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%).

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. / Science, Faculty of / Computer Science, Department of / Graduate

Research about ribonucleic acid (RNA) is gaining popularity as we widen our knowledge about its function. But to properly examine new structures, we need robust computational tools to analyse different properties. One of such tools is structural superposition, which is a method to align two structures over each other and quantify their similarity. This tool can be used on tertiary RNA structures for visual comparison, clustering or the assessment of their function. The aim of this thesis is to present a novel approach for achieving RNA superposition using information about secondary RNA structure and its link to trees. Tree edit distance algorithms are used to compare the trees, and a multitude of methods for generating the structural superposition from the calculated tree similarities is presented. The new method is aligned in the context of existing works, and its accuracy is compared to the best current approaches for structural superposition. The implementation can be accessed at https://github.com/gyfis/rawted.

No description available.

N6-threonylcarbamoyl adenosine (t6A) is a universally conserved tRNA modification found at position 37 of tRNAs which decode ANN codons. Structural studies have implicated its presence as a requirement for the disruption of a U-turn motif in certain tRNAs, leading to the formation of properly structured anticodon stem loop. This structure is proposed to enhance the base pairing between U36 of tRNA and A1 of the codon which aids in translational frame maintenance. Despite significant effort since its discovery in the 1970s the enzymes involved in its biosynthesis remained undiscovered. Bioinformatic analysis identified two proteins as likely candidates for t6A synthesis, YrdC and YgjD. Subsequent gene knockout experiments in yeast were consistent with their involvement in t6A biosynthesis in vivo. Furthermore, clustering between the bacterial genes ygjD, yeaZ and yjeE as well as the identification of a protein interaction network between YgjD, YeaZ, and YjeE suggested that YeaZ and YjeE might be involved in t6A biosynthesis. The genes encoding ygjD, yeaZ, yrdC and yjeE were cloned from E. coli and the recombinant protein was purified. Experiments analyzing the incorporation of [U-14C]-L-threonine and [14C]-bicarbonate (substrates previously indicated in its biosynthesis) into tRNA in the presence of these four proteins demonstrated the first reconstitution of the t6A pathway in vitro. LC-MS analysis verified the formation of t6A, and these proteins were renamed TsaD (YgjD), TsaB (YeaZ), TsaC (YrdC), and TsaE (YjeE). Biochemical characterization of this pathway suggested that the formation of t6A proceeds through an unstable threonylcarbamoyl adenosine monophosphate (TC-AMP) intermediate, which is produced by TsaC from its substrates CO2, L-threonine and ATP. To investigate this reaction in more detail a coupled assay was developed, enabling sensitive detection of turn over. TsaC is a promiscuous enzyme which readily accepts several amino acids as substrates. The formation of t6A from TC-AMP is catalyzed by TsaD, TsaB, and TsaE. Of these three proteins only TsaD is universally conserved suggesting it is the protein catalyzing the transfer of the threonylcarbamoyl moiety to A37 of tRNA. This transfer is not promiscuous as only TC-AMP serves as an efficient substrate for t6A formation. Structural investigation of these proteins are consistent with the formation of a single protein complex potentially alleviating issues with the reactivity and instability of TC-AMP.

The work presented explored the in vivo and in vitro synthesis of ribosomal RNA in the Gram positive, spore-forming bacterium Bacillus subtilis. The investigation began with a study of rRNA synthesis in B. subtilis during steady state growth and under nutritional shift-up conditions. The percent of transcription which is ribosomal RNA was measured by hybridization of pulse labeled RNA to a specific DNA probe carrying the 3' end of the 23S RNA gene. The fractional rate of ribosomal RNA synthesis increased with cellular growth rate, and showed a rapid increase after a nutritional shift up. RNA synthesis during infection with an amber mutant of bacteriophage SP01 was also examined. Infected cells continued to synthesize rRNA at the preinfection rate, but could not respond to media enrichment by increasing the percent rRNA-synthesis. The latter study suggested the existence of a specific RNA polymerase that transcribed ribosomal RNA genes. The conclusions from the in vivo study led to an analysis of rRNA transcription in vitro. The isolation of the putative ribosomal RNA specific RNA polymerase was attempted by affinity chromatography on cellulose complexed with plasmid DNA containing the promoter region of the B. subtilis rrnB rRNA operon, and by sedimentation through a glycerol gradient. No difference in activity profile was observed when transcription activity at the rRNA tandem promoters was compared to activity at a non-ribosomal promoter. Since in vivo analysis of the control of rRNA synthesis in Escherichia coli suggested that regulation occurs at the level of transcription initiation, in vitro transcription initiation at the B. subtilis rRNA promoters was investigated using the single round transcription assay. Initial rates of transcription were different at each of the two tandem promoters of the B. subtilis rrnB operon: the upstream promoter, PI, initiated slowly, while the downstream promoter, P2, initiated faster. In addition, transcription initiation at the two promoters appeared to be linked. The formation of a heparin resistant complex at the PI promoter affected the stability of the heparin resistant complex formed at the P2 promoter. The kinetics of transcription initiation at the tandem rRNA promoters were examined using the tau plot analysis. RNA polymerase had a high affinity for both rRNA promoters, but the rate of initiation at these promoters was relatively slow when compared to non-ribosomal promoters. Finally, transcription initiation on two artificial tandem promoter constructs was compared with initiation on the native tandem promoter construct. In general, PI was shown to have a positive effect on transcription from downstream promoters, but had specific effects on different promoters. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Escherichia coli was grown in the presence of amino acid analogues or in the absence of required amino acids. The tRNAs. were isolated and characterized. Numerous changes were observed in the total tRNA acceptance for particular amino acids although in no instance did these changes occur for amino acids corresponding to the adverse growth condition. The isoacceptor patterns for particular labelled aminoacyl-tRNAs were determined on the anion exchanger RPC-5. Novel isoacceptor tRNAs were observed under several growth conditions. Significant changes in tRNA isoacceptor distributions were noted. In certain instances it appeared that changes in total amino acid acceptance could be explained in terms of the increased or decreased synthesis of particular tRNA isoacceptors while for other tRNAs it seemed that changes occurred in the synthesis of all isoacceptors for a particular amino acid such that the relative amounts of isoacceptors remained constant even when total amino acid acceptance changed considerably. E. coli was grown over a wide temperature range, 17°C to 44°C, and the tRNA isolated and characterized. Novel tRNA isoacceptors were observed at both high and low growth temperatures for most but not all tRNAs. It was shown that the same isoacceptors could be formed at both extremes of temperature. Preliminary results suggest that the novel isoacceptors are formed as the result of a temperature aggravation of a nutritional problem at extremes of growth temperature. One of the novel tRNA isoacceptors formed under a variety of adverse growth conditions, tRNA3[sup Val] , was purified and partially characterized. The results are consistent with tRNA3[sup Val] being an undermodified precursor of the major isoacceptor tRNA₁[sup Val]. E. coli str[sup D] was grown and the tRNA isolated and characterized. Major differences in the amino acid acceptances for several tRNAs were observed. These changes were accomplished without any significant changes in the relative isoacceptor distributions as determined by RPC-5 chromatography. Gel electrophoretic analysis was performed on tRNA from cells grown at extremes of growth temperature. Significant differences were observed in the 5S region; there was an accumulation of material in cells grown at low temperature and a decrease of material in cells grown at high temperature. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

The advancement of Nucleic acids (DNA and RNA) sequencing technology has enabled many projects targeted towards the identification of genome structure and transcriptome complexity of organisms. The first conclusions of the human and mouse projects have underscored two important, yet unexpected, findings. First, while almost the entire genome is transcribed, only 5% of it encodes for proteins. Thereby, most transcripts are noncoding RNA. This includes both short RNA (<200 nucleotides (nt)) comprising piRNAs; microRNAs (miRNAs); endogenous Short Interfering RNAs (siRNAs) among others, and includes lncRNA (>200nt). Second, a significant portion of the mammalian genome (45%) is composed of Repeat Elements (REs). RE are mostly relics of ancestral viruses that during evolution have invaded the host genome by producing thousands of copies. Their roles within their host genomes have yet to be fully explored considering that they sometimes produce lncRNA, and have been shown to influence expression at the transcriptional and post-transcriptional levels. Moreover, because some REs can still mobilize within host genomes, host genomes have evolved mechanisms, mainly epigenetic, to maintain REs under tight control. Recent reports indicate that REs activity is regulated in somatic cells, particularily in the brain, suggesting a physiological role of RE mobilization during normal development. In this thesis, I focus on the analysis of ncRNAs, specifically REs; piRNAs; lncRNAs in human and mouse post-mitotic somatic cells. The main aspects of this analysis are: Using sRNA-Seq, I show that piRNAs, a class of ncRNAs responsible for the silencing of Transposable elements (TEs) in testes, are present also in adult mouse brain. Furthermore, their regulation shows only a subset of testes piRNAs are expressed in the brain and may be controlled by known neurogenesis factors. To investigate the dynamics of the transcriptome during cellular differentiation, I examined deep RNA-Seq and Cap Analysis of Gene Expression (CAGE) data from time-course progression program of primary human skeletal muscle cell differentiation. I contrasted this program with Duchenne Muscular Dystrophy (DMD) donors. I identified novel candidates, protein-coding genes and lncRNAs, that may be involved in myogenesis and reaffirmed known myogenic players. Using RNA-Seq data, I designed a novel pipeline to identify possible de novo insertion sites during muscular differentiation, which I have also tested on embryonic mouse cerebral cortex.

RNA binding proteins (RBPs) play crucial roles in regulating every stage of the mRNA life cycle and mediating non-coding RNA functions. Despite their importance, the specific roles of most RBPs remain unexplored because we do not know what specific RNAs most RBPs bind. Current methods, such as crosslinking and immunoprecipitation followed by sequencing (CLIP-seq), have expanded our knowledge of RBP-RNA interactions but are generally limited by their ability to map only one RBP at a time. To address this limitation, we developed SPIDR (Split and Pool Identification of RBP targets), a massively multiplexed method to simultaneously profile global RNA binding sites of dozens to hundreds of RBPs in a single experiment. SPIDR employs split-pool barcoding coupled with antibody-bead barcoding to increase the throughput of current CLIP methods by two orders of magnitude. SPIDR reliably identifies precise, single-nucleotide RNA binding sites for diverse classes of RBPs simultaneously. Using SPIDR, we explored changes in RBP binding upon mTOR inhibition and identified that 4EBP1 acts as a dynamic RBP that selectively binds to 5’-untranslated regions of specific translationally repressed mRNAs only upon mTOR inhibition. This observation provides a potential mechanism to explain the specificity of translational regulation controlled by mTOR signaling. SPIDR has the potential to revolutionize our understanding of RNA biology and both transcriptional and post-transcriptional gene regulation by enabling rapid, de novo discovery of RNA-protein interactions at an unprecedented scale.

Growth Hormone (GH), also known as somatotropin, is a protein hormone secreted from the anterior pituitary gland. GH action results in longitudinal bone growth in children and adolescents and continues later to control a variety of metabolic reactions in adults. DNA polymorphisms leading to a decrease in GH expression result in persons of short stature, whereas those leading to overexpression of GH produce either acromegaly or gigantism. In order to diagnose and treat patients with altered GH production, understanding the control of GH expression is thus clinically relevant. In order to understand how transcription of GH is determined by selective regulatory modulators, two rat somatotroph cell lines were studied that differed in their relative differentiation: MtT/S cells are considered as fully differentiated somatotrophs that express GH exclusively; MtT/Se cells are related somatotrophs with GH expression that is responsive to estrogen treatment. These cell lines were obtained from the Riken Cell Bank (Japan) and then grown and cultured in accordance with established protocols. After harvesting of cells from each line, total RNA was extracted using a rapid affinity method (Qiagen). After validation of the RNA integrity index of each RNA isolate (Affymetrix), they were sent to an off-site laboratory (NovoGene) for RNA-Seq analysis. Samples were examined in triplicate for comparison using standard procedures for adapter ligation, library construction, amplification and sequencing. In our previous work with single gene quantitative RT-PCR, we measured expression of 9 separate targets, primarily transcriptional controllers, in these cell lines. Now, the use of RNA-Seq quantifies the levels of all mRNAs in each sample without the need for specific primers or probes. Therefore, it is possible to find nearly all of the mRNAs that demonstrate changing abundance without requiring prior evidence of their role in the terminal differentiation of the MtT/S from MtT/Se cells. After extensive QC for validation, initial analysis of the data shows more than 36 million validated sequenced reads from each replicate sample with >96.39% mapping to the reference rat genome sequence. Basic analysis shows that 13,012 expressed genes were 87% similar, with 1,175 (9.0%) unique to MtT/Se cells and 484 (3.7%) genes selectively present in MtT/S cells. Of these, 329 genes were upregulated in expression while only 57 were reduced. For comparison with our previous study, we are now confirming differences in GH transcripts and the transcription factors/regulators previously measured, particularly the GH regulator Zn16, a protein with 16 zinc fingers known to bind to the GH promoter DNA. Further analysis of the RNA-Seq profile is focused on the unique and unidentified changes in expression that differentiate these cell lines. Analysis of the differentiation of MtT/S and MtT/Se cells will further understanding of GH regulation in the body.