A tuberculose (TB) é considerada uma das principais doenças infecciosas e apresenta fatores críticos como a relação com o HIV/AIDS, tratamento longo e a resistência a múltiplos fármacos. A enzima di-hidrofolato redutase das micobactérias (mtDHFR) é um alvo pouco explorado e apresenta grande potencial para o desenvolvimento de novos fármacos contra TB. Estudos preliminares obtiveram fragmentos com baixa afinidade à mtDHFR, entretanto com potencial para otimização. Com isso, o fragmento foi usado como protótipo para a proposição de 22 análogos. Os compostos foram planejados utilizando informações sobre ligantes e a estrutura tridimensional de mtDHFR, além do biososterismo como estratégia norteadora. Os ensaios de docking molecular com a mtDHFR revelaram que os análogos propostos tiveram escores interessantes e, além disso, a inserção de substituintes demonstrou favorecer a ligação à enzima, o que corroborou o planejamento. Com isso, sintetizou-se 22 análogos planejados e o protótipo MB872, por meio de protocolos de alquilação, hidrólise e cicloadição 1,3 dipolar para os compostos com anéis triazol e tetrazol. Os compostos foram obtidos com rendimentos de bom a ótimo (60 ~ 90%) e suas estruturas foram elucidadas por RMN 1H e 13C. Os resultados do ensaio de inibição enzimática corroboraram com os dados de docking, uma vez que a presença do grupo carboxílico revelou ser importante para a atividade. Além disso, alguns dos compostos revelaram atividades interessantes, entre 8 a 40 µM, sendo que o mais ativo apresentou IC50 de 7 µM. Ensaios de cinética enzimática com o análogo mais ativo indicou uma inibição não competitiva com o substrato natural da enzima, uma vez que os valores de Km se mantiveram constantes, enquanto Vmax decaiu (0,22 µM e 0,43 - 0,34 ΔFU/min, respectivamente). Os análogos sintetizados foram mandados para ensaio in vitro para avaliar a atividade frente a micobactéria. / Tuberculosis (TB) is an important infectious disease and presents critical factors such as the relationship with HIV / AIDS, long treatment and resistance to multiple drugs. The enzyme dihydrofolate reductase from mycobacteria (mtDHFR) is a poorly explored and presents great potential to be a target for new drugs against TB. Preliminary studies have obtained fragments with low affinity to mtDHFR, but with potential to become lead compounds. Therefore, the fragment was used as a prototype for 22 analogues proposed in this work. The compounds were designed using bioisosterism, information about ligands and the three-dimensional structure of mtDHFR. Molecular docking assays with mtDHFR revealed satisfactory scores for anlogues. Furthermore, the insertion of substituents seemed to increase the affinity with the enzyme. Thereby, twenty two analogues and prototype were synthesized using alkylation, hydrolysiss and 1,3-dipolar cycloaddition methods. The compounds were obtained in good yields (60 ~ 90%) and their structures were elucidated with 1H and 13C NMR spectroscopy. The enzymatic affinity assay corroborates docking results, because the presence of carboxyl group showed to be important for the activity. Furthermore, some of the compounds revealead interesting activities, ranging 8 to 40 µM. The most active showed IC50 of 7 µM and enzyme kinetics assays indicated noncompetitive inhibition with natural enzyme substrate. The synthesized analogs were sent for in vitro assay to assess mycobacteria activity.

Orientador: Orlando Necchi Junior / Banca: Carla Ferragut / Banca: Ciro Cesar Zanini Branco / Resumo: Vários estudos envolvendo flora e distribuição de comunidades de macroalgas lóticas já foram desenvolvidos na região noroeste do estado de São Paulo; entretanto, nunca foi realizada qualquer abordagem sobre macroalgas lóticas em fragmentos florestais remanescentes, aspecto mais relevante deste estudo. Foram testadas as seguintes hipóteses: (1) entre as regiões/biomas estudados no estado de São Paulo, o atual trabalho deve apresentar maior similaridade florística com Floresta Tropical, por ser mais próxima e composta também por Floresta Estacional Semidecidual; (2) características intrínsecas de cada corpo d'água devem exercer maior influência na riqueza e abundância das comunidades de macroalgas do que parâmetros mais gerais de cada fragmento, como forma, tamanho e matriz adjacente, e da ordem de grandeza do riacho e sua respectiva bacia de drenagem. Este trabalho teve como objetivo geral realizar o levantamento florístico e analisar a distribuição ecológica das comunidades de macroalgas lóticas de fragmentos florestais remanescentes de Floresta Estacional Semidecidual da região noroeste do estado de São Paulo (20o00'13"-21o37'14"S, 48o32'26"- 50o26'02"O). Foram amostrados 17 riachos pertencentes a 12 fragmentos, no intervalo de junho a agosto de 2007 e 2008, período mais favorável para a região (estação seca). Foram identificadas 16 espécies de macroalgas, pertencentes a 14 gêneros. A maioria das espécies (69%) foi encontrada em um único ponto. Cyanophyta e Chlorophyta foram os grupos predominantes (44 e 37,5%), seguidos por Rhodophyta (12,5%) e Heterokontophyta (6%). Phormidium retzii (Cyanophyta) foi a espécie mais frequente, ocorrendo em seis pontos de amostragem. Apenas duas espécies representaram novos registros para a região: Trichocoleus sociatus (Cyanophyta) e Vaucheria pseudogeminata (Heterokontophyta); a última representa... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Several studies involving the flora and distribution of lotic macroalgal communities have been carried out in the northwest region of São Paulo State, southeastern Brazil. However, the lotic macroalgae in remnant forest fragments have never been studied, the most relevant aspect of this study. The following hypotheses have been tested: 1) among the biomes/regions previously studied in São Paulo State, the flora of the northwest region is expected to reveal the highest floristic similarity with the Tropical Forest, since it is the nearest biome and also composed by semidecidual seasonal forest; 2) particular characteristics of each water body are expected to be more influential on the species richness and abundance of macroalgal communities than more general parameters of each forest fragment, such as surrounding matrix, stream order and position in the respective drainage basin. This study aimed at surveying the flora and describing the ecological distribution the of lotic macroalgal communities from remnant forest fragments composed of semidecidual seasonal forest in the northwest region of São Paulo State (20o00'13"-21o37'14"S, 48o32'26"-50o26'02"O). 17 streams were sampled in 12 forest fragments from June to August of 2007 and 2008 during the typical most favorable period in the region (dry season). 16 species have been surveyed, belonging to 14 genera. 69% of species were found in a single site. Cyanophyta and Chlorophyta were the predominant algal groups (44 amd 37.5%), followed by Rhodophyta (12.5%) and Heterokontophyta (6%). Phormidium retzii (Cyanophyta) was the most frequent species, occurring in six sampling sites. Only two species were new records for the region: Trichocoleus sociatus (Cyanophyta) and Vaucheria pseudogeminata (Heterokontophyta); the later represents the first report for Brazil. The regional flora was more similar (nine species in common, 56%)... (Complete abstract click electronic access below) / Mestre

Thesis (Ph. D.)--University of Florida, 2003. / Title from title page of source document. Includes vita. Includes bibliographical references.

Reasoning about temporal and spatial information is a common task in computer science, especially in the field of artificial intelligence. The topic of this thesis is the study of such reasoning from a computational perspective. We study a number of different qualitative point based formalisms for temporal reasoning and provide a complete classification of computational tractability for different time models. We also develop more general methods which can be used for proving tractability and intractability of other relational algebras. Even though most of the thesis pertains to qualitative reasoning the methods employed here can also be used for quantitative reasoning. For instance, we introduce a tractable and useful extension to the quantitative point based formalism STP. This extension gives the algebra an expressibility which subsumes the largest tractable fragment of the augmented interval algebra and has a faster and simpler algorithm for deciding consistency. The use of disjunctions in temporal formalisms is of great interest not only since disjunctions are a key element in different logics but also since the expressibility can be greatly enhanced in this way. If we allow arbitrary disjunctions, the problems under consideration typically become intractable and methods to identify tractable fragments of disjunctive formalisms are therefore useful. One such method is to use the independence property. We present an automatic method for deciding this property for many relational algebras. Furthermore, we show how this concept can not only be used for deciding tractability of sets of relations but also to demonstrate intractability of relations not having this property. Together with other methods for making total classifications of tractability this goes a long way towards easing the task of classifying and understanding relational algebras. The tractable fragments of relational algebras are sometimes not expressive enough to model real-world problems and a backtracking solver is needed. For these cases we identify another property among relations which can be used to aid general backtracking based solvers to finnd solutions faster. / Article I is a revised and extended version of the following three papers: 1. Mathias Broxvall and Peter Jonsson. Towards a Complete Classification of Tractability in Point Algebras for Nonlinear Time. In Proceedings of the 5th International Conference on Principles and Practice of Constraint Programming (CP-99), pp. 129-143, Alexandria, VA, USA, Oct, 1999. 2. Mathias Broxvall and Peter Jonsson. Disjunctive Temporal Reasoning in Partially Ordered Time Structures. In Proceedings of the Seventeenth National Conference on Artificial Intelligence (AAAI-2000), pp. 464-469, Austin, Texas, USA, Aug, 2000. 3. Mathias Broxvall. The Point Algebra for Branching Time Revisited. In Proceedings of the Joint German/Austrian Conference on Artificial Intelligence (KI-2001), pp. 106-121, Vienna, Austria, Sep, 2001. --- Article II is a revised and extended version of the following paper: Mathias Broxvall, Peter Jonsson and Jochen Renz: Refinements and Independence: A Simple Method for Identifying Tractable Disjunctive Constraints. In Proceedings of the 6th International Conference on Principles and Practice of Constraint Programming (CP-2000), pp. 114-127, Singapore, Sep, 2000.

<p>Abstract</p><p>DNA replication is an integral part of the cell cycle. Every time a cell divides, the entire genome has to be copied once and only once in a timely manner. In order to accomplish this, DNA replication begins at many points throughout the genome. These start sites are called origins of replication, and they are initiated in a temporal manner throughout S phase. How these origins are selected and regulated is poorly understood. Saccharomyces cerevisiae and Schizosaccharomyces pombe have autonomously replicating sequences (ARS) that can replicate plasmids extrachromosomally and function as origins in the genome. Metazoans, however, have shown no evidence of ARS activity.</p><p>DNA replication is a multistep process with several opportunities for regulation. Potential origins are marked with the origin recognition complex (ORC), a six subunit complex. In S. cerevisiae, ORC binds to the ARS consensus sequence (ACS), but no sequence specificity is seen in S. pombe or in metazoans. Therefore, factors other than sequence play a role in origin selection.</p><p>In G1, the pre-replicative (pre-RC) complex assembles at potential origins. This involves the recruitment of Cdc6 and Cdt1 to ORC, which then recruits MCM2-7 to the origin. In S phase, a subset of these pre-RC marked origins are initiated for replication. These origins are not fired simultaneously; instead, origins are fired in a temporal manner, with some firing early, some firing late, and some not firing at all.</p><p>The temporal firing of origins leads to wide regions of the genome being copied at different times during S phase. , which makes up the replication timing profile of the genome. These regions are not random, and several correlations between replication timing and both transcriptional activity and chromosomal landscape. Regions of the genome with high transcriptional activity tend to replicate earlier in S phase, and it is well know that the gene rich euchromatin replicates earlier than the gene poor heterochromatin. Additionally, areas of the genome with activating chromatin marks also replicate earlier than regions with repressive marks. Though many correlations have been observed, no single mark or transcriptional player has been shown to directly influence replication timing.</p><p>We mapped the replication timing profiles of three cell lines derived from Drosophila melanogaster by pulsing cells with the nucleotide analog bromodeoxyuridine (BrdU), enriching for actively replicating DNA labeled with BrdU, sequencing with high throughput sequencing and mapping the sequences back to the genome. We found that the X chromosome of the male cell lines replicated earlier than the X chromosome in the female cell line or the autosomes. We were then able to compare the replication timing profiles to data sets for chromatin marks acquired through the modENCODE (model organism Encyclopedia Of DNA Elements). We found that the early replicating regions of the male X chromosomes correlates with acetylation of lysine 16 on histone 4 (H4K16).</p><p>Hyperacetylation of H4K16 on the X chromosome in males is a consequence of dosage compensation in D. melanogaster. Like many organisms, D. melanogaster females have two X chromosomes while males have one. To compensate for this difference, males upregulate the genes on the X chromosome two-fold. This upregulation is regulated by the dosage compensation complex (DCC), which is restricted to the X chromosome. This complex includes a histone acetyl transferase, MOF, which acetylates H4K16. This hyperacetylation allows for increased transcription of the X chromosome. </p><p>We hypothesized that the activities of the DCC and the hyperacetylation of H4K16 also influences DNA replication timing. To test this, I knocked down components of the DCC (MSL2 and MOF) using RNAi. Cells were arrested in early S phase with hydroxyurea, released, and pulsed with the nucleotide analog EdU. The cells were arrested in metaphase and labeled for H4K16 acetylation and EdU. We found that male cells were preferentially labeled with EdU on the X chromosome, which corresponded with H4k16 acetylation. When the DCC was knocked down, H4K16 acetylation was lost along with preferential EdU labeling on the X chromosome. These results suggest that the DCC and H4K16 acetylation are necessary for early replication of the X chromosome. Additionally, early origin mapping of different cell lines showed that while ORC density does not differ between male and female cell lines, early origin usage is increased on the X chromosome of males, suggesting that this phenomenon is regulated at the level of activation, not pre-RC formation. Other experiments in female cell lines have been unclear about whether the DCC and subsequent H4K16Ac is sufficient for early X replication. However, these results are exciting because this is, to our knowledge, the first mark that has been found to directly influence replication timing.</p><p>In addition to these timing studies, I attempted to design a new way to map origins. A consequence of unidirectional replication with bidirectional replication fork movement is Okazaki fragments. These are short nascent strands on the lagging strand of replicating DNA. Because these fragments are small, we can isolate them by size and map them back to the genome. Okazaki density could tell us about origin usage and any directional preferences of origins. The process proved to be tedious, and although they mapped back with a higher density around ORC binding sites than randomly sheared DNA, little information about origin usage was garnered from the data. Additionally, the process proved difficult to repeat.</p><p>In these studies, we examined the replication timing program in D. melanogaster. We found that the male X chromosome replicates earlier in S phase, and this early replication is regulated by the DCC. However, it is unclear if the change in chromatin landscape directly influences replication or if the replication program is responding to other dosage compensation cues on the X chromosome. Regardless, we have found one the first conditions in which a mark directly influences the DNA replication timing program.&#8195;</p> / Dissertation

L'objectif de cette thèse est la recherche de nouvelles techniques de génération de contenu numérique pour des applications de jeu vidéo. Le manque de variété de terrains, d'objets et de détails affecte fortement le réalisme des paysages de synthèse.Dans ce contexte, un des principaux goulots d'étranglements est la modélisation des ressources graphiques permettant de créer les scènes. Afin de simplifier et d'accélérer cette tâche, nous présentons des méthodes permettant de générer automatiquement du contenu graphique pour créer de grands paysages à la fois complexes et originaux.Notre première approche permet de créer et d'éditer rapidement des variétés d'objets à partir d'un modèle initial fourni par un graphiste, sous la contrainte d'une représentation avec très peu de triangles. Nous présentons également une méthode de génération procédurale des variétés d'objets. Ensemble, ces méthodes permettent de créer aussi bien des variétés de formes naturelles que des ouvrages d'arts tels que des routes, des ponts et des tunnels capables de s'adapter automatiquement au relief d'un paysage.Nous proposons une autre méthode, s'appuyant sur une simulation physique et thermique, pour créer des paysages hivernaux évoluant au cours du temps en fonction des conditions climatiques. Cette approche permet de suivre l'évolution du manteau neigeux ainsi que l'épaisseur de la glace qui se forme en surface d'un lac.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 174-188).

DNA diagnostics are important technologies in molecular and cellular biology. By allowing identification of specific sequences, DNA-based diagnostics potentially provide more accurate and rapid results than protein- or antigen-based diagnostics, primarily because phenotypic changes come much later than changes in genotype. Despite this advantage, there are fewer diagnostic or imaging systems that target DNA than those targeting proteins, antibodies, or antigens.Each type of DNA-based diagnostic has its own, unique set of limitations; however, most can be attributed to issues related to sequence restriction, signal detection, specificity, or some combination thereof. For example, while PCR-based methods allow amplification and assessment of specific DNA sequences, they lack the ability to report information of specific cells, or cell types, within the heterogeneous pool of cells typically found in a tumor biopsy. In addition, none of the currently available DNA detection methods has the potential to be utilized in living cells, a disadvantage which limits the potential applications.The work presented here describes the design and development of a new methodology for the detection of specific double-stranded DNA sequences. This detection method is based on the concept that two inactive fragments of a reporter protein, each coupled to engineered zinc finger DNA-binding motifs, are able to reassemble and form an active complex in the presence of a predefined DNA sequence. This system, designated sequence-enabled reassembly (SEER), can achieve single base-pair specificity, and has the potential to be utilized in living cells.In this dissertation, we discuss the efforts from constructing to refining the system, as well as the future applications of SEER in diagnostics and therapeutics. Chapter I will provide an introduction to DNA detection methods, on which the principles of the SEER system are based. Chapter II describes the design and construction of an enzymatic SEER system, SEER-LAC, using beta-lactamase as the enzyme. In Chapter III, we outline the in vitro characterization of the SEER-LAC system, followed by its optimization in Chapter IV. Chapter V illustrates the efforts to develop SEER system for mammalian cell culture applications. In the final chapter, the future developments and applications of SEER are discussed.

This study adapted and developed methods of assessing and modelling biodiversity of butterflies and day-flying moths in habitat fragments, and determined those factors affecting their presence, abundance and species richness in a sample of 46 isolated urban remnants in south-west Western Australia. The specific objectives were to: (i) assess the effectiveness of transect–based sampling to quantify the species richness of habitat fragments; (ii) examine patterns of species richness in habitat fragments and quantify the detectability of each species recorded; (iii) review and rationalize the methods used to fit species–area–habitat models; and (iv) model species incidence, abundance and total richness of butterflies in urban habitat fragments and determine implications and priorities for their conservation. / These objectives were achieved and the principal findings of the research are: (i) The transect method provides an accurate assessment of butterfly species richness in isolates provided that the level of sampling (proportion of area surveyed) is adequate, that sufficient surveys are conducted during the flight season to ensure high levels of detectability, and that surveys are conducted at appropriate times and during suitable weather conditions. Although randomly placed transects are preferable, logistic constraints often dictate the use of existing pathways, roadsides or management tracks – which requires the use of longer transects but is more practical in urban remnants. / (i) The transect method provides an accurate assessment of butterfly species richness in isolates provided that the level of sampling (proportion of area surveyed) is adequate, that sufficient surveys are conducted during the flight season to ensure high levels of detectability, and that surveys are conducted at appropriate times and during suitable weather conditions. Although randomly placed transects are preferable, logistic constraints often dictate the use of existing pathways, roadsides or management tracks – which requires the use of longer transects but is more practical in urban remnants. / (iii) Almost a century of fitting species–area curves has failed to produce agreement on which function is the best model of the relationship. Many of the proposed functions are identical, special cases of others or have arisen from transcription errors. Empirical comparison of these functions requires methods suited to the distribution of species number such as the generalized linear model, method of maximum likelihood and the information-theoretic approach, and proper attention to covariates and their interactions. / (iv) Site area and vegetation condition were the dominant determinants of the presence, abundance and total species richness of resident butterflies and day-active moths in 46 urban habitat fragments in south-west Western Australia. Larger sites with more high quality (undisturbed) vegetation favoured 16 of 20 native species and only one benefited from disturbance. A further nine species not sufficiently widespread or abundant to enable individual analysis were collectively more prevalent in larger sites. Resource quality and quantity dominated the patterns of site occupancy, and increased site connectivity did not favour any species – results consistent with habitat resources, not metapopulation effects, determining current distribution patterns. As expected, the presence of non-resident species was unaffected by site area. The total number of resident species at each site reflected the collective responses of the individual species: increasing with area and declining with vegetation disturbance. The effects of area and vegetation quality were not simply additive: disturbance had a far greater impact on small remnants. This interaction is inconsistent with the area per se hypothesis: in the absence of disturbance there was no evidence of a species–area effect. / This study is the first comprehensive, quantitative assessment of the distribution and ecology of butterflies and day-flying moths in Australian urban habitat fragments and provides a baseline against which future changes in species distributions may be measured. The results have important implications for the conservation of butterflies and day-flying moths in the region. Maintenance of vegetation quality is of paramount importance and is vital in smaller remnants. Large remnants, being less susceptible to local extinctions, will be essential for the persistence of many species. Many functions have been proposed to model the species–area relationship but empirical comparisons have been hindered by methodological problems – this study conducted a re-examination of the relationship and presents an appropriate framework to compare functions. This study is also one of few to demonstrate and quantify the importance of interactions in explaining patterns of species richness and should stimulate future research into the importance of these effects.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.