Liberal human rights theory has informed Western political policy for decades. An ascending China challenges Western dominance in political theory and philosophy and forces Western theorists to respond. A comprehensive analysis of Western scholarship on human rights and the Confucian tradition makes it clear that there are many structural and systemic issues within this area of study. It also makes it clear that there have been many potentially useful observations and methodologies suggested throughout the literature that have been obscured. One such approach is applied that brings the political theory of John Rawls and Tu Weiming into conversation. As a result, a more nuanced understanding of the Chinese Confucian intellectual tradition in both Western and Chinese terms can be developed, while important questions are raised about human rights theory.

Liberal human rights theory has informed Western political policy for decades. An ascending China challenges Western dominance in political theory and philosophy and forces Western theorists to respond. A comprehensive analysis of Western scholarship on human rights and the Confucian tradition makes it clear that there are many structural and systemic issues within this area of study. It also makes it clear that there have been many potentially useful observations and methodologies suggested throughout the literature that have been obscured. One such approach is applied that brings the political theory of John Rawls and Tu Weiming into conversation. As a result, a more nuanced understanding of the Chinese Confucian intellectual tradition in both Western and Chinese terms can be developed, while important questions are raised about human rights theory.

The rate and accuracy of protein production is the main determinant of bacterial growth. Elongation Factor Tu (EF-Tu) provides the ribosome with aminoacylated tRNAs, and is central for its activity. In Salmonella enterica serovar Typhimurium, EF-Tu is encoded by the genes tufA and tufB. A bacterial cell depending on tufA499-encoded EF-Tu mutant Gln125Arg grows extremely slowly. We found evidence that this is caused by excessive degradation of mRNA, which is suggested to be the result of transcription-translation decoupling because the leading ribosome is ‘starved’ for amino acids and stalls on the nascent mRNA, which is thus exposed to Riboendonuclease RNase E. The slow-growth phenotype can be reversed by mutations in RNase E that reduce the activity of this enzyme. We found that the EF-Tu mutant has increased levels of ppGpp during exponential growth in rich medium. ppGpp is usually produced during starvation, and we propose that Salmonella, depending on mutant EF-Tu, incorrectly senses the resulting situation with ribosomes ‘starving’ for amino acids as a real starvation condition. Thus, RelA produces ppGpp which redirects gene expression from synthesis of ribosomes and favours synthesis of building blocks such as amino acids. When ppGpp levels are reduced, either by over-expression of SpoT or by inactivation of relA, growth of the mutant is improved. We suggest this is because the cell stays in a fast-growth mode. RNase E mutants with a conditionally lethal temperature-sensitive (ts) phenotype were used to address the long-debated question of the essential role of RNase E. Suppressor mutations of the ts phenotype were selected and identified, both in RNase E as well as in extragenic loci. The internal mutations restore the wild-type RNase E function to various degrees, but no single defect was identified that alone could account for the ts phenotype. In contrast, identifying three different classes of extragenic suppressors lead us to suggest that the essential role of RNaseIE is to degrade mRNA. One possibility to explain the importance of this function is that in the absence of mRNA degradation by RNase E, the ribosomes become trapped on defective mRNAs, with detrimental consequences for continued cell growth.

Determining how coordinated gene expression changes direct embryonic heart development is paramount to understanding the genetic causes and developmental origins of congenital cardiomyopathies. Towards this goal, I present an optimized protocol for mouse thiouracil tagging (TU-tagging), a novel transcriptomics methodology for defining dynamic and cell specific gene expression programs, and validate TU-tagging for cardiovascular research. I then apply related and additional high-resolution approaches to characterize how vascular endothelial growth factor (VEGF) signaling coordinates cell and gene expression dynamics underlying heart ventricle development. TU-tagging is a powerful approach to study dynamic gene expression programs of defined cell types while they are natively embedded within a complex organ. TU-tagging integrates genetic and chemical approaches to provide temporally controlled in vivo covalent labeling of cell type–specific RNA. Here, I describe two significant optimizations of the TU-tagging molecular biology and bioinformatics workflows that improve the method’s ability to identify differentially expressed genes and expand the technology’s utility. Next, using chemical inhibition of VEGF signaling in combination with high-resolution imaging and transcriptomic methods, I show that VEGF signaling directly promotes formation of the trabeculae that uniquely develop in the heart ventricle. By RNA-Seq, I identify VEGF-dependent target genes, including Gpr126 and Bmp10, which encode additional signaling proteins. I further show that myocardial Bmp10 expression and resulting endocardial Bmp10-driven pSMAD1/5/8 signaling is under sustained control by endocardial VEGF signaling. This continuous VEGF-BMP signaling interplay between endocardial and myocardial cells led me to examine the dynamic tissue arrangements between the two cell types during early stages of trabeculation. By extensive staining and high resolution imaging, I show that endocardial cells can be subdivided into three classes; 1) quiescent cavity cells that are well-separated from the myocardium, 2) proliferative and signal responsive transition zone/stalk cells that directly interact with myocardium to coordinate both cell types’ activities, and 3) CD34-expressing migratory tip-like cells uniquely found at the base of forming trabeculae. VEGF promotes trabeculation by 1) driving proliferation of endocardial transition zone/stalk cells and, secondarily, neighboring myocardium, and 2) directing the outward migration of endocardial tip cells that causes myocardial tissue to accumulate within individual and extensive ridge-like trabeculae. Defining these multiple roles of VEGF signaling during ventricle development reveals a novel conceptual framework for understanding trabeculation mechanisms and therefore processes likely to be disrupted in common congenital cardiomyopathies. This dissertation includes previously published and unpublished co-authored material. / 10000-01-01

Submitted by Roberth Novaes (roberth.novaes@live.com) on 2018-07-18T14:46:31Z No. of bitstreams: 1 LORENANascimento_DISSERTAÇÃO_COMPLETA_DEFINITIVA.pdf: 1753376 bytes, checksum: 751fbd325a853e63ab31c5b04508c211 (MD5) / Approved for entry into archive by Setor de Periódicos (per_macedocosta@ufba.br) on 2018-07-19T19:44:57Z (GMT) No. of bitstreams: 1 LORENANascimento_DISSERTAÇÃO_COMPLETA_DEFINITIVA.pdf: 1753376 bytes, checksum: 751fbd325a853e63ab31c5b04508c211 (MD5) / Made available in DSpace on 2018-07-19T19:44:57Z (GMT). No. of bitstreams: 1 LORENANascimento_DISSERTAÇÃO_COMPLETA_DEFINITIVA.pdf: 1753376 bytes, checksum: 751fbd325a853e63ab31c5b04508c211 (MD5) / FAPESB / Esta pesquisa tem por objetivo analisar quais são os condicionadores que atuam na escolha dos pronomes tu/você no português popular falado nas comunidades de Salvador e Amargosa, na Bahia. Através deste estudo, temos por proposta investigar o uso variável dos pronomes tu e você, através de inquéritos do Programa de Estudos do Português Popular Falado de Salvador (PEPP) e de inquéritos gravados em Amargosa entre os meses de julho e dezembro do ano 2016, a fim de aferir se o fenômeno constitui uma variação estável ou caminha para uma mudança linguística, além de investigar se o fenômeno em questão é marcado pela variação diatópica. Esse trabalho tem como base o modelo teórico-metodológico da Sociolinguística Quantitativa, constituído pelo sociolinguista William Labov. A coleta de dados foi realizada através de 12 inquéritos do PEPP e 12 inquéritos gravados em Amargosa. Os informantes são homens e mulheres em igual número, distribuídos em três faixas etárias (I: 15 a 24 anos; III: 45 a 55 anos; IV: 65 anos em diante). Após a transcrição das gravações, foram realizados o levantamento dos dados; a codificação, seguindo uma chave de codificação, e então, a análise estatística através do pacote de programas GoldVarb. Por fim, foram realizadas a análise e interpretação desses dados obtidos. Os resultados apontaram para uma preferência pelo pronome você em ambas as cidades, sendo que em Salvador, você se mostrou categórico nas escolhas dos falantes. Entre as onze variáveis inicialmente elencadas na pesquisa, as variáveis Tipo de Frase, Tipo de Discurso, Tipo de Referência, Faixa Etária e Escolaridade foram selecionadas pelo GoldVarb como as mais relevantes para a variação tu/você em Salvador e Amargosa. No município de Amargosa, os pronomes parecem estar fortemente influenciados pela variável Intimidade, que embora tenha sido observada no decorrer da análise, não foi avaliada nesse estudo. / This research aims to analyze which are the conditioners that act in the choice of the pronouns tu/você in the popular Portuguese spoken in the communities of Salvador and Amargosa, in Bahia. Through this study, we propose to investigate the variable use of the pronouns tu and você, through surveys of the Program of Studies of Popular Portuguese Spoken of Salvador (PEPP) and of surveys recorded in Amargosa between July and December of the year 2016, in order to verify if the phenomenon constitutes a stable variation or is going to a linguistic change, besides investigating if the phenomenon in question is marked by the diatopic variation. This work is based on the theoretical-methodological model of Quantitative Sociolinguistics, constituted by sociolinguist William Labov. Data collection was carried out through 12 PEPP surveys and 12 surveys recorded in Amargosa. The informants are men and women in equal numbers, divided into three age groups (I: 15 to 24 years, III: 45 to 55 years, IV: 65 years and on). After the transcription of the recordings, the data were collected; The coding, following a coding key, and then the statistical analysis through the GoldVarb program package. Finally, the analysis and interpretation of these data were performed. The results pointed to a preference for the pronoun você in both cities, and in Salvador, você were categorical in the choices of the speakers. Among the eleven variables initially listed in the research, the variables Type of Phrase, Speech Type, Reference Type, Age Range and Schooling were selected by GoldVarb as the most relevant for the variation in Salvador / Amargosa. In the municipality of Amargosa, pronouns appear to be heavily influenced by the variable Intimacy, which although it was observed during the analysis, was not evaluated in this study.

Genes encoding proteins that carry out essential informational tasks in the cell, in particular where multiple interaction partners are involved, are less likely to be transferable to a foreign organism. Here, we investigated the constraints on transfer of a gene encoding a highly conserved informational protein, translation elongation factor Tu (EF-Tu), by systematically replacing the endogenous tufA gene in the Escherichia coli genome with its extant and ancestral homologs. The extant homologs represented tuf variants from both near and distant homologous organisms. The ancestral homologs represented phylogenetically resurrected tuf sequences dating from 0.7 to 3.6 billion years ago (bya). Our results demonstrate that all of the foreign tuf genes are transferable to the E. coli genome, provided that an additional copy of the EF-Tu gene, tufB, remains present in the E. coli genome. However, when the tufB gene was removed, only the variants obtained from the gammaproteobacterial family (extant and ancestral) supported growth which demonstrates the limited functional interchangeability of E. coli tuf with its homologs. Relative bacterial fitness correlated with the evolutionary distance of the extant tuf homologs inserted into the E. coli genome. This reduced fitness was associated with reduced levels of EF-Tu and reduced rates of protein synthesis. Increasing the expression of tuf partially ameliorated these fitness costs. In summary, our analysis suggests that the functional conservation of protein activity, the amount of protein expressed, and its network connectivity act to constrain the successful transfer of this essential gene into foreign bacteria. IMPORTANCE Horizontal gene transfer (HGT) is a fundamental driving force in bacterial evolution. However, whether essential genes can be acquired by HGT and whether they can be acquired from distant organisms are very poorly understood. By systematically replacing tuf with ancestral homologs and homologs from distantly related organisms, we investigated the constraints on HGT of a highly conserved gene with multiple interaction partners. The ancestral homologs represented phylogenetically resurrected tuf sequences dating from 0.7 to 3.6 bya. Only variants obtained from the gammaproteobacterial family (extant and ancestral) supported growth, demonstrating the limited functional interchangeability of E. coli tuf with its homologs. Our analysis suggests that the functional conservation of protein activity, the amount of protein expressed, and its network connectivity act to constrain the successful transfer of this essential gene into foreign bacteria.

Life is anti-entropic and highly organized phenomenon with two characteristics reinforcing each other: homochirality and the stereospecific catalysis of chemical reactions. The exclusive presence of L-amino acids and R-sugars in living world well depict this. Hypothetically, the amino acids and sugars of reverse chirality could form a parallel kingdom which is highly orthogonal to the present world. The components from this mirror kingdom, such as protein or nucleic acid, will be much more resistant to the defensive mechanism of present living system, which could be of great value. Therefore, by gradually rewiring the present bio-machineries, we look to build a bridge leading us to the space of mirror-imaged biomolecules. We begin by investigating protein synthesis with mirror amino acid since most amino acids contain one chiral center to be inversed comparing to sugars. In this work, we analyzed three stages critical for the incorporation of D-amino acid into ribosomal protein synthesis: amino acylation, EF-Tu binding of amino acyl-tRNA and delivery bias, and ribosome catalyzed peptidyl transfer. We have demonstrated that the affinity between EF-Tu and amino acyl-tRNA plays critical role on D-amino acid incorporation, and built a platform aimed to select for ribosome tolerating D-amino acid better. / Chemistry and Chemical Biology

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by overexpression of the transcription factors OCT4, SOX2, KLF4, and c-Myc holds great promise for the development of personalized cell replacement therapies. In an attempt to minimize the risk of chromosomal disruption and to simplify reprogramming, several studies demonstrated that a reduced set of reprogramming factors is sufficient to generate iPSC. We recently showed that a reduction of reprogramming factors in murine cells not only reduces reprogramming efficiency but also may worsen subsequent differentiation. To prove whether this is also true for human cells, we compared the efficiency of neuronal differentiation of iPSC generated from fetal human neural stem cells with either one (OCT4; hiPSC1F-NSC) or two (OCT4, KLF4; hiPSC2F-NSC) reprogramming factors with iPSC produced from human fibroblasts using three (hiPSC3F-FIB) or four reprogramming factors (hiPSC4F-FIB). After four weeks of coculture with PA6 stromal cells, neuronal differentiation of hiPSC1F-NSC and hiPSC2F-NSC was as efficient as iPSC3F-FIB or iPSC4F-FIB. We conclude that a reduction of reprogramming factors in human cells does reduce reprogramming efficiency but does not alter subsequent differentiation into neural lineages. This is of importance for the development of future application of iPSC in cell replacement therapies.

Ribosomes translate the genetic information contained in mRNAs into protein by linking together amino acids with the help of aminoacyl-tRNAs. In bacteria, protein synthesis stalls when the ribosome reaches the 3'-end of truncated mRNA transcripts lacking a stop codon. Trans-translation is a conserved bacterial quality control process that rescues stalled ribosomes. Transfer-messenger RNA (tmRNA) and its protein partner SmpB mimic a tRNA by entering the A site of the ribosome and accepting the growing peptide chain. The ribosome releases the truncated mRNA and resumes translation on the tmRNA template. The open reading frame found on tmRNA encodes a peptide tag that marks the defective nascent peptide for proteolysis. A stop codon at the end of the open reading frame allows the ribosome to be recycled and engage in future rounds of translation.The entry of tmRNA into stalled ribosomes presents a challenge to our understanding of ribosome function because during the canonical decoding process, the ribosome specifically recognizes the codon-anticodon duplex formed between tRNA and mRNA in the A site. Recognition of proper base-pairing leads to conformational changes that accelerate GTP hydrolysis by EF-Tu and rapid accommodation of the tRNA into the ribosome for peptidyl transfer. The puzzle is that tmRNA enters stalled ribosomes and reacts with the nascent peptide in the absence of a codon-anticodon interaction. Instead, SmpB binding in the decoding center begins the rescue process, but it has been unclear how SmpB licenses tmRNA entry into stalled ribosomes. We analyzed a series of SmpB and ribosomal RNA mutants using pre-steady-state kinetic assays for EF-Tu activation and peptidyl transfer. Although the conserved 16S nucleotides A1492 and A1493 play an essential role in canonical decoding, they play little or no role in EF-Tu activation or peptidyl transfer to tmRNA. In contrast, a third nucleotide, G530, stacks with the side chain of SmpB residue His136, inducing conformational changes that lead to GTP hydrolysis by EF-Tu. A portion of the C-terminal tail forms a helix within the mRNA channel, monitoring the length of mRNA bound in the ribosome to avoid aborting productive protein synthesis. Helix formation in the mRNA channel is essential for accommodation and peptidyl transfer, but not for GTP hydrolysis. We show that conserved residues in the tail are essential for EF-Tu activation, accommodation, or translocation to the P site. Our findings lead to a clearer model of how the tmRNA-SmpB complex enters stalled ribosomes.

Silicon (Si) is known as beneficial element for graminaceous plants. The importance of Si for plant functioning of cereals was recently emphasized. However, about the effect of Si availability on biomass production, grain yield, nutrient status and nutrient use efficiency for wheat (Triticum aestivum L.), as one of the most important crop plants worldwide, less is known so far. Consequently, we assessed the effect of a broad range of supply levels of amorphous SiO2 on wheat plant performance. Our results revealed that Si is readily taken up and accumulated basically in aboveground vegetative organs. Carbon (C) and phosphorus (P) status of plants were altered in response to varying Si supply. In bulk straw biomass C concentration decreased with increasing Si supply, while P concentration increased from slight limitation towards optimal nutrition. Thereby, aboveground biomass production increased at low to medium supply levels of silica whereas grain yield increased at medium supply level only. Nutrient use efficiency was improved by Si insofar that biomass production was enhanced at constant nitrogen (N) status of substrate and plants. Consequently, our findings imply fundamental influences of Si on C turnover, P availability and nitrogen use efficiency for wheat as a major staple crop.