Respiratory syncytial virus (RSV) is the leading cause of respiratory illness in children worldwide. RSV has a negative sense RNA genome, which is the template for viral mRNA transcription and genome replication, and encodes a polymerase to carry out viral RNA synthesis. The promoters for RSV transcription and genome replication are found in a 44-nucleotide (nt), 3´-extragenic region called the leader (Le). Replication is initiated opposite the first nt of the Le, and transcription of the first gene begins at position +45, at a gene start (GS) sequence. However, transcription is also dependent on sequence within Le1-12. Interestingly, Le nucleotides 3-12 bear strong similarity to a GS signal. We hypothesized that this GS-like sequence is the recruitment site for transcribing polymerase. To test this hypothesis, we examined RNA synthesis events at the Le promoter. We identified a previously undescribed RNA initiation site at Le position +3 (Le+3) that was used frequently during RSV infection. Initiation at Le+3 led to the production of a small ~25 nt RNA. Le+3 initiation was shown to occur independently of replication initiation at +1, indicating it is a bona fide initiation site. Mutation of Le1-12 to increase similarity to a GS resulted in elongation of Le+3 RNA and a decrease in transcription initiation at the GS, demonstrating that the Le initiation sequence alters polymerase processivity and impacts downstream transcription events. Preliminary experiments to determine the function of the small RNA showed that it increased levels of viral RNA replication, suggesting it may be involved in influencing a switch from transcription to replication. These studies suggest a model for RSV transcription initiation, whereby the transcribing polymerase enters at the 3´–end of the genome, initiates RNA synthesis from Le+3 and generates a small RNA, and is then positioned to initiate transcription at the first GS. The small RNA that is generated may act as a feedback molecule to promote RNA replication. These findings provide a greater understanding of polymerase behavior at the promoter and may inform rational drug and vaccine design.

In this paper, an improved method [1] for the chemical synthesis of 2SeU-RNA was reported using a streamlined strategy employs 2'-O-Thiomopholine-4-carbothioate protecting group. And single step deprotection of the resulting oligoribonucleotide product using 1,2-diamines/toluene under anhydrous conditions would retain the Selenium atom introduced on the 2-possiton of the modified Uracil. The process is doable with most standard heterobase protection and deprotection, it greatly simplifies the synthesis of 2SeU-RNAs and can be applied to other Selenium modified RNAs synthesis. It makes the synthesis of RNA become as simple and efficient as the chemical synthesis of DNA. Furthermore, the design and synthesis of self-assembling 2SeU-RNA square are reported which enable further structure studies and application of unique 2SeU-RNAs.

O objetivo do presente estudo foi avaliar os efeitos da suplementação de levedura hidrolisada (fonte de nucleotídeos) em dietas de porcas em fase de lactação sobre o consumo de ração e peso corporal, composição bromatológicas do leite, concentração de RNA e nucleotídeos, e desempenho da progênie. Os nucleotídeos foram derivados de uma levedura hidrolisada (Hilyses®, ICC Brazil). Foram utilizadas 80 porcas (Agroceres PIC®) distribuídas em um delineamento inteiramente casualizado, com 4 tratamentos (0, 4, 8 e 12 kg/ton de Hilyses) e 20 repetições, sendo cada porca uma unidade experimental. As porcas foram alimentadas com as dietas experimentais 3 dias antes da parição, quando foram transferidas para a maternidade, até o desmame dos leitões aos 21,57 ± 0,88 dias de idade. O número de leitões por matriz (10,48 ± 0,26) e peso dos leitões (1,70 ± 0,04 kg) foram ajustados (equalizados) ao nascimento. Os parâmetros avaliados nas porcas foram peso ao parto (PPP, kg), peso ao desmame (PPD, kg), perda de peso (PPC, %) e consumo de ração (CR, kg). O número de leitões desmamados (NLD), peso do leitão ao desmame (PD, kg), peso da leitegada ao desmame (PLD, kg), ganho de peso da leitegada (GPL, kg), mortalidade (M, %) e produção de leite (PL = 1kg de GPL = 4kg de leite produzido) foram mensurados. Amostras de colostro e leite (aos 11 e 20 dias de lactação) foram coletadas para análises bromatológicas, RNA (mg/ml de leite) e nucleotídeos e nucleosídeos (g/ml leite). Os dados foram analisados pelo GLM (SAS), e as médias comparadas pelo teste de Tukey (P=0,05). A suplementação de nucleotídeos na dieta de porcas em fase de lactação não resultou em diferenças (P>0,05) no PPP, PPD, PPC e CR. Os leitões das porcas alimentadas com dietas suplementadas com levedura hidrolisada tiveram uma melhora (P<0,05) no NLD, PLD, GPL, M e PL quando comparados com as não suplementadas com a levedura hidrolisada. Não houve diferença (P>0,05) entre os tratamentos no PD. Em geral os níveis 8 e 12 kg/ton de inclusão de Hilyses mostraram os melhores resultados de desempenho dos leitões (6% e 4,5% maiores sobre o GPL) e menor M (41,7% e 53,5% comparados ao grupo controle). Os resultados das análises bromatológicas do colostro e leite de porcas tiveram uma composição similar entre os tratamentos, no entanto houve uma tendência de aumento na lactose com o aumento da inclusão de Hilyses. A concentração de RNA no leite (aos 11 e 20 dias de idade) foram maiores (P<0,05) nos tratamentos com suplementação da levedura hidrolisada. O total de nucleotídeos e nucleosídeos no leite foram afetados (P<0,05) pelos tratamentos aos 20 dias de lactação. Este estudo demonstrou que a suplementação para porcas lactantes teve um efeito positivo na qualidade do leite, e consequentemente, aumentou o ganho de peso da leitegada e o número de leitões desmamados (+3,5%). / The objective of this study was to evaluate effects of hydrolyzed yeast (nucleotide source) in farrowing sows diets on feed intake and body weight, milk bromatological composition, RNA and nucleotides, and progeny performance. The nucleotides were derived from a yeast source (Hilyses®, ICC Brazil).The trial was conducted with 80 sows (Agroceres PIC®) distributed in a completely randomized design, with 4 treatments (0, 4, 8, or 12 kg/MT Hilyses®) and20 replications of 1 sow in each per treatment. The sows were fed experimental diets starting 3 days before farrowing, when sows were transferred to the maternity unit, until weaning of piglets at 21 days of age. The number of piglets per sow (10.48±0.26) and piglet weight (1.70±0.04kg) was adjusted (equalized) at birth. The sow parameters were weight after farrowing (WF,kg), weight after weaning (WW,kg), weight loss (WL,%), and feed intake (FI,kg). The number of weaned piglets (NWP), piglet weight at weaning (PWW,kg), litter weight at weaning (LWW,kg), litter weight gain (LWG,kg), mortality (MORT,%), and milk production (MP,kg; 1kg of piglet weight = 4kg of milk) were measured. Samples of colostrum and milk (11, 20 days of lactation) were collected for laboratory bromatological analysis, RNA (mg/ml milk) and nucleotides and nucleosides (&#956;g/ml milk). The data were analyzed using the GLM (SAS), and means were compared by Tukeys test (P=0.05). Nucleotide supplementation in the diet of farrowing sows resulted in no difference (P>0.05) in WF, WW, WL, or FI. The piglets from sows fed diets supplemented with nucleotides had improved (P<0.05) NWP, LWW, LWG, MORT, and MP compared with unsupplemented diets. There were no differences (P>0.05) between treatments in PWW. In general, the 8kg/MT and 12kg/MT levels showed best piglet performance results (6% and 4.5% higher LWG than control group) and lower MORT (41.7% and 53.5% lower than control group). The results of bromatological analysis of colostrum and milk had a similar composition between the treatments, however, there was a numerical increase tendency in lactose with hydrolyzed yeast supplementation. Hilyses® supplementation gave no significant response (P>0.05) in Total RNA in colostrums, but the amount of RNA present in milk at 11 and 20 days of lactation significantly increased (P<0.05). The total of nucleotides and nucleosides in milk at 20 days of lactation period were affected (P<0.05) by treatments. This study demonstrated that supplementation of nucleotides to farrowing sows had a positive carryover effect on milk quality, which, consequently, increased the litter weight gain and the number of, weaned piglets (+3.5%).

Orientador: Danillo Pinhal / Resumo: Nos vertebrados, o coração é o primeiro órgão a se formar e adquirir função no embrião e todos os eventos subsequentes na vida do organismo dependem de sua atividade. Ao longo da evolução desses animais, incluindo o Homo sapies, o coração adquiriu traços morfofuncionais distintos, resultando em fenótipos característicos nas mais variadas espécies viventes. Entretanto a base molecular que sustenta a variação fenotípica permanece pouco conhecida. Uma vez que o genoma desses animais é relativamente conservado quanto à estrutura e função, é plausível pensar que as variações morfofuncionais resultem, em grande parte, da expressão diferencial de genes e seus reguladores. MicroRNAs (miRNAs) são pequenos RNAs não codificantes com importante função na regulação gênica pós-transcricional. Essas moléculas reconhecidamente atuam nosmais variados processos biológicos, incluindo as vias de biogênese e manutenção cardíaca. Contudo, até o momento, são escassos os dados disponíveis que tratam do impacto da atividade de miRNAs no desenvolvimento e evolução do coração de vertebrados. No presente estudo, com o objetivo de gerar novos conhecimentos acerca da base molecular da variabilidade fenotípica do coração, foi realizada a caracterização e análise comparativa das assinaturas de expressãodo conjunto total de miRNAs no coração de espécies representativas dos cinco grandes grupos de vertebrados. Para isso, os transcritos de miRNAs de amostras detecido cardíaco de 13 espécies distintas foram inv... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In vertebrates, heart is the first organ to form in the embryo and all subsequent events in the life of an organism depend on its function. Heart has acquired distinct morphological traits during vertebrate evolution, which leaded to distinct morphology traits in the living species. However, the molecular mechanisms that drive the differential morphology among these species remain uncharted. Taking into consideration the higher level of conservation of the genome among these species, we can infer that the differential expression of genes and regulators lead to the distinct pattern of heart morphology. MicroRNAs (miRNAs) are small molecules with an important role upon post-transcriptional regulation. These molecules have been shown essential for several cellular processes in vertebrates, including cardiac biology, but little is known about the roles of these small molecules in the development and evolution process of vertebrate’s heart. In the present study, we characterized the global expression of miRNAs in the heart of species from the five vertebrate groups, (fish, amphibians, reptiles, birds and mammals) and performed an evolutionary comparative analysis of miRNA expression signatures in order to bring novel insights about the molecular basis of heart phenotypic variability. RNA-seq was used to characterize miRNA expression profiles in the heart of 13 vertebrate species and deeply analyzed using bioinformatics. We found 32 miRNAs with conserved expression to all 13 specie... (Complete abstract click electronic access below) / Doutor

Finally, the fourth part of this thesis demonstrated the potential of plasma RNA integrity for noninvasive clinical diagnosis. Based on previous reports of elevated RNase activities in the circulation of cancer patients, it was hypothesized that plasma RNA integrity might serve as a useful tumor marker. Using nasopharyngeal carcinoma (NPC) as a disease model, it was found that plasma RNA in untreated NPC patients was of lower integrity than that in healthy individuals. Further analysis showed that patients undergoing radiotherapy had increased RNA integrity in the post-treatment plasma samples. These findings hence suggested that measurement of plasma RNA integrity may provide a feasible approach for noninvasive cancer detection. / Much recent interest has been focused on the clinical applications of cell-free circulating RNA for molecular diagnostics. Despite the rapid development of plasma RNA as a potential diagnostic tool, much of the biology of these molecular species remains enigmatic. This thesis aimed to investigate the integrity of cell-free RNA molecules in plasma and to study the effects of different preanalytical factors on this new biological parameter. Moreover, the possibility that plasma RNA integrity might serve as a useful clinical marker was explored. / The first part of this thesis was to develop a quantitative method for analyzing RNA integrity in plasma. One-step real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was used to amplify transcript sequences corresponding to multiple regions along a housekeeping gene, glyceraldehyde-3 phosphate dehydrogenase (GAPDH). It was demonstrated that 5' transcript fragments were predominant, when compared with those derived from the middle or 3' region, in the plasma of healthy individuals. This method was validated using two-step RT-PCR and serial dilution assays. / The potential generality of the underrepresentation of 3' mRNA fragments was further studied by using circulating placental RNA as a model system. Seven transcripts were analyzed in the plasma of pregnant women: the beta subunit of human chorionic gonadotropin (betahCG), tissue factor pathway inhibitor 2 (TFPI2), adrenomedullin (ADM), inhibin beta A subunit (INHBA), placenta-specific 1 (PLAC1), pregnancy-associated plasma protein A (PAPPA), and GAPDH. The second part of this thesis showed that for five of the seven genes, there appeared to be a greater abundance of transcript fragments arising from the 5' end than those from the 3' end in maternal plasma, and that for every gene under study, the 5'-specific assay had a higher rate of detection when compared to the 3'-specific one. Apart from biological significance, these data have implications for maximizing the sensitivity of fetal RNA detection in maternal plasma for future diagnostic use. / The results presented in this thesis not only have improved the current understanding of the biological nature of cell-free circulating RNA, but also have provided important information regarding the potential clinical utility of a new parameter, plasma RNA integrity, for the field of medical diagnostics. / To ensure accurate interpretation of the results on plasma RNA integrity, a number of preanalytical issues were investigated. In the third part of this thesis, several findings were described: (1) filtration of plasma samples did not change the observation that 5' end transcript was the predominant species; (2) time delay in the processing of plasma could lead to decreased RNA concentrations despite the lack of variation in plasma RNA integrity; and (3) repeated freezing and thawing of plasma samples, but not extracted RNA, could reduce RNA integrity significantly. / Wong Chi Kwan Blenda. / "August 2006." / Adviser: Yuk Ming Dennis Lo. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1453. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 148-184). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Alternative splicing of pre-mRNA is a critical mechanism for enabling genetic diversity, and is a carefully regulated process in neuronal differentiation. RNA binding proteins (RBPs) are developmentally expressed and physically interact with RNA to drive specific splicing changes. This work tests the hypothesis that RBP-RNA interactions are critical for regulating timed and coordinated alternative splicing changes during neurodevelopment and that these splicing changes are in turn part of major regulatory mechanisms that underlie morphological and functional maturation of neurons. I describe our efforts to identify functional RBP-RNA interactions, including the identification of previously unobserved splicing events, and explore the combinatorial roles of multiple brain-specific RBPs during development. Using integrative modeling that combines multiple sources of data, we find hundreds of regulated splicing events for each of RBFOX, NOVA, PTBP, and MBNL. In the neurodevelopmental context, we find that the proteins control different sets of exons, with RBFOX, NOVA, and PTBP regulating early splicing changes and MBNL largely regulating later splicing changes. These findings additionally led to the observation that CNS and sensory neurons express a variety of different RBP programs, with many sensory neurons expressing a less mature splicing pattern than CNS neurons. We also establish a foundation for further exploration of neurodevelopmental splicing, by investigating the regulation of previously unobserved splicing events.

Wang, Dakui. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 208-234). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

RNA molecules play important roles, including catalysis of chemical reactions and control of gene expression, and their functions largely depend on their folded structures. Since determining these structures by biochemical means is expensive, there is increased demand for computational predictions of RNA structures. One computational approach is to find the secondary structure (a set of base pairs) that minimizes a free energy function for a given RNA conformation. The forces driving RNA folding can be approximated by means of a free energy model, which associates a free energy parameter to a distinct considered feature. The main goal of this thesis is to develop state-of-the-art computational approaches that can significantly increase the accuracy (i.e., maximize the number of correctly predicted base pairs) of RNA secondary structure prediction methods, by improving and refining the parameters of the underlying RNA free energy model. We propose two general approaches to estimate RNA free energy parameters. The Constraint Generation (CG) approach is based on iteratively generating constraints that enforce known structures to have energies lower than other structures for the same molecule. The Boltzmann Likelihood (BL) approach infers a set of RNA free energy parameters which maximize the conditional likelihood of a set of known RNA structures. We discuss several variants and extensions of these two approaches, including a linear Gaussian Bayesian network that defines relationships between features. Overall, BL gives slightly better results than CG, but it is over ten times more expensive to run. In addition, CG requires software that is much simpler to implement. We obtain significant improvements in the accuracy of RNA minimum free energy secondary structure prediction with and without pseudoknots (regions of non-nested base pairs), when measured on large sets of RNA molecules with known structures. For the Turner model, which has been the gold-standard model without pseudoknots for more than a decade, the average prediction accuracy of our new parameters increases from 60% to 71%. For two models with pseudoknots, we obtain an increase of 9% and 6%, respectively. To the best of our knowledge, our parameters are currently state-of-the-art for the three considered models.

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 105-129). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--University of Chicago, Department of Biochemistry and Molecular Biology, 2001. / Includes bibliographical references. Also available on the Internet.