Le développement d'organismes photosynthétiques (piégeant le C02 en préservant l'eau douce et les terres cultivables sans ajout d'engrais) capables d'utiliser l'énergie solaire pour produire du dihydrogène (H2) passe par une meilleure compréhension du rôle de l'hydrogénase dans le métabolisme cyanobactérien. Le Laboratoire de Biologie et Biotechnologie des Cyanobatéries où j'ai travaillé durant ma thèse utilise une approche de "Biologie Intégrative" pour analyser le métabolisme qui conduit à la photo-production d’H2 chez la cyanobactérie modèle Synechocystis sp. PCC6803. Mon travail s'est focalisé sur l’analyse des réseaux de régulation amenant à la production d'H2 par l’hydrogénase bidirectionnelle à centre Ni-Fe (composée de 5 sous-unités) codée par l’opéron hox. Lorsque j’ai débuté ce travail, 2 activateurs de l’opéron hox avaient été identifiés: AbrB1 et LexA. Un article dont je suis co-premier auteur est paru (Dutheil et al. 2012 J Bact.), il décrit l'identification par l'utilisation de diverses approches d'un nouveau facteur de transcription de l'opéron hox: AbrB2 (homologue d'AbrB1). J'ai ainsi montré que l'expression de l’opéron hox était régulée négativement par AbrB2 en utilisant des fusions transcriptionnelles au gène rapporteur cat (introduites dans la souche sauvage ou dépourvues d'AbrB2) ainsi que des expériences de qRT-PCR. Par la technique de retard sur gel, nous avons confirmé une interaction directe entre AbrB2 et la région promotrice de l’opéron hox. En collaboration avec deux laboratoires du CEA, nous avons montré qu'un mutant dépourvu d’AbrB2 possède une activité hydrogénase augmentée, confirmant ainsi qu'AbrB2 est un régulateur négatif de la production d'H2.Dans un deuxième temps et en collaboration avec deux post-doc du laboratoire, nous avons mis en évidence le rôle de la cystéine unique d'AbrB2 dans le contrôle redox de son activité de régulation transcriptionnelle.Par la technique du retard sur gel,j’ai montré que cette cystéine n’est pas cruciale pour la fixation d'AbrB2 sur le promoteur hox, mais que par contre, la modification redox de celle-ci l’affecte de manière drastique. Dans le cadre de collaborations, nous avons identifié la modification post-traductionnelle qui peut avoir lieu sur la cysteine d'AbrB2 et il s’agit de la première fois, qu’un tel mécanisme de régulation est identifié pour cette famille de régulateur et chez les cyanobactéries. J’ai construit une souche portant l'allèle muté abrB2 Cys>Ser sur le chromosome et exprimé par le promoteur sauvage d’abrB2. J’ai montré grâce à cette construction et en utilisant diverses techniques (activité hydrogénase, qRT-PCR, Western blot et transcriptome) que la cystéine d'AbrB2 joue un rôle dans son activité de régulation qui est 60% moins bonne sur les 529 gènes cibles (directes ou indirectes) du régulateur muté. L’effet est également visible sur l’activité hydrogénase. Ce résultat a été complété par des tests de surexpression thermoinduite d’AbrB2 qui montrent que la mutation C34S affecte la stabilité de la protéine qui ne s’accumule pas autant que la sauvage dans les même conditions et dont la surexpression est létale. Un manuscrit dont je suis copremier auteur et décrivant ces résultats est en cours de finalisation et sera prochainement soumis à l’Intern. Journ. of Hydrogen Energy.L’ensemble de ces travaux permet de mieux comprendre les mécanismes biologiques liés à l’expression de l’hydrogénase bidirectionnelle et vont dans le sens d’un rôle important de celle-ci dans la détoxification des stress redox. La détermination des relations entre les différents régulateurs de l’hydrogénase et les possibles modifications post-traductionnelles de chacun de ces facteurs que j’ai mises en évidence traduisent une enzyme à la régulation complexe. Ces nouvelles connaissances permettent d’éclairer sous un angle nouveau la photoproduction d’H2 par les cyanobactéries et permettront peut-être d’élaborer des stratégies de production d’H2 efficace. / Developing photosynthetic organism (trapping CO2 while preserving fresh water and arable soils without adding fertilizers) able to use Sun light to produce dihydrogen (H2) is depending on a better understanding of the role of hydrogenase in the cyanobacterial metabolism. The Laboratoire de Biologie et Biotechnologie des Cyanobactéries (LBBC) where I worked during my thesis uses « Integrative Biology » approach to analyze the metabolism leading to H2 photoproduction by the model cyanobacterium Synechocystis sp. PCC6803. My work focused on analyzing the regulation network leading to H2 production by the bidirectionnal hydrogenase with Ni-Fe cluster (composed of 5 subunits) encoded by hox operon. When I started this work, two transcriptionnal activators were identified : AbrB1 and LexA. An article, of which I’m sharing first author position, is published (Dutheil et al. 2012 J Bact.), it describes the identification by different approachs of a new transcriptionnal factor of hox operon : AbrB2 (homologous to AbrB1). I showed that hox expression is negatively regulated by AbrB2 by using transcriptionnal fusion to cat reporter gene (introduced in the wild type background or the AbrB2-deleted one) and qRT-PCR experiments. By the electrophoretic mobility shift assay (EMSA) method, we confirmed a direct interaction between AbrB2 and the promoter region of hox operon. Collaborating with two CEA laboratories, we showed that a mutant lacking AbrB2 harbours an increased hydrogenase activity, validating that AbrB2 is a negative regulator of H2 production.In a second time of my thesis and colaborating with two post-doc of the laboratory, we evidenced the role of the unique cysteine of AbrB2 in redox-controlling the transcriptionnal regulator activity of the protein.Using the EMSA method, I showed that the cysteine is not crucial for AbrB2 fixing on hox promoter, but also that the redox modification occuring on this residue inhibits this same binding activity. Collaborating with other labs, we identified the post-translational modifications that may occur on AbrB2 cysteine and it is the first time that such a regulating mechanism is identified for this family of regulators and in cyanobacteria. I constructed a strand harbouring the abrB2C34S mutant allele on the chromosome and expressed by the abrB2 natural promoter. I showed with this construction and using diverse methods (hydrogenase activity, qRT-PCR, Western blot and transcriptome) that AbrB2 cysteine plays a role in its regulating activity : regulating activity is 60% less efficient towards the 529 target genes (either direct and indirect) of the mutated regulator. The effect is also seen on hydrogenase activity and hox genes. This result was completed by thermoinduced overexpression assays that show that C34S mutation of AbrB2 alters protein stability : the mutated protein accumulates less than wild type allele in the same conditions, which is lethal. A manuscript, of which I’m sharing first author position, and describing those results is being finalised and will be submitted soon to the IJHE (International Journal of Hydrogen Energy).Altogether, my results allow a better understanding of the biological mechanisms linked to bidirectionnal hydrogenase expression and agree with a possible role for hydrogenase in detoxifying redox stresses. The determination of the relationships between the different regulators of hydrogenase, and their possible post-translational modifications that I revealed, highlight an enzyme with complex regulation. This new knowledge brings an original outlook on hydrogen photoproduction by cyanobacteria and shall allow elaboration of efficient H2 production strategies.

A large acceptance polarimeter has been designed to measure recoil polarisation in pseudoscalar (Jπ=0−) meson photoproduction reactions. The device was installed at the MAMI facility at the Institut für Kernphysik in Mainz, Germany. A racetrack microtron provided a longitudinally polarised 1.5 GeV electron beam, which impinged on a 12 μm copper radiator, producing a beam of circularly polarised Bremsstrahlung photons with energies between 400-1400 MeV. The electrons were then momentum analysed in the Glasgow Photon Tagging Spectrometer to tag the photon energy with a resolution of ~4 MeV. The photons were incident on a liquid hydrogen target, and the reaction products were detected using the Crystal Ball and TAPS detectors. The beam-recoil polarisation observable Cx, which describes the fraction of circular polarisation transferred from the photon beam to the recoiling nucleon, was measured in the reactions γp→pπ0 and γp→pη from data taken in September-October 2008. The results for π0 production give a significant expansion of the world data set and are shown to be consistent with the few previous measurements taken at Jefferson Lab, USA, while the results for the η channel are a world first. The observed values for Cx are compared to the current solutions from the two leading partial wave analyses, SAID and MAID, with wide angular coverage up to a photon beam energy of 1400 MeV. Significant discrepancies in the prediction of Cx can be resolved by the new data.

The resonance spectrum of the nucleon gives direct information on the dynamics and interactions of its constituents. This offers an important challenge to the theoretical models of nucleon structure, including the emerging Lattice QCD predictions, conformal field theories and more phenomenological, QCD-based approaches. Although the various models predict different features for the excitation spectra, the experimental information is currently of too poor quality to differentiate between these models. Pion photoproduction from the nucleon is a powerful probe of the spectrum as most resonances are expected to couple to the pion decay channel. However, cross-sections alone are not sensitive enough to allow identification of the underlying excitation spectrum, as the resonances have energy widths larger than their separations. A major world effort is underway to additionally measure polarisation observables in the production process. For a model-independent analysis a “complete” set of single- and doublepolarisation observables needs to be measured in experiments involving polarised beams, targets and a means of determining recoil nucleon polarisation. In particular, the beam asymmetry is a critical observable for the constraint of partial wave analyses (PWA) used to extract the nucleon excitation spectrum from the data. Almost all of the available world data on the beam asymmetry has been taken on the proton, with the neutron dataset sparse, containing only three experiments at fixed angles and in a limited photon energy range. The lack of extensive data on the neutron is a major deficiency, as different resonances have very different electromagnetic couplings to the proton and neutron. As a result, the data from the two targets will have very different relative contributions from, and sensitivities to, the spectrum of nucleon resonances. Moreover, neutron data is essential for the separation of the isoscalar and isovector components of the reaction amplitudes. This thesis presents a very high statistics measurement of the photon beam asymmetry on the neutron with close-to-complete angular coverage and a wide range of invariant mass (1610 – 2320 MeV) extending over the third resonance region, where the excitation spectrum is particularly ill defined. The experiment was conducted at the Thomas Jefferson National Accelerator Facility (JLab) using a tagged, linearly polarised photon beam, a liquid deuterium target and the CEBAF Large Acceptance Spectrometer (CLAS). The quality and quantity of the data has allowed an invariantmass resolution of 10 MeV and an angular resolution of 0.1 in the cosine of the centre-of-mass pion production angle, θ. Good agreement is evident in the regions where there is kinematic overlap with sparse previous data. Comparison of the new data is made with the two main partial wave analyses, SAID andMAID. Significant discrepancy is observed at backward θ with SAID (across most of the energy range) and MAID (up to ∼ 1750 MeV) and also below ∼ 35◦ in θ with both analyses. This extensive new dataset will help significantly to constrain partial wave analyses and will be a crucial part of the current world effort to use meson photoproduction to tackle long-standing uncertainties in the fundamental excitation spectrum of the nucleon. As a first step towards this the refitting of the SAID partial wave analysis incorporating the new data was carried out and shows very significant changes in the properties of the magnetic P11, P13, D13, D35, F15, G17 and G19 partial waves.

Cette thèse présente une mesure des sections efficaces pour la production inclusive de jets à grand E_T (>= 21 GeV) en photoproduction, à partir d'un lot d'événements enregistrés par H1 en 1996 et 1997 et correspondant à une luminosité intégrée de 24.1 pb-1. Le domaine cinématique de la mesure est défini par Q^2 < 1 GeV^2 et 95 < W < 285 GeV. Les jets sont reconstruits dans le référentiel du laboratoire dans la région en pseudo-rapidité -1 < eta^jet < 2.5 avec l'algorithme inclusif k_T. Les mesures sont corrigées au niveau hadron et comparées à des calculs QCD au LO et NLO. Les distributions mesurées sont bien prédites, en normalisation et en forme, par les prédictions de QCD au NLO obtenues avec les différentes densités de parton (PDFs) du photon et du proton disponibles. Les corrections d'hadronisation appliquées aux calculs QCD améliore légèrement l'accord avec les données. La précision actuelle des résultats expérimentaux aussi bien que des prédictions théoriques ne permettent pas de distinguer entre les différentes PDFs. Une nouvelle mesure des sections efficaces à petit E_T (>=5 GeV), réalisée à partir d'un lot d'événements étiquetés (Q^2 < 0.01 GeV^2) « tout venants » correspondant à une luminosité intégrée de 0.47 pb-1 est également présentée. Le spectre en E_T obtenue en combinant les données à petit et grand E_T est bien décrite par les calculs QCD au NLO. Son ajustement par une loi de puissance inspirée de QCD donne un résultats consistant avec ceux obtenus précédemment dans des mesures similaires. Les calculs Monte-Carlos au LO, incluant une simulation des ordres supérieurs, de l'hadronisation et de l'événement sous-jacent dans les processus résolus, décrivent la forme des distributions mesurées. Finalement, les données sont comparées aux résultats similaires obtenus auprès des collisionneurs p-pbar.

Thirteen N* states have been well-established according to the Particle Data Group, but some relativized quark models predict that many more N* resonances exist. Diquark models predict that the N* spectrum is limited by a correlated quark-pair in the nucleon, but there is strong evidence for the existence of the [Special characters omitted.] (1900)** resonance, which is absent in diquark models. Measuring the spectrum of N* states will provide valuable information on the relevant degrees of freedom within the nucleons. Most of the experimental searches for the N* states have been conducted in the πN channel. Some models of baryon decays predict that most of the unobserved N* states couple somewhat weakly to the πN channel, and that some couple non-negligibly to the KY, K*Y , and KY* channels. Measurements of the cross sections and polarization observables of strangeness photoproduction reactions can provide additional information on the spectrum of N* states. These measurements can be used in coupled-channel partial-wave analyses that can provide simultaneous constraints on the N* resonance parameters from several channels. These analyses can also take into account hadronic rescattering, which is predicted to have a large effect on the measured cross sections. However, to determine the isospin decomposition of the photo-transition amplitudes to these channels, photoproduction measurements are necessary on both the proton and the neutron. Measurements of the differential cross sections of the γn [arrow right] K* (892) 0 Λ and γn [arrow right] K + Σ*(1385) - reactions have been performed using data from the Jefferson Lab Hall B CLAS g13 experiment. No experimental cross section data have yet been published on the γn [arrow right] K* (892) 0 Λ reaction, and the only published cross section data on the γn [arrow right] K + Σ*(1385) - reaction are limited to forward angles, where t -channel K + and K* + exchanges are predicted to dominate. These cross sections are compared against theoretical models to study the channel interactions that give rise to their distributions. These reactions also have the same final state particles ( K + π - pπ - ), so studies of their potential interference were performed as well. A measurement of the γn [arrow right] pπ - cross section was also performed, and the agreement with published results within the uncertainties validated the integrity of the data and procedures used in this analysis.

Symmetry tests and more precisely parity violation experiments using the properties of the weak interaction give us unique insight into the internal hadronic structure of matter. The Gº experiment at Jefferson Laboratory used parity violating electron scattering to probe the strange quark contribution to the electromagnetic nucleon form factors, (GMs and GEs) as well as the axial contribution, (GAe). The data taken during the experiment provide further information on the axial transition form factor of the N - $\Delta$ transition, (GANΔ), as well as the scale of the low energy constant (dΔ) characterizing the parity violating γNΔ coupling. The analysis of backward angle Gº data taken with a liquid deuterium target to deduce the parity violating asymmetry for pion photoproduction on the Δ resonance, and the first experimental constraint on the value of dΔ, are reported in this thesis. The results showed that dΔ = (8.3 ± 25.3) gπ where the uncertainty is dominated by statistics, and that 75 percent of the theory range would be excluded by this measurement at 1 sigma.

Symmetry tests and more precisely parity violation experiments using the properties of the weak interaction give us unique insight into the internal hadronic structure of matter. The Gº experiment at Jefferson Laboratory used parity violating electron scattering to probe the strange quark contribution to the electromagnetic nucleon form factors, (GMs and GEs) as well as the axial contribution, (GAe). The data taken during the experiment provide further information on the axial transition form factor of the N - $\Delta$ transition, (GANΔ), as well as the scale of the low energy constant (dΔ) characterizing the parity violating γNΔ coupling. The analysis of backward angle Gº data taken with a liquid deuterium target to deduce the parity violating asymmetry for pion photoproduction on the Δ resonance, and the first experimental constraint on the value of dΔ, are reported in this thesis. The results showed that dΔ = (8.3 ± 25.3) gπ where the uncertainty is dominated by statistics, and that 75 percent of the theory range would be excluded by this measurement at 1 sigma.

Chiral perturbation theory is a useful tool in the study of low energy reactions involving light particles. However the inclusion of heavy particles in chiral perturbation theory results in large contributions from loop diagrams which violate the standard power counting scheme. We review two methods, referred to as heavy baryon chiral perturbation theory and infrared regularization, which remove the high energy effects of the heavy particles and which therefore do not violate the power counting scheme. We then use these two methods to calculate the amplitude for pion photoproduction to fourth order and prove that the two amplitudes are equivalent.

Chiral perturbation theory is a useful tool in the study of low energy reactions involving light particles. However the inclusion of heavy particles in chiral perturbation theory results in large contributions from loop diagrams which violate the standard power counting scheme. We review two methods, referred to as heavy baryon chiral perturbation theory and infrared regularization, which remove the high energy effects of the heavy particles and which therefore do not violate the power counting scheme. We then use these two methods to calculate the amplitude for pion photoproduction to fourth order and prove that the two amplitudes are equivalent.

abstract: The nucleon resonance spectrum consists of many overlapping excitations. Polarization observables are an important tool for understanding and clarifying these spectra. While there is a large data base of differential cross sections for the process, very few data exist for polarization observables. A program of double polarization experiments has been conducted at Jefferson Lab using a tagged polarized photon beam and a frozen spin polarized target (FROST). The results presented here were taken during the first running period of FROST using the CLAS detector at Jefferson Lab with photon energies ranging from 329 MeV to 2.35 GeV. Data are presented for the E polarization observable for eta meson photoproduction on the proton from threshold (W=1500 MeV) to W=1900 MeV. Comparisons to the partial wave analyses of SAID and Bonn-Gatchina along with the isobar analysis of eta-MAID are made. These results will help distinguish between current theoretical predictions and refine future theories. / Dissertation/Thesis / Ph.D. Physics 2011