R. B. Cunninghame Graham’s contribution to the political and literary life of Scotland: party, prose, and the political aesthetic

This thesis examines the substantive and verifiable political and cultural legacy of the social reformer and author, Robert Bontine Cunninghame Graham (1852-1936), one of the most famous and controversial Scots of his generation. Much has been written about Graham before and after his death, in biographies, memoirs, and analyses of his large canon of essays, but there has been little analysis of his political beliefs and the influences that shaped them, and how these directed his efforts into the creation of the first party of labour in Britain, and his later involvement in the first nationalist party in Scotland. The thesis therefore seeks to uncover Graham’s philosophies through his political statements, and to extrapolate them, where possible, into the themes of his political campaigns and in his later literary works. It is thus the intention to transcend previous perspectives by looking at Graham not solely as a politician, nor an author, but as an eloquent, disquieted, principled, fervid moralist and contrarian, for whom politics and writing were fundamentally the same thing, the means by which his deeply felt moral anger could be expressed

Analysis of a Library of Escherichia coli Transporter Knockout Strains to Identify Transport Pathways of Antibiotics

Antibiotic resistance is a major global healthcare issue. Antibiotic compounds cross the bacterial cell membrane via membrane transporters, and a major mechanism of antibiotic resistance is through modification of the membrane transporters to increase the efflux or reduce the influx of antibiotics. Targeting these transporters is a potential avenue to combat antibiotic resistance. In this study, we used an automated screening pipeline to evaluate the growth of a library of 447 Escherichia coli transporter knockout strains exposed to sub-inhibitory concentrations of 18 diverse antimicrobials. We found numerous knockout strains that showed more resistant or sensitive phenotypes to specific antimicrobials, suggestive of transport pathways. We highlight several specific drug-transporter interactions that we identified and provide the full dataset, which will be a useful resource in further research on antimicrobial transport pathways. Overall, we determined that transporters are involved in modulating the efficacy of almost all the antimicrobial compounds tested and can, thus, play a major role in the development of antimicrobial resistance

Substrate and Inhibitor-Specific Conformational Changes in the Human Serotonin Transporter Revealed by Voltage-Clamp Fluorometry s

ABSTRACT The serotonin transporter (SERT) regulates neurotransmission by the biogenic monoamine neurotransmitter serotonin (5-HT, 5-hydroxytryptamine) in the central nervous system, and drugs inhibiting SERT are widely used for the treatment of a variety of central nervous system diseases. The conformational dynamics of SERT transport function and inhibition is currently poorly understood. We used voltage-clamp fluorometry to study conformational changes in human SERT (hSERT) during 5-HT transport and inhibitor binding. Cys residues were introduced at 12 positions in hSERT to enable covalent attachment of a rhodamine-based fluorophore. Transport-associated changes in fluorescence from fluorophore-labeled hSERT expressed in Xenopus oocytes could be robustly detected at four positions in hSERT: endogenous Cys109 in the top of transmembrane domain (TM) 1b, Cys substituted for Thr323 in the top of TM6, Ala419 in the interface between TM8 and extracellular loop (EL) 4, and Leu481 in EL5. The reporter positions were used for timeresolved measurement of conformational changes during 5-HT transport and binding of cocaine and the selective serotonin reuptake inhibitors fluoxetine and escitalopram. At all reporter positions, fluorescence changes observed upon substrate application were distinctly different from those observed upon inhibitor application, with respect to relative amplitude or direction. Furthermore, escitalopram, fluoxetine, and cocaine induced a very similar pattern of fluorescent changes overall, which included movements within or around TM1b, EL4, and EL5. Taken together, our data lead us to suggest that competitive inhibitors stabilize hSERT in a state that is different from the apo outward-open conformation as well as inward-facing conformations

Identification and Engineering of Transporters for Efficient Melatonin Production in Escherichia coli

Transporter discovery and engineering play an important role in cell factory development. Decreasing the intracellular concentration of the product reduces product inhibition and/or toxicity. Lowering intracellular concentrations is especially beneficial for achieving a robust strain at high titers. However, the identification of transporters for xenobiotic chemicals in the host strain is challenging. Here we present a high-throughput workflow to discover Escherichia coli transporters responsible for the efflux of the inhibitory xenobiotic compound melatonin. We took advantage of the Keio collection and screened about 400 transporter knockouts in the presence of a high concentration of melatonin. We found five transporters that when knocked out showed decreased tolerance to melatonin, indicating they are exporters of melatonin. We overexpressed these five genes individually in the production strain and found that one of them, yhjV, encoding a transporter with unknown substrates, resulted in a 27% titer increase in cultivation mimicking fed-batch fermentation. This study demonstrates how microbial cell factories can be improved through transporter identification and engineering. Further, these results lay the foundation for the scale-up of melatonin production in E. coli

Alinhamento interpessoal, representacional e morfossintático na Gramática Discursivo-Funcional

Este artigo se debruça sobre o mapeamento entre os Níveis Interpessoal, Representacional e Morfossintático da gramática, o chamado alinhamento, segundo o arcabouço da Gramática Discursivo-Funcional (GDF). Propõe uma tipologia das línguas baseada no que a sua organização morfossintática codifica: distinções pragmáticas (p.ex. em Tagalo), distinções semânticas (p.ex. em Achém), ou distinções inerentes à morfossintaxe (p.ex. em Inglês, Basco ou a língua Kham). A inclusão tanto do Sujeito como do Objeto e de línguas tanto acusativas como ergativas no tratamento do alinhamento morfossintático permitiu-nos abranger tipos tipologicamente mais variáveis e demonstrar o potencial da GDF para a análise contrastiva das línguas.<br>Within the framework of Functional Discourse Grammar (FDG), alignment concerns the relations between the Interpersonal, Representational and Morphosyntactic Levels of grammar. This article proposes a typology of languages based upon what we find to be encoded in their morphosyntactic organization: pragmatic distinctions (as in Tagalog), semantic distinctions (as in Acheh), or distinctions inherent to the morphosyntax (as in English, Basque and Kham). By including both subject and object, and both accusative and ergative languages in our treatment of morphosyntactic alignment, we provide a better coverage of typological variation and show the potential of FDG for cross-linguistic analysis

Primary to post-primary: Issues in school choice

This thesis examines the process of choosing a secondary school. Through the use of online surveys, the opinions of parents and students are explored. Along with the views of parents and students, principals are surveyed in order to examine their thoughts on the promotion and marketing of secondary schools. How do principals define marketing? What do they promote and where? What do they believe parents and students look for in a school? Parents and students were asked what they consider important factors to be in deciding on the school of choice, where information is sourced, and the perceived effectiveness of the information. The results of the principals’ survey have been compared with the parents and students. Principals tend to market aspects of their school that they believe that parents and students would look for. Although only one-third of surveyed principals have received specific training in marketing, principals are addressing common areas for both parents and students—the school curriculum, relationships between staff and pupils, and extra-curricular activities, with the latter being of more importance to students than to parents. School marketing activities are focused more on the perceived needs of the parent than the student, despite the study finding that the decision is a joint one in close to half of the participating families. Students are satisfied with the level of input they have into the decision-making process—they are not seeking to have more say. The majority of parents reported that they are satisfied they have the information required to make an informed decision as to which secondary school would be best for their child. Both parents and students were generally satisfied with the information found in school prospectuses and obtained at school open days

Primary to post-primary: Issues in school choice

This thesis examines the process of choosing a secondary school. Through the use of online surveys, the opinions of parents and students are explored. Along with the views of parents and students, principals are surveyed in order to examine their thoughts on the promotion and marketing of secondary schools. How do principals define marketing? What do they promote and where? What do they believe parents and students look for in a school? Parents and students were asked what they consider important factors to be in deciding on the school of choice, where information is sourced, and the perceived effectiveness of the information. The results of the principals’ survey have been compared with the parents and students. Principals tend to market aspects of their school that they believe that parents and students would look for. Although only one-third of surveyed principals have received specific training in marketing, principals are addressing common areas for both parents and students—the school curriculum, relationships between staff and pupils, and extra-curricular activities, with the latter being of more importance to students than to parents. School marketing activities are focused more on the perceived needs of the parent than the student, despite the study finding that the decision is a joint one in close to half of the participating families. Students are satisfied with the level of input they have into the decision-making process—they are not seeking to have more say. The majority of parents reported that they are satisfied they have the information required to make an informed decision as to which secondary school would be best for their child. Both parents and students were generally satisfied with the information found in school prospectuses and obtained at school open days

Intelligent host engineering for metabolic flux optimisation in biotechnology

Optimising the function of a protein of length N amino acids by directed evolution involves navigating a ‘search space’ of possible sequences of some 20(N). Optimising the expression levels of P proteins that materially affect host performance, each of which might also take 20 (logarithmically spaced) values, implies a similar search space of 20(P). In this combinatorial sense, then, the problems of directed protein evolution and of host engineering are broadly equivalent. In practice, however, they have different means for avoiding the inevitable difficulties of implementation. The spare capacity exhibited in metabolic networks implies that host engineering may admit substantial increases in flux to targets of interest. Thus, we rehearse the relevant issues for those wishing to understand and exploit those modern genome-wide host engineering tools and thinking that have been designed and developed to optimise fluxes towards desirable products in biotechnological processes, with a focus on microbial systems. The aim throughput is ‘making such biology predictable’. Strategies have been aimed at both transcription and translation, especially for regulatory processes that can affect multiple targets. However, because there is a limit on how much protein a cell can produce, increasing k(cat) in selected targets may be a better strategy than increasing protein expression levels for optimal host engineering

An Untargeted Metabolomics Strategy to Identify Substrates of Known and Orphan <i>E. coli</i> Transporters.

Transport systems play a pivotal role in bacterial physiology and represent potential targets for medical and biotechnological applications. However, even in well-studied organisms like Escherichia coli, a notable proportion of transporters, exceeding as many as 30%, remain classified as orphans due to their lack of known substrates. This study leveraged high-resolution LC-MS-based untargeted metabolomics to identify candidate substrates for these orphan transporters. Human serum, including a diverse array of biologically relevant molecules, served as an unbiased source for substrate exposure. The analysis encompassed 26 paired transporter mutant contrasts (i.e., knockout vs. overexpression), compared with the wild type, revealing distinct patterns of substrate uptake and excretion across various mutants. The convergence of candidate substrates across mutant scenarios provided robust validation, shedding light on novel transporter-substrate relationships, including those involving yeaV, hsrA, ydjE, and yddA. Furthermore, several substrates were contingent upon the specific mutants employed. This investigation underscores the utility of untargeted metabolomics for substrate identification in the absence of prior knowledge and lays the groundwork for subsequent validation experiments, holding significant implications for both medical and biotechnological advancements