aripo-Zika (AZ) vaccination by evaluating passive transfer of maternal antibodies, (ii) the optimal dosage regimen, (iii) anti-vector immunity to the ARPV backbone, and (iv) the effects of boosters on vaccine efficacy. We also evaluated AZ safety via a co-infection study. Our results show a near linear relationship between increased dose and immunogenicity, with 1011 genome copies being the most effective minimum dose administered. Inclusion of boosters further increased the immunogenicity of AZ. Additionally, prior immunization with AZ showed minimal effects on subsequent immunization with an ARPV-West Nile virus (AWN) vaccine candidate, confirming the applicability of the ARPV backbone to multiple flavivirus vaccine candidates. In vitro co-infection of ZIKV with ARPV, and ZIKV with AZ in African green monkey kidney cells (i.e., Vero-76) indicated ARPV and AZ remain incapable of replication in vertebrate cells, even in the presence of active ZIKV replication. Altogether, our data suggests that the ARPV platform is a safe and effective strategy for the development of flavivirus vaccines. / Master of Science in Life Sciences / Vaccines are one of the best tools available since their initial conception. Vaccines have collectively increased human lifespan and reduced the burden of disease in humans and animals worldwide. Vaccine research aims to create vaccines that have a perfect balance of safety and efficacy. The goal is to produce a vaccine that can generate a strong immune response against the virus(es) of interest, while causing the least harm or side effects from the vaccine. Insect-specific viruses are viruses that infect insect cells, but are unable to replicate in humans or other vertebrate cells. The Auguste Lab has created a chimeric vaccine using the genome of an insect-specific virus called Aripo-Zika virus (AZ) that is genetically related to Zika virus. A person vaccinated with AZ is expected to develop an immune response against Zika but would not have any disease or side effects associated with a Zika infection or virus replication. In order to determine if this vaccine would be safe and effective enough to advance to clinical trials in humans, we must first determine if it is safe in smaller animal models. My studies have five central aims. First, determine the lowest dose of AZ that can be given and still be protective against Zika disease in mouse models. Second, determine if boosters are necessary and if they increase protection. Third, determine if immunity derived from vaccination can be passed down from mother to pups. Fourth, determine if Zika virus and AZ can co-exist in the same cell line without AZ replication occurring. Lastly, determine if mice can be vaccinated with AZ and subsequently with another similar Aripo virus-based vaccine (i.e., Aripo-West Nile) without changing the effectiveness of the subsequent immunization. Our results showed that AZ is able to be passed from mother to pup, 1011 genome copies is the minimum protective dose, and boosters can increase the effectiveness of AZ. We also found that AZ does not replicate in vertebrate cells when it co-exists with ZIKV and subsequent vaccination with Aripo-West Nile does not seem affect the effectiveness of either vaccine.

Chez les bactéries à Gram-négatif, deux systèmes très bien conservés et essentiels à la survie de la cellule bactérienne ont été identifiés : les systèmes Tol et TonB. Ces deux systèmes utilisent la force proton motrice, issue de la membrane interne et transfert l’énergie associée pour le transport actif de molécules (TonB) ou nécessaire au maintien de l’intégrité membranaire (Tol). Ces 2 systèmes ont été détournés de leurs fonctions initiales et parasités par les colicines, leur conférant un rôle primordial dans le mécanisme d’import de la colicine. Une colicine est une bactériocine (toxine) produite par Escherichia coli pour tuer des souches apparentées. Ce sont des toxines spécifiques et hautement actives. Cependant E.coli a développé des mécanismes de protection afin de résister à l’action cytotoxique des colicines. Ces mécanismes de résistance consistent essentiellement à produire des protéines d’immunité, qui vont pour la plupart se fixer sur le domaine catalytique de la colicine et l’empêcher d’exercer son action létale. La bactérie Yersinia pestis, agent de la peste, possède une colicin-like bactériocine, la pesticine, dont l’activité est de dégrader le peptidoglycane. L’action de la pesticine est inhibée par une protéine d’immunité, Pim, localisée dans le périplasme. Le principal objectif de ce projet est de comprendre les mécanismes d’inhibition de la pesticine par sa protéine d’immunité, grâce à des données biochimiques et structurales, mais aussi d’apporter des solutions pour contourner ce problème de résistance. La structure de la pesticine révèle des homologies structurales avec le T4 lysozyme du bactériophage T4. Pour contourner le problème de la résistance bactérienne liée à la protéine d’immunité, une solution a été de fusionner le domaine de réception/translocation de la pesticine avec le T4 lysozyme. Nous avons ainsi pu créer et résoudre la structure tridimensionnelle d’une protéine chimère fonctionnelle, capable de se fixer sur FyuA (récepteur de la pesticine) et tuer une souche exprimant ce récepteur et dont l’activité létale n’est pas inhibée par Pim. / In Gram-negative bacteria, two essential systems for cell survival have been characterized: the Tol and TonB system. Both Ton and Tol systems are very well conserved in Gram-negative bacteria and coupled to the proton motive force across the inner membrane, acting as energy transducers for active transport (Ton) or maintenance of outer envelope integrity (Tol). Both systems have been embezzled from their primary function and hijacked by colicins as part of the colicin killing pathway. Colicin is a bacteriocin (toxin) produced by and toxic to some strains of Escherichia coli. Colicins are highly effective toxins. However E.coli could develop protective mechanisms to resist to colicin cytotoxic effect. These mechanisms essentially consist to produce an immunity protein. These proteins bind to colicin catalytic domain and inhibit its lethal activity. Yersinia pestis, plague agent, possesses its own colicin-like bacteriocin, Pesticin, which degrades murein. Pesticin activity is inhibited by an immunity protein, Pim, localized in the periplasm. The main goal of this project is to understand inhibition mechanisms between Pim and Pesticin by biochemical and structural data and to provide solution to overcome the resistance issue, since Pesticin was thought to be used as antimicrobial agent. The Pesticin structure has revealed that Pesticin share structural homologies with the T4 lysozyme from the bacteriophage T4. To overcome the resistance issue due to the immunity protein, one solution has been to fuse the Pesticin binding/translocation domain with the T4 lysozyme. Thus, we could engineered and solved the three-dimensional structure of a chimera protein, able to bind FyuA (Pesticin physiological receptor) and kill a FyuA expressing strain, in which the lethal activity is not affected by Pim.