No description available.

During early oogenesis in Xenopus laevis about half of the 5S ribosomal RNA and most of the transfer RNA produced is stored in a ribonucleoprotein (RNP) complex that sediments at 42S. The other half of the 5S RNA produced is stored in a separate 73 RNP particle. As ribosome production gets underway in mid-oogenesis both the 1|2S and 7S particles disappear, the 5S rRNA being incorporated into the ribosomes, the tRNA being released as a slower sedimenting particle. Heterogeneity in the composition of the 42S particle was observed with respect to both protein and RNA components. The proteins of the 423 particle, Mr 48000 (P48) and Mr 43000 (P43) appear to be cleaved to smaller proteins. A derivative of P43 of Mr 17000 (P17) possibly becomes a ribosomal protein. Several observations are consistent with the view that P43 may accompany 53 RNA to the nucleolus before its cleavage product (P17) becomes incorporated into the ribosome. The chemical and structural relationships of P48, P43 and the protein component of the 73 RNP particle, transcription factor IIIA were studied and it was established that they are the products of three different genes. The 423 particle proteins have also been shown to have a binding affinity for 53 RNA genes (P48), tRNA genes (P43) or ribosomal genes (P43). In vivo inhibition of 5S RNA transcription by an anti-P48 antibody or tRNA transcription by an anti-P43 antibody suggest that these proteins may have a role in the transcription of these genes. Interactions between P43 and tRNA genes were analysed in most detail and indicating a specific interaction between these two components. The results taken as a whole suggest a central role for 42S particle proteins in particular P43, in co-ordinating the formation of the protein translational machinary.

DNA double-strand breaks (dsb) are thought to be major radiation-induced lesions in biological end-points such as cell lethality and chromosome aberrations. Based on this notion, this project aimed to extend further the investigation of the role of dsb in radiation-induced mutagenesis. The initial part of the project involved optimising conditions for the mutation assay so as to select for 'true' tk-mutants in Chinese hamster cells, following treatment with X-rays. This was important, due to the insufficient previous mutation data involving this locus in Chinese hamster cells. Furthermore, the choice of the tk locus over the more commonly used hprt locus was based on existing evidence of its higher sensitivity, as found in mutation experiments with the L5178Y mouse lymphoma cell line (Evans et al, 1986). An Initial comparative study was carried out to measure the induced mutation frequency following X-ray irradiation in both the parent Chinese hamster Ovary (CHO KI) cell line and its X-ray- sensitive mutant (xrs 5) cell line. This mutant line was chosen because of its characteristic marked deficiency in dsb repair, yet normal ability to rejoin single-strand breaks (Kemp et al, 1984., Costa and Bryant, 1988). This allowed the study of the role of dsb in mutation induction. The enhanced mutation induction observed in xrs 5 over that in CHO KI cells suggested the Importance of dsb in radiation-induction mutagenesis. The next experimental strategy adopted involved the use of the DNA synthesis inhibitor, 9-D-arabinofuranosyladenine (ara A). The choice of this drug was based on previous work by Bryant and Blocher (1982) and Iliakis and Bryant (1983) who, using DNA unwinding and neutral velocity sedimentation, showed ara A to strongly inhibit dsb repair, Plateau-phase CHO KI cells were exposed to X-rays alone or in combination width ara A , the latter treatment showing an increased induction of mutations. This suggested the possible existence of dsb which are fixed as mutations in the absence of DNA polymerization, suggesting a sub-class of dsb which may be critical in the steps leading to the induction of a mutation. XV The third approach was to use restriction endonucleases (RE) which were introduced into cells by electroporation. This method unlike ionising radiation, induced 'pure' dsb. The use of this method was based on the work of Bryant (1984), who used RE to mimic radiation-induced damage in the induction of chromosomal aberrations. Two different types of RE were used: those which produce blunt- and those which produce cohesive-ended dsb. In all mutation experiments with these enzymes, blunt-ended dsb were found to be more effective in generating mutations compared to cohesive-ended dsb. This suggests a possible further resolution of type(s) of dsb that would be induced by radiation in the ability to induce mutations i.e dependent on the end-structure of the induced dsb. Blunt-ended dsb may thus represent the major type of critical pre-mutational lesions which may be fixed as a mutation, as a result of misrepair. Cohesive- ended dsb may be of lesser importance. Finally, a RE (Pvu II) which generates blunt-ended dsb was used to induce mutations at the hprt locus in Chinese hamster (V79) cells. DNA from mutant cells was analysed using Southern blot and PCR analysis of 3 exons in the hprt gene. Some of the mutants (5/15) showed large deletions (representing complete loss of the gene), a change similar to that observed in mutants Induced following treatment with ionising radiation (e.g. Thacker, 1986). However, the percentage of large deletion mutants (70%) observed in radiation- induced mutants was higher than that (~34%) obtained with RE- induced mutation data. This preliminary data on the analysis of RE- induced mutations suggests that blunt-ended dsb mimics radiation- induced pre-mutational lesions, resulting in some large genomic changes (e.g. large deletions). However, a larger number of RE- induced mutants would have to be analysed before a more accurate comparison between RE and ionising mutation data can be made. In summary, this study provides evidence for dsb as a major pre-mutational lesion in cells exposed to ionising radiation, and suggests the existence of a sub-class of dsb in relation to mutation induction. In addition, RE offer the possibility of gaining further understanding of the role of dsb in the origin of mutations such as those caused by deletions.

It has been demonstrated that administration of prednisolone to rats causes loss of RNA and protein in the gastrocnemius muscle and increases in RNA and protein levels in the liver. These changes have been shown to involve alterations in RNA turnover in the two tissues. In liver, rate of synthesis of ribosomal RNA was shown to be increased and its rate of breakdown decreased following administration of prednisolone. In muscle, the steroid caused net decreases in rates of synthesis and breakdown of ribosomal RNA. These prednisolone-induced alterations in RNA turnover were not the result of changes in the RNA precursor pool in the tissues. Prednisolone treatment was shown to cause an increase in the activity of the RNA polymerase believed to be responsible for ribosomal RNA synthesis in the liver. This enzyme activity in the muscle was decreased following administration of prednisolone. Ribonuclease activity was decreased in the liver and muscle of prednisolone-treated animals. These alterations of enzymic activities could explain the observed changes in RNA turnover in prednisolone-treated animals. More prednisolone binding occurred in the liver cytosol than occurred in the muscle cytosol. Prednisolone receptors in the muscle appeared to be of a smaller molecular size than receptors in the liver. The prednisolone-receptor complexes from muscle were less stable in a low ionic environment than complexes from the liver. These quantitative and qualitative differences may have dictated the response of each tissue to prednisolone. In the course of this work conditions have been established for isolating cytoplasmic RNA from rat gastrocnemius muscle in an essentially undegraded form and in good yield.

The technique of in situ hybridisation was optimised for the detection of viral RNA using radioactively-labelled single-stranded DNA and RNA probes, and applied to three areas of interest. Optimum hybridisation conditions were determined in vitro using cells infected with the single-stranded negative sense RNA paramyxoviruses. Transcription of RNA probes was the most rapid and efficient method of probe labelling, since electrophoretic purification was not required and large amounts of RNA were produced. However, their use for in situ hybridisation was problematic due to RNase contamination and low sensitivity. In contrast, DNA probes produced from M13 clones and oligonucleotide probes gave consistent hybridisation results and were preferred in subsequent studies for their ease of use, stability and sensitivity. The effect of virus-host interactions on the clearance of the paramyxovirus, SV5, in a mouse model was investigated by detection of viral RNA and protein in lung sections. Immunisation with purified SV5 proteins prior to infection provided protection against infection, indicated by a reduction in the level of viral RNA and protein, due to enhanced clearance of virus by primed T cells. X-irradiation of the host prior to infection resulted in prolonged or persistent infection in which RNA was detected up to 19 days post-infection. The potential of in situ hybridisation for detection of aetiological agents was demonstrated by investigation of the presence of measles virus in two chronic human diseases. Thus, measles virus RNA was detected in brain sections from a patient with subacute sclerosing panencephalitis and in the osteoclasts of bone sections from a patient with Paget's disease of bone. In situ hybridisation was used to analyse expression of the two immediate-early genes of herpesvirus saimiri, the 52K gene and the hinG gene. Differential expression was detected by hybridisation to mRNA using oligonucleotide probes, in productively-infected cells. The 52K gene was expressed asynchronously throughout the population in agreement with immunocytochemical detection of the 52K protein. In contrast, the hinG gene was expressed synchronously, with all cells showing similar levels of hybridisation, indicating a specific control mechanism for expression of the 52K gene, which differs from that of the hinG gene in requiring or being inhibited by additional factors. This may have relevance to the mechanism of establishment of latency in this virus.

Submitted by Marcio Filho (marcio.kleber@ufba.br) on 2017-06-02T12:54:45Z No. of bitstreams: 1 Dissertacao_Carlos_Eduardo_Tanajura_da_Silva.pdf: 4957398 bytes, checksum: 6c7b0143e1fa8db0895eb3ea9bd85d0b (MD5) / Approved for entry into archive by Vanessa Reis (vanessa.jamile@ufba.br) on 2017-06-08T11:03:34Z (GMT) No. of bitstreams: 1 Dissertacao_Carlos_Eduardo_Tanajura_da_Silva.pdf: 4957398 bytes, checksum: 6c7b0143e1fa8db0895eb3ea9bd85d0b (MD5) / Made available in DSpace on 2017-06-08T11:03:34Z (GMT). No. of bitstreams: 1 Dissertacao_Carlos_Eduardo_Tanajura_da_Silva.pdf: 4957398 bytes, checksum: 6c7b0143e1fa8db0895eb3ea9bd85d0b (MD5) / O Brasil destaca-se entre os maiores exportadores de grãos do mundo, sendo assim, cada vez mais surgem novos produtos e derivados destas Commodity. Os métodos para classificação desses produtos muitas vezes são custosos e demorados, quase sempre se valendo de técnicas de química analítica e métodos matemáticos como PCA (Principal Component Analysis), PCR (Principal Components Regression) ou PLS (Properties of Partial Least Squares) e RNA (Redes Neurais Artificiais) para aumentar sua eficiência. Devido à grande variedade de produtos são necessários métodos mais eficientes para qualificar, caracterizar e classificar estas substâncias, uma vez que o preço final deve refletir a excelência do produto que chega ao consumidor. Este trabalho propõe uma solução para classificação de óleos vegetais: Canola, Girassol, Milho e Soja colocados no mercado por diferentes marcas e fabricantes. O método de análise empregado é a fluorescência induzida por LED de amostras de óleo diluídas em heptano, com diferentes concentrações, sendo que a classificação dos espectros de fluorescência foi feita por RNA. Foram produzidas e caracterizadas 640 amostras, sendo 480 para treinamento da rede neural e 160 para sua validação. Para a classificação das amostras de fluorescência, os dados foram organizados em dois estudos, o primeiro com referência ao tipo das amostras, o segundo a titulação, este por final contento três arranjos dos dados e RNAs distintas. Na classificação do tipo das amostras, a rede conseguiu identificar 115 amostras, tendo acertado aproximadamente 72% destas amostras de validação. A classificação por titulação, utilizou a metade das amostras de fluorescência, o universo de treinamento passou a ter 240 amostras, as de validação 80. Para esse segundo estudo houve 3 arranjos desses dados, o resultado do primeiro arranjo teve 33 amostras classificadas com sucesso de 80, o segundo 49 e o terceiro 31.

No description available.

The factors that determine RNA structure formation, stability, and dynamics are inexorably linked to RNA function. The Hammerhead ribozyme (HHRz) has long served as a model for studying metal-dependent folding and catalysis in RNA. The HHRz consists of three helices meeting at a common junction of conserved nucleotides that form the active site of the ribozyme. Current models of metal-dependent HHRz function involve a requirement for divalent metals to globally fold the ribozyme at low metal concentrations, followed by a second metal-dependent process which activates the HHRz for catalysis. The exact role of metal ions in activating HHRz catalysis is still a subject of investigation. We used 2-aminopurine substitutions near the active site of the ribozyme to determine if this second metal-dependent process involves a conformational rearrangement in the core of the ribozyme. We find evidence for a conformational change beyond global folding in the core of the ribozyme that not only correlates with metal activated catalysis but is also sensitive to the identity of the metal ions used for folding. Though phosphorothioate substitutions indicate that a ground-state coordination of a catalytic metal to the scissile phosphate is required for efficient catalysis, our folding studies show that this coordination event is not absolutely required for folding of the HHRz core. To investigate possible roles for metal ions in general acid-base catalysis, we tested the pH dependence of the HHRz rate using a variety of metal ions. We find the pH dependent rate profile of the ribozyme is shifted by transition metal ions, whereas other group II metals show similar profiles to Mg

DEAD-BOX (DDX) RNA helicases are a large family of proteins characterized by the presence of a DEAD/H (Asp-Glu-Ala-Asp/His) motif. Their main function is to unwind double stranded RNA to promote downstream molecular events. They are involved in virtually all steps of RNA metabolism such as transcription, translation, RNA export and degradation, ribosome biogenesis and pre-mRNA splicing. The aim of my work was to investigate the functions of human DDX3X and DDX54 RNA helicases, particularly in in vitro cancer models. To provide insight into their molecular functions, I identified RNAs bound by DDX3X and DDX54 in breast cancer (MCF7) cells by performing iCLIP experiments. This generated two very distinct RNA binding profiles: DDX3X preferentially bound exonic regions of mRNAs encoding translational factors, whilst DDX54 preferentially bound non-coding RNAs and intronic regions of mRNAs encoding nuclear proteins. Further bioinformatic analysis identified a few discrete binding motifs within DDX3X target RNAs. One of these, within the human JUND transcript, was validated as a DDX3X binding site using electrophoretic mobility shift assays. Notably, the levels of proteins encoded by mRNAs bound by DDX3X were altered following knockdown of the helicase. These data highlight the importance of DDX3X in maintaining appropriate levels of certain proteins, which in turn may explain at least some of the changes in phenotype observed upon DDX3X knockdown. In this regard, I showed that knocking down DDX3X or DDX54 in MCF7 cells slowed cell proliferation by inducing a G1/S phase arrest. Furthermore, a CRISPR/Cas9 dropout screen in a leukaemia cell line (MLL-AF9) showed that both helicases are essential for growth of these cells. However, loss of DDX3X or DDX54 had little effect on proliferation of immortalized NIH3T3 cells indicating that their loss is not generally lethal. In subsequent CRISPR/Cas9 dropout screens in various other cancer cell lines, including some derived from solid tumours, DDX54 was found to be essential for growth of all cell lines. In contrast, DDX3X was required for proliferation of only a subset of these. Focusing on DDX3X, I identified that the integrity of the helicase's RNA binding domains is essential for growth and cell cycle progression of an acute myeloid leukaemia cell line (OCI-AML3). Overall, my findings shed mechanistic insight upon the role of DDX RNA helicases in cancer and identify DDX3X and DDX54 as potential targets for therapeutic intervention in certain cancers.

Orientadores: Iscia Teresinha Lopes-Cendes, Ivan de Godoy Maia / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-04T19:04:59Z (GMT). No. of bitstreams: 1 Pereira_TiagoCampos_D.pdf: 3895694 bytes, checksum: d999bfc92e9a2e2c757db34bbfc7d7fa (MD5) Previous issue date: 2005 / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular