40 research outputs found
Propriétés physico-chimiques et structurales de deux hémoprotéines de cyanobactérie thermophile
Photosynthesis converts solar energy into chemical energy. This process involves a large number of proteins and protein complexes. The first protein complex in the photosynthetic chain is Photosystem II within the oxidation of water takes place. PSII is composed of the D1 and D2 proteins. In the thermophile cyanobacterium Thermosynechococcus elongatus, three genes encoded three different D1 proteins. The first part of this thesis describes the development of proteomics tools based on 2D gel-electrophoresis to study the proteome of three different variants, each expressing a single different D1 protein. Very few differences were found. However, only one expressed the protein Tll0287. The second part of the thesis describes the characterization of Tll0287. It was characterized using different techniques: electronic absorption and Raman resonance spectroscopies and spectro-electrochemistry. Tll0287 has been previously identified as a c-type cytochrome, but it presents some unexpected characteristics. The UV-visible absorption and Raman resonance spectra of reduced Tll0287 show a pH dependence. The reduced and oxidized states each had two different forms of the heme. A switch of ligands from a cysteine to histidine was observed in the reduced state. Redox titration showed multiple midpoints at pH 10 and 5. Tll0287 was shown to fix a CO molecule at pH 7.6. These physical properties suggested that Tll0287 could be a sensor. The crystallographic studies reveal that Tll0287 does not have a classical c-type cytochrome fold and is similar to other known sensor proteins, strengthening the hypothesis that it is a sensor. Deletion mutants were constructed that will help to better understand the function of this new cytochrome. The third part describes a study of the PsbV2, another c-type cytochrome. In order to obtain sufficient quantities to carry out characterization of this protein, it was overexpressed in a homologous system using the promotor of the rubisco enzyme. The redox midpoint potential of PsbV2 was found to be very low, below -460 mV (vs SHE, pH 5). The UV-visible absorption spectrum of the reduced form was determined. The crystallographic structure of PsbV2 was solved and reveals an axial cysteine ligand. Although both Tll0287 and PsbV2 share this feature, their different structures and physico-chemical properties suggest that their functions are unlikely to be similar. A major contribution of this thesis is the characterization of a new c-type cytochrome sensor in cyanobacteria and the development of proteomic tools required to study it.La photosynthĂšse permet de convertir lâĂ©nergie solaire en Ă©nergie chimique. Ce processus met en jeu un grand nombre de protĂ©ines et complexes protĂ©iques. Le premier complexe de la chaĂźne photosynthĂ©tique est le photosystĂšme II oĂč a lieu lâoxydation de lâeau. Le PSII est composĂ© des protĂ©ines D1 et D2. Chez la cyanobactĂ©rie thermophile Thermosynechococcus elongatus, il y a trois gĂšnes qui codent trois protĂ©ines D1 diffĂ©rentes. La premiĂšre partie de la thĂšse dĂ©crit le dĂ©veloppement dâoutils protĂ©omiques basĂ©s sur les gels dâĂ©lectrophorĂšse 2D pour Ă©tudier le protĂ©ome des trois diffĂ©rents variants, qui expriment chacun une seule protĂ©ine pour D1. Peu de diffĂ©rences ont Ă©tĂ© trouvĂ©es. Toutefois, un seul des variants exprime Tll0287. La deuxiĂšme partie de la thĂšse dĂ©crit la caractĂ©risation de Tll0287 avec diffĂ©rents techniques : spectroscopies dâabsorption UV-visible ou de rĂ©sonance Raman et spectro-Ă©lectrochimie. Tll0287 a Ă©tĂ© identifiĂ© comme un cytochrome de type c, mais il prĂ©sente beaucoup de caractĂ©ristiques inattendues. Les spectres dâabsorption UV-visible et de rĂ©sonance Raman de Tll0287 rĂ©duit montrent une dĂ©pendance vis-Ă -vis du pH. Deux formes dâhĂšmes sont prĂ©sents dans chacun des Ă©tats oxydĂ© et rĂ©duit. Un changement du ligand cystĂ©ine a Ă©tĂ© observĂ© quand lâhĂšme est rĂ©duit. Les titrages redox prĂ©sentent de multiples potentiels Ă pH 10 et pH 5. Tll0287 peut fixer une molĂ©cule de CO Ă pH 7,6. Ces caractĂ©ristiques suggĂšrent que Tll0287 pourrait ĂȘtre une protĂ©ine senseur. De plus, la structure cristallographique montre que Tll0287 nâa pas un repliement classique dâun cytochrome de type c mais celui dâune protĂ©ine senseur. Les mutants de dĂ©lĂ©tion du gĂšne tll0287 ont Ă©tĂ© construits et aideront Ă comprendre la fonction de ce nouveau cytochrome. La troisiĂšme partie dĂ©crit lâĂ©tude de PsbV2 : un autre cytochrome de type c. Afin dâobtenir en quantitĂ© suffisante la protĂ©ine pour permettre sa caractĂ©risation, elle a Ă©tĂ© surexprimĂ©e dans un systĂšme homologue en utilisant le promoteur de lâenzyme de la rubisco. Le potentiel redox de PsbV2 a Ă©tĂ© dĂ©terminĂ©, comme Ă©tant trĂšs bas, infĂ©rieur Ă -460 mV (vs SHE, pH 5). Le spectre dâabsorption UV-visible de la forme rĂ©duite a Ă©tĂ© caractĂ©risĂ©. La structure cristallographique de PsbV2 a Ă©tĂ© rĂ©solue et a rĂ©vĂ©lĂ© une cystĂ©ine comme ligand axial et un repliement proche de celui de cytochromes connus de T.elongatus. Bien que Tll0287 et PsbV2 prĂ©sentent une cystĂ©ine comme ligand axial, leurs structures et leurs propriĂ©tĂ©s physico-chimiques suggĂšrent que leurs fonctions sont bien diffĂ©rentes. Une contribution majeure de cette thĂšse est la caractĂ©risation dâun nouveau senseur Ă hĂšme de type c chez les cyanobactĂ©ries et le dĂ©veloppement dâoutils nĂ©cessaires pour son Ă©tude
Replacing Face-To-Face Classes by Synchronous Online Technologies: The HOU Experience
AbstractSince 2009, HOU has been providing live virtual classes for various distance learningprograms.This paper will provide an opportunity to look at the issues involved in the use of thesemultimedia-enabled delivery approaches, the technology behind them, the logistics involved,and to provide an HOU perspective of the experiences encountered.The goal of research was to provide a systematic methods to implement the highlyinteractive live session. The additional goals was to design the portable hardware and easy touse software toolset as well as easy to follow guidelines on how to propel the lectures fromthe conventional dull chalk and talk and to minimise the number of staff required to give thelectures.Through a combination of surveys and feedback from lecturers and students, we are ableto better understand the obstacles and to continuously improve on the effectiveness of theseinteractive delivery approaches
What do Vietnamese executives understand corporate social responsibility?
The Corporate Social Responsibility (CSR) literature in developing countries is seriously meager. This paper explores CSR in the Vietnamese construction industry which has faced many scandals and directed attention toward the question of the responsibilities of these businesses. The study employs the CSR definition documented by Carroll (1979; 1991) to develop a framework for exploring executivesâ perceptions towards CSR. Using Carrollâs CSR pyramid and adopting in-depth interview as a method to collect data, the study critically examines the personal understanding of managers in nine companies in the construction industry. The findings show that although Vietnam is a developing country, managers are aware of the significance of environmental issue as a responsibility that businesses must address. Moreover, the managers also believe that corporate contributions to society, and corporate reputation and prestige, are expectations of society. Despite many breakthroughs in executivesâ understanding of CSR, they are not sufficiently and systematically aware of CSR and need a stronger supports, such as issuing appropriate policies, from government in adopting CSR in real business practice
A Novel Thermostable Cytochrome P450 from Sequence-Based Metagenomics of Binh Chau Hot Spring as a Promising Catalyst for Testosterone Conversion
Biotechnological applications of cytochromes P450 show difficulties, such as low activity,
thermal and/or solvent instability, narrow substrate specificity and redox partner dependence. In an
attempt to overcome these limitations, an exploitation of novel thermophilic P450 enzymes from
nature via uncultured approaches is desirable due to their great advantages that can resolve nearly
all mentioned impediments. From the metagenomics library of the Binh Chau hot spring, an open
reading frame (ORF) encoding a thermostable cytochrome P450âdesignated as P450-T3âwhich
shared 66.6% amino acid sequence identity with CYP109C2 of Sorangium cellulosum So ce56 was
selected for further identification and characterization. The ORF was synthesized artificially and
heterologously expressed in Escherichia coli C43(DE3) using the pET17b system. The purified enzyme
had a molecular weight of approximately 43 kDa. The melting temperature of the purified enzyme
was 76.2 âŠC and its apparent half-life at 60 âŠC was 38.7 min. Redox partner screening revealed that
P450-T3 was reduced well by the mammalian AdR-Adx4-108 and the yeast Arh1-Etp1 redox partners.
Lauric acid, palmitic acid, embelin, retinoic acid (all-trans) and retinoic acid (13-cis) demonstrated
binding to P450-T3. Interestingly, P450-T3 also bound and converted testosterone. Overall, P450-T3
might become a good candidate for biocatalytic applications on a larger scale
Safety and efficacy of fluoxetine on functional outcome after acute stroke (AFFINITY): a randomised, double-blind, placebo-controlled trial
Background
Trials of fluoxetine for recovery after stroke report conflicting results. The Assessment oF FluoxetINe In sTroke recoverY (AFFINITY) trial aimed to show if daily oral fluoxetine for 6 months after stroke improves functional outcome in an ethnically diverse population.
Methods
AFFINITY was a randomised, parallel-group, double-blind, placebo-controlled trial done in 43 hospital stroke units in Australia (n=29), New Zealand (four), and Vietnam (ten). Eligible patients were adults (aged â„18 years) with a clinical diagnosis of acute stroke in the previous 2â15 days, brain imaging consistent with ischaemic or haemorrhagic stroke, and a persisting neurological deficit that produced a modified Rankin Scale (mRS) score of 1 or more. Patients were randomly assigned 1:1 via a web-based system using a minimisation algorithm to once daily, oral fluoxetine 20 mg capsules or matching placebo for 6 months. Patients, carers, investigators, and outcome assessors were masked to the treatment allocation. The primary outcome was functional status, measured by the mRS, at 6 months. The primary analysis was an ordinal logistic regression of the mRS at 6 months, adjusted for minimisation variables. Primary and safety analyses were done according to the patient's treatment allocation. The trial is registered with the Australian New Zealand Clinical Trials Registry, ACTRN12611000774921.
Findings
Between Jan 11, 2013, and June 30, 2019, 1280 patients were recruited in Australia (n=532), New Zealand (n=42), and Vietnam (n=706), of whom 642 were randomly assigned to fluoxetine and 638 were randomly assigned to placebo. Mean duration of trial treatment was 167 days (SD 48·1). At 6 months, mRS data were available in 624 (97%) patients in the fluoxetine group and 632 (99%) in the placebo group. The distribution of mRS categories was similar in the fluoxetine and placebo groups (adjusted common odds ratio 0·94, 95% CI 0·76â1·15; p=0·53). Compared with patients in the placebo group, patients in the fluoxetine group had more falls (20 [3%] vs seven [1%]; p=0·018), bone fractures (19 [3%] vs six [1%]; p=0·014), and epileptic seizures (ten [2%] vs two [<1%]; p=0·038) at 6 months.
Interpretation
Oral fluoxetine 20 mg daily for 6 months after acute stroke did not improve functional outcome and increased the risk of falls, bone fractures, and epileptic seizures. These results do not support the use of fluoxetine to improve functional outcome after stroke
Epigenetic modulators as therapeutic targets in prostate cancer
Prostate cancer is one of the most common non-cutaneous malignancies among men worldwide. Epigenetic aberrations, including changes in DNA methylation patterns and/or histone modifications, are key drivers of prostate carcinogenesis. These epigenetic defects might be due to deregulated function and/or expression of the epigenetic machinery, affecting the expression of several important genes. Remarkably, epigenetic modifications are reversible and numerous compounds that target the epigenetic enzymes and regulatory proteins were reported to be effective in cancer growth control. In fact, some of these drugs are already being tested in clinical trials. This review discusses the most important epigenetic alterations in prostate cancer, highlighting the role of epigenetic modulating compounds in pre-clinical and clinical trials as potential therapeutic agents for prostate cancer management.info:eu-repo/semantics/publishedVersio
Physico-chemical and structural properties of two hemeproteins from thermophile cyanobacteria
La photosynthĂšse permet de convertir lâĂ©nergie solaire en Ă©nergie chimique. Ce processus met en jeu un grand nombre de protĂ©ines et complexes protĂ©iques. Le premier complexe de la chaĂźne photosynthĂ©tique est le photosystĂšme II oĂč a lieu lâoxydation de lâeau. Le PSII est composĂ© des protĂ©ines D1 et D2. Chez la cyanobactĂ©rie thermophile Thermosynechococcus elongatus, il y a trois gĂšnes qui codent trois protĂ©ines D1 diffĂ©rentes. La premiĂšre partie de la thĂšse dĂ©crit le dĂ©veloppement dâoutils protĂ©omiques basĂ©s sur les gels dâĂ©lectrophorĂšse 2D pour Ă©tudier le protĂ©ome des trois diffĂ©rents variants, qui expriment chacun une seule protĂ©ine pour D1. Peu de diffĂ©rences ont Ă©tĂ© trouvĂ©es. Toutefois, un seul des variants exprime Tll0287. La deuxiĂšme partie de la thĂšse dĂ©crit la caractĂ©risation de Tll0287 avec diffĂ©rents techniques : spectroscopies dâabsorption UV-visible ou de rĂ©sonance Raman et spectro-Ă©lectrochimie. Tll0287 a Ă©tĂ© identifiĂ© comme un cytochrome de type c, mais il prĂ©sente beaucoup de caractĂ©ristiques inattendues. Les spectres dâabsorption UV-visible et de rĂ©sonance Raman de Tll0287 rĂ©duit montrent une dĂ©pendance vis-Ă -vis du pH. Deux formes dâhĂšmes sont prĂ©sents dans chacun des Ă©tats oxydĂ© et rĂ©duit. Un changement du ligand cystĂ©ine a Ă©tĂ© observĂ© quand lâhĂšme est rĂ©duit. Les titrages redox prĂ©sentent de multiples potentiels Ă pH 10 et pH 5. Tll0287 peut fixer une molĂ©cule de CO Ă pH 7,6. Ces caractĂ©ristiques suggĂšrent que Tll0287 pourrait ĂȘtre une protĂ©ine senseur. De plus, la structure cristallographique montre que Tll0287 nâa pas un repliement classique dâun cytochrome de type c mais celui dâune protĂ©ine senseur. Les mutants de dĂ©lĂ©tion du gĂšne tll0287 ont Ă©tĂ© construits et aideront Ă comprendre la fonction de ce nouveau cytochrome. La troisiĂšme partie dĂ©crit lâĂ©tude de PsbV2 : un autre cytochrome de type c. Afin dâobtenir en quantitĂ© suffisante la protĂ©ine pour permettre sa caractĂ©risation, elle a Ă©tĂ© surexprimĂ©e dans un systĂšme homologue en utilisant le promoteur de lâenzyme de la rubisco. Le potentiel redox de PsbV2 a Ă©tĂ© dĂ©terminĂ©, comme Ă©tant trĂšs bas, infĂ©rieur Ă -460 mV (vs SHE, pH 5). Le spectre dâabsorption UV-visible de la forme rĂ©duite a Ă©tĂ© caractĂ©risĂ©. La structure cristallographique de PsbV2 a Ă©tĂ© rĂ©solue et a rĂ©vĂ©lĂ© une cystĂ©ine comme ligand axial et un repliement proche de celui de cytochromes connus de T.elongatus. Bien que Tll0287 et PsbV2 prĂ©sentent une cystĂ©ine comme ligand axial, leurs structures et leurs propriĂ©tĂ©s physico-chimiques suggĂšrent que leurs fonctions sont bien diffĂ©rentes. Une contribution majeure de cette thĂšse est la caractĂ©risation dâun nouveau senseur Ă hĂšme de type c chez les cyanobactĂ©ries et le dĂ©veloppement dâoutils nĂ©cessaires pour son Ă©tude.Photosynthesis converts solar energy into chemical energy. This process involves a large number of proteins and protein complexes. The first protein complex in the photosynthetic chain is Photosystem II within the oxidation of water takes place. PSII is composed of the D1 and D2 proteins. In the thermophile cyanobacterium Thermosynechococcus elongatus, three genes encoded three different D1 proteins. The first part of this thesis describes the development of proteomics tools based on 2D gel-electrophoresis to study the proteome of three different variants, each expressing a single different D1 protein. Very few differences were found. However, only one expressed the protein Tll0287. The second part of the thesis describes the characterization of Tll0287. It was characterized using different techniques: electronic absorption and Raman resonance spectroscopies and spectro-electrochemistry. Tll0287 has been previously identified as a c-type cytochrome, but it presents some unexpected characteristics. The UV-visible absorption and Raman resonance spectra of reduced Tll0287 show a pH dependence. The reduced and oxidized states each had two different forms of the heme. A switch of ligands from a cysteine to histidine was observed in the reduced state. Redox titration showed multiple midpoints at pH 10 and 5. Tll0287 was shown to fix a CO molecule at pH 7.6. These physical properties suggested that Tll0287 could be a sensor. The crystallographic studies reveal that Tll0287 does not have a classical c-type cytochrome fold and is similar to other known sensor proteins, strengthening the hypothesis that it is a sensor. Deletion mutants were constructed that will help to better understand the function of this new cytochrome. The third part describes a study of the PsbV2, another c-type cytochrome. In order to obtain sufficient quantities to carry out characterization of this protein, it was overexpressed in a homologous system using the promotor of the rubisco enzyme. The redox midpoint potential of PsbV2 was found to be very low, below -460 mV (vs SHE, pH 5). The UV-visible absorption spectrum of the reduced form was determined. The crystallographic structure of PsbV2 was solved and reveals an axial cysteine ligand. Although both Tll0287 and PsbV2 share this feature, their different structures and physico-chemical properties suggest that their functions are unlikely to be similar. A major contribution of this thesis is the characterization of a new c-type cytochrome sensor in cyanobacteria and the development of proteomic tools required to study it
Low-temperature photochemistry in photosystem II from Thermosynechococcus elongatus induced by visible and near-infrared light
The active site for water oxidation in photosystem II (PSII) consists of a Mn4Ca cluster close to a redox-active tyrosine residue (TyrZ). The enzyme cycles through five sequential oxidation states (S0 to S4) in the water oxidation process. Earlier electron paramagnetic resonance (EPR) work showed that metalloradical states, probably arising from the Mn4 cluster interacting with TyrZ·, can be trapped by illumination of the S0, S1 and S2 states at cryogenic temperatures. The EPR signals reported were attributed to S0TyrZ·, S1TyrZ· and S2TyrZ·, respectively. The equivalent states were examined here by EPR in PSII isolated from Thermosynechococcus elongatus with either Sr or Ca associated with the Mn4 cluster. In order to avoid spectral contributions from the second tyrosyl radical, TyrD·, PSII was used in which Tyr160 of D2 was replaced by phenylalanine. We report that the metalloradical signals attributed to TyrZ· interacting with the Mn cluster in S0, S1, S2 and also probably the S3 states are all affected by the presence of Sr. Ca/Sr exchange also affects the non-haem iron which is situated approximately 44ââ« units away from the Ca site. This could relate to the earlier reported modulation of the potential of QA by the occupancy of the Ca site. It is also shown that in the S3 state both visible and near-infrared light are able to induce a similar Mn photochemistry
D1 protein variants in Photosystem II from Thermosynechococcus elongatus studied by low temperature optical spectroscopy
International audienceIn Photosystem II (PSII) from Thermosynechococcus elongatus, high-light intensity growth conditions induce the preferential expression of the psbA3 gene over the psbA1 gene. These genes encode for the D1 protein variants labeled D1:3 and D1:1, respectively. We have compared steady state absorption and photo-induced difference spectra at 50 ms) QAâ yield was ⌠95% in D1:3, and ⌠60% in D1:1, relative to green light excitation. (iv) For the D1:1 variant, the quantum efficiency of photo-induced oxidation of side-pathway donors was lower. These effects can be correlated with amino acid changes between the two D1 variants. The effects on the pheophytin Qx band can be attributed to the hydrogen bond from Glu130 in D1:3 to the 131-keto of PheoD1, which is absent for Gln130 in D1:1. The reduced yield with red light in the D1:1 variant could be associated with either the Glu130Gln change, and/or the four changes near the binding site of PD1, in particular Ser153Ala. Photo-induced QAâ formation with far red light is assigned to the direct optical excitation of a weakly absorbing charge transfer state of the reaction centre. We suggest that this state is blue-shifted in the D1:1 variant. A reduced efficiency for the oxidation of side-pathway donors in the D1:1 variant could be explained by a variation in the location and/or redox potential of P+