Dissertations / Theses on the topic 'Xlsx'

Mutations in the Abnormal Spindle-like Microcephaly-associated (ASPM) gene are the most common cause of primary microcephaly, a rare condition characterized by a severe reduction of brain size at birth. Several studies allowed to identify ASPM as a centrosome and mitotic spindle protein that regulates cell division and spindle orientation. However, little is known about ASPM molecular mechanisms, especially in human neural cells relevant to the disease. In order to decipher the molecular mechanisms of action of ASPM in human corticogenesis, we used co-immunoprecipitation (coIP) followed by mass spectrometry to identify the interactors of ASPM in human HEK cells and human cortical progenitors differentiated from pluripotent stem cells engineered to tag the endogenous ASPM protein. We thus identified and validated 14 ASPM interactors of which 12 are newly reported, and seven are found specifically in neural cells, including the important spindle pole regulator Nuclear mitotic apparatus (NUMA). We then characterized the expression and localization of the identified proteins in human cortical progenitors differentiated from control and isogenic ASPM mutant cells. This revealed that many of the identified proteins are selectively located at the spindle pole, and that this selective localization is disrupted in mutant cells for several of the interactors, in particular the MAP7 domain-containing protein 1 (MAP7D1) and DnaJ homolog subfamily B member 6 (DNAJB6). Our data uncover some of the complex ASPM interactome relevant and specific to human brain development and microcephaly, and suggest that ASPM acts as a major molecular hub at the centrosome and mitotic spindle to control the patterns of cell division of cortical progenitors.
Doctorat en Sciences biomédicales et pharmaceutiques (Médecine)
info:eu-repo/semantics/nonPublished

The goal of this project was to streamline and automate a business system. This was achieved through the implementation of four subtasks. This report describes what parts of the system that needed to be updated and why. It also describes how the development was carried out and what results the project ultimately led to.   The project's tasks were to: Create a report generator designed to generate Excel documents. Develop a symbol generator where a user, through a web-based interface, could generate symbols. The symbols could then be used in the company's mobile application. Create an interface for a web service, and to develop an add-in for the modeling software Revit Architecture 2014.
Målet med detta projekt var att effektivisera och automatisera ett företagssystem. Detta skulle uppnås genom implementationen av fyra deluppgifter. Denna rapport beskriver vilka delar av systemet som behövde utvecklas och varför. Den beskriver även hur utvecklingen genomfördes samt vilka resultat projektet slutligen ledde till.   Projektets deluppgifter var att: Skapa en rapportgenerator för generering av Excel-dokument. Utveckla en symbolgenerator där man genom ett web-baserat gränssnitt kunde generera symboler avsedda att användas i företagets mobila applikation. Skapa ett gränssnitt för en web-tjänst samt utveckla ett add-in till modelleringsprogrammet Revit Architecture 2014.

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Financiadora de Estudos e Projetos
The ferroelectric titanate of lead modified with Lanthanum metal system (or PLT) is the most studied of the perovskite structure. It is known that ceramic bodies with specify compositions and adequately chemical preparations can present transparency. However, a quantitative or qualitative analysis of its electro-optics characteristics was not carried through. Therefore, in this work it was ceramic preparation of PLT and the characterization and analysis of concentration of Lanthanum metal and the conditions of processing influence in its microstructural, dielectric, ferroelectrics, optic, structural and electro-optics properties. In this work the solid state was used in the obtained of PLT, using the nominal formula (Pb1-xLax)TiO3, with 0.18_ x _ 0.23. Ceramic bodies had been firing by the uniaxial Hot Pressing technique. Heat treatments in conventional oven under O2 and PbO atmosphere, had been used for the improvement of the transmitance of the samples. In the DRX analysis it was observed formation of perovskite phase with tetragonal symmetry as principal in the powder with little variation of the tetragonalidade factor in function of the Lanthanum metal concentration. For the densitied ceramics, it was meet perovskite phase with tetragonal symmetry and an reduction of the tetragonalidade factor of with the increase of the Lanthanum metal concentration. The grain average size of the ceramic bodies linearly increased with the Lanthanum metal concentration, presenting distinct dependence for each composition in relation to the heat treatment. The dielectric and ferroelectrics properties had been affected by the high conductivity presented for the ceramic bodies with less than 20h of thermal treatment. Such conductivity, as well as the opacity and the yellow color observed in the samples, was associated with the presence of the phase PbO in the grain boundaries. However, high values of transmitance in the next infra-red and visible region and less dielectric losses in low frequencies could have been observed in samples with 20h thermal treatment, depending on the Lanthanum metal concentration. For x=0.23, the highest concentration of analyzed La, the ceramic bodies thermal treatments presented a reduction in the transmittance, probably due ix to the effect of light scattering of rich Lanthanum metal phases precipitated in the grain boundaries (even though it was not detected for DRX). For the first time, the electro-optic coefficients could have been considered for PLT ceramics. The results had shown the importance of thermal treatment after-hot pressing and the concentration of Lanthanum metal in the obtaining of the transparent ceramic bodies with structural, microstructural, electric and optic properties adjusted for the technological applications.
O sistema ferroelétrico titanato de chumbo modificado com lantânio (ou PLT) encontra-se entre os mais conhecidos com estrutura perovskita. Sabe-se, também, por um único trabalho na literatura, que corpos cerâmicos de PLT em composições especificas e preparados adequadamente podem apresentar transparência. Contudo uma análise quantitativa, ou mesmo qualitativa de suas características eletro-ópticas ainda não foi realizada. Por isso, neste trabalho propôs-se a preparação de cerâmicas de PLT para a caracterização e análise da influencia da concentração de lantânio e das condições de processamento em suas propriedades estruturais, microestruturais, dielétricas, ferroelétricas, ópticas e eletro-ópticas. Este trabalho utilizou o processo de síntese por reação de estado sólido na obtenção de pós cerâmicos do sistema ferroelétrico de titanato de chumbo modificado com lantânio, utilizando a fórmula nominal (Pb1-xLax)TiO3, com 0,18 _ x _ 0,23. Corpos cerâmicos foram densificados pela técnica de prensagem uniaxial a quente, tratamentos térmicos, em forno convencional em atmosfera de O2 e PbO, foram utilizados para melhora do grau de transmitância das amostras. Com a análise por difração de raios X observou-se a formação de fase perovskita com simetria tetragonal como majoritária, nos pós com pouca variação do fator de tetragonalidade em função da concentração de lantânio. Para as cerâmicas densificadas, encontrou-se a fase perovskita com simetria tetragonal e uma diminuição acentuada do fator de tetragonalidade com o aumento da concentração de lantânio. O tamanho médio de grão dos corpos cerâmicos aumentou linearmente com a concentração de lantânio, apresentando dependência distinta para cada composição em relação ao tempo de tratamento térmico. As propriedades dielétricas e ferroelétricas foram fortemente afetadas pela alta condutividade apresentada pelos corpos cerâmicos densificados com menos de 20h de tratamento térmico. Tal condutividade, assim como a opacidade e a cor avermelhada observada nas amostras, foi associada à presença da fase de PbO em forma amorfa nos contornos de grão. Contudo altos valores de transmitância na região do visível e infravermelho próximo e menores perdas dielétricas em baixas freqüências puderam ser observadas em amostras com tratamento térmico de 20h, dependendo da concentração de lantânio. Para x=0,23, a mais alta concentração de La analisada, os corpos cerâmicos tratados termicamente apresentaram uma redução na transmitância provavelmente devido ao efeito de espalhamento de luz de fases ricas em lantânio precipitado nos contornos de grão (embora não detectadas por difração de raios X). Pela primeira vez, os coeficientes eletro-ópticos de cerâmicas de PLT puderam ser estimados. Os resultados obtidos mostraram a importância de tratamento térmico pós-prensagem a quente e da concentração de lantânio na obtenção corpos cerâmicos de PLT transparentes com propriedades estruturais, microestruturais, elétricas e ópticas adequadas a aplicações tecnológicas.

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CAPES
Resíduos de tiro (GSR) são evidências físicas de grande valor forense em casos envolvendo o uso de armas de fogo. Mas apesar de sua importância, existe uma carência metodológica para a caracterização do GSR proveniente de munição ambiental (NTA). Adicionalmente, o tamanho micrométrico das partículas geradas torna sua detecção em uma cena de crime ou em um suspeito uma tarefa difícil, podendo originar falsos-negativos. Neste sentido, tem sido proposto o uso de materiais luminescentes para a marcação química e óptica de munições. O presente trabalho aborda o desenvolvimento de marcadores luminescentes baseados na rede metal-orgânica (MOF) [Ln(BTC)] (em que BTC = ácido trimésico), desde a sua síntese, aplicação e avaliação da toxicidade, assim como seu uso na codificação de munição e sua caracterização junto aos resíduos orgânicos de disparo (OGSR). Por isto, este trabalho é dividido em cinco capítulos. No primeiro são discutidos aspectos relativos à síntese da MOF [Eu(BTC)] e à sua influência nas propriedades estruturais, térmicas e espectroscópicas dessa rede. Para tal, os métodos de síntese hidrotermal convencional e hidrotermal assistido por micro-ondas (MW), com diferentes tempos de síntese e proporções metal:ligante, são analisados. Com os métodos adotados, a MOF [Eu(BTC)] foi obtida com elevada cristalinidade, estabilidade térmica e luminescência. Adicionalmente, sínteses mais curtas e proporção metal:ligante 1:1 produziram redes com maior pureza de fase. Neste caso, o método assistido por micro-ondas se destacou devido ao rápido tempo de síntese. No segundo capítulo é apresentada a eficiência dessa MOF como marcador luminescente de munições. Com esta rede, além da detecção visual dos resíduos luminescentes (LGSR), técnicas como espectroscopia de emissão e microscopia eletrônica de varredura acoplada a espectroscopia por dispersão de energia (MEV/EDS) foram usadas para caracterizar e confirmar a presença do LGSR nos resíduos coletados, tanto da arma como das mãos do atirador. No terceiro capítulo são apresentados os dados relativos à avaliação da toxicidade oral aguda e subaguda da MOF [Eu(BTC)] em ratos. Para avaliação da toxicidade aguda, o protocolo 423 da OECD (Organisation for Economic Co-operation and Development) foi utilizado. Como resultado, nenhum sinal de toxicidade foi observado até mesmo para a dosagem mais elevada, e a dose letal média (DL50) foi estimada em 5000 mg/kg. Com isto, a MOF [Eu(BTC)] foi classificada na categoria menos tóxica do GHS (Globally Harmonized System). A toxicidade subaguda foi avaliada através da administração por 7 dias consecutivos de 300 mg/kg e, apesar de observados sinais graves de toxicidade, estes foram atribuídos à aspiração do material particulado para o pulmão, e não a toxicidade do marcador. Estes resultados atribuem a MOF [Eu(BTC)] uma elevada margem de segurança para aplicação como marcador de GSR. No quarto capítulo é proposto um método de codificação de munição baseado no uso de marcadores co-dopados [Y1-xLnx(BTC)] com os íons Eu3+, Sm3+, Tb3+ e/ou Yb3+. Após a realização de disparos, o marcador utilizado em cada munição foi identificado por MEV/EDS em função da composição química dos resíduos em 100% dos casos (sem que o analista tivesse conhecimento do marcador presente em cada munição). Além disso, foi possível estabelecer correlações atirador-arma-local do disparo através da composição dos resíduos em 89% dos casos. E no último capítulo é abordada a caracterização simultânea dos resíduos orgânicos e luminescentes por meio da microscopia Raman. Com a metodologia proposta, através de uma única medida foi possível determinar o tipo de marcador e pólvora utilizados, fornecendo um código muito seguro para caracterização e classificação inequívoca dos resíduos como provenientes de disparo de arma de fogo. Além disso, o LGSR atuou como sonda luminescente ajudando a coletar o OGSR em distâncias acima daquelas normalmente abordadas na literatura.
Gunshot residue (GSR) are physical evidences of great forensic value in firearms-related crimes. Despite its importance, there is a methodologic need for the characterization of GSR from non-toxic ammunition (NTA). In addition, the micrometric size of the generated particles makes its detection in a crime scene or on a suspect a hard task, which may produce false-negatives. In this sense, the use of luminescent materials has been proposed for chemical and optical marking of ammunition. This work presents the development of luminescent markers based on the metal-organic framework (MOF) [Ln(BTC)], since its synthesis, application and toxicity evaluation, as well as its ability to encode ammunitions and its simultaneous characterization with the organic gunshot residue (OGSR). Hence, this work is divided in five chapters. In the first chapter, aspects related to the synthesis of the MOF [Eu(BTC)] and its influence on the structural, thermal and spectroscopic properties are discussed. To this end, conventional hydrothermal synthesis and microwave(MW)-assisted hydrothermal synthesis, with different reaction times and metal:ligand proportions are analyzed. With the chosen methods, the MOF [Eu(BTC)] was obtained with high crystallinity, thermal stability and luminescence. However, faster synthesis and 1:1 metal:ligand proportion produced frameworks with higher phase purity. In this case, the MW-assisted method stood out due to the faster reaction time. In the second chapter, the efficiency of the MOF [Eu(BTC)] as a luminescent marker for ammunition is presented. With this framework, besides visual detection of the luminescent residues (LGSR), techniques as emission spectroscopy and scanning electron microscopy with energy dispersive X-ray spectroscopy (MEV/EDS) were used to characterize and confirm the presence of LGSR in the residues collected, both on the gun and in the shooter’s hand. In the third chapter, data related to the evaluation of acute and subacute oral toxicity of the MOF [Eu(BTC)] in rats is presented. For evaluation of acute oral toxicity, the OECD (Organisation for Economic Co-operation and Development) 423 guideline was used. As a result, no signs of toxicity was observed even at the highest dose, and the median lethal dose (LD50) estimated was 5000 mg/Kg. There by, the MOF [Eu(BTC)] was classified in the less toxic category of GHS (Globally Harmonized System). The subacute toxicity was evaluated by consecutive administration of 300 mg/Kg for 7 days and, despite severe toxicity signs were observed, they were attributed to the aspiration of the particulate material into the lungs, but not to the marker’s toxicity. These results give a high safety margin for application of this MOF as a GSR marker. In the fourth chapter an ammunition encoding method is proposed based on the use of the co-doped markers [Y1-xLnx(BTC)] with the ions Eu3+, Sm3+, Tb3+ and/or Yb3+. After shots, the markers used in each ammunition was identified by SEM/EDS due the chemical composition of the residues, in 100% of the cases (without the analyst’s knowledge of which marker was present in each ammunition). Furthermore, shooter-weapon-location correlations was established by the composition of the residues in 89% of the cases. And in the last chapter the simultaneously characterization of the organic and luminescent residues was performed by Raman microscopy. With the proposed methodology, the type of marker and gunpowder used were identified by a single run, providing a security code for its characterization and the unequivocal classification of the residues as coming from firearm discharge. Furthermore, the LGSR acted as a luminescent probe, helping to collect the OGSR further than those reported in the literature.

The following document is a meta-commentary on the article, "La misogynie à visage féminin: Hircan's Role as Marguerite's Anti-feminist Voice in the Heptaméron (VII & XLIX)," co-authored by Dr. Robert J. Hudson and myself, which will shortly be submitted for publication. It contains an annotated bibliography of all our primary and secondary sources and an account of writing the article. Our article examines what Marguerite de Navarre, the sixteenth-century French Renaissance author of the Heptaméron (a collection 72 nouvelles, all supposedly true stories being told by a group of ten devisants to one another), intended by her inclusion of the misogynist, Hircan. As we demonstrate, current scholarships views Marguerite as one of the first authors to create a space for women in literature, and further, that the Heptaméron was meant to serve the didactic purpose of forming young ladies' perspectives and behavior. Given this, Hircan, whose debasing views on women are shared in each of his stories and interlocutory commentaries, seems an odd devsiant for Marguerite to create; and so, we ask, why did she include him? We conclude that Hircan serves as Marguerite's straw man for the worst aspects of sixteenth-century French society, allowing her to subvert him and demonstrate how Hircan (and by extension, French society's) views towards women ought to be considered inappropriate. To support our reading, we start by explaining the historical context, demonstrating that the attitudes Hircan represents did indeed exist and were prevalent. We then show how Marguerite undermines Hircan: first, by making him so grotesque that the reader finds his views repugnant, and second, in allowing other devisants—especially Parlamente and Oisille—to use superior arguments to overturn his perspectives. Finally, we demonstrate how Marguerite uses Hircan's own tales against him, by having his fellow devisants interpret his stories completely differently from his womanizing and debasing purposes—instead find praise for women in them.

The importance of Automatic Speech Recognition (ASR) Systems, whose job is to generate text from audio, is increasing as the number of applications of these systems is rapidly going up. However, when it comes to training ASR systems, the process is difficult and rather tedious, and that could be attributed to the lack of training data. ASRs require huge amounts of annotated training data containing the audio files and the corresponding accurately written transcript files. This annotated (labeled) training data is very difficult to find for most of the languages, it usually requires people to perform the annotation manually which, apart from the monetary price it costs, is error-prone. A supervised training task is impractical for this scenario.  The Arabic language is one of the languages that do not have an abundance of labeled data, which makes its ASR system's accuracy very low compared to other resource-rich languages such as English, French, or Spanish. In this research, we take advantage of unlabeled voice data by learning general data representations from unlabeled training data (only audio files) in a self-supervised task or pre-training phase. This phase is done by using wav2vec 2.0 framework which masks out input in the latent space and solves a contrastive task. The model is then fine-tuned on a few amounts of labeled data. We also exploit models that have been pre-trained on different languages, by using wav2vec 2.0, for the purpose of fine-tuning them on Arabic language by using annotated Arabic data.   We show that using wav2vec 2.0 framework for pre-training on Arabic is considerably time and resource-consuming. It took the model 21.5 days (about 3 weeks) to complete 662 epochs and get a validation accuracy of 58%.  Arabic is a right-to-left (rtl) language with many diacritics that indicate how letters should be pronounced, these two features make it difficult for Arabic to fit into these models, as it requires heavy pre-processing for the transcript files. We demonstrate that we can fine-tune a cross-lingual model, that is trained on raw waveforms of speech in multiple languages, on Arabic data and get a low word error rate 36.53%. We also prove that by fine-tuning the model parameters we can increase the accuracy, thus, decrease the word error rate from 54.00% to 36.69%.

Les contraintes environnementales préconisent le remplacement des substances à base de plomb présentes dans les équipements électroniques. Dans ce cadre, nous avons synthétisé par voie sol-gel des solutions solides de (La1-xLnx)2Ti2O7 (Ln = Pr, Nd, Sm) sous forme massive et en couches minces sur des substrats de SrTiO3-Nb orientés (100) et (110). Sous forme de céramiques, les propriétés piézoélectriques des solutions solides à structure pérovskite en feuillet sont révélées. Sous forme de couches minces, l’investigation par diffraction de rayons X (DRX) en mode θ/θ montre que les films présentaient une orientation préférentielle (012) lorsqu’ils sont déposés sur des substrats de SrTiO3 orientés (100) et (001) lorsqu’ils sont déposés sur des substrats orientés (110). Des mesures par DRX haute résolution et des cartographies de l’espace réciproque révèlent l’existence d’orientations supplémentaires sur ces films. Ces orientations sont (025) pour les films déposés sur SrTiO3(100) et (212) pour les films déposés sur SrTiO3(110). Les mesures électriques réalisées à l’échelle macroscopique laissent présager le caractère ferroélectrique des films. Les études menées à l’échelle locale en microscopie à force piézoélectrique (PFM) confirment la ferroélectricité de tous les films. Cette microscopie met en évidence l’existence de domaines ferroélectriques dans lesquels les vecteurs de polarisation présentent des composantes dans et hors du plan du film, en bon accord avec les mesures en DRX HR.Ces résultats prometteurs placent ces solutions solides comme des candidats potentiels dans le cadre du remplacement des substances à base de plomb dans les équipements électroniques
The environmental constraints advocate the replacement of lead-based substances found in electronic equipments. In this context, we have synthesized (La1-xLnx)2Ti2O7 (Ln = Pr, Nd, Sm) solid solutions as bulks and thin films deposited on SrTiO3-Nb substrates oriented (100) or (110). In ceramics’ form, ferroelectric and piezoelectric properties of these layered perovskite solid solutions are found. In thin films’ form, the investigation by θ/θ X-ray diffraction (XRD) shows a (012) preferred orientation for the films deposited on (100)-SrTiO3 substrates and (001) for (110)-SrTiO3 substrates’ films. The measurements by high resolution XRD and reciprocal space mappings reveal the existence of supplementary orientations on these films, which are (025) for films deposited on (100)-SrTiO3 substrates and (212) for films deposited on (110)-SrTiO3 substrates. The macroscopic electrical measurements suggest a ferroelectric nature in these samples. The local electrical measurements performed by piezoelectric force microscopy (PFM) confirm the ferroelectricity in all films. This microscope reveals the existence of ferroelectric domains which the polarization vectors have components in and out of film’s plane, in good agreement with HR-XRD results. These promising results place these solid solutions as potential candidates under the replacement of lead-based substances in electronic equipments

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CNPQ – Conselho Nacional de Desenvolvimento Científico e Tecnológico
O médico e político Carlos Barbosa Gonçalves experienciou uma carreira política de relevo durante a Primeira República. Neste período, ocupou os cargos de deputado estadual, vice-presidente e presidente do estado e senador. No entanto, acreditamos que o sucesso político experimentado durante o período republicano só foi possível pela ascensão do Partido Republicano Rio-grandense (PRR) ao poder – ocasionada pela Proclamação da República – e também porque Barbosa possuía os requisitos necessários para tanto. Sendo assim, o objetivo deste trabalho é investigar quais recursos (econômicos, políticos, sociais e simbólicos) Barbosa herdou e empenhou-se em adquirir para que pudesse ocupar a posição de líder político local de Jaguarão, bem como ter sucesso em outras esferas políticas durante o período republicano. Para que isso fosse possível, analisamos – através de inúmeros tipos de fontes – diferentes momentos, tanto dos Gonçalves da Silva, quanto de Barbosa. Com relação aos antecedentes familiares, investigamos as relações da família com Jaguarão e a Guerra dos Farrapos, como também a situação econômica familiar. Quando tratamos especificamente de Barbosa, o acompanhamos nos estudos realizados no Rio de Janeiro, na propaganda republicana em Jaguarão e também a atuação médica.
The doctor and politician Carlos Barbosa Gonçalves experienced a political career of relief during the First Republic. During this period, he held the positions of state deputy, state president and senator. However, we believe that the political success experienced during the republican period was only possible by the rise of the Rio-grandense Republican Party (PRR) to power – occasioned by the Proclamation of the Republic – and also because Barbosa had the necessary requirements for it. Thus, the objective of this work is to investigate which resources (economic, political, social and symbolic) Barbosa inherited and committed himself to acquire so that he could occupy the position of local political leader of Jaguarão, as well as to succeed in other political spheres during the republican period. For this to be possible, we analyzed – through many types of sources – different moments, both from Gonçalves da Silva and Barbosa. Regarding the family history, we investigated the family's relations with Jaguarão and the Farrapos War, as well as the familiar economic situation. When we deal specifically with Barbosa, we accompany him in his studies in Rio de Janeiro, in republican propaganda in Jaguarão, and also in medical practice.

This master thesis was oriented to study of the development application possibilities in programming language ABAP in the information system mySAP. There were studied integration possibilities of SAP system with Adobe PDF forms and application MS Excel. Advantages and disadvantages of these two technologies were compared. The theoretical part of diploma thesis describes also the technology of SAP systems based on NetWeaver platform and the main product mySAP. The practical part describes a concrete business scenario of purchase order process where is also invoice verification list included. The result of the work is analysis, design and implementation of concrete solution for "invoice verification list" generating in SAP system. A development SAP VUT application was used for implementation and testing.

In natural environments, bacterial physiology is frequently characterized by slow metabolic rates and complex cellular heterogeneities. The opportunistic pathogen Pseudomonas aeruginosa provides one such example; P. aeruginosa forms untreatable chronic biofilm infections of the cystic fibrosis lung, where oxygen limitation can lead to states of metabolic dormancy. To better understand the biology of these states, in vitro experiments must be adapted to better recapitulate natural settings. However, low rates of protein turnover and cellular or phenotypic complexity make these systems difficult to study using established methods. Here we adapt the bioorthogonal noncanonical amino acid tagging (BONCAT) method for time- and cell-selective proteomic analysis to the study of P. aeruginosa. Analysis of proteins synthesized in an anoxic dormancy state led to the discovery of a new type of transcriptional regulator which we designated SutA. We performed detailed analyses of SutA’s role in transcription under slow growth states and we elucidated the structural basis for its regulatory behavior. Additionally, we used cell-selective targeting of BONCAT labeling to measure the dynamic proteomic response of an antibiotic-tolerant biofilm subpopulation. Overall this work shows the utility of selective proteomics as applied to bacterial physiology and describes the broad biological insight obtained from that application.

Overall, this thesis encompasses three main directions: the study of neural circuits in sleep (Chapter 2), the development and testing of tools for measuring neuromodulator release (Chapter 3), and methods for in vivo characterization of gene delivery vehicles (Chapter 5).

The role of the neuromodulator serotonin in sleep has been debated for over 60 years. Until recently, the serotonergic system was widely thought to be part of the arousal system and promote wakefulness. In Chapter 2, we investigate the function of serotonin-producing neurons in murine and zebrafish sleep with tools featuring superior specificity and precision compared to previously employed techniques. Our results demonstrate that the serotonergic raphe are sleep-promoting and required for sleep homeostasis. Intriguingly, serotonergic neurons in mice can have opposing effects on sleep depending on the firing mode.

The release of serotonin from neurons can be regulated by the frequency of neuronal firing and can occur at classical synapses, varicosities, soma, and dendrites. Further examination of the complex signaling mechanism of serotonin would benefit from tools capable of measuring the release of serotonin in vivo with long-term stability and high spatiotemporal resolution. To this end, we developed and characterized iSeroSnFR, an intensity-based genetically encoded serotonin indicator. In Chapter 3, we demonstrate that iSeroSnFR can detect serotonin release in freely behaving mice during fear conditioning, social interaction, and sleep-wake transitions.

Adeno-associated viruses (AAVs) have been extensively used as gene delivery vehicles in basic neuroscience and gene therapy. However, optimization of transduction efficiency and target specificity remain a key challenge to overcome. Several AAV vector engineering approaches have been devised for this purpose and yield large collections of candidates that require further in vivo characterization. However, conventional characterization methods fall short with regard to in-depth cell type tropism analysis and/or high-throughput capabilities. In Chapter 5, we address this shortcoming with single-cell RNA sequencing technologies based on the Drop-seq method. We established an experimental and computational pipeline that allows us to profile the viral tropism of multiple AAV variants in parallel across numerous complex cell types.

Bio-orthogonal non-canonical amino acid tagging (BONCAT) is an analytical method that allows the selective analysis of the subset of newly synthesized cellular proteins produced in response to a biological stimulus. In BONCAT, cells are treated with the non-canonical amino acid L-azidohomoalanine (Aha), which is utilized in protein synthesis in place of methionine by wild-type translational machinery. Nascent, Aha-labeled proteins are selectively ligated to affinity tags for enrichment and subsequently identified via mass spectrometry. The work presented in this thesis exhibits advancements in and applications of the BONCAT technology that establishes it as an effective tool for analyzing proteome dynamics with time-resolved precision.

Chapter 1 introduces the BONCAT method and serves as an outline for the thesis as a whole. I discuss motivations behind the methodological advancements in Chapter 2 and the biological applications in Chapters 2 and 3.

Chapter 2 presents methodological developments that make BONCAT a proteomic tool capable of, in addition to identifying newly synthesized proteins, accurately quantifying rates of protein synthesis. I demonstrate that this quantitative BONCAT approach can measure proteome-wide patterns of protein synthesis at time scales inaccessible to alternative techniques.

In Chapter 3, I use BONCAT to study the biological function of the small RNA regulator CyaR in Escherichia coli. I correctly identify previously known CyaR targets, and validate several new CyaR targets, expanding the functional roles of the sRNA regulator.

In Chapter 4, I use BONCAT to measure the proteomic profile of the quorum sensing bacterium Vibrio harveyi during the time-dependent transition from individual- to group-behaviors. My analysis reveals new quorum-sensing-regulated proteins with diverse functions, including transcription factors, chemotaxis proteins, transport proteins, and proteins involved in iron homeostasis.

Overall, this work describes how to use BONCAT to perform quantitative, time-resolved proteomic analysis and demonstrates that these measurements can be used to study a broad range of biological processes.

Organisms including bacteria, insects, and mammals make decisions to alter aspects of their development based on signals from the environment. The roundworm Caenorhabditis elegans can escape environmental collapse by halting reproductive growth and entering the stress-resistant dauer larval stage. Dauer larvae are spore-like and have specialized behaviors for finding and stowing onto carrier animals for dispersal. The decision to enter dauer is an anticipatory decision that is based on the inputs of food, pheromone, and temperature.

Here, I show that touch is an overlooked input into the dauer entry decision. Using quantitative dauer entry assays on CRISPR knock-ins and existing mutants in mechanosensation, I demonstrate that gentle, harsh, and piezo touch promote dauer entry. By measuring pheromone sensation and signal tranmission in mechnanosensation-defective mutants, I show that mechanosensation likely inputs into the decision in parallel with pheromone. Further confirmation that touch promotes dauer entry is provided using direct mechanical stimulation of C. elegans, and I provide a plausible role for touch in sensing dauer-promoting weather and crowding conditions.

Using RNA-seq, I also show that 8,042 genes are differentially expressed between dauer and reproductive development. Within this dataset, we observed the striking up-regulation of 64 neuropeptide genes (encoding 215 peptides) during dauer. By comparison, the entire human genome contains 97 neuropeptide genes (encoding 270 peptides). In particular, we observed coordinated up-regulation of the FMRFamide-like neuropeptides (FLPs). Using sbt-1 mutants to knock down neuropeptide processing, we demonstrate that peptidergic signaling promotes the dauer entry decision, promotes vigorous waving during the dauer-specific nictation behavior (carrier animal-hitchhiking), and is necessary for switching from repulsion to CO₂ (a carrier animal cue) in non-dauers to CO₂ attraction in dauers. By testing individual neuropeptides using CRISPR knockouts and existing strains, we show that 7 FLPs promote dauer entry while 4 FLPs inhibit. I therefore propose plausible roles for these FLPs in acting downstream of and/or modulating the sensation of food, pheromone, temperature, and touch inputs. We also demonstrate that FLP-10/FLP-17, which are expressed in the CO₂-sensing BAG neuron, promote CO₂ chemotaxis and nictation in dauers. These findings reveal that neuropeptides can alter decision-making and behavior during C. elegans dauer entry. Through a meta-analysis, we discovered similar up-regulation of FLPs in the dauer-like infective juveniles of diverse parasitic nematodes, suggesting that this may be an ancient mechanism for expanding the behavioral repertoire of nematodes.

Further utilizing our RNA-seq dataset, I identified several markers for conveniently tracking and manipulating the dauer entry decision. These include col-183 (which tracks dauer fate in the hypodermis), ets-10 (neurons and intestine), nhr-246 (intestine and muscle), and led-1 (reproductive fate in hypodermis). Using condition shift experiments, we demonstrate that the dauer markers label animals during dauer-commitment. We show that these markers can be used to manipulate the entry decision by driving the reproduction-promoting gene daf-9/Cytochrome P450 under the control of the dauer-commitment markers. We further demonstrate that the markers can be used to track tissue coordination and its breakdown in partial dauer mutants, and propose strategies for using the markers to identify the intercellular signals that coordinate the dauer entry decision.

I have discovered that the C. elegans dauer entry decision is more complex than previously realized, I have shown that C. elegans dauers obtain new behaviors through FLP signaling, and I have engineered tools for conveniently tracking and manipulating the dauer entry decision. My findings may illuminate how animals make robust decisions in uncertain environments, and have implications for how densely information and behaviors can be packed into a nervous system.

Early T-cell development converts multipotent precursors to committed pro-T cells, silencing progenitor genes while inducing T-cell genes. However, both the underlying steps of developmental progression and the regulations involved have remained obscure. Although some of the expressions of important regulators in early T-cell development have been studied in bulk populations, the nature of heterogeneity in this constantly refreshed developmental continuum makes it difficult to understand the developmental trajectories that the cells have undergone using bulk analysis, both in natural conditions and under gene perturbations.

Combining droplet-based single cell RNA sequencing (scRNA-seq), deep-sequenced whole-transcript scRNA-seq, and seqFISH for key regulatory genes, we established regulatory phenotypes of sequential ETP subsets; confirmed initial co-expression of progenitor- with T-cell specification genes; defined stage-specific relationships between cell-cycle and differentiation; and generated a pseudotime model from ETP to T-lineage commitment, supported by RNA velocity and transcription factor perturbations. This model was validated by developmental kinetics of ETP subsets at population and clonal levels. The results imply that multilineage priming is integral to T-cell specification in natural developing pro-T cells in the thymus.

Moreover, we examined the functional implications of some of the transcription factors (TFs) through bone marrow (BM) derived ex-vivo differentiation systems. Using scRNA-seq, Cell Hashing, and a pool-based CRISPR/Cas9 perturbation system, we established the normal and perturbed developmental trajectories before and after the T-lineage commitment stages. Our analysis revealed that, without the essential lineage commitment TF, Bcl11b, the developing early T cells immediately realized the lack of the essential regulator around the proliferating late DN2a stage. But instead of pushing the developmental path backwards to resemble the earlier stage of uncommitted cells, cells lacking Bcl11b underwent a diverging route of accumulation of 'non-T' genes that are not naturally expressed in earlier stages, potentially leading to the eventual loss of Notch responses. Our results also revealed the complex regulations by TFs that set up the earliest T-lineage progression and commitment conditions. The SCENIC analysis suggested that Gata3 and Tcf7, despite both being important regulatory factors for T-lineage progression, have very different regulatory roles in controlling proliferation and suppressing myeloid lineages. Furthermore, pseudotime analysis also showed that some of the stem and progenitor genes and 'multilineage' associated genes expressed by early pro-T cells potentially hold back the T-lineage differentiation speed. In summary, our study leveraged both in vivo thymic pro-T cells' developmental trajectory obtained through single-cell analysis and ex-vivo derived T cells for internal-controlled perturbations, and revealed some profound roles of TFs in regulating early T-cell differentiation processes.

Cells within biological systems are constantly adjusting their protein synthesis in response to various environmental changes. To study the rapid cellular regulations in complex biological systems, global proteomic profiling provides important information on system-level regulations, yet physiological properties characteristic of individual cellular subpopulations could be hidden under the characterization. Instead, cell-selective proteomic profiling allows researchers to reveal the heterogeneities in biological systems with phenotypically and even genetically distinct subpopulations under different microenvironments.

Chapter 1 describes the development of bioorthogonal noncanonical amino acid tagging (BONCAT) for proteomic profiling with resolution in both space and time: its initial role is protein labeling with temporal resolution via pulse-addition of noncanonical amino acid, which could be recognized by endogenous aminoacyl tRNA-synthetase (aaRS), into systems of interest; later on, mutant aaRSs are identified through mutant synthetase library screening, which allows for efficient incorporation of various types of noncanonical amino acids that could hardly be activated by endogenous machineries. The identification and exploitation of mutant aaRSs allow sensitive cellular selectivity during protein labeling. With unprecedented spatiotemporal resolution of BONCAT, and the advancement in high-resolution mass spectrometry and computational algorithms, BONCAT is a powerful technique for selective proteomic profiling to study physiological regulations in a wide range of complex biological systems. Chapter 2 describes the application of the BONCAT method in cell-selective proteomic profiling in Pseudomonas aeruginosa biofilms. In this work, we targeted an iron-starved subpopulation in biofilms and compared its proteomic profile with that of the entire system. Key gene and pathway regulations in the subpopulation are found through the analysis of the proteomic data, which suggest that iron-starved cells shift their priority towards housing keeping pathways, adapt an energy- and resources-saving mode to cope with their harsh local environmental conditions, and get prepared to disperse for better survival. Analysis of poorly studied proteins highly upregulated in the subpopulation led to the discovery of a previously uncharacterized protein (PA14_52000) that is potentially related to iron acquisition. The transposon insertion mutant PA14_52000::tn showed significantly enhanced pyoverdine production in rich medium and reduced biofilm formation.

Chapter 3 describes the study of physiological regulations in Bacillus subtilis K-state subpopulation via BONCAT. A subset of B. subtilis cells, typically 10% - 20% of the entire population, enter K-state in a stochastic manner. With the low level of K-state entry rate and high randomness, we challenged BONCAT to specifically capture gene and pathway regulations in K-state cells and compared the proteomic profiling with that of the entire population. Regardless of the difficulties of selective protein labeling inherent in the system, our results indicate that BONCAT has high specificity and resolution in proteomic profiling for minor subpopulations and proteins with low overall absolute abundance. We found multiple pathways and genes characteristic of K-state regulated differentially from the entire population, either significantly up- or down-regulated. Proteins that are uncharacterized or previously known for functions irrelevant of K-state are highly abundant in the subpopulation, providing new insight toward their alternative functions critical for K-state cells and future investigation directions of K-state study.

碩士
國立臺灣大學
漁業科學研究所
89
Homeobox genes are important for establishing body plan and determining cell-type or cell-lineage throughout the metazoans. The homeobox genes of the Xlox family are essential for the early morphogenesis of the midgut region and cellular differentiation during embryonic development. Since homeobox sequences and expression patterns of Xlox family homeobox genes are highly conserved in organisms as diverse as leeches to vertebrates, these genes provide an excellent tool to study the evolution of endoderm-derived gut structure among animal phyla. In this study, I identified the homeobox sequences of Xlox family genes from two species of polychaete, Neanthes glandicincta and Hydroides elegans, using the GenomeWalker technique. Phylogenetic analyses of the Xlox homeodomain amino acid sequence show that there are 3 major clades: vertebrates, leeches, and a mixture of marine invertebrates, including amphioxus, starfish, and polychaetes. These results suggest that the Xlox homeodomain sequences of amphioxus, starfish, and polychaetes are more ancestral than those of vertebrates and leeches. Moreover, the Xlox homeodomain sequences of leeches evolved faster than that of polychaetes, implying that the complexity of the leech’s gut structure may be related to the changes of the Xlox genes of the Annelida.

碩士
國立虎尾科技大學
電子工程系碩士班
102
In this study, (Ba1-xLnx)Y2ZnO5 (Ln = Eu, Pr) phosphors were synthesized using solid-state reaction method. The crystal structure and photoluminescence proper¬ties were then investigated. The XRD results shows that the crystal structure of (Ba1-xLnx)Y2ZnO5 (Ln = Eu, Pr) phosphors calcined 1250 oC for 12 h can be attributed to the orthorhombic structure for BaY2ZnO5. The FE-SEM results show that there are no obvious differences for surface morphology of (Ba1-xPrx)Y2ZnO5 phos¬phors. By 457 nm excitation, the emission spectra show that there are some emission bands appear in the range of 490 ~ 520 nm, 530 ~ 560 nm , 615 ~ 635 nm and 670 ~ 690 nm, respectively corresponding to the 3P0→3H2,4,5,6 、3P1?3H4 and 1D2→3H4,5 electron transition of the Pr3+ ion. The optimal PL intensities is obtained for (Ba1-xPrx)Y2ZnO5 phosphor of x=0.005, and which has a CIE chomaticity coordinates of (x = 0.343, y = 0.278) locating in the green region. In addition, for (Ba1-xEux)Y2ZnO5 system, the dominant transitions are 5D3→7FJ, 5D2→7FJ for low Eu3+ doping concentration. The intensities of the emission from 5D3 and 5D2 decrease with increasing the Eu3+ concentration, and which is due to the nonradiative cross-relaxation process between the Eu3+ ions. The (Ba1-xEux)Y2ZnO5 phosphor emit orange red luminescence when the x＞0.07. The optimal PL intensity is obtained when the Eu3+ ion concentration is 20 mol%, and which has a CIE chomaticity coordi¬nates of (0.095, y = 0.662) locating in the orange red region. The Asym¬metry ratio (5D0→7F2/5D0→7F1) is about 1.16, which is lower than that for Eu3+ion substitute Y3+ ion. Keywords: phosphors, Pr3+ ion, solid-state reaction, photoluminescence

碩士
國立成功大學
資源工程學系碩博士班
92
Potassium niobate (KNbO3) crystal is a ferroelectric material with a perovskite-type crystal structure. The system of KNbO3 is orthorhombic in room temperature. The phase transition temperature of pure KNbO3 is -10℃, 225℃, and 425℃ from rhombohedral to tetragonal, and then to cubic. The dielectric properties of KNbO3 have changes from paraelectricity without spontaneous polarization to ferroelectricity with spontaneous polarization, when the system structure of KNbO3 changes from cubic to tetragonal. The opposite displacement between the ions causes the change of crystal structure and produce the situation of this spontaneous polarization. According to the crystal chemistry theory, La3+ was doped into KNbO3 with perovskite structure. (K1-xLax/3)NbO3 solid solution was synthesized by La3+ substituting for part sites of K+. Therefore, (K1-xLax/3)NbO3 powders was synthesized by solid state reaction in this study. It has better piezoelectric properties around the boundary of solid-state region. But, the biggest solid-state region was not clearly determined in this study and it may be more then 0.2 lanthanum addition. The component of (K1-xLax/3)NbO3 with best properties is (K0.95La0.5/3)NbO3. Relative density can get up to 99% at 1100℃ for 0.3 h. The relative dielectric constant is 1191 and quality factor is 44.1, when the frequency is 1 kHz. In addition, poling mechanism of (K1-xLax/3)NbO3 were determined according to the relative dielectric constant and quality factor were measured in 1 kHz and 1 MHz.

碩士
國立臺灣大學
漁業科學研究所
85
The polymerase chain reaction was carried out in order to survey the homeobox-gene fragments of sea urchin, Tripneustes gratilla, and starfish Archaster typicus. A total eight sequences were identified. The results of comparative analysis with known homeobox sequences suggested that six sequences were of the Hox type. One of the six sequences may be the anterior- type group gene, four may be the medial-type ones, and two may be the posterior-type ones. These results suggested that echinoderms may have a single cluster of Hox-Complex. Possibly, only one anterior-type group gene exists in echinoderms, which is different from the hemichordates and the cephalochordates that are close relatives of echinoderms. This difference is probably due to the lack of cephalic structures in the echinodermae body form. Echinoderms have at least two posterior- type homeobox genes, implying that the tandem duplication of the ancestral gene of this group occurred in echinoderms as other deuterostomes. Besides, two sequences were of the Xlox family of the sea urchin and the starfish. In addition, the expression of the Xlox family homeobox gene in the starfish embryos was detected by reverse transcription polymerase chain reactions. The expression of mRNA of the Xlox family gene in the starfish embryos was detected during two periods from egg to cleavage and from gastrula to early bipinnaria. A maternal effect role in the early development and a role in the enteroceol formation or the differentiation of the gut cells in the later period are proposed for the Xlox family gene.

Este trabalho surgiu no ambito do estágio final do Mestrado de Matemática e Aplicações e cujo objetivo principal é apresentar todas as fases na empresa Centroproduto. São expostas sugestões de melhorias a este processo. Inicialmente foram realizado testes de software da empresa, atividade que despendeu mais tempo. Posteriormente foi desempenhada a técnica de Web Scraping, uma técnica de recolha de informações de websites para o marketing da empresa onde a descoberta de a biblioteca ‘Selenium’ do permitiu introduzir tarefas automatizadas na empresa. Mais tarde foram elaboradas especificações do comportamento esperado, isto é, tabelas com as indicações que o grupo de testes deve proceder. Por fim, foram desenvolvidas diversas tarefas automatizadas que antes eram efetuadas manualmente
This work came under the internship of the Masters in Mathematics and Applications and the principal aim is to explain all of the tasks at the company Centroproduto. Will be exposed all of the improvements done on the company software tests. This was the activity that make more time of the internship. After that was developed the Web Scraping technic wich includes collect of websites information to the marketing sector at the Centroproduto. In this tasks was very important the discover of the Selenium library, that allowed to automatized company daily tasks. Later was created some behavic especifications which means tables with group tests indications. At least was developed different automatized tasks that where only did manualy before
Mestrado em Matemática e Aplicações