Handling light safely: the function of higher plants antenna systems analyzed by reverse genetic

L\u2019obiettivo di questa tesi \ue8 comprendere le basi molecolare dei meccanismi che regolano la raccolta della luce e della fotoprotezione nelle piante superiori. Uno dei principali meccanismi di fotoprotezione del PSII \ue8 il non photochemical quenching (NPQ), esso risponde alle variazioni dell'intensit\ue0 luminosa lavorando come valvola di sicurezza per ridurre la sovra-eccitazione nel fotosistema: dissipando sotto forma di calore l\u2019energia assorbita in eccesso. Allo scopo di identificare tale meccanismo biofisico, i partner coinvolti e il sito nel quale avviene, \ue8 stato isolato e caratterizzato un mutante di Arabidopsis thaliana privo delle antenne monomeriche del PSII, denominato NoM. Quest\u2019ultimo inoltre \ue8 stato incrociato con delle linee mutanti, le quali impediscono, rispettivamente, la sintesi di zeaxantina e luteina o l'accumulo di PSBS. I risultati ottenuti suggeriscono che NPQ \ue8 catalizzato da 2 meccanismi indipendenti: la risposta pi\uf9 rapida viene attivata e catalizzata all'interno delle antenne monomeriche e dipende dalla formazione del radicale cationico, mentre le antenne maggiori (trimeri LHCII) sono responsabili della componente pi\uf9 lenta, nella quale la zeaxantina risulta essere cruciale. Successivamente \ue8 stata esaminata la fotoprotezione del PSI, a tale scopo sono stati utilizzati una serie di mutanti di Arabidopsis alterati nella biosintesi dei carotenoidi e delle xantofille. In particolare \ue8 stato analizzato il mutante szl1, che presenta un\u2019alterata composizione delle xantofille e un contenuto di \u3b2-carotene inferiore rispetto al wild type, il mutante npq1 incapace di sintetizzare zeaxantina e il mutante szl1-npq1 che combina le caratteristiche di entrambi. In condizioni di eccesso di luce, analisi su piante/complessi isolati PSI-LHCI hanno rivelato che la zeaxantina non ha effetto nel proteggere il PSI, mentre il \u3b2-carotene svolge un ruolo fondamentale riducendo la formazione di clorofilla tripletto (3Chl*), la quale pu\uf2 interagire con l\u2019ossigeno e formare ROS. Infine per chiarire la funzione specifica delle antenne del PSI (LHCI), \ue8 stato isolato un mutante di Arabidopsis privo del sistema LHCI (\u394Lhca), esso \ue8 caratterizzato da un alterato stato redox del plastochinone, indice di uno sbilanciamento nell\u2019assorbimento di energia tra PSI e PSII e da un ridotto tasso di crescita in tutte le condizioni testate, soprattutto sottoponendo le piante ad un regime di luce pulsata. La mancanza di LHCI non viene compensata da un sovraccumulo di altri prodotti genici della famiglia LHC, dimostrando quindi che PSI-LHCI \ue8 un sistema stabile, privo delle propriet\ue0 dinamiche di PSII. Il mutante compensa la ridotta dimensione dell'antenna incrementando fortemente la migrazione dei trimeri LHCII dal PSII al PSI, come strategia volta ad alleviare lo squilibrio nell'assorbimento dell\u2019energia di eccitazione tra i due fotosistemi. LHCII assorbe alle stesse lunghezze d'onda quando legato al PSI o al PSII; quindi l'assenza di forme spettrali in grado di assorbire fotoni nel rosso lontano, caratteristico delle antenne LHCI, riduce ulteriormente la capacit\ue0 di raccolta della luce nel mutante in quanto vi \ue8 una competizione tra PSII e PSI per gli stessi fotoni. Tutti questi risultati indicano un ruolo fondamentale dell'LHCI nell'ottimizzazione del flusso elettronico lineare, in particolare in condizioni di luce limitante, e nell'adattamento delle piante a condizioni di luce sempre mutevoli, tipiche dell\u2019ambiente naturale.The aim of this thesis is to understand the molecular basis of the mechanisms regulating light harvesting and photoprotection in higher plants. NPQ, the major photoprotective mechanisms of PSII, responds to variations in light intensity, working as a safety valve to reduce the excitation pressure in the photosystem. To identify the site(s) of quenching within the PSII antenna system, the partners in quenching reactions and the biophysical mechanism by which the quenching reactions are initiated, we constructed and characterize NoM mutant, lacking all monomeric LHC proteins while retaining LHCII trimers. Further, we introduced the npq1, and lut2 mutations, and found that lack of monomeric antennae changed the xanthophyll-dependence of the residual NPQ activity. We concluded that NPQ is catalyzed by 2 independent mechanisms with the fastest activated response catalyzed within monomeric LHC proteins depending on the formation of radical cation, while LHCII was responsible for the slowly component, which does not depend on lutein, while zeaxanthin was essential. Then we investigated the role of LHCI in the photoprotection of PSI, by focusing on carotenoid-protein interaction. We analyzed the mutants szl1, with a lower carotene content, and npq1, with suppressed zeaxanthin formation. Despite zeaxanthin bound LHCI under excess light, it neither enhanced photoprotection of the supercomplex. Instead, szl1 plants displayed a far stronger photoinhibition of PSI than WT plants and a greater level of 3Chl* originating in the LHCI moiety. We conclude that a crucial mechanism is mediated by carotenes bound to LHCI, which is instrumental in reducing the yield of harmful 3Chl*. To elucidate the specific function of LHCI, the phenotype of an Arabidopsis mutant devoid of the LHCI system (\u394Lhca) was studied over a range of conditions. Lack of LHCI could not be compensated by over-accumulation of other LHC gene products, thus showing that PSI-LHCI is a stable system, lacking the dynamic properties of PSII. Capacity of building a trans-thylakoid \u394pH gradient, redox balance of photosynthetic membrane and growth rate were all impaired in the mutant, especially under conditions of rapidly changing light. Mutant compensates for decreased antenna size by strongly enhancing LHCII binding to PSI with respect to the WT, as a strategy aimed at alleviating the imbalance in the excitation energy between PSs. Indeed, by comparing the excitation energy transfer efficiency properties of WT and mutant PSI-LHCII complexes, we found that LHCII is a very good antenna when bound to PSI core complex, independently on the presence of LHCI. However, under conditions of fluctuating light, the redox unbalance displayed by \u394Lhca plants reveals that the depletion of LHCI cannot be entirely compensated, despite greatly enhancing the transition to state II. LHCII absorbs the same wavelengths when bound to PSI or PSII; therefore, besides lower pigment content, absence of far-red spectral forms further disadvantage light-harvesting capacity of PSI-LHCII, due to the \u201cshading\u201d effect by the abundant PSII-LHCII antenna system. The hypothesis was confirmed by comparing growth of WT and mutant plants under lamps either enriched or devoid of far-red wavelengths. All these results point to a crucial role of the LHCI in optimizing linear electron flow, particularly in limiting light conditions, and in the adaptation of plants to life under ever-changing light conditions, typical of canopies

Loss of LHCI system affects LHCII re-distribution between thylakoid domains upon state transitions

LHCI, the peripheral antenna system of Photosystem I, includes four light-harvesting proteins (Lhca1-Lhca4) in higher plants, all of which are devoid in the Arabidopsis thaliana knock-out mutant \u394Lhca. PSI absorption cross-section was reduced in the mutant, thus affecting the redox balance of the photosynthetic electron chain and resulting in a more reduced PQ with respect to the wild type. \u394Lhca plants developed compensatory response by enhancing LHCII binding to PSI. However, the amplitude of state transitions, as measured from changes of chlorophyll fluorescence in vivo, was unexpectedly low than the high level of PSI-LHCII supercomplex established. In order to elucidate the reasons for discrepancy, we further analyzed state transition in \u394Lhca plants. The STN7 kinase was fully active in the mutant as judged from up-regulation of LHCII phosphorylation in state II. Instead, the lateral heterogeneity of thylakoids was affected by lack of LHCI, with LHCII being enriched in stroma membranes with respect to the wild type. Re-distribution of this complex affected the overall fluorescence yield of thylakoids already in state I and minimized changes in RT fluorescence yield when LHCII did connect to PSI reaction center. We conclude that interpretation of chlorophyll fluorescence analysis of state transitions becomes problematic when applied to mutants whose thylakoid architecture is significantly modified with respect to the wild type

Advances in Thick GEM-like gaseous electron multipliers. Part I: atmospheric pressure operation

Thick GEM-like (THGEM) gaseous electron multipliers are made of standard printed-circuit board perforated with sub-millimeter diameter holes, etched at their rims. Effective gas multiplication factors of 100000 and 10000000 and fast pulses in the few nanosecond rise-time scale were reached in single- and cascaded double-THGEM elements, in atmospheric-pressure standard gas mixtures with single photoelectrons. High single-electron detection efficiency is obtained in photon detectors combining THGEMs and semitransparent UV-sensitive CsI photocathodes or reflective ones deposited on the top THGEM face; the latter benefits of a reduced sensitivity to ionizing background radiation. Stable operation was recorded with photoelectron fluxes exceeding MHz/mm2. The properties and some potential applications of these simple and robust multipliers are discussed.Comment: 41 pages, 27 figures. Submitted to Nucl. Instr. and Meth. A, Dec 21, 200

Review of Power Conversion and Conditioning Systems for Stationary Electrochemical Storage

This paper deals with the power conversion system architectures to interface a stationary electrochemical storage installation with the network. Theoretical justifications about the conversion system layouts and control, used for actual Italian installations, are given. This paper aims at giving the power energy society an overview of actual possibilities of static conversion of d.c. battery sources

Um estudo sobre as opiniões de consumidores não especializados, no município de São Paulo, acerca da utilização do software livre e do software proprietário

The aim of this study was to construct a scale to assess attitudes and opinions on free software and proprietary software, to analyze the opinions of students of Higher Education Institutions (IES) about the use of Free Software (SL) and Proprietary Software (SP), representing two types of software on the market of information technology. To conduct this study was used a literature review and a field survey with 400 respondents by the trial samples, which provided grants and theoretical models to compare these two types of software. Thus it was possible to identify nine factors for the comparison between the SL and SP from the view of students of courses of administration, accounting and others, who knew the two types of software, but were not experts in the subject and were established in the municipality of São Paulo. The overall results showed advantages for Free Software in relation to Software Owner. The literature review and field survey allowed the identification of discrepancies between some factors in the opinions of the authors and students, which can be explained by the bias of the works consulted and also by preference of students from the sample by a type of software or other. Keywords: Consumer behavior, Free Software, Proprietary Software.O objetivo deste estudo foi construir uma escala de atitudes para analisar as opiniões sobre o software livre e o software proprietário, bem como analisar a opinião de alunos de Instituição de Ensino Superior (IES) acerca da utilização do Software Livre (SL) e do Software Proprietário (SP), duas modalidades de softwares representativas no mercado de tecnologia da informação. Para conduzir este estudo foram realizados revisão bibliográfica e levantamento de campo com 400 entrevistados, por amostras de julgamento, os quais forneceram subsídios e modelos teóricos para comparar esses dois tipos de software. Dessa forma, foi possível identificar nove fatores de comparação entre o SL e o SP, a partir da opinião dos alunos dos cursos de Administração, Ciências Contábeis e outros, que conheciam os dois tipos de softwares, porém não eram especialistas no assunto e estavam estabelecidos no município de São Paulo. Os resultados gerais apontaram vantagens para o Software Livre em relação ao Software Proprietário. A revisão bibliográfica e o levantamento de campo permitiram a identificação de algumas discrepâncias entre opiniões de autores e alunos, que pode ser explicada pela tendenciosidade das obras consultadas e também pela preferência compartilhada dos alunos da amostra por um tipo de software ou outro.   

Characterization of LAPPD timing at CERN PS testbeam

Large Area Picosecond PhotoDetectors (LAPPDs) are photosensors based on microchannel plate technology with about 400 cm$^2$ sensitive area. The external readout plane of a capacitively coupled LAPPD can be segmented into pads providing a spatial resolution down to 1 mm scale. The LAPPD signals have about 0.5 ns risetime followed by a slightly longer falltime and their amplitude reaches a few dozens of mV per single photoelectron. In this article, we report on the measurement of the time resolution of an LAPPD prototype in a test beam exercise at CERN PS. Most of the previous measurements of LAPPD time resolution had been performed with laser sources. In this article we report time resolution measurements obtained through the detection of Cherenkov radiation emitted by high energy hadrons. Our approach has been demonstrated capable of measuring time resolutions as fine as 25-30 ps. The available prototype had performance limitations, which prevented us from applying the optimal high voltage setting. The measured time resolution for single photoelectrons is about 80 ps r.m.s.Comment: 35 pages, 23 figure

LHCII can substitute for LHCI as an antenna for photosystem i but with reduced light-harvesting capacity

Light-harvesting complexes (LHCs) are major constituents of the antenna systems in higher plant photosystems. Four Lhca subunits are tightly bound to the photosystem I (PSI) core complex, forming its outer antenna moiety called LHCI. The Arabidopsis thaliana mutant δLhca lacks all Lhca1-4 subunits and compensates for its decreased antenna size by binding LHCII trimers, the main constituent of the photosystem II antenna system, to PSI. In this work we have investigated the effect of LHCI/LHCII substitution by comparing the light harvesting and excitation energy transfer efficiency properties of PSI complexes isolated from δLhca mutants and from the wild type, as well as the consequences for plant growth. We show that the excitation energy transfer efficiency was not compromised by the substitution of LHCI with LHCII but a significant reduction in the absorption cross-section was observed. The absence of LHCI subunits in PSI thus significantly limits light harvesting, even on LHCII binding, inducing, as a consequence, a strong reduction in growth

Near-Infrared Observations of BL Lacertae Host Galaxies

Multi-band near-infrared images of twelve BL Lacertae objects were obtained with the 2.5m telescope at the Las Campanas Observatory in order to determine the properties of their underlying host galaxies. Resolved emission was clearly detected in eight of the lowest redshift targets (up to z~0.3), and was modeled with a de Vaucouleurs r^{1/4} surface brightness law. We find that the morphologies match the elliptical galaxy profiles well, and that the BL Lac objects reside in large and luminous, but otherwise normal hosts -- consistent with previous studies done predominantly at optical wavelengths. The median absolute K-band magnitude of the galaxies in this study is -26.2, the average half-light radius is 4.2 +/- 2.3 kpc, and their average integrated R-K color is 2.7 +/- 0.3 mag. These are well within the range of values measured previously in the H-band by Kotilainen et al. and Scarpa et al. in a comparable number of targets. Taking their data together with our results, we find a best-fit K-band Kormendy relation of \mu_{e} = 4.3 log_{10} (r_{e}/kpc) + 14.2 mag arcsec^{-2}, virtually identical to that obtained for normal ellipticals. Finally, the near-infrared colors determined for five galaxies (average J-K = 0.8 +/- 0.3 mag) are the first such measurements for BL Lac hosts, and match those expected from old stellar populations at the BL Lac redshifts.Comment: 10 pages, includes 4 figures and 3 tables. ApJ, accepte

A vertical facility based on raster scan configuration for the x-ray scientific calibrations of the ATHENA optics

The ATHENA X-ray observatory is a large-class ESA approved mission, with launch scheduled in 2028. The technology of Silicon Pore Optics (SPO) was selected since 2004 as the baseline for making the X-ray Mirror Assembly. Up to 700 mirror modules to obtain a nested Wolter like optics. The maximum diameter of the shells will be 2.5 m while the focal length is 12 m. The requirements for on-axis angular resolution and effective area at 1 keV are 5 arcsec HEW and 1.4 m2, while the field of view will be 40 arcmin in diameter (50 % vignetting). While in this moment there an on-going effort aiming at demonstrating the feasibility of a so large optics with so stringent scientific requirements, an important aspect to be considered regards the scientific calibrations of the X-ray optics. In this respect, the Point Spread Function and effective area have to be correctly measured and calibrated on-ground at different energies across the entire field of view, with a low vignetting. The approach considered so far foresees the use of a long (several hundreds of meters) facility to allow a full illumination with low divergence of the entire optics module (or at least of large sections of it). The implementation of similar configurations in a completely new facility to be realized in Europe (friendly called "super Panter") or the retrofitting existing facilities like the XRCF at NASA/MSFC are being considered. In both cases the costs and the programmatic risks related to the implementation of these huge facilities, with their special jigs for the alignment of the ATHENA optics, represent important aspects to be considered. Moreover, the horizontal position of the optics to be used in full illumination facilities would determine gravitational deformations, not easy to be removed with actuators or by modeling. In this talk we will discuss a completely different concept, based on the mount of the optics in vertical position and the use of a raster scan of the ATHENA optics with a small (a few cm2 wide) highly collimated (1 arcsec or so) white beam X-ray. This system will allow us to operate a much compact system. The use of a vertical configuration will imply smaller gravitational deformations, that can be controlled with actuators able to compensate them. A proper camera system with a sufficient energy resolution will be able to grant a correct measurement of both PSF and effective area of the Mirror Assembly within the calibration requirements and in a reasonable integration time. Moreover, it may allow us also to perform end-to-end tests using the two flight focal plane instruments of ATHENA. The cost and risks for the implementation would be much lower than for the full illumination systems. The conceptual configuration and preliminary expected performance of the facility will be discussed

Improving PARSEC models for very low mass stars

Many stellar models present difficulties in reproducing basic observational relations of very low mass stars (VLMS), including the mass-radius relation and the optical colour-magnitudes of cool dwarfs. Here, we improve PARSEC (PAdova-TRieste Stellar Evolution Code) models on these points. We implement the T- \ufffd relations from PHOENIX BT-Settl model atmospheres as the outer boundary conditions in the PARSEC code, finding that this change alone reduces the discrepancy in the mass-radius relation from 8 to 5 per cent. We compare the models with multiband photometry of clusters Praesepe and M67, showing that the use of T- \ufffd relations clearly improves the description of the optical colours and magnitudes. But anyway, using both Kurucz and PHOENIX model spectra, model colours are still systematically fainter and bluer than the observations. We then apply a shift to the above T- \ufffd relations, increasing from 0 at Teff = 4730 K to ~14 per cent at Teff = 3160 K, to reproduce the observed mass-radius relation of dwarf stars. Taking this experiment as a calibration of the T- \ufffd relations, we can reproduce the optical and near-infrared colour-magnitude diagrams of low-mass stars in the old metal-poor globular clusters NGC 6397 and 47 Tuc, and in the intermediate-age and young solar-metallicity open clusters M67 and Praesepe. Thus, we extend PARSEC models using this calibration, providing VLMS models more suitable for the lower main-sequence stars over a wide range of metallicities and wavelengths. Both sets of models are available on PARSEC webpage. \ufffd 2014 The Authors