Research in drug development meets many challenges including lengthy, complex and costly procedures to identify novel pharmacotherapies. In our lab, we developed a method for fast screening of small molecules that interact with monoamine transports – dopamine and serotonin (DAT, SERT). These membrane proteins play important roles in brain neurotransmission responsible for cognition, motion and pleasure. Dysfunction in dopaminergic and serotonergic systems result in neurological disorders such as depression, Attention Deficit Hyperactivity Disorder (ADHD), schizophrenia and addiction. DAT and SERT are responsible for uptake of dopamine (DA) or serotonin (5HT) into the synapse and they limit neurotransmitter signaling. Drugs that mimic or antagonize actions of endogenous neurotransmitters (DA and 5HT) increase the concentrations of DA and/or 5HT either by blocking the transporter (blockers) or by competing uptake with neurotransmitter (substrate). The uptake of substrates is associated to an inward current that depolarizes the cell membrane. Voltage-gated calcium channels (CaV) can respond to small changes in membrane potential. In our method, we combined permanent cell line expressing the human dopamine transporter (hDAT) or the human serotonin transporter (hSERT) (FlpIn TREx expression system) with transient transfection of CaV. This system works as a tightly electrically coupled system. Cells challenged with substrate of the transports produce detectable Ca2+ signal while monoamine transporter blockers can inhibit these Ca2+ signals. The novelty of this method relies on the ability to discriminate between substrate and blockers of monoamine transporters. Preliminary experiments measuring our optimized cell system in a Flex Station 3 plate reader suggest that the co-expression of a voltage-gated Ca2+ channel, a monoamine transporter and a genetically encoded Ca2+ sensor constitute a rapid screening biosensor to identify active drugs at monoamine transporters. Our novel methodology can rapidly assess drug-effect profile on monoamine transporters and benefit development of new psychotherapeutics for treatment of mental illnesses. It can also be used to characterize mechanism of action of emerging drug of abuse, as well as to discover small molecules with novel drug-effect profile useful in basic neuroscience research.

Climate change and eutrophication, accelerated by anthropogenic activities, have impacted aquatic ecosystems worldwide. These impacts have stimulated the expansion of cyanobacterial blooms which pose severe threats to ecosystem functioning, environmental health, and the economy. However, the long-term effects of environmental change on bloom-forming cyanobacteria are not well understood as traditional paleolimnological approaches are of limited use in the reconstruction of cyanobacterial dynamics through time. Here, sediment DNA (sedDNA) was used to investigate long-term cyanobacterial trends using sediments from two experimental (fertilized L227 and acidified L223) and two reference (L224 and L442) lakes in the Experimental Lakes Area, Canada. First, to determine whether taxonomic bias might arise from the cyanobacterial sediment record, I performed a 1-year incubation experiment comparing the degradation rates of selected cyanobacterial genes under contrasting environmental conditions. Based on first-order linear decay models, Synechococcus sp. (Synechococcales) decayed the slowest under cold, anoxic conditions, followed by Trichormus (Nostocales), then Microcystis (Chroococcales), suggesting differential preservation of DNA. I then compared the quantitative performance of droplet digital polymerase chain reaction (ddPCR) and high-throughput sequencing (HTS) for the analysis of sedDNA and found that the ddPCR results were more consistent with the known history of the lakes. Furthermore, ddPCR showed that cyanobacterial abundance increased over the past century in all study lakes, but the greatest increase was observed in experimentally fertilized L227. HTS revealed shifts in the cyanobacterial community towards Nostocales dominance and a decrease in alpha diversity in response to phosphorus-only additions. An increase in abundance of the mcyE gene (indicative of microcystin producing taxa) was uniquely observed in L227 when nitrogen additions ceased. Heating degree days were important in explaining variation in the cyanobacterial community composition in all lakes, but nutrients had a greater influence on the L227 community. When sediment data were compared to historical surface water phytoplankton records, moderate to strong correlations between the two archives were found, validating the use of sedDNA. This research demonstrated that sedDNA can elucidate cyanobacterial trends at the community, population, and species level over multidecadal timescales in response to environmental change.

Taxon composition and biodiversity analyses are known powerful parameters for environmental site status and environment diagnosis. Many ecological studies assess taxon composition through traditional species identification and use bioindicator species to evaluate environmental conditions. The recent breakthrough in bulk sample sequencing combined with DNA barcoding has created a new era for environmental monitoring. Metabarcoding approaches are more robust in studying alpha, and beta diversity compare to the DNA barcoding and the conventional method of species identification, particularly for rare and cryptic species. Here we built upon ecological studies of bioindicator species and transferred the traditionally named taxa to DNA-based approaches. We developed a small customized DNA database for biodiversity assessment and taxonomic identification of environmental DNA samples using high-throughput amplicon sequences. It contains macroinvertebrate species that are known as indicators of specific environmental conditions. By implementing this small database into the KRAKEN algorithm for the first time, we were able to assess environmental biodiversity compared to other popular methods of taxonomic classification, especially in polluted environments where the taxonomic composition globally change by the presence of anthropogenic drivers. Our method is incredibly faster, and it requires significantly less computational power in contrast to common homology-based techniques. To evaluate our approach, we have also studied the importance of database’s size and the depth of sequencing in taxonomic classification of high-throughput DNA sequences. / Thesis / Master of Science (MSc) / We developed a small customized DNA database for biodiversity assessment and taxonomic identification of environmental DNA samples using high-throughput amplicon sequences. It contains macroinvertebrate species that are known as indicators of specific environmental conditions. By implementing this small database into the KRAKEN algorithm for the first time, we were able to assess environmental biodiversity compared to other popular methods of taxonomic classification, especially in polluted environments where the taxonomic composition globally change by the presence of anthropogenic drivers. Our method is incredibly faster, and it requires significantly less computational power in contrast to common homology-based techniques.

<p>The Gram-positive, soil dwelling bacteria of the genus <em>Streptomyces</em> produce greater than 50% of the clinically relevant antibiotics in use today. Thanks to the falling price of DNA sequencing, <em>Streptomyces</em> genomes are revealing that they encode more secondary metabolites (potential antibiotics) than they produce under standard laboratory conditions. By heterologously overexpressing the known pleiotropic regulators of antibiotic expression from <em>Streptomyces coelicolor</em> in several other <em>Streptomyces</em> species it has been shown that the secondary metabolite profile of these species can be influenced. While present-day methods of introducing genes (conjugation) and screening for antibiotics work well on a small scale, the low throughput nature of these protocols stand as a barrier to testing this hypothesis on a larger scale. The focus of the research presented here was to develop high throughput (HTP) methods of engineering and screening <em>Streptomyces</em>. With these two technologies in place, an attempt was to made to introduce three plasmids (pSET152-<em>ermE*</em>p-null, pSET152-<em>ermE</em>*p-<em>atrA</em> and pSET152-<em>ermE</em>*p-<em>lsr2_NTD</em>) into 120 wild-isolate <em>Streptomyces</em> species from the Wright Actinomycete Collection. Exconjugants were successfully obtained for all three plasmids in 48 species of <em>Streptomyces</em> and were screened for increased antimicrobial activity using a HTP, <em>lux</em>-based bioassay. Numerous strains showed increased antimicrobial activity but WAC00206, WAC00230 and WAC00263 with pSET152-<em>ermE</em>*p-<em>lsr2_NTD</em>showed the most promising improvement in antimicrobial activity. These hits have been designated as high priority for future investigation. These results suggest that HTP conjugation and the <em>lux</em>-based bioassay are powerful methods for introducing plasmids into and screening engineered streptomycetes.</p> / Master of Science (MSc)

Les travaux présentés dans cette thèse se situent dans le cadre de la synthèse de la parole à partir du texte et, plus précisément, dans le cadre de la synthèse paramétrique utilisant des règles statistiques. Nous nous intéressons à l'influence des descripteurs linguistiques utilisés pour caractériser un signal de parole sur la modélisation effectuée dans le système de synthèse statistique HTS. Pour cela, deux méthodologies d'évaluation objective sont présentées. La première repose sur une modélisation de l'espace acoustique, généré par HTS par des mélanges gaussiens (GMM). En utilisant ensuite un ensemble de signaux de parole de référence, il est possible de comparer les GMM entre eux et ainsi les espaces acoustiques générés par les différentes configurations de HTS. La seconde méthodologie proposée repose sur le calcul de distances entre trames acoustiques appariées pour pouvoir évaluer la modélisation effectuée par HTS de manière plus locale. Cette seconde méthodologie permet de compléter les diverses analyses en contrôlant notamment les ensembles de données générées et évaluées. Les résultats obtenus selon ces deux méthodologies, et confirmés par des évaluations subjectives, indiquent que l'utilisation d'un ensemble complexe de descripteurs linguistiques n'aboutit pas nécessairement à une meilleure modélisation et peut s'avérer contre-productif sur la qualité du signal de synthèse produit. / The work presented in this thesis is about TTS speech synthesis and, more particularly, about statistical speech synthesis for French. We present an analysis on the impact of the linguistic contextual factors on the synthesis achieved by the HTS statistical speech synthesis system. To conduct the experiments, two objective evaluation protocols are proposed. The first one uses Gaussian mixture models (GMM) to represent the acoustical space produced by HTS according to a contextual feature set. By using a constant reference set of natural speech stimuli, GMM can be compared between themselves and consequently acoustic spaces generated by HTS. The second objective evaluation that we propose is based on pairwise distances between natural speech and synthetic speech generated by HTS. Results obtained by both protocols, and confirmed by subjective evaluations, show that using a large set of contextual factors does not necessarily improve the modeling and could be counter-productive on the speech quality.

Submitted by Lucas Alexandre Ramos null (magrelolukas@hotmail.com) on 2016-09-12T11:55:17Z No. of bitstreams: 1 Monografia_Final.pdf: 4956502 bytes, checksum: b3c792e3df597c4fabe2093c7ea8b357 (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-09-14T17:56:52Z (GMT) No. of bitstreams: 1 ramos_la_me_bauru.pdf: 4956502 bytes, checksum: b3c792e3df597c4fabe2093c7ea8b357 (MD5) / Made available in DSpace on 2016-09-14T17:56:52Z (GMT). No. of bitstreams: 1 ramos_la_me_bauru.pdf: 4956502 bytes, checksum: b3c792e3df597c4fabe2093c7ea8b357 (MD5) Previous issue date: 2016-08-12 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Atualmente, dada a difusão dos computadores, a tarefa de se reconhecer padrões visuais está sendo cada vez mais automatizada, em especial para tratar a vasta e crescente quantidade de imagens digitais existentes. Aplicações de diversas áreas como biometria, recuperação de imagens baseada em conteúdo e diagnóstico médico, se valem do processamento de imagens, bem como de técnicas de extração e análise de características das mesmas, a fim de identificar pessoas, objetos, gestos, textos, etc. As características básicas que são utilizadas para a análise de imagens são: cor, textura e forma. Recentemente, foi proposto um novo descritor de formas denominado HTS (Hough Transform Statistics), o qual se baseia no espaço de Hough para representar e reconhecer objetos em imagens por suas formas. Os resultados obtidos pelo HTS sobre bases de imagens públicas têm mostrado que este novo descritor, além de apresentar altas taxas de acurácia, melhores do que muitos descritores tradicionais propostos na literatura, é rápido, pois tem um algoritmo de complexidade linear. O objetivo deste trabalho foi explorar as possibilidades de representação multiescala do HTS e, assim, propor novos descritores de formas. Escala é um parâmetro essencial em Visão Computacional e a teoria de espaço-escala refere-se ao espaço formado quando se observa, simultaneamente, os aspectos espaciais de uma imagem em várias escalas, sendo a escala a terceira dimensão. Os novos métodos multiescala propostos foram avaliados sobre várias bases de dados e seus desempenhos foram comparados com o desempenho do HTS e com os principais descritores de formas encontrados na literatura. Resultados experimentais mostraram que os novos descritores propostos neste trabalho são mais rápidos e em alguns casos também mais precisos. / Currently, given the widespread of computers through society, the task of recognizing visual patterns is being more and more automated, in particular to treat the large and growing amount of digital images available. Applications in many areas, such as biometrics, content-based image retrieval, and medical diagnostic, make use of image processing, as well as techniques for the extraction and analysis of their characteristics, in order to identify persons, objects, gestures, texts, etc. The basic features that are used for image analysis are: color, texture and shape. Recently, it was proposed a new shape descriptor called HTS (Hough Transform Statistics), which is based on the Hough space to represent and recognize objects in images by their shapes. The results obtained by HTS on public image databases have shown that this new shape descriptor, besides showing high accuracy levels, better than many traditional shape descriptors proposed in the literature, is fast, since it has an algorithm of linear complexity. In this dissertation we explored the possibilities of a multiscale and scale-space representation of this new shape descriptor. Scale is a key parameter in Computer Vision and the theory of scale-space refers to the space formed when observing, simultaneously, special aspects of an image at several scales, being the scale the third dimension. The multiscale HTS methods were evaluated on the same databases and their performances were compared with the main shape descriptors found in the literature and with the monoscale HTS. Experimental results showed that these new descriptors are faster and can also be more accurate in some cases. / FAPESP: 2014/10611-0

L’objectif de cette thèse est l’étude et la conception d’une plateforme de synthèse de parole expressive.Le système de synthèse — LIPS3, développé dans le cadre de ce travail, incorpore deux éléments : un module linguistique et un module de synthèse paramétrique par apprentissage statistique (construit à l’aide de HTS et de STRAIGHT). Le système s’appuie sur un corpus monolocuteur conçu, enregistréet étiqueté à cette occasion.Une première étude, sur l’influence de la qualité de l’étiquetage du corpus d’apprentissage, indique que la synthèse paramétrique statistique est robuste aux erreurs de labels et d’alignement. Cela répond au problème de la variation des réalisations phonétiques en parole expressive.Une seconde étude, sur l’analyse acoustico-phonétique du corpus permet la caractérisation de l’espace expressif utilisé par la locutrice pour réaliser les consignes expressives qui lui ont été fournies. Les paramètres de source et les paramètres articulatoires sont analysés suivant les classes phonétiques, ce qui permet une caractérisation fine des phonostyles.Une troisième étude porte sur l’intonation et le rythme. Calliphony 2.0 est une interface de contrôlechironomique temps-réel permettant la modification de paramètres prosodiques (f0 et tempo) des signaux de synthèse sans perte de qualité, via une manipulation directe de ces paramètres. Une étude sur la stylisation de l’intonation et du rythme par contrôle gestuel montre que cette interface permet l’amélioration, non-seulement de la qualité expressive de la parole synthétisée, mais aussi de la qualité globale perçue en comparaison avec la modélisation statistique de la prosodie.Ces études montrent que la synthèse paramétrique, combinée à une interface chironomique, offre une solution performante pour la synthèse de la parole expressive, ainsi qu’un outil d’expérimentation puissant pour l’étude de la prosodie. / The subject of this thesis was the study and conception of a platform for expressive speech synthesis.The LIPS3 Text-to-Speech system — developed in the context of this thesis — includes a linguistic module and a parametric statistical module (built upon HTS and STRAIGHT). The system was based on a new single-speaker corpus, designed, recorded and annotated.The first study analyzed the influence of the precision of the training corpus phonetic labeling on the synthesis quality. It showed that statistical parametric synthesis is robust to labeling and alignment errors. This addresses the issue of variation in phonetic realizations for expressive speech.The second study presents an acoustico-phonetic analysis of the corpus, characterizing the expressive space used by the speaker to instantiate the instructions that described the different expressive conditions. Voice source parameters and articulatory settings were analyzed according to their phonetic classes, which allowed for a fine phonostylistic characterization.The third study focused on intonation and rhythm. Calliphony 2.0 is a real-time chironomic interface that controls the f0 and rhythmic parameters of prosody, using drawing/writing hand gestures with a stylus and a graphic tablet. These hand-controlled modulations are used to enhance the TTS output, producing speech that is more realistic, without degradation as it is directly applied to the vocoder parameters. Intonation and rhythm stylization using this interface brings significant improvement to the prototypicality of expressivity, as well as to the general quality of synthetic speech.These studies show that parametric statistical synthesis, combined with a chironomic interface, offers an efficient solution for expressive speech synthesis, as well as a powerful tool for the study of prosody.

Limitadores de corrente supercondutores resistivos (LCSR) são dispositivos com características elétricas próximas ao ideal quando ocorre a transição do estado supercondutor para o estado normal, limitando a corrente de falta através da inserção rápida de uma resistência na rede. A viabilidade técnica e econômica destes limitadores aumentou após o desenvolvimento dos supercondutores de alta temperatura crítica HTS. A primeira geração de fitas HTS (BSCCO) consiste em multifilamentos de material supercondutor embutidos em uma matriz de prata. A densidade de corrente crítica típica é de aproximadamente 140 A/mm2. Contudo, a resistência alcançada pelo LCSR quando atinge o estado normal não é suficiente para limitar a corrente de falta, sendo necessários longos comprimentos de fitas (~5 km) para que a corrente seja limitada. O recente desenvolvimento de fitas supercondutoras de YBCO com filmes finos texturizados e com substrato de alta resistividade superou o desempenho das fitas de BSCCO para esta aplicação. Com uma densidade de corrente crítica de 136 A/mm2, as fitas de YBCO são bem conhecidas pelo seu alto valor de índice n (aproximadamente 30), rápida resposta de transição após falta e alta resistência elétrica no estado normal. Neste trabalho foram analisados o comportamento elétrico e magnético das fitas de BSCCO e YBCO (em amostras curtas de 0,2 m) utilizando diferentes técnicas de caracterização das propriedades das fitas HTS. A partir destes resultados, medidas com pulsos de corrente DC e AC com intensidades de 3 a 7 vezes a corrente crítica do sistema (I=720 A a 1700 A) com duração de 1 a 5 ciclos da rede em 60 Hz (16 a 80 ms) foram realizados de forma a obter as características do tempo de recuperação em um elemento contendo 4 fitas em paralelo (0,4 m). Os resultados permitiram o projeto, construção e medidas em corrente AC de um protótipo LCSR (Ipico = 2 kA) contendo 16 elementos sob condições normais de operação da rede (220 V - 60 Hz). / Resistive Superconducting fault current limiter (SFCL) are devices with electrical behavior near the ideal when it changes its state from the superconducting to the normal state, limiting the fault current by the insertion of a fast transition resistance in the grid. The technical and economical feasibility of these limiters arose after the development of the high critical temperature superconductors HTS. First generation HTS tapes (BSCCO) consist of multifilamentary composite tapes embedded in a silver matrix. The typical critical current density is approximately 14 kA/cm2. However, the resistance reached by the SFCL when normal state occurs is not high enough to limit the fault current, making necessary long lengths of tapes (~5 km) for limiting purposes. The recently development of coated conductors composites with high resistivity metal substrate have succeeded the BSCCO tapes which are based on YBCO textured film. Upon carrying a critical current density of 13.6 kA/cm2, YBCO tapes are well known for their high n-index value (approximately 30), fast transition response after fault, and high electrical resistance in the normal state. In this work, we will present the electrical and magnetic performance of BSCCO and YBCO tapes (short samples of 0.2 m) using several techniques to characterize the properties of the HTS tapes. It was also performed analysis with DC and AC currents peaks with strength of 3 to 7 times the critical current (I=720 A to 1700 A) lasting 1 to 5 cycles (16 to 80 ms) in order to verify the recovery characteristics in an element with 4 tapes in a parallel connection. And with those characteristics we designed and constructed and measured in AC current a SFCL prototype (Ipeak = 2 kA) consisting in 16 elements to use in the grid (220 V - 60 Hz) under operational conditions.

Limitadores de corrente supercondutores resistivos (LCSR) são dispositivos com características elétricas próximas ao ideal quando ocorre a transição do estado supercondutor para o estado normal, limitando a corrente de falta através da inserção rápida de uma resistência na rede. A viabilidade técnica e econômica destes limitadores aumentou após o desenvolvimento dos supercondutores de alta temperatura crítica HTS. A primeira geração de fitas HTS (BSCCO) consiste em multifilamentos de material supercondutor embutidos em uma matriz de prata. A densidade de corrente crítica típica é de aproximadamente 140 A/mm2. Contudo, a resistência alcançada pelo LCSR quando atinge o estado normal não é suficiente para limitar a corrente de falta, sendo necessários longos comprimentos de fitas (~5 km) para que a corrente seja limitada. O recente desenvolvimento de fitas supercondutoras de YBCO com filmes finos texturizados e com substrato de alta resistividade superou o desempenho das fitas de BSCCO para esta aplicação. Com uma densidade de corrente crítica de 136 A/mm2, as fitas de YBCO são bem conhecidas pelo seu alto valor de índice n (aproximadamente 30), rápida resposta de transição após falta e alta resistência elétrica no estado normal. Neste trabalho foram analisados o comportamento elétrico e magnético das fitas de BSCCO e YBCO (em amostras curtas de 0,2 m) utilizando diferentes técnicas de caracterização das propriedades das fitas HTS. A partir destes resultados, medidas com pulsos de corrente DC e AC com intensidades de 3 a 7 vezes a corrente crítica do sistema (I=720 A a 1700 A) com duração de 1 a 5 ciclos da rede em 60 Hz (16 a 80 ms) foram realizados de forma a obter as características do tempo de recuperação em um elemento contendo 4 fitas em paralelo (0,4 m). Os resultados permitiram o projeto, construção e medidas em corrente AC de um protótipo LCSR (Ipico = 2 kA) contendo 16 elementos sob condições normais de operação da rede (220 V - 60 Hz). / Resistive Superconducting fault current limiter (SFCL) are devices with electrical behavior near the ideal when it changes its state from the superconducting to the normal state, limiting the fault current by the insertion of a fast transition resistance in the grid. The technical and economical feasibility of these limiters arose after the development of the high critical temperature superconductors HTS. First generation HTS tapes (BSCCO) consist of multifilamentary composite tapes embedded in a silver matrix. The typical critical current density is approximately 14 kA/cm2. However, the resistance reached by the SFCL when normal state occurs is not high enough to limit the fault current, making necessary long lengths of tapes (~5 km) for limiting purposes. The recently development of coated conductors composites with high resistivity metal substrate have succeeded the BSCCO tapes which are based on YBCO textured film. Upon carrying a critical current density of 13.6 kA/cm2, YBCO tapes are well known for their high n-index value (approximately 30), fast transition response after fault, and high electrical resistance in the normal state. In this work, we will present the electrical and magnetic performance of BSCCO and YBCO tapes (short samples of 0.2 m) using several techniques to characterize the properties of the HTS tapes. It was also performed analysis with DC and AC currents peaks with strength of 3 to 7 times the critical current (I=720 A to 1700 A) lasting 1 to 5 cycles (16 to 80 ms) in order to verify the recovery characteristics in an element with 4 tapes in a parallel connection. And with those characteristics we designed and constructed and measured in AC current a SFCL prototype (Ipeak = 2 kA) consisting in 16 elements to use in the grid (220 V - 60 Hz) under operational conditions.

No description available.