Prokaryotic cells such as Escherichia coli use glucose as their preferred carbon source. In the absence of glucose, these cells resort to other sources to generate glucose and this process of de novo synthesis of glucose is termed gluconeogenesis. Phosphoenolpyruvate carboxykinase (Pck) is one of the three enzymes important in regulating gluconeogenesis. It converts oxaloacetic acid (OAA) from the Krebs cycle to phosphoenolpyruvate (PEP), a glycolytic intermediate. The Pck structural gene (pckA) is regulated by catabolite repression. There is a 100-fold induction of pckA-lacZ fusions at the onset of stationary phase concurrent with induction of glycogen synthesis. Mutants affecting the expression of pckA were analysed to shed some light on the mechanism of its genetic regulation.<p>Spontaneous mutants isolated with Pck- (lack of PEP carboxykinase activity) and Suc- (inability to utilise succinate as carbon source) phenotypes were previously characterised as atpG mutants defective in the ã subunit of ATP synthase.<p>In this work we find by reverse transcriptase and real time quantitative PCR that levels of pckA mRNA are normal in the atpG mutants and that the defects in expression of pckA are therefore likely at the level of translation, protein assembly and/or protein degradation. As expected, ATP synthase activity and proton pumping in inside-out membrane vesicles were defective in these atpG mutants. It is likely that one of these defects is affecting regulation or expression of the pckA gene. It was observed that atpG mutants were defective in calcium-dependent transformation although they could be made competent for electroporation. The atpG mutants were also defective for growth of P1 bacteriophage although they could serve as recipients for P1-dependent generalised transduction. These latter phenotypes are also likely due to defects in energy metabolism.

La surexpression rétrovirale du facteur de transcription HOXB4 résulte en une expansion sélective des cellules souches hématopoïétiques (CSH) in vitro et in vivo et ce, sans induire de leucémie. Par contre, la demi-vie intracellulaire de la protéine est de seulement une heure et le fait que la protéine disparaît du milieu de culture après environ 4 heures représente un obstacle majeur à l’utilisation clinique de la protéine HOXB4. Trois mutants HOXB4 ayant une substitution d`un seul acides aminés (AA) parmi les 31 premiers AA ont démontré une augmentation de la stabilité de la protéine. Nous avons donc évalué l’effet de HOXB4 et de ses trois mutants sur la production de cellules progénitrices myéloïdes. L’expression ectopique de HOXB4 sauvage (s-HOXB4) et HOXB4 mutant (m-HOXB4) a un effet comparable sur la fréquence des cellules progénitrices myéloïdes en essai clonogénique. Par contre, la capacité de prolifération des cellules progénitrices myéloïdes qui surexpriment s-HOXB4 et 1423 m-HOXB4 a été supérieure à celle des cellules contrôles (GFP seul) et des deux autres mutants. De plus, malgré le fait que toutes les variantes de HOXB4 confèrent une capacité d’autorenouvellement similaire aux cellules progénitrices multipotents (GEMM), la production des progéniteurs granulocytaires (CFU-G) est compromise lorsque les cellules surexpriment 1426 et 1427 m-HOXB4. D’autre part, la densité cellulaire des colonies myéloïdes qui surexpriment ces deux mutants est diminuée, ce qui suggère que ces mutations ont non seulement augmenté sa stabilité, mais potentiellement affecté certaines fonctions biologiques de s-HOXB4. Enfin, 1423 m-HOXB4 semble n’avoir perdu aucune fonction de s-HOXB4 dans nos évaluations clonogéniques in vitro, ce qui fait de ce mutant une molécule intéressante pour des applications cliniques d’expansion des cellules progénitrices hématopoïétiques. / Over-expression of the homeobox transcription factor HOXB4 results in a selective expansion of HSC in vitro and in vivo. However, the intracellular half-life of the protein is only one hour, which represents an important obstacle for its clinical use. Three HOXB4 mutants with single amino acid (AA) substitution in the first 31 AA have demonstrated increased intracellular stability of the protein. We evaluated the effect of wild type (wt) and mutant (m) HOXB4 on expansion and differentiation of myeloid progenitors. Ectopic expressions of wt- and m- HOXB4 had a comparable effect on the frequency of myeloid progenitors in semi-solid assay. However, the proliferative capacity of myeloid progenitors expressing wt-HOXB4 and 1423 m-HOXB4 was considerably better than that of control (GFP only) and other two mutants. Additionally, while all HOXB4 variants conferred similar self-renewal capacity to multipotent (GEMM) progenitors, the production of single-lineage granulocytic progenitors (CFU-G) was severely compromised when cells were expressing 1426 and 1427 m-HOXB4. Conversely, the cellularity of myeloid colonies was also diminished with these mutants suggesting, that both mutations affect certain biologic function(s) of wt-HOX4 in addition to increasing its stability. On the other hand, 1423 m-HOXB4 did not seem to lose any wt-HOXB4 functions tested in in vitro assays making this mutant an interesting target for further investigation.

The arbuscular mycorrhizal (AM) association results from a successful interaction between the genomes of the two symbiotic partners. In this context, the aim of my research was to better characterize the role of the late stage symbiosis-related pea genes PsSym36, PsSym33 and PsSym40 in the functional AM (i) by investigating the effect of mutations in the three genes on fungal and plant gene responses and (ii) by creating conditions for the localization of two of the genes, PsSym36 and PsSym40, on the pea genetic map for future map-based cloning. The expression of a subset of ten fungal and eight plant genes,previously reported to be activated during mycorrhiza development, was compared in Glomus intraradices-inoculated roots of wild type and Pssym36, Pssym33 and Pssym40 mutant pea plants. Most of the fungal genes were down-regulated in roots of the Pssym36 mutant where arbuscule formation is defective, and several were upregulated with more rapid fungal development in roots of the Pssym40 mutant. Microdissection of mycorrhizal PsSym40 roots corroborated preferential expression of the three G. intraradices genes SOD, DESAT and PEPISOM in arbuscule-containing cells. Inactivation of PsSym36 also resulted in down regulation of plant genes whilst mutation of the PsSym33 and PsSym40 genes affected plant gene responses in a more time-dependent way. Results thus indicate an implication of the investigated pea SYM genes in the modulation of plant and fungal molecular interactions linked to signaling, nutrient exchange or stress response regulation during AM symbiosis formation and functioning. Conditions for localization of the PsSym36 and PsSym40 genes on the pea genetic map were developed for their future map-based cloning. Based on the molecular markers obtained, it was possible to conclude that localization of the PsSym40 gene most likely resides outside the linkage groups I, II, III or V of the genetic map of pea.

No description available.

This work was conducted to identified the best conditions of conservation (temperature and atmosphere composition) for apples cultivars Fuji, Fuji Suprema and Fuji Kiku. The experiment consited of the following treatments: storage at -0,5ºC without atmosphere modification (RA); 1kPa of O2+<0,5kPa of CO2 at +0,5ºC; 0,8kPa of O2+<0,5kPa of CO2 at -0,5ºC; 1,2kPa of O2+<0,5kPa of CO2 at -0,5ºC; 1kPa of O2+ 2kPa of CO2 at -0,5ºC. The experimental arragment was completely radomized with 4 replicates of 23 fruits. Fruit quality was evaluated after 8 months of cold storage and after 7 days of shelf life at 20ºC. After 8 months of cold storage, and 7 days of shelf life at 20ºC, the fruit firmness, solids soluble concentration (SSC); tritable acidity (TA); totals polyphenols; fruits with diseases and physiological disorders; respiration and ethylene production were evaluated. At harvest the colour of the skin; reductor sugars; anthocyanins and vitamine C were evaluated moreover the variables listed before. The Fuji Suprema cv. had maximum value of anthocyanins and parameter a* of colour measurment. The Fuji Kiku cultivar showed the higher levels of firmness, SST, AT and vitamine C. Before the cold storage and shelf life period, the treatments 1,2kPa of O2 + < 0,5kPa of CO2, and 1kPa of O2 + 2kPa of CO2 showed the best results in relation of the quality especially in deleyed softening in Fuji. The CA treatments preserved the firmness in the other cultivars. SST were no afected by the AC treatments while AT were preserved in this storage conditions. The 1kPa of O2+ 2kPa of CO2 showed the lowest values of respiration and ethylene production. The Fuji cultivar and their mutant Fuji Kiku were sensible to the higher levels of CO2 (2kPa), evidencied for the most incidens of internal breakdow. The total polyphenols stained high and increase in some CA treatment for all the cultivars. No difference beteween -0,5 and + 0,5 º C, of temperature in relation of the quality, in the 1kPa of O2 + < 0,5kPa of CO2 for any of the cultivars evaluated were found. / O presente trabalho teve como objetivo identificar as melhores condições de conservação, em relação à temperatura e composição da atmosfera, de armazenamento que permitam a manutenção da qualidade das maçãs das cultivares Fuji, Fuji Suprema e Fuji Kiku. Os tratamentos avaliados foram: armazenamento a -0,5ºC sem modificação da atmosfera (AR); 1kPa de O2+<0,5kPa de CO2 a +0,5ºC; 0,8kPa de O2+<0,5kPa de CO2 a -0,5ºC; 1,2kPa de O2+<0,5kPa de CO2 a -0,5ºC; 1kPa de O2+ 2kPa de CO2 a -0,5ºC. O delineamento experimental utilizado foi o inteiramente causalizado, com 4 repetições sendo que cada unidade experimental constava de 23 frutos. Após 8 meses de armazenamento refrigerado, e 7 dias de exposição dos frutos à temperatura de 20ºC, foram avaliadas a firmeza da polpa; sólidos solúveis totais (SST), acidez titulável (AT); polifenóis totais; frutos com podridões e distúrbios fisiológicos; produção de etileno e respiração. No momento de colheita, além das variáveis anteriores, foram medidas a cor da epiderme; açúcares redutores; antocianinas totais e vitamina C. A Fuji Suprema apresentou os maiores valores de antocianinas totais e maior valor do parâmetro a* da cor, diferenciando-se estatisticamente das outras, enquanto que a cultivar Fuji Kiku mostrou os maiores valores de firmeza da polpa, SST, AT e vitamina C. De acordo com os resultados obtidos após o período de conservação e vida de prateleira, os tratamentos de 1,2kPa de O2 + < 0,5kPa de CO2, e 1kPa de O2 + 2kPa de CO2 apresentarão os maiores valores em firmeza da polpa em Fuji. Enquanto que os tratamentos de AC permitiram manter a firmeza nas outras cultivares. Não houveram diferenças nos tratamentos de atmosfera controlada (AC), para as variáveis SST mas estes tratamentos permitiram manter a AT. O tratamento de 1kPa de O2 + 2 kPa de CO2, determinou uma menor respiração e produção de etileno nas diferentes cultivares. A cultivar Fuji e sua mutante Fuji Kiku apresentaram susceptibilidade a altos níveis de CO2 (2kPa) manifestando uma maior incidência de degenerescência da polpa. Os polifenóis totais mantiveram-se elevados e inclusive aumentaram em alguns dos tratamentos de AC. Não houve diferenças entre as temperaturas de -0,5 e + 0,5, em relação a qualidade, no tratamento de 1kPa de O2 + < 0,5kPa de CO2 em nenhuma das cultivares.

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Muscle contraction is a highly fine-tuned process that requires the precise and timely construction of large protein sub-assemblies to form sarcomeres, the individual contractile units. Mutations in many of the genes encoding constituent proteins of this macromolecular machine result in defective functioning of the muscle tissue, and in humans, often lead to myopathic conditions like cardiomyopathies and muscular dystrophies, which affect a considerable number of people the world over. As more information regarding causative mutations becomes available, it becomes imperative to explore mechanisms of muscle development, maintenance and pathology. In striated muscles, contraction is regulated by the thin filament-specific tropomyosin (Tm) – troponin (Tn) complex (Ca2+-binding troponin-C, inhibitory troponin-I and tropomyosin-binding troponin-T). These troponin subunits are present in 1:1:1 ratio on thin filaments, with 1 Tm-Tn complex present on every 7th actin molecule. This stoichiometry is tightly regulated, and disturbances have been associated with functional defects. Each of these proteins has multiple isoforms, whose expression is controlled both spatially and temporally. The expression of specific combination of isoforms confers specific contractile properties to each muscle subtype. Drosophila melanogaster has been a preferred model of choice to study various aspects of muscle development for decades. In this study, the Indirect Flight Muscles (IFMs) of Drosophila have been used to investigate developmental and functional roles of two temporally regulated isoforms of a vital structural and regulatory component of the sarcomere – Troponin T (TnT). On a larger scale, whole genome expression profiles of mutants that are null for major myofbrillar proteins have also been discussed. IFMs serve as an excellent model system to address these questions, owing to the extreme ease of genetic manipulability in this system, and high degree of homology between mammalian and Dipteran cytoskeletal proteins. Chapter 1 covers basics of muscle biology, and the role of TnT in muscle contraction. Phenomena responsible for generating diversity in genes encoding muscle proteins – alternative splicing and isoform switching – have also been discussed. These mechanisms are highly conserved, as are patterns of TnT splicing and isoform expression across phyla. Mutations leading to altered splicing patterns lead to myopathic conditions, and the importance of model systems to study muscle biology has been emphasized. The advantages of studying Drosophila IFMs and a comprehensive overview of IFM development has been covered. The resources and experimental tools used have been described in Chapter 2. Two isoforms of TnT are alternatively spliced in the Drosophila thorax – one containing alternative exon 10a (expressed in adult IFMs and jump muscle); and one containing alternative exon 10b (expressed in pupae and newly eclosed flies). These exons are spliced in a mutually exclusive manner, and defects in splicing have been reported to cause uncontrolled, auto-destructive contractions. In Chapter 3, a splice mutant of TnT, up1, has been discussed, with respect to its developmental profile. Transgenic rescue experiments with two separate isoforms demonstrate rescue at the structural as well as functional level. Transgenic over-expression, however, leads to functional abnormalities, highlighting the importance of stoichiometry in multi-protein complexes. In Chapter 4, molecular signals that bring about the developmentally regulated TnT isoform switch are discussed. A splicing factor, Muscleblind, has been transgenically knocked down in normal and mutant IFMs to study effects on muscle function. Chapter 5 looks at whole genome transcriptional alterations in muscles null for either actin or myosin. All significant expression changes have been classified into categories based on different biological processes, and an attempt to differentiate generic muscle responses from filament-specific responses has been made. In conclusion, the studies have highlighted the importance of TnT isoform switching, and that extended expression of a pupal stage-specific isoform can partially compensate for loss of the adult isoform. Also, in the absence of major myofibrillar proteins, stress response pathways like heat shock response and protein degradation pathways are activated, along with a subset of metabolic responses that are unique to the thin or thick filament systems.

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Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-04-18T13:33:01Z No. of bitstreams: 2 Dissertação - Cláudio Antônio de Araújo - 2015.pdf: 6483664 bytes, checksum: bf12aa2fbdc30e9456d8036d9cc24fd1 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-04-18T13:34:40Z (GMT) No. of bitstreams: 2 Dissertação - Cláudio Antônio de Araújo - 2015.pdf: 6483664 bytes, checksum: bf12aa2fbdc30e9456d8036d9cc24fd1 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-04-18T13:34:40Z (GMT). No. of bitstreams: 2 Dissertação - Cláudio Antônio de Araújo - 2015.pdf: 6483664 bytes, checksum: bf12aa2fbdc30e9456d8036d9cc24fd1 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-12-04 / Traditionally, mutation testing is used for test set and/or test criteria evaluation once it is considered a good fault model. Since static analyzers, in general, report a substantial number of false positive warnings, Objective: This paper uses mutation testing for evaluating an automated static analyzer. The intention of this study is to define a prioritization approach of static warnings based on their correspondence with mutations. Method: We used mutation operators as a fault model to evaluate the direct correspondence between mutations and static warnings. The main advantage of using mutation operators is that they generate a large number of programs containing faults of different types, which can be used to decide the ones most probable to be detected by static analyzers. Results: The results obtained for a set of open-source programs indicate that: 1) correspondence exists when considering specific mutation operators such that static warnings may be prioritized based on their correspondence level with mutations; 2) correspondence exists when considering specific warning categories such that, assuming we perform static analysis considering these warning categories, mutation operators may be prioritized based on their correspondence level with warnings. Conclusion: It is possible to provide an incremental testing strategy aiming at reducing the cost of both static analysis and mutation testing using the correspondence information. On the other hand, knowing that Mutation Test has a high application cost, we identified mutations of some specific mutation operators, which an automatic static analyzer is not able to detect. Therefore, this information can used to prioritize the order of applying mutation operators incrementally considering, firstly, those with no correspondence with static warnings. / Considerando que: 1) analisadores estáticos automatizados são ferramentas que emitem avisos, sem que seja necessário a execução do produto de software correspondente, alertando sobre a presença de possíveis defeitos no código. Uma das críticas a tais ferramentas é a grande quantidade de avisos falsos positivos emitidos, isto é, avisos relatados que não correspondem a defeitos reais, mas demandam tempo de análise por parte do desenvolvedor; 2) tradicionalmente, o Teste de Mutação tem sido utilizado para avaliar (e melhorar) a qualidade de conjuntos de casos de teste e/ou de critérios de teste, uma vez que é considerado um bom gerador de defeitos de software. Objetivo: O objetivo do presente trabalho é investigar a correspondência entre avisos estáticos e mutações e, com isso, verificar quais avisos estão mais relacionados a esses possíveis defeitos (mutações) e, assim, possivelmente, serem avisos verdadeiros positivos. Método: Os operadores de mutação são utilizados neste trabalho como um modelo de defeitos para avaliar a correspondência entre mutações e avisos estáticos. A principal vantagem da utilização de operadores de mutação é que eles geram um grande número de programas com defeitos de diferentes tipos. Esses tipos de defeitos são usados em estudos experimentais para investigar a capacidade dos analisadores estáticos em detectá-los. Resultados: Os resultados obtidos com estudos experimentais para um conjunto de sistemas de código aberto indicam que existe correspondência quando são considerados alguns operadores de mutação da μJava e alguns tipos de avisos da FindBugs. Conclusão: Os resultados obtidos podem ser utilizados de duas maneiras distintas: Primeiro, é fornecida uma abordagem de análise incremental dos avisos, de acordo com o grau de correspondência com mutações. Segundo, com o objetivo de reduzir o custo do Teste de Mutação é fornecida uma abordagem de priorização incremental para análise dos mutantes dos operadores cujas mutações são menos “percebidas” pela FindBugs.

O cádmio (Cd) é um metal pesado encontrado naturalmente no meio ambiente em níveis da ordem de 0,5 mg.L-1. No entanto, atividades humanas têm aumentado sua concentração na biosfera, por meio de efluentes industriais não tratados. Essa elevação pode causar estresse oxidativo nas plantas, conferindo aberrações cromossômicas, danos ao DNA e alterações nos microtúbulos das células. Pesquisas têm avaliado os efeitos do Cd em diversas espécies de plantas. Entretanto, o tomateiro ainda não tem sido alvo de tais pesquisas mesmo sendo considerada uma planta modelo em outras áreas. Dessa forma, justifica-se a condução de experimentos que avaliem a genotoxicidade do Cd nesta espécie através de abordagens baseadas na citogenética. Este estudo foi conduzido para avaliar efeitos genotóxicos do Cd em células meristemáticas de raízes de tomateiro do cultivar Micro-Tom através da investigação de aberrações cromossômicas, grau de danos causados ao DNA e anormalidades das fibras do fuso mitótico e do citoesqueleto. Evidenciou-se que a partir da dose mínima de Cd empregada (variou de 0,018 a 13 mg.L-1), foram observadas aberrações cromossômicas, tais como quebras, perdas, adesões e pontes. Ainda empregando-se as mesmas doses de Cd, constataram-se danos ao DNA por meio do ensaio de cometa. Porém, de acordo com a dose de 3,7 mg.L-1 de Cd, os microtúbulos de ?-tubulinas sofreram modificações em sua estrutura que alteram a organização do citoesqueleto celular, do fuso mitótico e da citocinese. Em geral, o tomateiro apresentou sensibilidade em baixas concentrações de Cd, o que possibilita considerá-lo como uma importante planta modelo com potencial para estudos de genotoxicidade aplicados ao monitoramento ambiental e testes de compostos tóxicos. / Cadmium (Cd) is a heavy metal found naturally in the environment at low levels (around 0.5 mg.L-1). Some human activities have increased its concentration in the biosphere due to untreated industrial effluents. This increase may induce oxidative stress in plants and potentially causing chromosomal aberrations, DNA damage and changes in the cell microtubules. Researchers have evaluated the Cd effects in several plant species. However, tomato has not been the target of these researches even though it is considered as a plant model in others areas. Therefore, experiments are needed to assess the genotoxicity of Cd in this plant species through cytogenetical approaches. The present work was carried out to assess the Cd genotoxic effects on root-meristem cells of Micro-Tom based on chromosomal aberrations, DNA damage and abnormal mitotic spindle fibers and cytoskeleton. Low Cd concentrations (ranging from 0.018 to 13 mg.L-1) were able to induce chromosomal aberrations such as breaks, losses, stickiness and bridges. At the same concentrations, DNA damage was evidenced using the comet assay. Concentrations of 3.7 mg.L-1 Cd induced structural modifications of the microtubules of ?-tubulin that altered the organization of the cytoskeleton, the mitotic spindle fibers and cytokinesis. In general, tomato presented sensitivity to low concentrations of Cd. These results suggest that tomato is also a good model for studies of genotoxicity to be applied on environmental monitoring and toxic compounds assays.