Site-directed mutagenesis of the D2 protein in the green alga 'Chlamydomonas reinhardtii'

The D2 polypeptide together with the D1 polypeptide are key components of the photosystem II (PS II) complex which is involved in photosynthetic oxygen evolution. This thesis describes the use of site-directed mutagenesis to introduce specific amino acid changes in the D2 protein of Chlamydomonas reinhardtii. The mutations were constructed at positions D2-Leu205 and D2-Thr2. Plasmids carrying mutated copies of the psbD gene (which encodes D2) were introduced into the chloroplast genome of C. reinhardtii by means of a particle gun. In order to identify the transformants, a selectable marker encoding resistance to spectinomycin, was inserted either upstream or downstream of the psbD gene. Genetic characterisation of the mutants was carried out using Southern blotting, PCR and DNA sequencing. D2-Leu205 is thought to be analogous to residue Tyr210 of the M subunit of the reaction centre (RC) of the purple photosynthetic bacterium Rhodobacter sphaeroides. Previous mutagenesis experiments have shown that the replacement of M-Tyr210 by leucine reduces the rate of the primary charge separation in purple bacterial RCs. Urns, the mutation Leu205Tyr was constructed in order to study whether the rate of primary charge separation is increased in this mutant. The Leu205Tyr mutant could grow photoautotrophically at wild type rates. However, its light-saturated PS II activity was reduced to ~62% compared to the wild type. PS II RCs isolated from a "wild type” control transformant and from the Leu205Tyr mutant showed in ultrafast transient absorption measurements that the rate of formation of the radical pair was slowed down in the mutant (lifetime of 40±7 ps compared to 21±2 ps for the wild type PS II RCs). Also the quantum yield of charge separation was reduced by about 50%. Fluorescence and thermoluminescence measurements in whole cells were also consistent with a reduced quantum yield of charge separation in PS II. Mutations Thr2Ala and Thr2Ser were constructed in order to study the role of D2 phosphorylation in C. reinhardtii. Biochemical characterisation of the Thr2Ala and Thr2Ser mutants indicated that both strains could grow photoautotrophically at wild type rates. Moreover, oxygen evolution, fluorescence and thermoluminescence measurements suggested that these site-directed mutations have only minor effects on PS II function. In addition to these experiments, attempts were carried out to find out whether D2 can be phosphorylated in C. reinhardtii. These attempts included in vivo labelling of whole cells with [32P]-orthophosphate, in vitro labelling of thylakoid membranes with [y-32P]-ATP, pulse-chase labelling of cells with [uC]-acetate and the use of a monospecific antibody against phosphothreonine. The data obtained from these experiments did not provide any indication that D2 can be phosphorylated in C. reinhardtii.Open acces

Proteome analysis of Phytophthora cinnamomi, the causal agent of Dieback

Phytophthora cinnamomi is a nasty pathogen that causes significant environmental and agricultural destruction. In this thesis, proteomic analysis was used to obtain a biochemical snapshot of the organism to understand its contributors to pathogenicity. The mode of action of phosphite, the only chemical used to combat dieback disease was also elucidated as there is a growing pressure of chemical resistance

Interpretation of the Expected Value of Perfect Information and Research Recommendations:A Systematic Review and Empirical Investigation

calculations are increasingly performed to guide and underpin research recommendations. An EVPI value that exceeds the estimated cost of research forms a necessary (although not sufficient) condition for further research to be considered worthwhile. However, it is unclear what factors affect researchers ’ recommendations and whether there is a notional threshold of positive returns below which research is not recommended. The objectives of this study were to explore whether EVPI and other factors have a bearing on research recommendations and to assess whether there exists a threshold EVPI below which research is typically not recommended. Methods. A systematic lit-erature review was undertaken to identify applied EVPI calculations in the health care field. Study characteris-tics were extracted, including funder, location, diseas

Building solids inside nano-space: from confined amorphous through confined solvate to confined ‘metastable’ polymorph

The nanocrystallisation of complex molecules inside mesoporous hosts and control over the resulting structure is a significant challenge. To date the largest organic molecule crystallised inside the nano-pores is a known pharmaceutical intermediate – ROY (259.3 g mol1). In this work we demonstrate smart manipulation of the phase of a larger confined pharmaceutical – indomethacin (IMC, 357.8 g mol1), a substance with known conformational flexibility and complex polymorphic behaviour. We show the detailed structural analysis and the control of solid state transformations of encapsulated molecules inside the pores of mesoscopic cellular foam (MCF, pore size ca. 29 nm) and controlled pore glass (CPG, pore size ca. 55 nm). Starting from confined amorphous IMC we drive crystallisation into a confined methanol solvate, which upon vacuum drying leads to the stabilised rare form V of IMC inside the MCF host. In contrast to the pure form, encapsulated form V does not transform into a more stable polymorph upon heating. The size of the constraining pores and the drug concentration within the pores determine whether the amorphous state of the drug is stabilised or it recrystallises into confined nanocrystals. The work presents, in a critical manner, an application of complementary techniques (DSC, PXRD, solid-state NMR, N2 adsorption) to confirm unambiguously the phase transitions under confinement and offers a comprehensive strategy towards the formation and control of nano-crystalline encapsulated organic solids

Are acceptance and mindfulness-based interventions 'value for money'? : evidence from a systematic literature review

Objectives: Acceptance and mindfulness‐based interventions (A/MBIs) are recommended for people with mental health conditions. Although there is a growing evidence base supporting the effectiveness of different A/MBIs for mental health conditions, the economic case for these interventions has not been fully explored. The aim of this systematic review was to identify and appraise all available economic evidence of A/MBIs for the management of mental health conditions. Methods: Eight electronic bibliographic databases (MEDLINE, MEDLINE In‐Process & Other Non‐Indexed Citations, EMBASE, Web of Science, NHS Economic Evaluation Database (EED), Database of Abstracts of Reviews of Effects (DARE), Health Technology Assessment (HTA) database, and EconLit) were searched for relevant economic evaluations published from each database's inception date until November 2017. Study selection, quality assessment, and data extraction were carried out according to published guidelines. Results: Ten relevant economic evaluations presented in 11 papers were identified. Seven of the included studies were full economic evaluations (i.e., costs and effects assessed), and three studies were partial economic evaluations (i.e., only costs were considered in the analysis). The A/MBIs that had been subjected to economic evaluation were acceptance and commitment therapy (ACT), dialectical behaviour therapy (DBT), mindfulness‐based cognitive therapy (MBCT), and mindfulness‐based stress reduction (MBSR). In terms of clinical presentations, the evaluation of cost‐effectiveness of A/MBIs has been more focused on depression and emotional unstable personality disorder with three and four economic evaluations, respectively. Three out of seven full economic evaluations observed that A/MBIs were cost‐effective for the management of mental health conditions. Nevertheless, the heterogeneity of included populations, interventions, and economic evaluation study types limits the extent to which firm conclusions can currently be made. Conclusion: This first substantive review of economic evaluations of A/MBIs indicates that more research is needed before firm conclusions can be reached on the cost‐effectiveness of A/MBIs for mental health conditions

Comparative sub-cellular proteome analyses reveals metabolic differentiation and production of effector-like molecules in the dieback phytopathogen Phytophthora cinnamomi.

Phytopathogenic oomycetes pose a significant threat to global biodiversity and food security. The proteomes of these oomycetes likely contain important factors that contribute to their pathogenic success, making their discovery crucial for elucidating pathogenicity. Phytophthora cinnamomi is a root pathogen that causes dieback in a wide variety of crops and native vegetation world-wide. Virulence proteins produced by P. cinnamomi are not well defined and a large-scale approach to understand the biochemistry of this pathogen has not been documented. Soluble mycelial, zoospore and secreted proteomes were obtained and label-free quantitative proteomics was used to compare the composition of the three sub-proteomes. A total of 4635 proteins were identified, validating 17.7% of the predicted gene set. The mycelia were abundant in transporters for nutrient acquisition, metabolism and cellular proliferation. The zoospores had less metabolic related ontologies but were abundant in energy generating, motility and signalling associated proteins. Virulence-associated proteins were identified in the secretome such as candidate effector and effector-like proteins, which interfere with the host immune system. These include hydrolases, cell wall degrading enzymes, putative necrosis-inducing proteins and elicitins. The secretome elicited a hypersensitive response on the roots of a model host and thus suggests evidence of effector activity

Effectiveness and cost effectiveness of cardiovascular disease prevention in whole populations: modelling study

Objective To estimate the potential cost effectiveness of a population-wide risk factor reduction programme aimed at preventing cardiovascular disease

Gene validation and remodelling using proteogenomics of Phytophthora cinnamomi, the causal agent of Dieback

Phytophthora cinnamomi is a pathogenic oomycete that causes plant dieback disease across a range of natural ecosystems and in many agriculturally important crops on a global scale. An annotated draft genome sequence is publicly available (JGI Mycocosm) and suggests 26,131 gene models. In this study, soluble mycelial, extracellular (secretome), and zoospore proteins of P. cinnamomi were exploited to refine the genome by correcting gene annotations and discovering novel genes. By implementing the diverse set of sub-proteomes into a generated proteogenomics pipeline, we were able to improve the P. cinnamomi genome annotation. Liquid chromatography mass spectrometry was used to obtain high confidence peptides with spectral matching to both the annotated genome and a generated 6-frame translation. Two thousand seven hundred sixty-four annotations from the draft genome were confirmed by spectral matching. Using a proteogenomic pipeline, mass spectra were used to edit the P. cinnamomi genome and allowed identification of 23 new gene models and 60 edited gene features using high confidence peptides obtained by mass spectrometry, suggesting a rate of incorrect annotations of 3% of the detectable proteome. The novel features were further validated by total peptide support, alongside functional analysis including the use of Gene Ontology and functional domain identification. We demonstrated the use of spectral data in combination with our proteogenomics pipeline can be used to improve the genome annotation of important plant diseases and identify missed genes. This study presents the first use of spectral data to edit and manually annotate an oomycete pathogen