9 research outputs found
Supercharging of a family-5 glycoside hydrolase Cel5A from Thermobifida fusca to improve cellulose hydrolysis
No full textThe feasibility of producing useful fuels and chemicals from lignocellulosic biomass hinges on the efficiency of biomass deconstruction catalyzed by different enzymes such as cellulases and hemicellulases. Enzymatic hydrolysis is hindered by biomass recalcitrance caused by the tight crystalline cellulose structure as well as the structural organic biopolymer lignin. Lignin forms a protective covering over core cellulose and hemicellulose restricting access to enzymes while also being a major source of irreversible non-productive binding. Thermochemical pretreatment is often used to disrupt the lignin coating in order to increase exposure of cellulose and hemicellulose, but delocalized lignin remains present and still available for irreversible binding of cellulases/hemicelluases. As such, current industrial processes require high enzyme loadings to supplement low activity on crystalline cellulose, as well as enzyme lost to non-productive binding. Engineering strategies, such as enzyme supercharging which refers to the process of introducing positive or negatively charged amino acids in solvent exposed areas to create highly negative/positive net charge, have been useful in the past to combat biomass recalcitrance. Past work has focused on improving thermal stability of cellulases, as well as altering surface interactions in order to reduce binding to lignin but has not exhibited higher activity on highly crystalline cellulose. This thesis focuses on a supercharged library consisting of a family 5 glycoside hydrolase (GH5) Cel5A and its native family 2a carbohydrate binding module CBM2a from the thermophilic microbe Thermobifida fusca. The library consists of 33 enzymes in total, including the wild-type (WT), with mutations being made on both the CBM2a binding module and Cel5A catalytic domain (CD) spanning a net charge range of -51.3 to 36.9. To gather preliminary activity data on the entire library, each design was expressed in E. coli as small-scale cultures, and the cell lysate was screened using numerous assays. Activity data was collected using several substrates including soluble para-nitrophenol cellobioside (pNP-C), pretreated ammonia fiber expansion (AFEX) corn stover biomass, and crystalline cellulose. Lysate screening enabled the reduction of the library size from 33 mutants in total to 16 that showed activity either similar or greater compared to the wild type. From this reduced library, four constructs with CBM and two constructs with CD mutations were expressed and purified on a large scale. The purified mutants were used to validate the cell lysate screening and characterized further. Three notable mutants, one negatively supercharged, and two positively supercharged stood out compared to the wild-type enzyme. All three constructs have exhibited up to a 2-fold increase in activity on crystalline cellulose-I, and both positively supercharged enzymes have shown a five degree increase in their optimal hydrolysis temperature. Overall, this work provides insights on how supercharging can be used to tune surface interactions to decrease non-productive binding, increase activity on crystalline cellulose, as well as show how supercharging can impact thermal resilience of cellulase enzymes.M.S.Includes bibliographical reference
Untitled in english
No full textNos últimos anos vem se tornando mais corrente o uso de fórmulas baseadas no repique elástico, as quais originam-se da lei de Hooke, como a proposta por Chellis (1961), ou de modelos desenvolvidos a partir da Teoria da Equação da Onda, como aspropostas por Uto et al. (1985b) e por Matsuo et al. (1989). No entanto, apesar do significativo aumento do uso dessas fórmulas em obras correntes, observa-se que os resultados obtidos ainda não são suficientemente satisfatórios, visto queprocura-se utilizar coeficientes de segurança menores, o que exige maior precisão na avaliação da capacidade de carga em estacas cravadas. A realização de ensaios de carregamento estático ou dinâmico por amostragem representa uma boa soluçãopara avaliar a qualidade do estaqueamento, porém, desde que a variabilidade natural do maciço de solo não gere diferenças substanciais entre as estacas, caso contrário, o número de amostras ensaiadas pode ser insuficiente para representar ouniverso total de estacas de uma obra. Essa insuficiência pode ser minorada com a aplicação de fórmulas dinâmicas em uma amostragem maior que a ensaiada e, de preferência, na totalidade das estacas de uma determinada obra. Para tanto, énecessário que as fórmulas utilizadas sejam suficientemente confiáveis. Neste trabalho, apresenta-se um estudo sobre as fórmulas dinâmicas de Chellis (1961) e de Uto et al. (1985b), no qual avalia-se a confiabilidade dessas fórmulas eapresentam-se sugestões de modificações dasmesmas, objetivando-se a obtenção de resultados mais precisos. Realizou-se, inicialmente, uma pesquisa sobre o tema em estudo englobando, dentre outros assuntos, as fórmulas dinâmicas baseadas norepique elástico e os métodos de análise de capacidade de carga fundamentados na Teoria da Equação da Onda. Paralelamente, reuniram-se diversos relatórios de ensaios de carregamento dinâmico e compararam-se os resultados fornecidos pelo MétodoCase com os valores ) calculados pelas referidas fórmulas de cravação. Analisaram-se os principais parâmetros envolvidos, o que permitiu extrair conclusões importantes à compreensão das diferenças advindas da utilização das fórmulas estudadas emcomparação com os resultados do Método Case. Desse estudo constatou-se que a dispersão dos resultados está relacionada, dentre outros fatores, ao valor de deformação elástica atribuída ao solo, à resultante da superposição da ondas de tensão aolongo da estaca e ao chamado \'descarregamento\', no qual o deslocamento máximo (repique) ocorre antes do retorno das ondas refletidas na ponta da estaca, portanto, sem incluir a parcela de deformação elástica do solo. Por fim, foram propostasmodificações às fórmulas de Chellis (1961) e de Uto et al. (1985b), a partir da comparação dos valores de resistência mobilizada, calculados através das referidas fórmulas com aqueles fornecidos pelo Método Case, obtendo-se uma melhoraexpressiva nos resultados alcançados em comparação com os inicialmente obtidos.In the last years the use of formulas based on elastic rebound has become more current. These equations are based on Hooke\'s law as the one proposed by Chellis (1961), or derived from Wave Equation Theory, as the one presented by Uto et al.(1985b) and by Matsuo et al. (1989). However, despite the increasing use of these formulas meaningful in current buildings, it can be observed that the results are still not sufficiently satisfactory, noticing that, more and more, smaller safetyfactors are tryed to be used, what requires a better accuracy in the evaluation of the bearing capacity of driven piles. Performing static and dynamic load tests on selected piles represents a good solution to evaluate the quality of pilling,however, only if the variability of the natural ground doesn\'t bring substantial differences among the piles, otherwise, the number of tested samples can be insufficient to represent the complete universe of piles. This insufficiency can beovercome by applying the dynamic formulas in a sampling bigger than the one tested and, preferably, in the totality of piles of the building. However, it is necessary that the applied formulas can provide sufficiently reliable results. In thisresearch, a study about the dynamic formulas by Chellis (1961) and Uto el al. (1985b) is presented, in which reliability of these formulas is evaluated and suggestions are presented to modify them, aiming to obtain more accurate results.Initially, the research focused issuesrelated to the theme in a study including, among other issues, the dynamic formulas based on the elastic rebound and the methods of analysis of the bearing capacity based on the Wave Equation Theory.Several results of dynamic load tests were obtained and the results from the Case Method evaluation were compared to the values calculated by the referred dynamic formulas. The main envolved parameters were analyzed, permitting to comprehend the differences resulting from the use of those formulas, in comparison with Case Method. From this study it could be noticed that the scattering of the results are related, among other factors, to the adopted parameter of the elasticdeformation of the soil under the pile tip, to the superposition of the stress waves through the pile and to the so called \'unloading\', in which the maximum displacement (rebound) occurs before the reflected waves has returned from the pile tip,therefore, without including the register of a significant part elastic soil deformation. Finally, the author proposed modifications on the Chellis (1961) and Uto et al. (1985b) formulas, whose results were tested making comparisons of thecalculated mobilized resistance values and the values obtained by applying the Case Method. A significant improvement could be obtained by using this modifications, when compared with those resulting from the use of the original formulas
Good at heart: Developing a tertiary perinatal cardiac service; the first eight years of experience
No full textGenome scan of a second wave of NIMH genetics initiative bipolar pedigrees: chromosomes 2, 11, 13, 14, and X
No full textGenetics of recurrent early‐onset depression (GenRED): Design and preliminary clinical characteristics of a repository sample for genetic linkage studies
No full textThis is an initial report on a six-site collaborative project, Genetics of Recurrent Early-Onset Depression (GenRED). This is a study of a large sample of families with recurrent major depressive disorder (DSM-IV) beginning by the age 30 in probands or 40 in relatives. Evidence suggests that early onset and recurrence of depressive episodes predict substantially increased risk of depression in first-degree relatives compared with the general population, suggesting that susceptibility genes might be mapped with this phenotype. The projected sample of 800-1,000 affected sibling pairs (ASPs) and other relatives will be studied using genome scan methods. Biological materials and blinded clinical data will be made available through the NIMH cell repository program. The sample should have good-to-excellent power to detect a locus associated with a 24% or greater population-wide increase in risk to siblings. We describe 838 affected individuals from the first 305 families containing 434 independent ASPs, or 613 ASPs counting all possible pairs. The mean age at the onset was 18.5 years, with a mean of 7.3 episodes and longest episode of 655 days. Almost all subjects had experienced at least 4 weeks of depression with five or more additional symptom criteria. Frequencies of symptoms and psychiatric and medical comorbid are provided. Substance use was more common in males, and panic disorder in females. Within pairs of affected siblings, correlations were significant for age at onset, substance abuse/dependence, panic disorder, obsessive-compulsive disorder and nicotine initiation and persistence. We replicated previously reported associations among comorbid panic disorder and social phobia, chronicity of depression and suicidal behavior. This suggests comparability of our cases to those in earlier large family studies. This dataset should prove useful for genetic studies of a highly familial form of major depressive disorder
Genomewide Significant Linkage to Recurrent, Early-Onset Major Depressive Disorder on Chromosome 15q
No full textA genome scan was performed on the first phase sample of the Genetics of Recurrent Early-Onset Depression (GenRED) project. The sample consisted of 297 informative families containing 415 independent affected sibling pairs (ASPs), or, counting all possible pairs, 685 informative affected relative pairs (555 ASPs and 130 other pair types). Affected cases had recurrent major depressive disorder (MDD) with onset before age 31 years for probands or age 41 years for other affected relatives; the mean age at onset was 18.5 years, and the mean number of depressive episodes was 7.3. The Center for Inherited Disease Research genotyped 389 microsatellite markers (mean spacing of 9.3 cM). The primary linkage analysis considered allele sharing in all possible affected relative pairs with the use of the Zlr statistic computed by the ALLEGRO program. A secondary logistic regression analysis considered the effect of the sex of the pair as a covariate. Genomewide significant linkage was observed on chromosome 15q25.3-26.2 (Zlr=4.14, equivalent LOD = 3.73, empirical genomewide P=.023). The linkage was not sex specific. No other suggestive or significant results were observed in the primary analysis. The secondary analysis produced three regions of suggestive linkage, but these results should be interpreted cautiously because they depended primarily on the small subsample of 42 male-male pairs. Chromosome 15q25.3-26.2 deserves further study as a candidate region for susceptibility to MDD
The potter’s skill : perceptions of workmanship in the English ceramic industries, 1760-1800
No full textThis thesis focuses on perceptions of workmanship in the English porcelain
and earthenware industries between 1760 and 1800. Research by Berg and
Clifford has demonstrated a new interest in and valuation of workmanship
by contemporaries in the eighteenth century. Yet little is known of what
contemporaries understood workmanship to mean, or be. This thesis
argues that understandings of workmanship affected both the consumption
and production practices of eighteenth-century contemporaries. It does so
by concentrating on six groups of people – industrial tourists, consumers,
retailers, designers, manufacturers and workers. It demonstrates the
different ways in which contemporaries perceived hand skills and tacit
knowledge by examining a range of sources such as letters, prints, trade
cards, travel accounts and objects.
This thesis concludes that meanings of ‘workmanship’ - that combination of
effort, work and skill - were shifting in the second half of the eighteenth
century. For those not employed in manufacturing, reading manuals,
seeing production in action and handling objects all challenged their ideas
of workmanship. These experiences encouraged contemporaries to
question the meaning of innovative products and the manufacturing
techniques used to make them. Similarly, in manufacturing the
development of the design process and the demands of novelty and
standardisation forced manufacturers, designers and modellers to ask how
to achieve ‘excellent workmanship’. At the same time, workers understood
and valued their work in different terms – as a hard-won, social and
physical skill. This thesis argues that for eighteenth-century contemporaries
‘workmanship’ was a complex idea, under challenge from developments in
production and consumption. In so doing it moves the interlinked history
of manufacturing and consumption away from the extant debates of
economic historians and into a different sub-disciplinary space, namely
cultural history; a space that has tended to neglect the cultural aspects of
production
Genome-wide scan and conditional analysis in bipolar disorder: evidence for genomic interaction in the National Institute of Mental Health genetics initiative bipolar pedigrees
No full textThe molecular genetics of bipolar affective disorder : South African populations, endophenotypes, and environmental influence
No full textIncludes bibliographical references.The identification of the genetic variants underpinning bipolar disorder (BPD) has been impeded by a complex pattern of inheritance that may include by genetic heterogeneity, genetic epistasis, gene-environment interactions, incomplete penetrance and variable expressivity. In this thesis three strategies were employed to ameliorate these confounding factors. The first strategy was to focus on a theoretically genetically-homogeneous population with BPD. A unique South African sample including 190 individuals of the relativity reproductively-isolated Afrikaner population yielded promising evidence of linkage to chromosome 1 q31-32 and weaker peaks at lOq23 and 13q32, regions previously implicated in the disorder. A family-based analysis suggested that the 3' variable number tandem repeat (VNTR) variant of the dopamine transporter gene (DAT) is associated with bipolar-spectrum illness in the 132-strong sample of British ancestry. The second strategy was to carry out genetic linkage and association analyses using quantitative traits (elldophenotypes) that were closely associated with BPD. As part of this process a variety of personality traits were evaluated in the cohort, and anxiety related, novelty-seeking, hyperthymic, and cyclothymic personality traits were found to aggregate in participants with BPD and to a lesser extent repeated unipolar illness (MDE-R). These traits were therefore used as quantitative markers or endophenotypes of BPD. The quantitative linkage analysis indicated that a variant in the region of 13q32 may influence the development of novelty-seeking-related traits in the largest Afrikaner pedigree, while the personality trait, ""Stability"", was weakly linked to 4p16 in the total sample. The catechol-o-methytransferase (COM1) Va1l58Mct and the Brain Derived Neurotrophic Factor (BDNF) Va1l66Met polymorphisms were associated with mood-labile-cyclothymic and hyperthymic·-novelty-seeking traits, respectively. the DA T VNTR and the Notch4 exonic repeat variants were associated with a broad range of ""pathological"" personality traits in the sa11lples of British and Afrikaner origin, respectively. The sample was also evaluated with a battery of neuropsychological tasks and the BPD 1 and MDE-R groups displayed both verbal and visual memory recall deficits while the BPD 1 sample also suffered from recognition memory deficits. The neurocognitive trait, ""Memory"" was therefore used as a second endophenotype generating potential linkage signals on IOq23 and 22q 11. The exonic 48bp VNTR polymorphism in the dopamine 4 receptor (DRD4) gene was associated with '""Memory"" performance. As a third strategy, a potentially important aetiological factor, childhood trauma, was measured, and used to test for gene-environment interactions between the various candidate genes and bipolar-illness or BPD-related endophenotypes in the cohort. BPD and M DE-R individuals displayed significantly higher levels of emotional and physical abuse, and the former variable was also associated with the development of anxiety-related and unstable personality traits. A functional variant of the COM1 gene was found to interact with abuse to predispose to anxiety-related, unstable cyclothymic and novelty-seeking related personality traits. The combination of childhood abuse and possession of low-activity MAO-A gene variants was also associated the development of more anxious and unstable personality traits. All interaction between sexual abuse and the B])NF gene modulated performance on verbal and visual memory tasks. A similar interaction between the ApoE gene and sexual abuse was observed. Although a number of theoretical obstacles remain to be resolved, the analyses of isolated populations coupled with the use of endophenotypes and the testing or gene environment interactions, holds out great promise for the eventual elucidation of the genetic basis of hi polar affective disorder
