Viróides e virusóides: relíquias do mundo de RNA

Até meados do século XX, os vírus eram considerados os representantes mais simples da escala biológica. A descoberta dos RNAs satélites e dos viróides por volta de 1970 foi surpreendente, pois comprovou-se a existência de uma nova classe de moléculas auto-replicativas ainda mais simples, denominada agentes sub-virais. Há indícios de que os viróides e virusóides (que formam uma classe de RNAs satélites), teriam feito parte do "Mundo de RNA" (que precedeu o mundo atual baseado no DNA e proteínas), podendo ser considerados fósseis moleculares dessa era antiga. A simplicidade desses agentes sub-virais e o fato de que a molécula de RNA deve interagir diretamente com fatores do hospedeiro para o desenvolvimento do seu ciclo infeccioso colocam esses patógenos como um modelo para o estudo de processos metabólicos celulares. Nos últimos anos, tem-se observado um volume grande de publicações visando elucidar aspectos da interação viróide/hospedeiro, como os mecanismos da patogênese, movimento dos viróides nas plantas hospedeiras, silenciamento gênico e atividades das ribozimas. Mudanças recentes ocorridas na taxonomia desses patógenos com a criação de famílias, gêneros e espécies, além da descoberta de novos viróides, também têm sido verificadas. A presente revisão visa atualizar o leitor quanto aos recentes avanços nas pesquisas com viróides, principalmente na taxonomia, filogenia e em vários aspectos moleculares da interação viróide/hospedeiro. Estão incluídas também algumas características dos virusóides e sua relação evolutiva com os viróides.

The Significance of Hydrogen Bonding Interactions in the Cleavage of RNA

RNA is essential for all living organisms. It has important roles in protein synthesis, controlling gene expression as well as catalyzing biological reactions. Chemically RNA is a very stable molecule, although in biological systems many agents catalyze the cleavage of RNA, such as naturally occurring enzymes and ribozymes. Much effort has been put in the last decades in developing highly active artificial ribonucleases since such molecules could have potential in the therapeutic field and provide tools for molecular biology. Several potential catalysts have emerged, but usually detailed cleavage mechanism remains unresolved. This thesis is aimed at clarifying mechanistic details of the cleavage and isomerization of RNA by using simpler nucleoside models of RNA. The topics in the experimental part cover three different studies, one concerning the mechanism of catalysis by large ribozymes, one dealing with the reactivity of modified and unmodified RNA oligonucleotides and one showing an efficient catalysis of the cleavage and isomerization of an RNA phosphodiester bond by a dinuclear metal ion complex. A review of the literature concerning stabilization of the phosphorane intermediate of the hydrolysis and isomerization of RNA phosphodiester bond is first presented. The results obtained in the experimental work followed by mechanistic interpretations are introduced in the second part of the thesis. Especially the significance of hydrogen bonding interactions is discussed.

Novel CO2 Regulated Proteins in Synechocystis PCC 6803

The large biodiversity of cyanobacteria together with the increasing genomics and proteomics metadata provide novel information for finding new commercially valuable metabolites. With the advent of global warming, there is growing interest in the processes that results in efficient CO2 capture through the use of photosynthetic microorganisms such as cyanobacteria. This requires a detailed knowledge of how cyanobacteria respond to the ambient CO2. My study was aimed at understanding the changes in the protein profile of the model organism, Synechocystis PCC 6803 towards the varying CO₂ level. In order to achieve this goal I have employed modern proteomics tools such as iTRAQ and DIGE, recombinant DNA techniques to construct different mutants in cyanobacteria and biophysical methods to study the photosynthetic properties. The proteomics study revealed several novel proteins, apart from the well characterized proteins involved in carbon concentrating mechanisms (CCMs), that were upregulated upon shift of the cells from high CO₂ concentration (3%) to that in air level (0.039%). The unknown proteins, Slr0006 and flavodiiron proteins (FDPs) Sll0217-Flv4 and Sll0219-Flv2, were selected for further characterization. Although slr0006 was substantially upregulated under C_i limiting conditions, inactivation of the gene did not result in any visual phenotype under various environmental conditions indicating that this protein is not essential for cell survival. However, quantitative proteomics showed the induction of novel plasmid and chromosome encoded proteins in deltaslr0006 under air level CO₂ conditions. The expression of the slr0006 gene was found to be strictly dependent on active photosynthetic electron transfer. Slr0006 contains conserved dsRNA binding domain that belongs to the Sua5/YrdC/YciO protein family. Structural modelling of Slr0006 showed an alpha/beta twisted open-sheet structure and a positively charged cavity, indicating a possible binding site for RNA. The 3D model and the co-localization of Slr0006 with ribosomal subunits suggest that it might play a role in translation or ribosome biogenesis. On the other hand, deletions in the sll0217-sll218- sll0219 operon resulted in enhanced photodamage of PSII and distorted energy transfer from phycobilisome (PBS) to PSII, suggesting a dynamic photoprotection role of the operon. Constructed homology models also suggest efficient electron transfer in heterodimeric Flv2/Flv4, apparently involved in PSII photoprotection. Both Slr0006 and FDPs exhibited several common features, including negative regulation by NdhR and ambiguous cellular localization when subjected to different concentrations of divalent ions. This strong association with the membranes remained undisturbed even in the presence of detergent or high salt. My finding brings ample information on three novel proteins and their functions towards carbon limitation. Nevertheless, many pathways and related proteins remain unexplored. The comprehensive understanding of the acclimation processes in cyanobacteria towards varying environmental CO₂ levels will help to uncover adaptive mechanisms in other organisms, including higher plants.

Georeferenced database generation with the purpose of hydrologic molding in reservoirs of the hydrographic basin of Jaguaribe river in the state of Ceará, Brazil

The edafoclimatic conditions of the Brazilian semiarid region favor the water loss by surface runoff. The state of Ceará, almost completely covered by semiarid, has developed public policies for the construction of dams in order to attend the varied water demand. Several hydrological models were developed to support decisive processes in the complex management of reservoirs. This study aimed to establish a methodology for obtaining a georeferenced database suitable for use as input data in hydrological modeling in the semiarid of Ceará. It was used images of Landsat satellite and SRTM Mission, and soil maps of the state of Ceará. The Landsat images allowed the determination of the land cover and the SRTM Mission images, the automatic delineation of hydrographic basins. The soil type was obtained through the soil map. The database was obtained for Jaguaribe River hydrographic basin, in the state of Ceará, and is applicable to hydrological modeling based on the Curve Number method for estimating the surface runoff.

Detection of rabies virus nucleoprotein-RNA in several organs outside the Central Nervous System in naturally-infected vampire bats

Rabies is a neurological disease, but the rabies virus spread to several organs outside the central nervous system (CNS). The rabies virus antigen or RNA has been identified from the salivary glands, the lungs, the kidneys, the heart and the liver. This work aimed to identify the presence of the rabies virus in non-neuronal organs from naturally-infected vampire bats and to study the rabies virus in the salivary glands of healthy vampire bats. Out of the five bats that were positive for rabies in the CNS, by fluorescent antibody test (FAT), viral isolation in N2A cells and reverse transcription - polymerase chain reaction (RT-PCR), 100% (5/5) were positive for rabies in samples of the tongue and the heart, 80% (4/5) in the kidneys, 40% (2/5) in samples of the salivary glands and the lungs, and 20% (1/5) in the liver by RT-PCR test. All the nine bats that were negative for rabies in the CNS, by FAT, viral isolation and RT-PCR were negative for rabies in the salivary glands by RT-PCR test. Possible consequences for rabies epidemiology and pathogenesis are discussed in this work.

Chromatoid body mediated RNA regulation in mouse male germline

Male germ cell differentiation, spermatogenesis is an exceptional developmental process that produces a massive amount of genetically unique spermatozoa. The complexity of this process along with the technical limitations in the germline research has left many aspects of spermatogenesis poorly understood. Post-meiotic haploid round spermatids possess the most complex transcriptomes of the whole body. Correspondingly, efficient and accurate control mechanisms are necessary to deal with the huge diversity of transcribed RNAs in these cells. The high transcriptional activity in round spermatids is accompanied by the presence of an uncommonly large cytoplasmic ribonucleoprotein granule, called the chromatoid body (CB) that is conjectured to participate in the RNA post-transcriptional regulation. However, very little is known about the possible mechanisms of the CB function. The development of a procedure to isolate CBs from mouse testes was this study’s objective. Anti-MVH immunoprecipitation of cross-linked CBs from a fractionated testicular cell lysate was optimized to yield considerable quantities of pure and intact CBs from mice testes. This protocol produced reliable and reproducible data from the subsequent analysis of CB’s protein and RNA components. We found that the majority of the CB’s proteome consists of RNA-binding proteins that associate functionally with different pathways. We also demonstrated notable localization patterns of one of the CB transient components, SAM68 and showed that its ablation does not change the general composition or structure of the CB. CB-associated RNA analysis revealed a strong accumulation of PIWI-interacting RNAs (piRNAs), mRNAs and long non-coding RNAs (lncRNAs) in the CB. When the CB transcriptome and proteome analysis results were combined, the most pronounced molecular functions in the CB were related to piRNA pathway, RNA post-transcriptional processing and CB structural scaffolding. In addition, we demonstrated that the CB is a target for the main RNA flux from the nucleus throughout all steps of round spermatid development. Moreover, we provided preliminary evidence that those isolated CBs slice target RNAs in vitro in an ATPdependent manner. Altogether, these results make a strong suggestion that the CB functions involve RNA-related and RNA-mediated mechanisms. All the existing data supports the hypothesis that the CB coordinates the highly complex haploid transcriptome during the preparation of the male gametes for fertilization. Thereby, this study provides a fundamental basis for the future functional analyses of ribonucleoprotein granules and offers also important insights into the mechanisms governing male fertility.

Primary and Revision Hip Replacement. A University Hospital Database and Registry Study

Large-headed total hip arthroplasty (THA) and hip resurfacing arthroplasty (HRA) with metal-on-metal (MoM) bearings became popular during the last decade. Recently, it has become evident that the large-head MoM hip implants are associated with increased revision rates despite their theoretical advantages. The purpose of this study was to evaluate the early results of primary MoM hip replacements and of acetabular revisions. I analyzed retrospectively the results of four MoM implant designs and the survival rate of acetabular revisions with impaction bone grafting, as documented in the Turku University Hospital database. Further, I evaluated the correlation between femoral head size and dislocation rate, and used the Finnish Arthroplasty Register data to compare the survival of three large-head MoM THAs to analogous HRAs. The early results for the Magnum M2A–ReCap THA were good. A larger head size decreased the risk of dislocation. Articular surface replacement (ASR) THA yielded inferior results compared to analogous HRA. For two other designs the results were similar. The R3–Synergy THA yielded inferior results compared to the reference implants. The survival of acetabular reconstructions with impaction bone grafting was inferior compared to previous reports. In conclusion, the early results of the Biomet ReCap–Magnum design were promising, and large head sizes decreased the dislocation rate. The survival of different MoM hip implant designs varied. The survival of new designs and techniques may be inferior to those reported by the clinics where implants are developed. An important caveat is that early promising results of new devices may rapidly worsen. New implants need to be introduced in a controlled fashion to the market; here, arthroplasty registers are a valuable tool that needs to be used.

Improving reporting management with relational database management system

Data management consists of collecting, storing, and processing the data into the format which provides value-adding information for decision-making process. The development of data management has enabled of designing increasingly effective database management systems to support business needs. Therefore as well as advanced systems are designed for reporting purposes, also operational systems allow reporting and data analyzing. The used research method in the theory part is qualitative research and the research type in the empirical part is case study. Objective of this paper is to examine database management system requirements from reporting managements and data managements perspectives. In the theory part these requirements are identified and the appropriateness of the relational data model is evaluated. In addition key performance indicators applied to the operational monitoring of production are studied. The study has revealed that the appropriate operational key performance indicators of production takes into account time, quality, flexibility and cost aspects. Especially manufacturing efficiency has been highlighted. In this paper, reporting management is defined as a continuous monitoring of given performance measures. According to the literature review, the data management tool should cover performance, usability, reliability, scalability, and data privacy aspects in order to fulfill reporting managements demands. A framework is created for the system development phase based on requirements, and is used in the empirical part of the thesis where such a system is designed and created for reporting management purposes for a company which operates in the manufacturing industry. Relational data modeling and database architectures are utilized when the system is built for relational database platform.

RNA and DNA aptamers as potential tools to prevent cell adhesion in disease

Recent research has shown that receptor-ligand interactions between surfaces of communicating cells are necessary prerequisites for cell proliferation, cell differentiation and immune defense. Cell-adhesion events have also been proposed for pathological conditions such as cancer growth, metastasis, and host-cell invasion by parasites such as Trypanosoma cruzi. RNA and DNA aptamers (aptus = Latin, fit) that have been selected from combinatorial nucleic acid libraries are capable of binding to cell-adhesion receptors leading to a halt in cellular processes induced by outside signals as a consequence of blockage of receptor-ligand interactions. We outline here a novel approach using RNA aptamers that bind to T. cruzi receptors and interrupt host-cell invasion in analogy to existing procedures of blocking selectin adhesion and function in vitro and in vivo.

Molecular characterization of DDX26, a human DEAD-box RNA helicase, located on chromosome 7p12

DEAD-box proteins comprise a family of ATP-dependent RNA helicases involved in several aspects of RNA metabolism. Here we report the characterization of the human DEAD-box RNA helicase DDX26. The gene is composed of 14 exons distributed over an extension of 8,123 bp of genomic sequence and encodes a transcript of 1.8 kb that is expressed in all tissues evaluated. The predicted amino acid sequence shows a high similarity to a yeast DEAD-box RNA helicase (Dbp9b) involved in ribosome biogenesis. The new helicase maps to 7p12, a region of frequent chromosome amplifications in glioblastomas involving the epidermal growth factor receptor (EGFR) gene. Nevertheless, co-amplification of DDX26 with EGFR was not detected in nine tumors analyzed.

Low variation in ribosomal DNA and internal transcribed spacers of the symbiotic fungi of leaf-cutting ants (Attini: Formicidae)

Leaf-cutting ants of the genera Atta and Acromyrmex (tribe Attini) are symbiotic with basidiomycete fungi of the genus Leucoagaricus (tribe Leucocoprineae), which they cultivate on vegetable matter inside their nests. We determined the variation of the 28S, 18S, and 5.8S ribosomal DNA (rDNA) gene loci and the rapidly evolving internal transcribed spacers 1 and 2 (ITS1 and ITS2) of 15 sympatric and allopatric fungi associated with colonies of 11 species of leafcutter ants living up to 2,600 km apart in Brazil. We found that the fungal rDNA and ITS sequences from different species of ants were identical (or nearly identical) to each other, whereas 10 GenBank Leucoagaricus species showed higher ITS variation. Our findings suggest that Atta and Acromyrmex leafcutters living in geographic sites that are very distant from each other cultivate a single fungal species made up of closely related lineages of Leucoagaricus gongylophorus. We discuss the strikingly high similarity in the ITS1 and ITS2 regions of the Atta and Acromyrmex symbiotic L. gongylophorus studied by us, in contrast to the lower similarity displayed by their non-symbiotic counterparts. We suggest that the similarity of our L. gongylophorus isolates is an indication of the recent association of the fungus with these ants, and propose that both the intense lateral transmission of fungal material within leafcutter nests and the selection of more adapted fungal strains are involved in the homogenization of the symbiotic fungal stock.

The RNA interference revolution

The discovery of double-stranded RNA-mediated gene silencing has rapidly led to its use as a method of choice for blocking a gene, and has turned it into one of the most discussed topics in cell biology. Although still in its infancy, the field of RNA interference has already produced a vast array of results, mainly in Caenorhabditis elegans, but recently also in mammalian systems. Micro-RNAs are short hairpins of RNA capable of blocking translation, which are transcribed from genomic DNA and are implicated in several aspects from development to cell signaling. The present review discusses the main methods used for gene silencing in cell culture and animal models, including the selection of target sequences, delivery methods and strategies for a successful silencing. Expected developments are briefly discussed, ranging from reverse genetics to therapeutics. Thus, the development of the new paradigm of RNA-mediated gene silencing has produced two important advances: knowledge of a basic cellular mechanism present in the majority of eukaryotic cells and access to a potent and specific new method for gene silencing.