Biochemical and molecular characterisation of purine transporters of Trypanosoma brucei brucei

This thesis describes the elucidation and comparison of substrate recognition relationships for the H2 nucleobase transporter of T. b. brucei and the main nucleobase transporter of human erythrocytes. Using standard transport kinetics, application of the Cheng-Prusoff equation and a derivation of the Nernst equation, it was possible to determine the Gibbs free energy (DG°) for the interactions of purine analogues with each transporter, which allowed predictions about the nature of the interactions that are essential for uptake. A range of unusual tricyclic and "fleximer" purine analogues was also assayed for ability to interact with the various purine transporters in T. b. burcei and human erythrocytes. This provided further insights into the extent that the substrate-binding pocket can accommodate unusual and large substrates. Some of the purine analogues used for the substrate-recognition study also displayed limited trypanocidal activity in vitro. More importantly, these results provide a foundation for the design and development of purine nucleobase analogues with anti-trypanosomal action that are efficiently and selectively accumulated by the parasite. One of the main aims of the project was to clone and characterise nucleobase transporters from T. b. brucei. The initial functional complementation strategy in nucleobase-transport deficient trypanosomes proved unsuccessful due to the presence of an additional, previously uncharacterised purine transporter in the trypanosome selection background and other technical obstacles. Homology searching of the T. b. brucei genome database led to the identification of a sequence with substantial similarity to the Adenosine Transporter 1 (TbAT1) gene previously shown to be responsible for the P2 nucleoside transport activity. The AT-like sequence was cloned, functionally expressed in heterologous systems (Saccharomyces cerevisiae and Xenopus oocytes), and characterised as the high-affinity Nucleobase Transporter 1 (TbNBT1). This also marked the first time a nucleobase-specific transporter had been cloned and characterised from any protozoan

Spaces for play:Listening to children's voices

This paper explores young children’s voices about their play spaces in one Scottish primary school. 45 children (ages 5-7 years) participated, choosing from a range of creative methods (e.g., InPhoTours, drawing, mapping) to share their voices. Using a ‘Playful Research Ethics Framework’, a developmentally appropriate framework which involved the use of visual aids, puppets, songs, Makaton symbols and discussions as well as attention to any cues of disengagement, this research aimed at achieving children’s ongoing informed assent. Four themes were identified: (a) the ‘whole child’ in the space, (b) space and relationships, (c) function of space, and (d) impact (or lack) of children’s voices about space. Children expressed differences in ownership, creativity and imagination in indoor and outdoor spaces. Further, despite perceiving there to be a lack of agency, children were willing to share their voices. There are implications for both practice and research in terms of adults willing to effectively listen to children’s voices and acting on them. This study makes original and significant contributions which have the potential to impact research and practice with young children internationally

Spaces for play:Listening to children's voices

This paper explores young children’s voices about their play spaces in one Scottish primary school. 45 children (ages 5-7 years) participated, choosing from a range of creative methods (e.g., InPhoTours, drawing, mapping) to share their voices. Using a ‘Playful Research Ethics Framework’, a developmentally appropriate framework which involved the use of visual aids, puppets, songs, Makaton symbols and discussions as well as attention to any cues of disengagement, this research aimed at achieving children’s ongoing informed assent. Four themes were identified: (a) the ‘whole child’ in the space, (b) space and relationships, (c) function of space, and (d) impact (or lack) of children’s voices about space. Children expressed differences in ownership, creativity and imagination in indoor and outdoor spaces. Further, despite perceiving there to be a lack of agency, children were willing to share their voices. There are implications for both practice and research in terms of adults willing to effectively listen to children’s voices and acting on them. This study makes original and significant contributions which have the potential to impact research and practice with young children internationally

Design and synthesis of a series of truncated neplanocin fleximers

In an effort to study the effects of flexibility on enzyme recognition and activity, we have developed several different series of flexible nucleoside analogues in which the purine base is split into its respective imidazole and pyrimidine components. The focus of this particular study was to synthesize the truncated neplanocin A fleximers to investigate their potential anti-protozoan activities by inhibition of S-adenosylhomocysteine hydrolase (SAHase). The three fleximers tested displayed poor anti-trypanocidal activities, with EC50 values around 200 μM. Further studies of the corresponding ribose fleximers, most closely related to the natural nucleoside substrates, revealed low affinity for the known T. brucei nucleoside transporters P1 and P2, which may be the reason for the lack of trypanocidal activity observed

Limited diversity associated with duplicated class II MHC-DRB genes in the red squirrel population in the United Kingdom compared with continental Europe

The red squirrel (Sciurus vulgaris) population in the United Kingdom has declined over the last century and is now on the UK endangered species list. This is the result of competition from the eastern grey squirrel (S. carolinensis) which was introduced in the 19th century. However, recent evidence suggests that the rate of population decline is enhanced by squirrelpox disease, caused by a viral infection carried asymptomatically by grey squirrels but to which red squirrels are highly susceptible. Population genetic diversity provides some resilience to rapidly evolving or exotic pathogens. There is currently no data on genetic diversity of extant UK squirrel populations with respect to genes involved in disease resistance. Diversity is highest at loci involved in the immune response including genes clustered within the major histocompatibility complex (MHC). Using the class II DRB locus as a marker for diversity across the MHC region we genotyped 110 red squirrels from locations in the UK and continentalEurope. Twenty four Scvu-DRB alleles at two functional loci; Scvu-DRB1 and Scvu- DRB2, were identified. High levels of diversity were identified at both loci in the continental populations. In contrast, no diversity was observed at the Scvu-DRB2 locus in the mainland UK population while a high level of homozygosity was observed at the Scvu-DRB1 locus. The red squirrel population in the UK appears to lack the extensive MHC diversity associated with continental populations, a feature which may have contributed to their rapid decline

Quantitative Genetics of the Maize Leaf Microbiome

The degree to which the genotype of an organism can affect the composition of its associated microbial communities ("microbiome") varies by organism and habitat, and in many cases is unknown. We analyzed the metabolically active bacteria of maize leaves across 300 diverse maize lines growing in a common environment. We performed comprehensive heritability analysis for 49 community diversity metrics, 380 bacterial clades, and 9,042 predicted metagenomic functions. We find that only a few bacterial clades (5) and diversity metrics (2) are significantly heritable, while a much larger number of metabolic functions (200) are. Many of these associations appear to be driven by the Methylobacteria in each sample. Among these heritable metabolic traits, Fisher's exact test identifies significant overrepresentation of traits relating to short-chain carbon metabolism, secretion, and nitrotoluene degradation. Genome-wide association analysis identified a small number of associated loci for these heritable traits, including two that affect multiple traits. Our results indicate that while most of the maize leaf microbiome composition is driven by environmental factors and/or stochastic founder events, a subset of bacterial taxa and metabolic functions is nonetheless significantly impacted by host genetics. Additional work will be needed to identify the exact nature of these interactions and what effects they may have on their host

The Trypanocide Diminazene Aceturate Is Accumulated Predominantly through the TbAT1 Purine Transporter: Additional Insights on Diamidine Resistance in African Trypanosomes

Resistance to diminazene aceturate (Berenil) is a severe problem in the control of African trypanosomiasis in domestic animals. It has been speculated that resistance may be the result of reduced diminazene uptake by the parasite. We describe here the mechanisms by which [(3)H]diminazene is transported by Trypanosoma brucei brucei bloodstream forms. Diminazene was rapidly accumulated through a single transporter, with a K(m) of 0.45 ± 0.11 μM, which was dose dependently inhibited by pentamidine and adenosine. The K(i) values for these inhibitors were consistent with this transporter being the P2/TbAT1 adenosine transporter. Yeast expressing TbAT1 acquired the ability to take up [(3)H]diminazene and [(3)H]pentamidine. TbAT1-null mutants had lost almost all capacity for [(3)H]diminazene transport. However, this cell line still displayed a small but detectable rate of [(3)H]diminazene accumulation, in a nonsaturable manner. We conclude that TbAT1 mediates [(3)H]diminazene transport almost exclusively and that this explains the observed diminazene resistance phenotypes of TbAT1-null mutants and field isolates

"Reporting The Media":The Occupational Subculture Of The Sports Journalist

Lipoic acid (LA) is an essential cofactor of alpha-keto acid dehydrogenase complexes (KADHs) and the glycine cleavage system. In Plasmodium, LA is attached to the KADHs by organelle-specific lipoylation pathways. Biosynthesis of LA exclusively occurs in the apicoplast, comprising octanoyl-[acyl carrier protein]: protein N-octanoyltransferase (LipB) and LA synthase. Salvage of LA is mitochondrial and scavenged LA is ligated to the KADHs by LA protein ligase 1 (LplA1). Both pathways are entirely independent, suggesting that both are likely to be essential for parasite survival. However, disruption of the LipB gene did not negatively affect parasite growth despite a drastic loss of LA (> 90%). Surprisingly, the sole, apicoplast-located pyruvate dehydrogenase still showed lipoylation, suggesting that an alternative lipoylation pathway exists in this organelle. We provide evidence that this residual lipoylation is attributable to the dual targeted, functional lipoate protein ligase 2 (LplA2). Localisation studies show that LplA2 is present in both mitochondrion and apicoplast suggesting redundancy between the lipoic acid protein ligases in the erythrocytic stages of P. falciparum.Publisher PDFPeer reviewe