Biochemical characterisation of the cyanobacterial Hik2-Rre1 two-component regulatory system.

PhDTwo-component signal transduction systems (TCS) consist of a sensor histidine kinase and a response regulator. TCS are ubiquitous in prokaryotes, but found only in some eukaryotes. TCS mediate adaptation to various environmental changes in bacteria, plants, fungi, and protists. Histidine kinase 2 (Hik2) is a sensor histidine kinase found in all cyanobacteria. The Hik2 homologue known as Chloroplast Sensor Kinase is found in algae and plants, where it is encoded by the nuclear genome and it is targeted to chloroplasts. CSK couples the redox state of the photosynthetic electron transport chain to chloroplast gene transcription. This thesis describes biochemical characterisation of the signal transduction mechanism of Hik2 and its response regulator (Rre) partners in order to clarify the Hik2-Rre two-component signal transduction pathway. Results presented in this thesis illustrate that the autophosphorylation activity of the full-length Hik2 protein is specifically inhibited by sodium ions. An autophosphorylation event of a histidine kinase is the result of homodimerisation and is followed by trans or cis-autophosphorylation of each monomer on its conserved histidine residue. Chemical crosslinking revealed that the Hik2 protein exists predominantly as a phosphorylated (autokinase active) monomer, tetramer, and higher-order oligomeric complexes. The functions of these different oligomeric states of Hik2 are also discussed. From a previous study, which was based on an observation from a yeast two-hybrid assay, Hik2 was proposed to form a two-component pair with Rre1 and RppA. However, no further evidence was presented to support either direct interaction or direct phosphotransfer activity of the Hik2-Rre pair. This thesis confirms interaction of Hik2-Rre1 and Hik2-RppA two-component ! %! pairs using an in vitro pull-down assay and phosphotransfer kinetics. Finally, a model is proposed for the Hik2 based two-component signal transduction pathway

Role of a rhomboid protease in a CO2-concentrating mechanism

Photosynthetic efficiency of some of the most important global crop plants is limited by the low affinity for CO2 of ribulose bisphosphate carboxylase–oxygenase (Rubisco) and the wastage of energy from photosynthesis via the release of fixed CO2 in a side reaction with oxygen. Experimental increases in the CO2 concentration around C3 plants did enhance the level of photosynthesis and yield, so it is of interest to investigate the crop-improvement potential of the CO2-concentrating mechanism (CCM) utilised by cyanobacteria to overcome the limitations of Rubisco. Utilising this mechanism of improving Rubisco efficiency and thus crop yield is attractive as it does not necessarily require major changes to leaf anatomy. The cyanobacterial CCM allows these photosynthetic prokaryotes to increase CO2 concentration up to 1000-fold near carboxysomes, protein-walled compartments of localised Rubisco, and increased CO2 levels also activate Rubisco and repress photorespiration. Within a complex network of CCM regulation is control at the level of transcription for genes encoding CO2-uptake and bicarbonate transport. Amongst the regulatory components are LysR-type transcription factors acting as activators or repressors, and the protease FtsH2. We report here how a rhomboid protease also plays a pivotal role in transcriptional control of CCM genes in Synechocystis sp. PCC6803

The role of social capital in the internationalization process of Egyptian and UK SMEs: a co-evolutionary perspective

There is increasing evidence of the positive role that the social capital plays in facilitating the internationalisation of SMEs. Network social capital serves as a key platform for SMEs to access resources and competencies they lack. Yet surprisingly little is known about how network social capital co-evolves with the international growth and survival of SMEs. Moreover, little is known about how SMEs from different institutional contexts rely on their network attachments to cope with the challenges presented by the external environment. This study aims to address these gaps through providing a comprehensive analysis of internationalisation patterns, network social capital development and how these two fields may co-evolve over time and compares well-established SMEs from two national contexts (UK and Egypt) which contrast in their cultures and levels of institutional development. This study relies on comparative multiple case studies and adopts a mixed methods approach to data analysis to investigate co-evolutionary patterns and inter-country differences respectively. The study contributes to the International Entrepreneurship literature through offering a comprehensive/dynamic understanding of internationalisation and by indicating key differences in the behaviour of SMEs from emerging and developed economies. It concludes that the co-evolutionary perspective usefully highlights the dynamic interaction between SMEs and their contexts. This study relies on comparative multiple case studies consisting of twenty UK and twenty Egyptian SMEs. These case studies investigate different internationalisation and networking patterns and identify co-evolutionary patterns of internationalisation and associated networking behaviour over time. A mixed methods approach to data analysis was used to investigate co-evolutionary patterns and inter-country differences respectively. The study contributes to the International Entrepreneurship [IE] literature through offering a comprehensive and dynamic understanding of internationalisation and by indicating key differences in the behaviour of SMEs from emerging and developed economies. It concludes that the co-evolutionary perspective usefully highlights the dynamic interaction between SMEs and their contexts. It shows that SMEs not only adapt to changes in their external environment but in some cases are able to influence the environment to their favour. The thesis closes with implications and recommendations for Egyptian and UK policy makers and managers

Potential of new isolates of Dunaliella Salina for natural β-Carotene production

The halotolerant microalga Dunaliella salina has been widely studied for natural β-carotene production. This work shows biochemical characterization of three newly isolated Dunaliella salina strains, DF15, DF17, and DF40, compared with D. salina CCAP 19/30 and D. salina UTEX 2538 (also known as D. bardawil). Although all three new strains have been genetically characterized as Dunaliella salina strains, their ability to accumulate carotenoids and their capacity for photoprotection against high light stress are different. DF15 and UTEX 2538 reveal great potential for producing a large amount of β-carotene and maintained a high rate of photosynthesis under light of high intensity; however, DF17, DF40, and CCAP 19/30 showed increasing photoinhibition with increasing light intensity, and reduced contents of carotenoids, in particular β-carotene, suggesting that the capacity of photoprotection is dependent on the cellular content of carotenoids, in particular β-carotene. Strong positive correlations were found between the cellular content of all-trans β-carotene, 9-cis β-carotene, all-trans α-carotene and zeaxanthin but not lutein in the D. salina strains. Lutein was strongly correlated with respiration in photosynthetic cells and strongly related to photosynthesis, chlorophyll and respiration, suggesting an important and not hitherto identified role for lutein in coordinated control of the cellular functions of photosynthesis and respiration in response to changes in light conditions, which is broadly conserved in Dunaliella strains. Statistical analysis based on biochemical data revealed a different grouping strategy from the genetic classification of the strains. The significance of these data for strain selection for commercial carotenoid production is discussed

Photosystem stoichiometry adjustment is a photoreceptor-mediated process in Arabidopsis

Plant growth under spectrally‐enriched low light conditions leads to adjustment in the relative abundance of the two photosystems in an acclimatory response known as photosystem stoichiometry adjustment. Adjustment of photosystem stoichiometry improves the quantum efficiency of photosynthesis but how this process perceives light quality changes and how photosystem amount is regulated remain largely unknown. By using a label‐free quantitative mass spectrometry approach in Arabidopsis here we show that photosystem stoichiometry adjustment is primarily driven by the regulation of photosystem I content and that this forms the major thylakoid proteomic response under light quality. Using light and redox signaling mutants, we further show that the light quality‐ responsive accumulation of photosystem I gene transcripts and proteins requires phytochrome B photoreceptor but not plastoquinone redox signaling as previously suggested. In far‐red light, the increased acceptor side limitation might deplete active photosystem I pool, further contributing to the adjustment of photosystem stoichiometry

Case Study in Refractory Non-Hodgkin's Lymphoma: Successful Treatment with Plerixafor

The present case study describes our experience in treating a young woman diagnosed with a relapsing case of diffuse large cell lymphoma, who was heavily pre-treated with chemotherapy and radiotherapy. Our only chance to improve her survival was by using high-dose chemotherapy, followed by peripheral stem cell rescue. Unfortunately, in this patient, collecting sufficient stem cells for bone marrow transplantation proved to be very difficult since she had already been heavily treated with chemotherapy and radiotherapy. Currently, granulocyte colony-stimulating factor (G-CSF) alone or G-CSF plus chemotherapy are the most commonly used treatments for stem cell mobilization. However, 5–30% of patients do not respond to these agents. Plerixafor is a new hematopoietic stem cell-mobilizing drug that antagonizes the binding of chemokine stromal cell-derived factor-1α to CXC chemokine receptor 4. It is indicated in combination with G-CSF to mobilize hematopoietic stem cells to the peripheral blood for collection and subsequent autologous transplantation in patients with non-Hodgkin's lymphoma and multiple myeloma [Kessans et al.: Pharmacotherapy 2010;30:485–492; Jantunen: Expert Opin Biol Ther 2011;11:1241–1248]. Based on our findings, we consider plerixafor to be a very efficient and practical solution to mobilize and collect stem cells among all patients in such a situation, enabling us to proceed to autologous bone marrow transplantation and peripheral stem cell rescue in order to improve the patients’ overall survival

Oligomeric states in sodium ion-dependent regulation of cyanobacterial histidine kinase-2

IMI thanks Queen Mary University of London for a graduate teaching studentship. LW thanks the China Scholarship Council (CSC) and Queen Mary University of London for financial support. SP held a Leverhulme Trust early-career post-doctoral research fellowship. JN is grateful for the continued support of the JST CREST Grant Number JPMJCR13M4, Japan. JFA acknowledges the support of research grant F/07 476/AQ and fellowship EM-2015-068 of the Leverhulme Trust

The influence of photoperiod and light intensity on the growth and photosynthesis of Dunaliella salina (chlorophyta) CCAP 19/30

The green microalga Dunaliella salina survives in a wide range of salinities via mechanisms involving glycerol synthesis and degradation and is exploited for large amounts of nutraceutical carotenoids produced under stressed conditions. In this study, D. salina CCAP 19/30 was cultured in varying photoperiods and light intensities to study the relationship of light with different growth measurement parameters, with cellular contents of glycerol, starch and carotenoids, and with photosynthesis and respiration. Results show CCAP 19/30 regulated cell volume when growing under light/dark cycles: cell volume increased in the light and decreased in the dark, and these changes corresponded to changes in cellular glycerol content. The decrease in cell volume in the dark was independent of cell division and biological clock and was regulated by the photoperiod of the light/dark cycle. When the light intensity was increased to above 1000 μmol photons m−2 s−1, cells displayed evidence of photodamage. However, these cells also maintained the maximum level of photosynthesis efficiency and respiration possible, and the growth rate increased as light intensity increased. Significantly, the intracellular glycerol content also increased, >2-fold compared to the content in light intensity of 500 μmol photons m−2 s−1, but there was no commensurate increase in the pool size of carotenoids. These data suggest that in CCAP 19/30 glycerol stabilized the photosynthetic apparatus for maximum performance in high light intensities, a role normally attributed to carotenoids

Transcriptional Control of Photosynthesis Genes: The Evolutionarily Conserved Regulatory Mechanism in Plastid Genome Function

Chloroplast sensor kinase (CSK) is a bacterial-type sensor histidine kinase found in chloroplasts—photosynthetic plastids—in eukaryotic plants and algae. Using a yeast two-hybrid screen, we demonstrate recognition and interactions between: CSK, plastid transcription kinase (PTK), and a bacterial-type RNA polymerase sigma factor-1 (SIG-1). CSK interacts with itself, with SIG-1, and with PTK. PTK also interacts directly with SIG-1. PTK has previously been shown to catalyze phosphorylation of plastid-encoded RNA polymerase (PEP), suppressing plastid transcription nonspecifically. Phospho-PTK is inactive as a PEP kinase. Here, we propose that phospho-CSK acts as a PTK kinase, releasing PTK repression of chloroplast transcription, while CSK also acts as a SIG-1 kinase, blocking transcription specifically at the gene promoter of chloroplast photosystem I. Oxidation of the photosynthetic electron carrier plastoquinone triggers phosphorylation of CSK, inducing chloroplast photosystem II while suppressing photosystem I. CSK places photosystem gene transcription under the control of photosynthetic electron transport. This redox signaling pathway has its origin in cyanobacteria, photosynthetic prokaryotes from which chloroplasts evolved. The persistence of this mechanism in cytoplasmic organelles of photosynthetic eukaryotes is in precise agreement with the CoRR hypothesis for the function of organellar genomes: the plastid genome and its primary gene products are Co-located for Redox Regulation. Genes are retained in plastids primarily in order for their expression to be subject to this rapid and robust redox regulatory transcriptional control mechanism, whereas plastid genes also encode genetic system components, such as some ribosomal proteins and RNAs, that exist in order to support this primary, redox regulatory control of photosynthesis genes. Plastid genome function permits adaptation of the photosynthetic apparatus to changing environmental conditions of light quantity and quality

Goodbye Hartmann trial: a prospective, international, multicenter, observational study on the current use of a surgical procedure developed a century ago

Background: Literature suggests colonic resection and primary anastomosis (RPA) instead of Hartmann's procedure (HP) for the treatment of left-sided colonic emergencies. We aim to evaluate the surgical options globally used to treat patients with acute left-sided colonic emergencies and the factors that leading to the choice of treatment, comparing HP and RPA. Methods: This is a prospective, international, multicenter, observational study registered on ClinicalTrials.gov. A total 1215 patients with left-sided colonic emergencies who required surgery were included from 204 centers during the period of March 1, 2020, to May 31, 2020. with a 1-year follow-up. Results: 564 patients (43.1%) were females. The mean age was 65.9 ± 15.6 years. HP was performed in 697 (57.3%) patients and RPA in 384 (31.6%) cases. Complicated acute diverticulitis was the most common cause of left-sided colonic emergencies (40.2%), followed by colorectal malignancy (36.6%). Severe complications (Clavien-Dindo ≥ 3b) were higher in the HP group (P < 0.001). 30-day mortality was higher in HP patients (13.7%), especially in case of bowel perforation and diffused peritonitis. 1-year follow-up showed no differences on ostomy reversal rate between HP and RPA. (P = 0.127). A backward likelihood logistic regression model showed that RPA was preferred in younger patients, having low ASA score (≤ 3), in case of large bowel obstruction, absence of colonic ischemia, longer time from admission to surgery, operating early at the day working hours, by a surgeon who performed more than 50 colorectal resections. Conclusions: After 100 years since the first Hartmann's procedure, HP remains the most common treatment for left-sided colorectal emergencies. Treatment's choice depends on patient characteristics, the time of surgery and the experience of the surgeon. RPA should be considered as the gold standard for surgery, with HP being an exception