No description available.

Rotavirus (RV) is a viral pathogen that causes severe, watery diarrhea and vomiting in the young of humans and other animals. RV infections result in over 200,000 pediatric deaths around the world each year, especially in developing nations. Within the infected host cell, RV forms inclusion bodies, called viroplasms, where many stages of viral replication occur. The RV polymerase, known as VP1, must localize to viroplasms during infection where it replicates the virus' RNA genome. The work described in this dissertation focused on identifying region(s) of VP1 essential for its viroplasmic localization and its function as a polymerase. We found that a single amino acid change in a region of the polymerase called the N-terminal domain negatively impacted its capacity to localize to viroplasms during infection as well as its enzymatic activity in a test tube. Follow up studies using VP1 proteins from divergent strains and a mutant containing only the N-terminal domain of VP1 provided more insight into polymerase localization determinants. In total, our work suggests that the VP1 N-terminal domain plays an important role in localizing the polymerase to viroplasms via interactions with other viral proteins and supporting its function as a polymerase. / Ph. D. / Rotavirus (RV) is a viral pathogen that causes severe, watery diarrhea and vomiting in the young of humans and other animals. RV infections result in over 200,000 pediatric deaths around the world each year, especially in developing nations. Within the infected host cell, RV forms inclusion bodies, called viroplasms, where many stages of viral replication occur. The RV polymerase, known as VP1, must localize to viroplasms during infection where it replicates the virus’ RNA genome. The work described in this dissertation focused on identifying region(s) of VP1 essential for its viroplasmic localization and its function as a polymerase. We found that a single amino acid change in a region of the polymerase called the N-terminal domain negatively impacted its capacity to localize to viroplasms during infection as well as its enzymatic activity in a test tube. Follow up studies using VP1 proteins from divergent strains and a mutant containing only the N-terminal domain of VP1 provided more insight into polymerase localization determinants. In total, our work suggests that the VP1 N-terminal domain plays an important role in localizing the polymerase to viroplasms via interactions with other viral proteins and supporting its function as a polymerase.

O lodo de esgoto é associado a altas concentrações de poluentes inorgânicos em amplitudes que podem inviabilizar o uso direto em solo agrícola, de acordo com os limites máximos normatizados pela legislação, cujo destino final são aterros sanitários que, apesar de ser uma alternativa de menor impacto ambiental, é uma prática onerosa e finita. Entretanto, esse material é rica fonte matéria orgânica, macro e micronutrientes para as plantas como boro, cobre, cobalto, ferro, manganês, molibdênio, níquel e zinco para as plantas, comumente deficientes em solos tropicais. Em nosso estudo, exploramos esse subproduto como matriz orgânica para a formulação de um fertilizante organomineral (FOM) como alternativa à adubação mineral convencional para a cultura da soja em solos de Cerrado, além de possíveis alterações na comunidade microbiana do solo. O experimento foi conduzido em casa de vegetação utilizando dois Latossolos Vermelhos distróficos de texturas distintas (argiloso e franco-argilo-arenoso), diferentes doses de aplicação de FOM (40%, 60%, 80%, 100% e 120%) em três formas físicas (granulada, peletizada e farelada), em comparação com a adubação mineral convencional com (M+Mi+S) e sem (M+S) os micronutrientes B e Zn, além do tratamento sem adubação (CT). O delineamento utilizado foi de blocos inteiramente casualizados. As plantas foram conduzidas até os 60 dias após o plantio (DAP) e foram avaliados os parâmetros fitotécnicos pertinentes a massa seca da parte aérea (MSPA), massa seca de raiz (MSR), altura de plantas, diâmetro do colmo e número de nódulos ativos. A análise química da parte aérea (macro e micronutrientes) foi utilizada para cálculo do teor de nutrientes acumulados no tecido vegetal. Para avaliar a atividade microbiana dos solos utilizou-se a respiração basal do solo (RBS), carbono da biomassa microbiana (C-BM) e atividade das enzimas urease, fosfatase ácida e β-glucosidase. Em ambos os solos, tanto o FOM em menores doses (40% e 60%) quanto os tratamentos minerais, apresentaram desempenhos fitotécnicos semelhantes entre si, bem como o acúmulo de nutrientes no tecido vegetal das plantas, exceto para os elementos P, K e B. Não foram observadas diferenças significativas no desenvolvimento das plantas e nos atributos microbiológicos que possam ser atribuídas as formas físicas do FOM. Embora a RBS tenha indicado aumento na atividade microbiana dos solos a partir da adição de doses crescentes de FOMs, isso não refletiu nos demais parâmetros fitotécnicos ou microbiológicos avaliados, indicando que a planta foi a principal moduladora do comportamento da comunidade microbiana e da dinâmica de nutrientes. Adicionalmente, as análises enzimáticas mostraram-se pouco efetivas diante de condições de estresse observadas neste estudo para gerar respostas em função dos tratamentos. Conclui-se que a utilização de lodo de esgoto como matriz orgânica na formulação de FOMs pode ser uma alternativa agronomicamente viável e sustentável à adubação mineral convencional para a fertilização da soja em solos de Cerrado. / Sewage sludge is associated with high concentrations of inorganic pollutants in amplitudes that may make direct use in agricultural soil unfeasible, in accordance with the maximum limits established by legislation, the final destination of which are sanitary landfills, which, despite being a lower impact alternative is an onerous and finite practice. However, this material is rich source organic matter, macro and micronutrients for plants such as boron, copper, cobalt, iron, manganese, molybdenum, nickel and zinc for plants, commonly deficient in tropical soils. In our study, we explored this by-product as an organic matrix for the formulation of an organomineral fertilizer (FOM) as an alternative to the conventional mineral fertilization for soybean cultivation in Cerrado soils, as well as possible changes in the soil microbial community. The experiment was conducted in a greenhouse using two different dystrophic Red Latosols of different textures (clayey and sandy loam), different doses of FOM application (40%, 60%, 80%, 100% and 120%) in three (M + Mi + S) and without (M + S) the micronutrients B and Zn, besides the treatment without fertilization (CT). The design was completely randomized blocks. The plants were harvested up to 60 days after planting (DAP) and the relevant plant breeding parameters were evaluated for aerial dry mass (MSPA), dry root mass (DMW), plant height, shoot diameter and number of nodules active. The chemical analysis of aerial part (macro and micronutrients) was used to calculate the nutrient content accumulated in the vegetal tissue. Soil microbial biomass (C-BM) and activity of the enzymes urease, acid phosphatase and β-glucosidase were used to evaluate soil microbial activity. In both soils, FOM at lower doses (40% and 60%) and mineral treatments presented similar phytotechnical performances as well as nutrient accumulation in the plant tissue of the plants, except for the elements P, K and B. No significant differences were observed in the development of the plants and in the microbiological attributes that can be attributed to the physical forms of FOM. Although RBS indicated an increase in microbial activity of the soils from the addition of increasing doses of FOMs, this did not reflect the other phytotechnical or microbiological parameters evaluated, indicating that the plant was the main modulator of microbial community behavior and nutrient dynamics. In addition, the enzymatic analyzes showed little effectiveness in the stress conditions observed in this study to generate responses depending on the treatments. It is concluded that the use of sewage sludge as an organic matrix in the formulation of FOMs can be an agronomically viable and sustainable alternative to conventional mineral fertilization for soybean fertilization in Cerrado soils.

<p>This thesis work consisted of the development of a virtual instrument that automates enzyme activity measurements and spectrum measurements with the spectrophotometer UVmini-1240. The purpose was to expand the functionality of the instrument, to eliminate the human error and to decrease the amount of time spent on measurements. A PC was connected to the UVmini-1240 via a RS-232C interface and the cell position and temperature was regulated with a CPS-240A controller. The new interface allows all the parameters to be set in the same place. It allows a visualization of the continuous monitoring of the sample absorbance and the option to save the data for post-processing. Also a module for measuring the spectrum of a sample in the wavelength range of 190 nm to 1100 nm is included. The graphical programming language LabView was used to develop the virtual instrument. This thesis work also contained measurement series of the catalase enzyme activity. These were carried out to determine the best storage temperature for the catalase solution and to determine the optimal surrounding temperature for the highest activity in the catalase solution. The conclusions were that the activity does not change considerably the first week of storage, not matter the temperature, and that the activity goes down when the surrounding temperature reaches above 30° C. These measurements were part of a bigger project to develop an ultrasonic method for measuring enzyme activity at the Institute of Acoustics at C.S.I.C in Madrid.</p>

This thesis work consisted of the development of a virtual instrument that automates enzyme activity measurements and spectrum measurements with the spectrophotometer UVmini-1240. The purpose was to expand the functionality of the instrument, to eliminate the human error and to decrease the amount of time spent on measurements. A PC was connected to the UVmini-1240 via a RS-232C interface and the cell position and temperature was regulated with a CPS-240A controller. The new interface allows all the parameters to be set in the same place. It allows a visualization of the continuous monitoring of the sample absorbance and the option to save the data for post-processing. Also a module for measuring the spectrum of a sample in the wavelength range of 190 nm to 1100 nm is included. The graphical programming language LabView was used to develop the virtual instrument. This thesis work also contained measurement series of the catalase enzyme activity. These were carried out to determine the best storage temperature for the catalase solution and to determine the optimal surrounding temperature for the highest activity in the catalase solution. The conclusions were that the activity does not change considerably the first week of storage, not matter the temperature, and that the activity goes down when the surrounding temperature reaches above 30° C. These measurements were part of a bigger project to develop an ultrasonic method for measuring enzyme activity at the Institute of Acoustics at C.S.I.C in Madrid.

No description available.

Polyketide biosynthesis is a field that has had tremendous advances in the past 50 years. The understanding of the mechanisms is updated as investigations delve into domain interactions of these microbial natural products. Although numerous polyketides are known, similarities in the sequence of product generation can be used as templates for further exploration of enzymatic activity. The focus of studies recently has been towards developing protocols to manipulate the natural products resulting in medicinally important manufactured products. This investigation examined the mechanism of the acyl carrier protein (ACP) module involved in biosynthesis. / text

Klotho (KL), an anti-aging protein, attracted interest in the aging field because of the dramatic phenotype of KL deficient mice and its connection to signaling pathways implicated in aging. The KLVS variant consists of the F352V (KLV) and C370S (KLS) substitutions. It was detected in genome wide association studies (GWAS) that linked it to alterations in longevity and disease risk. The molecular mechanism(s) underlying these associations are unknown. To understand how KL increases the risk of age-related diseases, the studies in this dissertation investigated whether expression of the KLVS variant, when compared to wildtype (KLWT), displays differences in processing, protein-protein interactions and enzymatic activity. Differences in processing were evaluated by studying changes in shedding, half-life and plasma membrane localization of KL variants. The decrease in KLV shedding, as measured by the intracellular: extracellular ratio, were explained by a decreased half-life. This decreased half-life is potentially due to decreased KLV plasma membrane localization, which is attenuated by co-expression of dominant negative dynamin, suggesting a role of endocytosis in these differences. To assess whether there are changes in KLVS protein-protein interactions, differences in dimerization were measured by Blue Native gel electrophoresis and cross-linking. KLV dimerization was increased while KLS and KLVS variants decreased dimerization. Co-immunoprecipitation of tagged KL assessed whether these changes were due to alterations in homodimerization. The presence of KLVS in dimers decreased the levels of immunoprecipitated KL suggesting KLVS decreases homodimerization. Changes in heterodimerization of KLVS with fibroblast growth factor receptor (FGFR) 1c were also investigated through co-immunoprecipitation. KLVS increased heterodimerization with FGFR1c. Addition of FGF23, for which KL is a co-receptor, showed that KLVS increases FGF signaling downstream of FGFR1c. To determine differences in enzymatic activity of KLVS, 4-metylumbelliferyl-beta-D-glucuronide was used to measure alterations in glucuronidase activity. Results showed that KLVS had decreased enzymatic activity compared to KLWT. These findings are the first to show that KLVS leads to differences in function as demonstrated by decreased homodimerization and enzymatic activity and increased heterodimerization with FGFR1c. Given the association of KLVS with disease and longevity, these results suggest that these functions are integral in KL's anti-aging role in humans.

In all sequenced genomes, a large fraction of predicted genes encodes proteins of unknown biochemical function and up to 15% of the genes with ‘‘known’’ function are mis-annotated. Several global approaches are being employed to predict function, including sequence similarity searches, analysis of gene expression, protein interaction, and protein structure. Enzymes comprise a group of target proteins that require experimental characterization for accurate functional annotations. Here I applied enzyme genomics to identify new enzymes by screening individually purified proteins for enzymatic activity under relaxed reaction conditions, which allowed me to identify the subclass or sub-subclasses of enzymes to which the unknown protein belongs. Further biochemical characterization of proteins was facilitated by the application of secondary screens with natural substrates (substrate profiling). Application of general enzymatic screens and substrate profiling greatly sped up the identification of biochemical function of unknown proteins and the experimental verification of functional predictions produced by other functional genomics approaches. As a test case, I used this approach to characterize the members of the haloacid dehalogenase (HAD)-like hydrolase superfamily, which consists mainly of uncharacterized enzymes, with a few members shown to possess phosphatase, beta-phosphoglucomutase, phosphonatase, and dehalogenase activities. Low sequence similarity between the members of the HAD superfamily precludes the computational prediction of their substrates and functions. Using a representative set of 80 phosphorylated substrates I characterized the phosphatase activities of 21 soluble HADs from Escherichia coli and seven soluble HADs from Saccharomyces cerevisiae. E. coli HADs show broad and overlapping substrate specificity against a wide range of phosphorylated metabolites. The yeast enzymes were more specific, and one protein also showed protein phosphatase activity. Comparison of HAD substrate profiles from two model organisms showed several “functional niches” that are occupied by HADs, which include hydrolysis of nucleotides, phosphoglycolate, phosphoserine, and pyridoxal phosphate. I proposed the cellular function for a number of HADs from both organisms based on substrate specificities. The physiological relevance of the phosphatase activity with the preferred substrate was validated in vivo for one of the HADs, E. coli YniC.

The rehabilitation of degraded soils, such as those associated with post-mining sites, requires knowledge of the soil ecosystem and its physical, chemical, and biological composition in order for rehabilitation efforts to fulfil the long-term goal of reconstructing a stable ecosystem for rehabilitated mine soil. This study addresses the need for appropriate assessment criteria to determine the progress of rehabilitation and subsequently the success of management practices. Significant contributions made by this investigation included the establishment of minimum and maximum values for microbial community measurements from two case studies of rehabilitated coal discard sites. Furthermore, it was shown that there was no relationship between changes in microbial community function and structure and the rehabilitation age of the sites. Following this, the considerable impact of management practices on microbial communities was illustrated. The first part of the study investigated the temporal changes in microbial community function and structure in a chronosequence of rehabilitated coal discard sites aged 1 to 11 years. The most important observation made during the investigation of the microbial communities in the different aged soil covers of the rehabilitated coal discard sites, was that there was no relationship between rehabilitation age and microbial activity or abundance of certain microbial groups. What was responsible for a clear differentiation between sites and a shift in microbial community attributes was the management practices applied. A comparison of two chronosequences of rehabilitated coal discard sites was achieved by an application of the 'space-for-time' hypothesis. Sites of different ages and at separate locations ('space') were identified to obtain a chronosequence of ages ('time'). The two chronosequences included sites aged 1 to 11 years (chronosequence A) and 6 to 17 years (chronosequence B), respectively. Sites in the same chronosequence were managed identically, while there was a distinct difference in management practices applied to each chronosequence. The long-term effect of the different management regimes on the soil microbial community function and structure was investigated. Again, there was no relationship between rehabilitation age and microbial community measurements. Fluctuations of selected microbial properties occurred in both chronosequences and similar temporal trends existed over the rehabilitation periods. However, the less intensively managed chronosequence (8) seemed more stable (less fluctuation occurred) over the rehabilitation period than the more intensively managed chronosequence (A). It was therefore concluded that the microbial communities in the less managed sites maintained their functional and structural integrity within bounds in the absence of management inputs or disturbance. While there was similarity in the trends over time for individual microbial community measurements, the seemingly more stable conditions in chronosequence 6 are important in terms of the goal of rehabilitation. / Thesis (Ph.D. (Environmental Science)--North-West University, Potchefstroom Campus, 2007