Skeletal muscle possesses remarkable plasticity in responses to altered mechanical load. An established murine model used to increase mechanical load on a muscle is the surgical removal of the gastrocnemius and soleus muscles, thereby placing a functional overload on the plantaris muscle. As a consequence, there is hypertrophic growth of the plantaris muscle. We used this model to study the molecular mechanisms regulating skeletal muscle hypertrophy. Aged skeletal muscle demonstrates blunted hypertrophic growth in response to functional overload. We hypothesized that an alteration in gene expression would contribute to the blunted hypertrophic response observed with aging. However, the difference in gene expression was modest, with cluster analysis showing a similar pattern of expression between the two groups. Despite ribosomal protein gene expression being higher in the aged group, ribosome biogenesis was significantly lower in aged compared with young skeletal muscle in response to the hypertrophic stimulus (50% versus 2.5-fold, respectively). The failure to fully up-regulate pre-47S ribosomal RNA (rRNA) expression in old skeletal muscle undergoing hypertrophy indicated ribosomal DNA transcription by RNA polymerase I was impaired. Contrary to our hypothesis, the findings of the study suggest that impaired ribosome biogenesis was a primary factor underlying the blunted hypertrophic response observed in old skeletal muscle rather than dramatic differences in gene expression. As it appears ribosomal biogenesis may limit muscle hypertrophy, we assessed the dynamic changes in global transcriptional output during muscle hypertrophy, as the majority of global transcription is dedicated to ribosome biogenesis during periods of rapid growth. Metabolic labeling of nascent RNA using 5-ethynyl uridine permitted the assessment of cell type specific changes in global transcription and how this transcription is distributed within the myofiber. Using this approach, we demonstrate that myofibers are the most transcriptionally active cell-type in skeletal muscle, and furthermore, myonuclei are able to dramatically upregulate global transcription during muscle hypertrophy. Interestingly, the myonuclear accretion that occurs with hypertrophy actually results in lower transcriptional output across nuclei within the muscle fiber relative to sham conditions. These findings argue against the notion that nuclear accretion in skeletal muscle is necessary to increase the transcriptional capacity of the cell in order to support a growth response.

No description available.

The Sigma-54 Promoter Database contains computationally predicted sigma-54 promoters from over 60 prokaryotic species. Organisms from all major phyla were analysed and results were made available online at http://www.sigma54.ca. This database is particularly unique due to its inclusion of intragenic regions, grouping of data by COG and COG category, and the ability to summarize results either by phylum or database-wide. ClusterMine360 (http://www.clustermine360.ca/) is a database of microbial polyketide and nonribosomal peptide gene clusters. It takes advantage of crowd-sourcing by allowing members of the community to make contributions while automation is used to help achieve high data consistency and quality. The database currently has over 200 gene clusters from over 185 compound families. It also features a unique sequence repository containing over 10,000 PKS/NRPS domains. The sequences are filterable and downloadable as individual or multiple sequence FASTA files. This database will be a useful resource for members of the PKS/NRPS research community enabling them to keep up with the growing number of sequenced gene clusters and rapidly mine these clusters for functional information.

S:Als darrers 10 anys, el nostre coneixement de l'evolució dels animals ha estat objecte d'una revolució degut a l'aplicació de la Biologia Molecular al camp de la Filogènia, que ha rebutjat algunes hipòtesis antigues i n'ha proposat de noves. Un dels canvis més importants és el que subdivideix a tots els animals amb simetria bilateral (els Bilateria) en tres grups: els Lophotrochozoa, els Ecdysozoa y els Deuterostomata. Malgrat el gran cabdal d'informació que han suposat, les dades moleculars no han estat capaces de resoldre totes les relacions entre els fílums. Al començament, això era degut al l'ús de pocs marcadors moleculars, que no portaven prou informació i que patien artefactes metodològics com el Long Branch Atraction. L'objectiu d'aquest projecte és obtenir una filogènia molecular dels bilaterals el més resolta possible, mitjançant l'aplicació de dues aproximacions. La primera fa us de la gran quantitat de dades disponibles dels gens nuclears del RNA ribosomal (18S i 28S) a l'hora que es minimitza l'impacte dels artefactes filogenètics; s'ha obtingut una matriu de 104 representants dels bilaterals, pertanyents a 28 fílums i amb 3.700 parells de bases de longitud. Aquesta matriu s'ha analitzat mitjançant mètodes d'inferència filogenètica de tipus probabilístic (Maxima Versemblança i Inferència Bayesiana), tot fent servir models evolutius sofisticats i compartimentant les anàlisis pels grups problemàtics. La segona aproximació pretén construir una matriu dels bilaterals que maximitzi tant el nombre de marcadors com el de fílums representats. S'han seleccionat 13 gens candidat i recollit mostres per un total de 96 espècies de 31 fílums. S'han obtingut un total de 135 seqüències, que junt amb seqüències de les bases de dades, s'han fet servir per construir una matriu de 90 representants de 27 fílums amb 8.880 parells de bases de longitud i un 40% de missing data. Aquesta matriu s'ha analitzat amb mètodes d'inferència probabilístics.Els resultats principals dels dos estudis son: 1) es comprova la monofilia dels tres grans grups de bilaterals, així com la posició d'Acoela i Nemertodermatida com a bilaterals basals, 2) s'obté una filogènia resolta dels deuteròstoms, mentre que la filogènia dels ecdisozous resulta en una tricotomia i 3) s'obté una nova filogènia dels lofotrocozous, a on cal destacar als gnatostomulats i gastrotrics com els fílums més basals, la posició dels platihelmints com a grup germà de la resta d'animals espirals i la parafilia dels espirals degut a la posició de braquiòpodes i foronidis com a grup germà dels mol·luscs, 4) la filogènia obtinguda situa a Xenoturbella dins dels deuteròstoms, tot i que amb un suport molt baix, i no es pot descartar una posició més basal dins dels bilaterals, 5) els Chaetognatha pertanyen als ecdisozous, tot i que la posició exacta dins dels mateixos és dubtosa, i 6) la filogènia obtinguda ens ha permès especular sobre l'evolució de determinades característiques del patró corporal, mostrant la gran plasticitat de la seva aparició i la prevalença de l'adquisició independent d'algunes d'elles. / "Bilaterian molecular phylogeny: a multigenic approach"TEXT:Resolving the relationships among animal phyla is a key biological problem that remains to be solved. Morphology has been unable to determine the relationships among most phyla. During the past decade studies based on a limited number of markers established new hypotheses such as the existence of three bilaterian superclades (Deuterostomia, Ecdysozoa and Lophotrochozoa) but left major questions unresolved.The aim of this project is to obtain a well resolved bilaterian phylogeny applying two approaches. The first uses the great quantity of information available for 18S and 28S genes, while overcoming their pitfalls by combining several methods suggested in previous studies. A matrix was built, comprising 104 bilaterian representatives for 28 phyla and it is 3,700 base pairs long. The methods used include Maximum Likelihood and Bayesian Inference, the application of models with rate-heterogeneity across sites, wide taxon sampling, and compartmentalized analyses for each problematic clade. The second approach is based on maximizing the number of markers and the number of representatives. 13 genes were chosen as candidates and tissues for 96 species belonging to 31 phyla were sampled. 135 sequences were obtained, and used together with databases sequences to build a matrix 8,880 base pairs long, with 90 representatives for 27 bilaterian phyla and bears 40% of missing data.The results obtained show 1) the monophyly of the three superclades and the basal position of a paraphyletic Acoelomorpha, 2) a well resolved deuterostomates phylogeny, while ecdysozoan relationships turn up as a tricotomy, 3) a new lophotrochozoan phylogeny is obtained with gnathostomulids and gastrotrichs as the most basal phyla, flatworms as sistergroup to spiralians animals, and spiralians are paraphyletic due to the position of brachiopods and phoronids as sistergroup to molluscs, 4) Xenoturbella is shown within deuterostomates, though a more basal position cannot be rejected, 5) Chaetognatha belong to Ecdysozoa, though its exact position cannot be solved, and 6) the obtained phylogeny was used for a speculative discussion on morphological traits evolution, showing their great evolving plasticity.

No description available.

Ribosomal RNAs (rRNA) undergo several post-transcriptional modifications inside the cell. These modifications can be (1) RNA- independent (enzyme only) and (2) guide RNA-mediated. In the latter mechanism, a group of small, metabolically stable, non-coding RNAs, present as ribonucleoprotein (RNP) particles, modify ribosomal RNAs inside the cell. One of the highly abundant rRNA modifications is pseudouridine (Y) formation. In Archaea and Eukarya, pseudouridine synthases, with the help of small RNAs, form pseudouridines at functionally important regions in rRNA. Cbf5, the pseudouridine synthase, three other core proteins, and a box H/ACA RNA form the ribonucleoprotein complex in sRNP-mediated rRNA pseudouridylation. Certain Ys in rRNAs are evolutionarily conserved from Bacteria to human. Among those, two Ys are present in helix 69 of rRNA and one in helix 90. We successfully deleted Cbf5 in Haloferax volcanii, a haloarchaeon, and showed that the deleted strain was viable. It was the first report where Cbf5 deletion was achieved, because deletion or mutation of cbf5 or of its homologs is lethal in eukaryotes. We also found that the cbf5 deleted strain was unable to produce the three highly conserved Ys in rRNA of H. volcanii (position 1940, 1942 in helix 69, and 2605 in helix 90), whereas the tRNA Ys were intact. To identify the specific structural features of Cbf5 involved in rRNA Ψ formation, we used a cbf5 deleted strain which was complemented with a plasmid borne copy of the gene. Using the crystal structure of Pyrococcous furiosus Cbf5 as template, we created a homology model of H. volcanii Cbf5 (HvCbf5) and identified several residues and motifs/domains of HvCbf5 that might be important to the protein's enzymatic activity. By using an in vivo mutational approach, we confirmed some previously predicted and certain unidentified residues/motifs/domains that serve as positive determinants of rRNA Ys1940, 1942, and 2605 formation inside the cell. A box H/ACA RNA, sR-h45, was bioinformatically predicted before. We confirmed its presence as a double hairpin RNA inside the cell whose level goes down in the absence of Cbf5. We identified that sR-h45 is the guide RNA for sRNP-mediated Ys at the three above mentioned rRNA positions in H. volcanii. Each hairpin of this RNA can independently modify the substrate, both in vivo and in vitro. To characterize the structure of sR-h45, we have used a sR-h45 deleted strain where the function of sR-h45 was complemented with a plasmid-borne copy of the gene. By a combination of in vivo and in vitro mutagenic approaches, we determined specific nucleotides/structures of this RNA, involved in binding to the core proteins and also to the substrate RNA. We also identified that one hairpin of sR-h45 can modify two successive positions (1940 and 1942) in rRNA.

Ribosomal protein genes are crucial for ribosome biogenesis. The ribosome itself is responsible for protein synthesis and hence cellular growth and development. Intertwining network of proteins in conjugation with cellular environment such as nutrition and growth factors collectively regulate expression of the ribosomal protein genes. DNA microarray analysis has implicated the role of 26S proteasome in transcriptional regulation of the ribosomal protein genes tying protein degradation to protein synthesis pathway. To determine the mechanism as to how the 26S proteasome promotes transcription of the ribosomal protein genes a series of experiments were performed. The results reveal that the 19S subcomplex of the 26S proteasome is recruited to the promoters of the ribosomal protein genes in a TOR (Target of Rapamycin)-dependent manner. TOR signals environmental cues and controls the expression of the ribosomal protein genes. Thus recruited 19S proteasome subcomplex promotes transcriptional initiation via facilitation of the recruitment of co-activator NuA4 (Nucleasome acetyltransferase of histone H4) complex to activator Rap1. NuA4 enhances PIC (Pre-initiation complex) formation at the core promoter, but it is not clearly understood how does it do so. Researches have identified two different forms of TBP: TAF (TBP associated factor)-dependent form of TBP and TAF-independent form of TBP. This work shows that impaired association of NuA4 interferes with TFIID recruitment, but recruits TAF-independent form of TBP to the core promoter. This recruitment of TBP is dependent on SAGA (Spt-Ada-Gcn5-acetyltransferase). Like ribosomal protein genes, antisense transcription is also enhanced by TAFs. However, it remains unknown whether NuA4 also promotes TAF-regulated antisense transcription. The results illustrate that like ribosomal protein genes, transcription of GAL10 antisense is also promoted by NuA4 HAT (histone acetyl transferase). NuA4 HAT is recruited to the 3’-end of the GAL10 coding sequence, acetylates histone H4 and promotes GAL10 antisense transcription. This work also reveals the roles of other chromatin regulatory factors in controlling antisense transcription. Collectively, these results significantly advance our current understanding of the regulatory mechanisms of ribosomal protein genes’ expression and antisense transcription. The ribosome and antisense are involved in virtually all the biological processes. Aberrant expression of the ribosomal protein genes and antisense transcripts are associated with numerous human disorders including cancers and cardiovascular diseases. Therefore, analyses of their regulatory processes provide valuable information toward understanding the etiology of numerous human diseases with potential therapeutic interventions.

PEREIRA, Júlio Otávio Portela. Diversidade genética da abelha sem ferrão melipona quinquefasciata baseada no sequênciamento das regiões its1 e 18s do dna ribossômico nuclear. 2006. 141 f. Tese (doutorado em zootecnia)- Universidade Federal do Ceará, Fortaleza-CE, 2006. / Submitted by Elineudson Ribeiro (elineudsonr@gmail.com) on 2016-04-08T20:31:16Z No. of bitstreams: 1 2006_tese_joppereira.pdf: 2085821 bytes, checksum: da8ccd544517a5841c9efa06abf9dacd (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-05-25T22:48:38Z (GMT) No. of bitstreams: 1 2006_tese_joppereira.pdf: 2085821 bytes, checksum: da8ccd544517a5841c9efa06abf9dacd (MD5) / Made available in DSpace on 2016-05-25T22:48:38Z (GMT). No. of bitstreams: 1 2006_tese_joppereira.pdf: 2085821 bytes, checksum: da8ccd544517a5841c9efa06abf9dacd (MD5) Previous issue date: 2006 / The research was carried out from January 2003 to March 2006, at the Department of Animal Science and Department of Biology in the Federal University of Ceará, in Fortaleza, Ceará. Bee samples were collected in the states of Ceará, Piauí and Goiás, Brazil. The objective of this work was to study the genetic variability of populations of the stingless bee Melipona quinquefasciata, which occurs naturally in the plateau of Araripe (South of Ceará), in the plateau of Ibiapaba (West of Ceará), in Canto do Burití, Piauí and Luziânia-Goiás (Center-Western, Brazil). Our aims were to contribute to the correct taxonomic classification of the Northeastern population and to give initial support to future work on the rational breeding of this species. Meliponiculture in rational hives for honey production will stimulate the change of the present predatory actions into a productive bee rearing activity which is ecologically sustainable. The regions of nuclear ribosomal DNA 18S and partial ITS-1 were extracted and sequenced and the following aspects were determined: nucleotid composition, matrixes of genetic distances, multiple alignments and cladograms. Results showed a high degree of similarity among the samples: 0,008 to region 18S, and 0,015 to partial ITS-1. The sequences’ size found to region 18S varied: from 1823 to 1869, and to ITS-1 from 491 to 572. The cladogram made to 18S presented a single clade. However, when external samples (M. quadrifasciata, M. subnitida, M. scutellaris, M. mandaçaia), were added to ITS-1, three groups were formed reflecting the described subgenus by the morphological taxonomy. Distance among the localities where samples were colleted was not significantly correlated to the dissimilarity of the bees to 17 18S. Nevertheless, there was a correlation with partial ITS-1, which contained the Piauí sample. Our conclusions are: (i) the bee samples from Ceará and Piauí cannot be distinguished, in molecular terms, from the bee samples of Goiás, suggesting they are the same species, although presenting some level of variability among the populations; (ii) the results reflect the taxonomy based in morphological aspects for Melipona quinquefasciata; (iii) the geographical distance suggested some level of alteration in the genoma of bees which inhabit in the three studied regions; (iv) the region partial ITS-1 of the nuclear ribosomal DNA, even in small bee samples, can help to solve taxonomic doubts at the subgenus level, in Melipona. / A pesquisa foi desenvolvida no período de janeiro de 2003 a março de 2006, nos Departamentos de Zootecnia, e Biologia da Universidade Federal do Ceará, localizada no município de Fortaleza, estado do Ceará. As amostras de abelhas foram coletadas nos estados do Ceará, Piauí e Goiás. O objetivo desta tese foi estudar a diversidade genética de populações da abelha sem ferrão Melipona quinquefasciata, ocorrendo naturalmente na Chapada do Araripe (Sul do Ceará), na Chapada da Ibiapaba (Oeste do Ceará), na cidade de Canto do Burití-PI, e na cidade de Luziânia-GO (Centro-Oeste do Brasil), visando contribuir para a correta classificação taxonômica da população nordestina e dar subsídios iniciais para trabalhos de criação racional desta espécie, em colméias para produção de mel, motivando então que a atual ação extrativista e predatória, reverta-se numa atividade zootécnica produtiva e ecologicamente sustentável. As regiões do DNA ribossômico nuclear 18S e ITS-1 parcial foram extraídas e seqüenciadas, podendo-se verificar os seguintes aspectos: Composição nucleotídica, matrizes de distância genética, múltiplos alinhamentos e cladogramas. Os resultados mostraram alto grau de similaridade entre as amostras, 0,008 para região 18S e 0,015 para o ITS-1 parcial. O tamanho das seqüências correspondente à região 18S, foram de 1823 a 1869, do ITS-1 491 a 572. O cladograma gerado para o 18S, apresentou um único clado, porém, o ITS-1 quando acrescido de amostras externas (M. quadrifasciata, M subnitida, M scutellaris, M. mandaçaia), derivou-se em três grupos, refletindo os subgêneros descritos pela taxonomia morfológica. A distância entre as áreas não se correlacionou significativamente com a dissimilaridade das abelhas para o 18S, porém houve correlação com o ITS-1 parcial, que agregou a amostra oriunda do estado do Piauí. Conclui-se (i) que as abelhas amostradas nos estados do Ceará e Piauí são indistinguíveis em termos moleculares das abelhas do estado de Goiás, sugerindo tratar-se da mesma espécie, embora apresentando algum nível de variabilidade entre as populações; (ii) os resultados encontrados refletem a taxonomia por dados morfológicos, para a espécie Melipona quinquefasciata; (iii) o distanciamento geográfico sugeriu algum grau de alteração no genoma das abelhas que nidificam nos três estados estudados; (iv) a região ITS-1 parcial do DNA ribossômico nuclear, mesmo em pequenas amostragens de abelhas, pode ajudar a resolver dúvidas taxonômicas ao nível de subgêneros, em Melipona.

The Sigma-54 Promoter Database contains computationally predicted sigma-54 promoters from over 60 prokaryotic species. Organisms from all major phyla were analysed and results were made available online at http://www.sigma54.ca. This database is particularly unique due to its inclusion of intragenic regions, grouping of data by COG and COG category, and the ability to summarize results either by phylum or database-wide. ClusterMine360 (http://www.clustermine360.ca/) is a database of microbial polyketide and nonribosomal peptide gene clusters. It takes advantage of crowd-sourcing by allowing members of the community to make contributions while automation is used to help achieve high data consistency and quality. The database currently has over 200 gene clusters from over 185 compound families. It also features a unique sequence repository containing over 10,000 PKS/NRPS domains. The sequences are filterable and downloadable as individual or multiple sequence FASTA files. This database will be a useful resource for members of the PKS/NRPS research community enabling them to keep up with the growing number of sequenced gene clusters and rapidly mine these clusters for functional information.

The non-ribosomal peptide synthase (NRPS) blue pigment synthase (BpsA) has been shown in several heterologous hosts to mediate the production of the blue pigment indigoidine from two molecules of L-glutamine. Activation of BpsA is mediated by transfer of a coenzyme A (CoA) by a 4′-phosphopantetheinyl transferase (4′-PPTase). In this thesis, I explored heterologous co-expression of BpsA and the Pseudomonas aeruginosa 4′-PPTase (PaPcpS) and their co- localization to either cytoplasm or chloroplast stroma of the green model microalga Chlamydomonas reinhardtii. The alga represents a potentially sustainable production host for indigoidine, as it is able to grow using CO2 as a sole carbon source and (sun)light for its energy. Both heterologous proteins (BpsA and PaPcpS) could be expressed as full-length fusion proteins with either the mVenus yellow fluorescent reporter or spectinomycin resistance (aadA) selection marker in both subcellular localisations. Dual transformants were identified and subjected to multiple growth conditions to determine whether indigoidine was produced. Under no condition tested was indigoidine detected, indicating that either activation of BpsA or the catalysis of L-glutamine to indigoidine was not occurring in alga. Future work will be required to determine whether it is possible to activate the BpsA in C. reinhardtii. However, this represents the first documented example of expression of a heterologous NRPS in a eukaryotic alga and may serve as foundational work for other target NRPS expression projects.