Protein arginine methyltransferases (PRMTs) are enzymes that catalyze the methylation of protein arginine residues, resulting in the formation of monomethylarginine, and/or asymmetric or symmetric dimethylarginines. Although understanding of the PRMTs has grown rapidly over the last few years, several challenges still remain in the PRMT field. Here, we describe the development of two techniques that will be very useful in investigating PRMT regulation, small molecule inhibition, oligomerization, protein-protein interaction, and substrate specificity, which will ultimately lead to the advancement of the PRMT field. Studies have shown that having an N-terminal tag can influence enzyme activity and substrate specificity. The first protocol tackles this problem by developing a way to obtain active untagged recombinant PRMT proteins. The second protocol describes a fast and efficient method for quantitative measurement of AdoMet-dependent methyltranseferase activity with protein substrates. In addition to being very sensitive, this method decreases the processing time for the analysis of PRMT activity to a few minutes compared to weeks by traditional methods, and generates 3000-fold less radioactive waste. We then used these methods to investigate the effect of truncating the NT of human PRMT1 variant 1 (hPRMT1-V1) on enzyme activity, protein-protein interactions, and substrate specificity. Our studies show that the NT of hPRMT1-V1 influences enzymatic activity and protein-protein interactions. In particular, methylation of a variety of protein substrates was more efficient when the first 10 amino acids of hPRMT1v1 were removed, suggesting an autoinhibitory role for this small section of the N-terminus. Likewise, as portions of the NT were removed, the altered hPRMT1v1 constructs were able to interact with more proteins. Overall, my studies suggest the the sequence and length of the NT of hPRMT1v1 is capable of enforcing specific protein interactions.

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Although enhanced biological phosphorus removal (EBPR) and complete biological nutrient removal (BNR) systems can be operated successfully by experienced operators, the accuracy of design and strength of the scientific background need to be reinforced to enable accurate modeling and economically optimal design. One way to accomplish this would be through a better understanding of the biochemical mechanisms and microbial population dynamics that determine the reliability and efficiency of EBPR, and the utilization of this information to improve the design and operation of BNR plants. Such knowledge will also contribute to better structure of modeling tools that are used for design and educational purposes. The current body of knowledge is limited to observational studies that lack detailed biochemical explanations backed with a series of well planned experiments, and this has introduced uncertainties and inaccuracies into the biochemical and design models. Therefore, this study mainly covers a biochemical survey of the underlying metabolisms of active populations in BNR sludges. BNR biomass with biological phosphorus removal (BPR) capability was cultivated in continuous flow reactor (CFR) systems, configured as either University of Cape Town (UCT) and anoxic/oxic (A/O) systems. Following an acclimation period at 20°C, low temperature stress (5°C) was imposed on one UCT system for investigation of the response of the microbial consortium responsible from EBPR activity under cold temperature. Once a stable population with EBPR capabilities is established in each system, activities of ten enzymes that are hypothesized to be taking part in the EBPR metabolism were measured. These enzymes were selected among those that take part in major known pathways of bacterial energy and growth metabolism. Also, 13C-NMR was used as a tool to monitor the flux of labeled carbon in and out of pools of cellular storage; i.e. glycogen and polyhydroxyalkanoates (PHA). Combining the gathered information, accurate mass balances of carbons and reducing equivalents were calculated, eventually leading to determination of the biochemical pathways utilized by the EBPR consortium. Additionally, anaerobic stabilization of COD, a long debated but empirically established phenomenon, was addressed during the study. Considering the pathways proposed to be operative under different conditions imposed on the EBPR systems, a biochemical explanation for the occurrence of COD stabilization in wastewater treatment systems that incorporate anaerobic zones was proposed. Accordingly, depending on the pathways actively used by a microbial consortium, electrons stored in NADH and FADH2 can either be transferred to the terminal electron acceptor, oxygen, or they can be incorporated into storage polymers such as glycogen for future use. Such differences in metabolism reflect in the quantity of the oxygen consumed in the aerobic reactors. Thus, the correct incorporation of anaerobic stabilization of COD into process design would reduce design aeration requirements and result in economic savings during both construction and operation. / Ph. D.

The work developed in this thesis aimed to increase understanding about the variability and stability in eleven biotypes of Pochonia chlamydosporia, a facultative parasitic fungus with potential as a biological control agent against root-knot (Meloidogyne spp.), false root-knot (Naccobus spp.) and cyst nematodes (Heterodera and Globodera, spp.). Differences in performance were assessed by measuring saprophytic and parasitic growth using in vitro bioassays. Information on virulence (in vitro) was collected for a range of biotypes with the objective to relate in vitro parasitic growth with rhizosphere colonisation ability and secretion of extracellular enzymes. Results showed differences between biotypes in their ability to colonise the rhizosphere of plants, parasitise nematode eggs and to produce a range of extracellular enzymes but no significant relationships were found between saprophytic or parasitic growth and enzyme production. For the first time, the specific activity of protease, chitinase, esterase and lipase enzyme production by eleven biotypes of the fungus was examined. Enzymatic activity was shown to vary with the biotype and type of enzyme assayed and biotypes could be ranked according to their similarities in enzyme production A novel bioassay to estimate egg parasitism using liquid media highlighted the importance of nutrition in infection processes and suggested that all biotypes are able to infect large numbers of eggs rapidly if the conditions are favourable. The assay reliably detected fungal infection in nematode eggs within 48 hours and provided a simple, rapid assay to test the effect of specific nutrients at controlled concentrations on the infection process. Differences in infection rates between biotypes observed in previous tests on agar were not detected in the new assay in which nematode eggs and fungal conidia were added in suspension. Internal colonisation of individual whole Meloidogyne spp. eggs by P. chlamydosporia was observed using microscopy studies. The destruction of nematode eggs infected with the fungus within seven days, was confirmed. The in vitro formation of appressoria was studied in a range of P. chlamydosporia biotypes. for the first time. Biotypes were found to differ in their ability to produce appressoria but this ability was not related to differences in virulence (in vitro) against nematode eggs. Cont/d.

Fluorescent labeling is a method used for visualization of various types of biomolecules including proteins and protein complexes. However, the effect of protein labeling on protein structure and functions has not been investigated so far. The goal of the diploma thesis was to examine an influence of NHS-fluorescein binding on structure and function of human carbonic anhydrase I (hCA-I). The particular aims of this work were to prepare recombinant 15N-hCA-I which was used for NMR structure analysis of carbonic anhydrase upon fluorescent labeling. Furthermore, enzyme activity was measured in order to find out a correlation between the concentration of NHS- fluorescein and protein function. In addition, the reaction mixtures were systematically analyzed by ESI FT-ICR mass spectrometry. The analysis revealed experimental conditions for fluorescent labeling of human carbonic anhydrase I with minimal effect on protein structure and function. The results of this study show that the calculation of molar excess of NHS-fluorescein cannot rely on a simple procedure provided by manufacturer. However, due to decrease of enzyme activity upon fluorescent labeling, it is better to take into count the influence of NHS-fluorescein concentration on the relative enzymatic activity. Moreover, the calculation of molar...

Na última década, a análise da estrutura proteica baseada em teoria de redes/grafos tem emergido. A abstração da estrutura tridimensional proteica em forma de uma rede, leva em consideração os resíduos de aminoácidos e suas interações através do espaço, e apresenta um conjunto de conexões e propriedades mais complexas do que aquelas visualizadas apenas com a estrutura covalente. A análise da estrutura proteica identificou que as proteínas pertencem às redes de classes de \"mundo pequeno\" (small-world) e \"sem escala\" (scale-free), o que significa que seus resíduos de aminoácidos são altamente agregados e que existem poucas conexões entre 2 resíduos quaisquer da proteína. A identificação dos resíduos com alto grau de conexão, chamados centrais (\"resíduos hubs\"), é feita pela determinação do caminho mais curto que conecta um dado resíduo aos demais compreendidos nesta rede. A remoção destes resíduos centrais (hubs) afeta a integridade da rede de forma mais contundente diferentemente da remoção de resíduos que não são centrais. Até o momento estes \"resíduos hubs\" ainda não foram experimentalmente correlacionados com as propriedades enzimáticas de proteínas. Para tal finalidade, a estrutura terciária de uma β-glicosidase de Spodoptera frugiperda (Sfβgli) foi analisada como uma rede. Após calcular-se os caminhos médios entre todos os pares de aminoácidos da β-glicosidase, encontrou-se 11 resíduos centrais (\"resíduos hubs\"). Alinhamento de sequências e comparações estruturais indicaram alta conservação destes \"resíduos hubs\". Nosso objetivo foi produzir esta β-glicosidase mutando-se a maioria dos \"resíduos hubs\" e 3 aminoácidos não centrais (\"não hubs\"), expressar estes mutantes em E. coli, determinar suas propriedades enzimáticas como atividade catalítica e preferência pelo substrato e verificar a estabilidade destes mutantes em experimentos de inativação térmica. Os resultados obtidos sugerem que mutações nos \"resíduos hubs\" não afetam as propriedades catalíticas, contudo as enzimas com mutações nos \"resíduos hubs\" apresentaram uma menor estabilidade térmica. Estes resultados sugeriram que os \"resíduos hubs\" são relevantes na difusão da energia cinética (vibração) introduzida na estrutura desta β-glicosidase pelo seu aquecimento / In recent years, graph-theoretic approaches have established that protein structures can be modeled as complex networks of interacting residues. Proteins structures can be represented as small-world and scale-free networks that are usually highly clustered with few links connecting any pair of nodes. The identification of nodes with high connection degrees, called hubs, is made by determining the shortest path linking one amino acid to the further nodes comprising the network. Targeted removal of the hubs has greater affect on the integrity of the network structure in contrast to a random removal of amino acid residues comprising the network. Nevertheless these hubs had not previously been correlated with enzymatic properties. The tertiary structure of β-glycosidase from S. furgiperda (Sfβgly) was analyzed as a network. After calculating the averaged paths between all pairs of amino acid residues of Sfgly, we defined 11 hubs, which have the highest centrality on the network. Sequence alignment and structural comparison showed that these hubs residue are conserved among β-glycosidases. Our goal was to mutate most hubs and 3 ´non-hubs´ residues from Sfβgly, express these mutant enzymes in E. coli, test their enzymatic properties as catalytic efficiency and substrate preference, and verify the thermal stability of these mutants. The results implied that mutations in these hubs do not cause changes in catalytic properties although enzymes containing mutations in hubs showed lower thermal stability. Based on that, it was suggested that hub residues are important in the diffusion of kinetic energy (vibrations) introduced in the Sfβgly structure by heating

Existe uma grande preocupação com relação aos efeitos a longo prazo que muitos poluentes químicos possam ter sobre a saúde e o ambiente. Tal é o número de diferentes poluentes e a complexidade de suas interações que seus efeitos não podem ser prontamente definidos em programas de monitoramento que determinam a presença de poluentes no ambiente. Recentemente, pesquisas identificaram um grupo de enzimas conhecidas coletivamente como proteínas antioxidantes, que são induzidas em resposta aos poluentes, como os metais pesados e parecem proteger as células contra os possíveis danos causados por estes agentes. É possível que a análise da indução e acumulação de proteínas antioxidantes e/ou de defesa, possa fornecer um sistema biológico versátil de monitoramento dos efeitos da poluição. E assim, o estudo da atividade enzimática poderá vir a se configurar como um critério de avaliação da fitotoxicidade de metais pesados em plantas. Com esse propósito, foi desenvolvido um trabalho com o objetivo de estudar as alterações de crescimento e bioquímicas causadas pelo efeito do Cádmio (Cd) em arroz. Experimentos foram montados utilizando 8 diferentes genótipos de arroz da espécie Oryza sativa: F222, IAC202, Caipó, J199, IAC165, J79, F133, F35 e mais duas espécies: Arg2 (Oryza latifolia) e Oryza glumaepapulo nos quais foram submetidas a diferentes concentrações de Cd em solução nutritiva (0mM, 0,05mM, 0,5mM) durante um período de 3 e 7 dias para avaliação do efeito desse metal pesado no comprimento do sistema radicular. Foi realizado um segundo ensaio utilizando a cultivar IAC165, submetida a diferentes concentrações de CdCl2 (0mM, 0,01mM, 1,0mM). Nestas concentrações, coletas de folhas e raízes foram feitas em períodos de tratamentos constituídos de 0, 3, 6, 9, 12, 15, 18, 21, 24, 30, 36, 42, 48, 54, 60, 66, 72horas. Com relação ao crescimento radicular do 1° ensaio foram identificados três padrões de resposta: um primeiro, cuja resposta à menor dosagem (0,05 mM de CdCl2) é significativa e esta se mantém na dosagem seguinte (0,5 mM); um segundo, onde também é significativa a resposta à menor dosagem, mas esta se intensifica significativamente com o aumento da dosagem e um terceiro, no qual as linhagens mostraram-se resistentes às dosagens testadas, não sendo afetadas em seu padrão de crescimento de raízes no tempo desse estudo. O parâmetro bioquímico analisado no 2° ensaio foi relativo aos níveis de atividade de enzimas antioxidantes, como Catalase e Glutationa Redutase. que mostraram padrões semelhantes de resposta tanto para Catalase como para Glutationa Redutase, observando-se elevação das atividades dessas enzimas antioxidantes em folhas e raízes, sendo o aumento de atividade da Glutationa Redutase nas raízes altamente significativo, sugerindo que a síntese de Glutationa reduzida possa estar estimulada para subseqüente síntese de fitoquelatinas. / The response of a group of rice genotypes to cadmium exposure was tested using the root growth as a parameter during the time length of the treatment. Three distinct response patterns were identified: a significant initial (0.01 mM) dosage effect, which was maintained with the increase of CdCl2 concentration to 1 mM; a significant initial effect, which was intensified with the increase of CdCl2 concentration, and a group of varieties resistant ot the CdCl2 concentration tested. Significant interactions between dosage and varieties were observed. Enzymatic assays for catalase and gluthatione reductase were also carried out for in plants exposed to CdCl2. Similar response patterns for both enzymes were observed. The activities of catalase and gluthatione reductase in leaves and roots were increased and in the case of gluthatione reductase in roots, such an increase was highly significant, suggesting that the synthesis of reduced gluthatione may be stimulated for subsequent synthesis of phytochelatins.

The relationship between biodiversity and ecosystem functioning is important in maintaining agroecosystem sustainability. Plant-microbe symbioses, such as exists between the grass tall fescue (Schedonorus arundinaceum) and the asexual fungal endophyte Epichloë coenophiala, can be utilized to enhance agroecosystem functions, such as herbivore resistance. “Novel” E. coenophiala strains that vary in the production of mammal- and insect-toxic compounds have been identified, inserted into tall fescue cultivars, and are planted in pastures globally. Novel fungal endophyte-tall fescue associations may have divergent ecosystem function effects. This study assessed effects of different fescue-endophyte symbiotic combinations on pasture ecosystem function, including aboveground (fescue biomass, plant species richness, alkaloid synthesis, arthropod abundance) and belowground (soil microbial biomass, soil enzyme activity, trace gas fluxes) parameters. Results showed no significant effects of increasing symbiotic diversity within a fescue stand on aboveground measurements, bar arthropod abundance and alkaloid synthesis. Most soil parameters quantified had significant symbiotic diversity effects. For example, soil microbial biomass decreased whereas soil enzyme activity increased with increasing symbiotic diversity. Overall, our results suggested that increasing symbiotic diversity had weak to moderate effects on aboveground processes and stronger effects on certain belowground processes, indicating that symbiotic diversity can impact ecosystem functions and warrants further research.

<p>Reverse transcriptase (RT) is a crucial enzyme for retrovirus replication, and its presence in the virion is indispensable for infectivity. This thesis illustrates the use of RT activity assays as tools for quantitation and characterization of different retroviruses, particularly HIV. </p><p>A non radioactive assay, using microtiter plates, for the RT of Moloney murine leukemia virus (MMuLV) was developed. Assay conditions for MMuLV and HIV-1 RT, together with isozyme specific RT activity blocking antibodies, were shown useful for discrimination between RTs from different retrovirus genera. RT activity assay for HIV-1 was found to quantitate different subtypes more equally efficient than p24 antigen assays did.</p><p>Viral load (VL), the amount of HIV particles in the blood, is an important marker of the clinical status of an infected person. A method for VL determination based on RT activity (ExaVir Load) was developed. After plasma pretreatment, to inactivate cellular DNA polymerases, virions in patient plasma were immobilized on a gel, which was washed to remove disturbing factors. The virions were lysed with a detergent containing buffer and the lysate eluted. Finally, the RT activity in the lysate was determined and found to correlate strongly to VL by RNA according to a PCR based standard method (Roche Amplicor 1.5). The second version of the method was able to measure VL down to approximately 400 HIV-1 RNA copies/ml. The usefulness of RT from the VL procedure for determination of susceptibility towards anti-HIV drugs was demonstrated, and the results were in agreement with genotypic data. </p><p>Due to its technical simplicity, and ability to detect a broad range of HIV-1 subtypes, ExaVir Load and the drug susceptibility application are interesting for clinical use, particularly but not only in resource limited settings. The concept is also potentially useful for research purposes, e.g. in combination with specific RT assay conditions.</p>