Thesis (Ph. D.)--University of Chicago, Committee on Genetics. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph.D.) - University of Queensland, 2005. / Includes bibliography.

Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisor: Stanley Roux. Vita. Includes bibliographical references.

All fossil fuels contain varying levels of sulfur compounds which are undesirable because they cause environmental pollution, corrosion, acid rain and lead to health problems. There is strict international legislation for the permissible levels of sulfur compounds in fossil fuels. The aim of this research therefore was the biocatalyst development for biodesulfurisation using two approaches. In the first approach, Rhodococcus erythropolis IGTS8-5 and IGTS8-5G were immobilised in porous coke particles and tested in repeated cycles successfully. Both bacterial strains grew well in the chemically defined medium with glucose as the main carbon and energy source and the model sulfur compound dibenzothiophene (DBT) as the sole sulfur source. 0.8 g of cells was immobilized on 250 g of coke particles without refreshing the medium over 72 h while 1.8 g of cells were immobilised on 250 g of coke when the media was refreshed every 24 hours for 120 h after the initial immobilisation batch of 72h. The latter, were used repeatedly in twelve consequtive batch desulfurisation cycles during which the biodesulfurisation activity progressively decreased from over 95% removal of 100 ppm DBT to around 45% removal. DBT removal is often expressed in terms of 2-hydroxybiphenyl which is the end product of biodesulfurisation. The biodesulfurisation activityof immobilised bacteria was equivalent to 310 umol 2-HBP h-1g-1 dry cell weight during the first hour. Freely suspended cells on the other hand exhibited biodesulfurisation activity equivalent to 91 umol 2-HBP h-1g-1 dry cell weight. Unfortunately, after the first 24 h, the activity of the immobilised cells decreased to 12 umol 2-HBP h-1g-1 dry cell weight. Use of plant cell cultures for biodesulfurisation is the other novel aspect of this work. Armoracia rusticana (horse radish) cell culture was chosen as the novel biocatalyst since this plant is a well known source of peroxidase enzyme which is involved in the biodesulfurisation metabolism according to the literature on bacterial biodesulfurisation. Arabidospsis thaliana (thale cress) was also used since its genome is completely sequenced and it is a model organism in genomics studies. Our results indicate that cell suspensions of both plants did show biodesulfurisation activity by reducing the level of sulfur compounds, mainly DBT and other three derivatives from both aqueous and oil phase. When compared to the bacteria, in terms of DBT consumption, the activity of A. rusticana was calculated as 55 umol DBT h-1 g-1 DCW and 65 umol DBT h-1 g-1 DCW for A. thaliana while in bacteria it was 91 umol DBT h-1 g-1 DCW for IGTS8-5 and 73 umol DBT h-1 g-1 DCW for IGTS8-5G. Transcriptomics analysis of the plant cell cultures after exposure to the DBT when compared to control cultures showed alterations in gene expression levels several of which were related to sulfur metabolism and transmembrane transporters of sulfate.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-08-23T14:10:57Z No. of bitstreams: 1 texto completo.pdf: 1554288 bytes, checksum: 92b63f252bea1ea664382e7aee322330 (MD5) / Made available in DSpace on 2017-08-23T14:10:57Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1554288 bytes, checksum: 92b63f252bea1ea664382e7aee322330 (MD5) Previous issue date: 2016-02-22 / Fundação de Amparo à Pesquisa do Estado de Minas Gerais / O crescimento e o desenvolvimento vegetal são controlados por uma complexa rede metabólica controlada por fatores ambientais, incluindo a disponibilidade de água, nutrientes e luz, e por várias vias de sinalização. Uma das mais importantes vistas de sinalização, conservada em eucariotos, é a kinase Target of Rapamycin (TOR). Várias linhas de evidência demonstram que TOR exerce um papel fundamental no balanço de carbono e nitrogênio, agindo como um regulador essencial do metabolismo central, controlando o crescimento e a produção de biomassa. O amido é a principal forma de armazenamento de carbono e seu conteúdo é correlacionado negativamente com crescimento. Linhas transgênicas com expressão reduzida do gene TOR ou dos componentes do complexo apresentam um claro fenótipo de acúmulo de amido. No entanto, ainda não é elucidado se o acúmulo de amido é devido a um aumento na síntese, decréscimo na degradação ou ambos. Neste trabalho, plântulas de Arabidopsis tratadas com o inibidor químico específico de TOR AZD-8055, que age no sítio de ligação do ATP no domínio quinase de TOR, mostraram um fenótipo de acúmulo de amido logo após 4 horas de tratamento e este acúmulo é devido a um aumento nas taxas de síntese deste polímero. Além disso, plântulas onde TOR estava inibida apresentaram um aumento em torno de 30-40% em seu conteúdo de amido em comparação ao controle. Análise de perfil metabólico em plântulas com inibição de TOR apresentaram mudanças nos níveis de sacarose, frutose, glicose, maltose, manose e fosfato inorgânico. Todos estes metabólitos estão correlacionados direta ou indiretamente com o metabolismo de amido. Uma correlação entre o teor de manose, fosfato inorgânico e aumento de amido foi notável em plantas tratadas com AZD-8055. Análise de expressão gênica de subunidades da AGPase apresentou mudanças significativas nos pontos 18 e 24 horas após o tratamento com o inibidor. Atividade catalítica máxima da AGPase foi determinada nos pontos 2, 4, 6, 10 e 24 horas após a administração de AZD-8055. No entanto, a atividade só foi estatisticamente diferente entre tratamentos nos pontos 4 e 24 horas. Immunoblotting para esta enzima mostrou que após 4 horas de inibição da via TOR, o conteúdo de AGPase em sua forma dimérica tendeu a ser maior em plantas controle. Além disso, o teor total de AGPase e seu conteúdo na forma monomérica foram maiores em plantas tratadas com AZD-8055 após 6 horas de tratamento na luz. Sabendo que várias enzimas relacionadas ao metabolismo do amido são reguladas pelo potencial redox da célula, os níveis de glutationa foram mensurados para verificar o potencial redox celular. Plantas tratadas com AZD-8055 apresentaram mudanças na quantidade total de glutationa, pincipalmente na sua forma reduzida e o potencial redox da célula tendeu a ficar mais reduzido. Em conjunto, estes resultados indicam a participação de TOR no metabolismo do amido, mas o exato mecanismo por trás disso ainda está longe de ser elucidado. / Plant growth and development are maintained by a complex network controlled by environmental factors including the availability of water, light and nutrients and by several signaling pathways. One of the most important signaling pathways, conserved in eukaryotes, is the kinase Target of Rapamycin (TOR). Various lines of evidence point out that TOR plays a fundamental role in carbon and nitrogen balance, acting as an essential regulator on central metabolism by controlling growth and biomass production. Starch is the major form of carbon storage and its content is negatively correlated with growth. Transgenic lines with reduced expression of TOR gene or components of the TOR complex present a clear starch excess phenotype. However, it remained to be elucidated whether the accumulation of starch is due to increased synthesis, impaired degradation or both. In this work, Arabidopsis seedlings treated with the specific ATP-competitive inhibitor of TOR kinase AZD-8055 showed a starch excess phenotype right after 4 hours of treatment and the accumulation of starch was proved to be due to an augmentation in the rate of starch synthesis. Furthermore, TOR- inhibited plants presented an average increase of 20-30% in their starch content at the end of day when compared to control. Metabolite profiling analysis showed that TOR– inhibited plants exhibited broad changes in the levels of sucrose, fructose, glucose, maltose, mannose and orthophosphate, which are associated directly or indirectly with starch metabolism. In addition, a correlation between the amount of mannose, orthophosphate and increased starch content was noticed in AZD-treated plants. Gene expression analysis of AGPase subunits showed significant changes only from 18 and 24h after treatment. Although TOR inhibited plants displayed higher content of the active form of AGPase (monomer), enzymatic activity assays revealed that changes in AGPase activity might occur as secondary effect of TOR inhibition and might be not related to the starch excess phenotype observed 4 hours after AZD-treament. Since several enzymes related to starch metabolism are subject to redox regulation, the levels of glutathione were measured to verify the redox environment of the cells. TOR- inhibited plants showed changes in the pools of glutathione, mainly in its reduced form, and the redox state of the cells tended to be more reduced. Together, these results indicate the participation of TOR signalling on starch metabolism but the mechanistic behind this process need further studies.

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2017-08-22T17:21:48Z No. of bitstreams: 1 texto completo.pdf: 730467 bytes, checksum: e44535c0266d0e38f30c95a7b0afa815 (MD5) / Made available in DSpace on 2017-08-22T17:21:48Z (GMT). No. of bitstreams: 1 texto completo.pdf: 730467 bytes, checksum: e44535c0266d0e38f30c95a7b0afa815 (MD5) Previous issue date: 2016-04-08 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Neste trabalho é abordado a importância dos transportadores de adenilatos no metabolismo vegetal sob condições de estresse por meio de análises de expressão in silico e em plantas deficientes no transportador ADNT1 sob condições de hipóxia. A análise de expressão gênica in silico revelou aumento na expressão de dois transportadores principalmente, AAC3 que transporta ATP mitocondrial em troca de ADP citosólico na membrana interna da mitocôndria e ATPNC2 que transporta ATP citosólico em troca de ADP peroxissomal em diferentes situações de estresse (osmótico, UV-B, calor, salinidade, injúria, seca e frio), sendo os únicos transportadores a apresentarem aumento na expressão tanto na parte aérea quanto em raiz. A análise de coexpressão revelou que em condições de estresse o transportador AAC3 é coexpresso com diversos genes envolvidos com a degradação de proteínas e genes de respostas a estresse. Outros dois transportadores que foram coexpressos com um número considerável de genes sob condições de estresse, foram o transportador ATBT1 e o transportador ATPNC1, ambos foram coexpressos com 118 genes cada (r 0.65). Nota-se que o transportador ATBT1 foi coexpresso principalmente com genes envolvidos com a síntese de proteína e genes envolvidos com a regulação transcricional, processamento e degradação de RNA. Do mesmo modo, o transportador ATPNC1, foi co-expresso com genes envolvidos em diferentes processos metabólicos, com destaque para degradação de proteínas e metabolismo de aminoácidos, que podem ser utilizados como substratos alternativos para produção de ATP. Quanto ao ADNT1 observou- se que as plantas deficientes no transportador apresentaram um menor teor de amido ao final do estresse por hipóxia, indicando que o transportador ADNT1 é importante no metabolismo do carbono. Adicionalmente foi observado a importância do transportador ADNT1 em tecidos heterotróficos, verificou-se que este tem participação direta na germinação e crescimento radicular. / This paper discussed the importance of adenylates transporters in plant metabolism under stress conditions through in silico expression analysis and deficient plants in ADNT1 carrier under hypoxic conditions.Gene expression in silico analysis revealed increased expression of two conveyors mainly AAC3 carrying mitochondrial ATP exchange ADP cytosolic the inner membrane of mitochondria and ATPNC2 carrying ATP cytosolic in exchange for ADP peroxisomal in different stress situations (osmotic, UV-B, heat, salinity, injury, drought and cold), and the only carriers to submit increased expression both in the shoot and in the root. The coexpression analysis revealed that under stress conditions the AAC3 carrier is coexpressed with several genes involved in the degradation of proteins and gene responses to stress. Two other carriers that were co-expressed with a considerable number of genes under stress conditions, were the carrier ATBT1 and ATPNC1 carrier, both were co-expressed 118 genes each (r 0.65). Note that if the carrier was coexpressed ATBT1 mainly genes involved in protein synthesis and genes involved in transcriptional regulation, RNA processing and degradation. Similarly, the carrier ATPNC1, was co-expressed with genes involved in various metabolic processes, particularly protein degradation and metabolism of amino acids which can be used as alternative substrates for ATP production. As to ADNT1 it was observed that the plants deficient in the carrier had a lower starch content at the end of the hypoxic stress, indicating that the ADNT1 carrier is important in carbon metabolism. In addition it was observed the importance of ADNT1 carrier in heterotrophic tissues, it has been found that this has a direct interest in germination and root growth. / Não foi localizado o currículo lattes do autor.

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2016-04-26T10:59:04Z No. of bitstreams: 1 texto completo.pdf: 469789 bytes, checksum: 8216c1bd8e647fbcb93561781c7f23dc (MD5) / Made available in DSpace on 2016-04-26T10:59:04Z (GMT). No. of bitstreams: 1 texto completo.pdf: 469789 bytes, checksum: 8216c1bd8e647fbcb93561781c7f23dc (MD5) Previous issue date: 2014-11-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Apesar da conhecida associação entre o selênio (Se) e o metabolismo do carbono, as vias regulatórias que conectam o controle do crescimento com o metabolismo primário de plantas cultivadas com Se, não são, ainda conhecidas. Assim, este trabalho objetivou investigar a ação do Se no metabolismo primário em Arabidopsis thaliana. Para tanto, plantas de Arabidopsis thaliana foram cultivadas em solo suplementado com selenato de sódio, à concentração de 50 μM. O Sepromoveu uma redução significativa no crescimento das plantas de Arabidopsis. Os níveis de glicose tanto na parte aérea quanto no sistema radicular foram reduzidos por solução de Se. Além disso, o Se promoveu incremento significativo nos níveis de nitrato tanto na parte aérea como na raiz quando comparado com as plantas controle. No entanto, os níveis de aminoácidos totais, sacarose e proteínas permaneceram estáveis nas plantas tratadas com Se, em comparação com as do plantas controle. O conteúdo de amido foi alterado em plantas tratadas com Se. A exposição das plantas à solução de Se levou a um aumento na atividade da AGPase durante o final do período noturno. Contrariamente ao metabolismo do amido, o Se não promoveu alterações significativas na atividade da redutase do nitrato. Com o intuito de se investigar mais detalhadamente o efeito do Se no metabolismo de carboidratos, plantas de Arabidopsis Col-0 e dos mutantes pgm e sex1-4 foram tratadas com Se. Sob a ação do Se, a massa seca da parte aérea e da raiz bem como a taxa de crescimento relativo das plantas Col-0 e sex1-4 foram reduzidas, independente do fotoperíodo utilizado. O tratamento com Se não reduziu a razão parte aérea: raiz das plantas Col-0 e sex1-4. Interessante, a massa seca da roseta, a massa seca da raiz, a razão parte aérea: raiz e a taxa de crescimento relativo das plantas do mutante pgm não foram afetadas pelo tratamento com Se, sob condição de dia curto ou de dia longo. Ademais, menores acúmulos de macro e micronutrientes foram acompanhados de uma redução no crescimento das plantassex1-4cultivadas com Se. Assim, um desbalanço entre o metabolismo mineral e o crescimento parece ocorrer nas plantas com redução na degradação de amido. Em conjunto, as informações obtidas oferecem uma melhor compreensão dos mecanismos fisiológicos e metabólicos associados ao Se em plantas. / Despite the known association between selenium (Se) andthe carbon metabolism, the regulatory pathways that connect growth regulation with primary metabolism in plants cultivated with Se are still unknown. Hence, this study aimed to investigate the action of Seon the primary metabolism. For this purpose, Arabidopsis thaliana plants were cultivated in soil supplemented with 50 μM sodium selenate.Se caused a significant reduction ingrowth ofArabidopsis plants. Glucose levels in both shoot and root were reduced by Se. In addition, Se promoted a significant increase in nitrate levels in both shoot and roots, when compared to the control plants. However, levels of total amino acids, sucrose and proteins remained constant in plants treated withSe, when compared to the control plants. Starch content was also altered in plants treated with Se. The exposure of plants toSeled to an increase in AGPase activity during the late night period. By contrastto starch metabolism, Se did not cause significant changes in nitrate reductase activity. In order to further investigate the effect ofSe in carbohydrates metabolism, Col-0 Arabidopsis plants and pgmand sex1-4 mutants were treated with Se. In presence of Se, dry weight of shoot and root as well as the relative growth rate of Col-0 and sex1-4 plants were reduced,regardless of photoperiod used. The treatment with Se did not reduce the shoot-to- root ratio of Col-0 and sex1-4 plants. Interestingly,rosette and root dry weight, shoot-to-root ratio and relative growth rate of pgmplants were not affected by treatment withSe under both short- or long-day conditions. In addition, lower accumulation of macronutrients and micronutrients were accompanied by a growth reduction of sex1-4 plants cultivated with Se. Collectively, the results presented here show that an imbalance between mineral metabolism and growth seems to occur in plants with decreased starch breakdown in presence of Se. Altogether, the gathered information offers a better understanding of the physiological and metabolic mechanisms associated with Se in plants.

fósforo (P) e o nitrogênio (N) são nutrientes geralmente limitantes ao crescimento e desenvolvimento vegetal. Respostas aclimatativas à sua limitação referem-se às alterações do desenvolvimento radicular e mobilização, transporte, assimilação e metabolismo destes nutrientes. Para que este processo seja desencadeado, é necessária expressão de genes intimamente relacionados com a percepção e transdução do sinal de deficiência de P e N. Os genes envolvidos neste processo ainda são pouco conhecidos. Neste estudo objetivou-se averiguar o papel dos genes mutados nas rotas de aclimatação à limitação de Pi e assimilação de N através da identificação das modificações em nível de expressão gênica nos mutantes p9, p23 e p37, bem como através da avaliação da resposta dos mutantes p9 e p37 ao etileno. Os três mutantes são complementares e ineficientes em utilizar organofosfatos como fonte de P e p23 e p37 são resgatados quando o N é retirado do meio. Há interação P-N em relação à expressão de genes das rotas de aclimatação à limitação de P (AtACP5 e AtPT2), de assimilação de N (NRT1.1, NIA1 e NIA2) e na modulação das raízes laterais (ARF8). Os mutantes têm alteração na expressão dos genes que codificam transportadores de N e P de alta afinidade, assimilação de N, especialmente NIA2, e no caso de p9 e p23, ainda, ARF8. Na ausência de Pi e/ou N, houve rápida exaustão do centro quiescente nas raízes primárias em p23 e p37, assim, os genes mutados devem fazer parte de uma rota que medie as respostas do crescimento radicular em função da disponibilidade de Pi e N. Os mutantes p9 e p37 apresentam deficiência na sinalização ao etileno, sendo possível que p9 possua alteração na homeostase hormonal, e que modificação nos níveis de auxinas e citocininas afetem a síntese de etileno. / Phosphorus (P) and nitrogen (N) are nutrients highly required by plants, and limit plant growth and development. The main acclimation responses to P and N starvation include changes in root development, mobilization, transport, assimilation and metabolism of these nutrients. For properly acclimation, the expression of genes closely related to the perception and signal transduction of P and N deficiencies must work accurately. Which genes are involved in this process is still unclear, therefore, this study aimed to identify changes at the expression level in the p9, p23 and p37 mutants in an attempt to identify the role of the mutated genes in the acclimation pathway to Pi starvation. Furthermore, we aimed to verify the P-N interaction and the response of the mutants p9 and p37 to ethylene. The three mutants are inneficient in using organophosphates as the only source of P, and p9 and p23 recover the COL phenotype in the absence of N. Interaction between P and N was observed in the expression of genes involved in Pdeficiency acclimation, namely AtACP5 and AtPT2, as well as in the N assimilation, NRT1.1, NIA1 and NIA2, and ARF8, involved in lateral root modulation. It was observed that the mutated gene in p9, p23 and p37 affects genes that encode high affinity N transporters, genes involved in N assimilation, especially NIA2. And p9 and p23 also has the regulatory circuit that acts on the modulation of lateral roots affected. A rapid depletion of QC in primary roots of p23 and p37 was observed in the absence of Pi and/or N. It suggests that the mutated genes are involved in a pathway mediating the root growth in response to Pi and N availability. The mutants, p9 and p37, have some kind of deficiency in ethylene signaling. It is also possible that p9 is affected in its hormonal homeostasis, and changes in auxin and cytokinin levels affect the ethylene synthesis.

Metabolic flux analysis (MFA) is a powerful technique for quantifying the intracellular fluxes in central carbon metabolism. It relies on detection of stable isotope labelling from metabolites such as amino acids derived from protein. Current standard techniques are, however, unable to distinguish between different cell types in heterogeneous tissue. The aim of the thesis was to address this problem by developing and validating a strategy using green fluorescent protein (GFP) with cell type specific expression as a reporter protein for investigating the fluxes in specific cell types in the Arabidopsis thaliana root. The fundamental difficulty in applying a reporter protein strategy in a multicellular organism arises from the limited amount of recombinant protein expressed by the cells. The main novel contributions of the work in this thesis are threefold. First, a robust protocol for purification of GFP from the roots of Arabidopsis seedlings and for detection of reliable mass isotopomer distributions from the amino acids derived from GFP are described. Secondly, the reporter protein strategy is validated in this biological system with a focus on showing the data obtained by the use of the reporter protein is equal to that normally obtained from the total protein fraction. To expand on this, stable isotope labelling in isolated root hair cells is explored. These cells are easily isolated and show potential as a model system for cell type specific metabolism. Finally, the experimental data provide evidence for the feasibility of measuring data from specific cell types with appropriate mass spectrometric techniques. Analysis of cell type specific gene expression in this system suggests differences in the primary metabolism of different cell types cannot be ruled out without further investigation. Based on small scale in silico modelling described in this thesis, new solutions capable of providing data on sub-populations of cells are required, if central metabolism of the cell types differs significantly.

Accumulation of a given metal in plants depends on a delicate and precise balance of various biological processes. Some plants have developed strategies that allow them to tolerate heavy metals in extreme conditions without suffering toxicity. This research focuses on the characterization of two metal transporters, a member of the P1B-type (ATPase) transporter family (HMA3) and a member the Natural Resistance Associated Macrophage Protein (NRAMP) Nramp1 family. These transporters have proposed roles in ion homeostasis and mineral nutrition. The work here sought to determine if these transporters might have characteristics that suggest a role in heavy metal transport and tolerance in metal hyperaccumulating plants. These proteins are very well conserved among different taxa. Nonetheless, as little as a single amino acid change has the potential to modify their capacity to take up non essential metals such as Cd, or Pb, and/or increase affinity for other mineral nutrients. These transporters were cloned from a non-accumulator (Arabidopsis thaliana L) and two ecotypes (Prayon and Ganges) of the hyperaccumulator Noccaea caerulescens (formerly= Thlaspi caerulescens). The full cDNA of an ortholog of either Nramp1 or HMA3 was expressed in yeast in order to provide a heterologous model to elucidate how polymorphisms between the orthologs might translate into functional differences between the protein sequences. A comparison of the HMA3 sequences to each other, or the Nramp1 sequences to each other, demonstrated that major motifs and domains in each protein were highly conserved but that there were numerous single amino acid polymorphisms. Few of these polymorphisms corresponded to positions in a protein that are known to be critical for transporter function. However, metal accumulation, tolerance and cell growth assays showed that the Nramp1 and HMA3 genes from Arabidopsis encoded proteins with the expected broad selectivity for divalent ion transport. In contrast, the genes from the Thlaspi ecotypes encoded proteins that showed more selectivity for ion transport. The Thlaspi ecotypes showed high selectivity for cadmium but the accumulation of other elements differed between the Thlaspi orthologs. These results suggest that the polymorphisms present in the Thlaspi sequences have produced differences in the transport characteristics of both the HMA3 and the Nramp1 transporters.