Tesis sobre el tema "Lhc protein"

Die pflanzliche Light-harvesting complex-Proteinfamilie besteht aus Proteinen mit vielfältigen Funktionen. Dabei ist die Funktion der Light-harvesting-like 3-Proteine (LIL3) sowie der One-helix-Proteine (OHPs) weitestgehend unbekannt. Im Rahmen dieser Arbeit wurde gezeigt, dass LIL3 nicht nur mit der Geranylgeranyl-Reduktase (CHLP), sondern auch mit der Protochlorophyllid-Oxidoreduktase (POR) interagiert. Sowohl CHLP als auch POR werden über die Interaktion zu LIL3 an die Thylakoidmembran gebunden und dadurch stabilisiert. Beide Enzyme liefern die direkten Vorstufen für den von der Chlorophyll-Synthase (CHLG) katalysierten finalen Chlorophyll-Syntheseschritt. Neben der Bestätigung der bereits früher gezeigten Chlorophyllbindung von LIL3 konnte eine Affinität zu den späten Intermediaten der Chlorophyllbiosynthese Proto IX, MgP, MgPMME und Pchlid nachgewiesen werden. Die größte Affinität bestand dabei gegenüber dem Substrat von POR, Pchlid. Basierend auf diesen Erkenntnissen wird LIL3 als Regulator der späten Chlorophyllbiosynthese-Schritte vorgeschlagen: LIL3 transportiert Substrate zwischen den Enzymen und ermöglicht durch die Bindung von CHLP und POR die Synthese der Chlorophyll-Edukte in räumlicher Nähe. Dadurch wird die Versorgung von CHLG mit dessen Edukten favorisiert. Beide OHP-Varianten (OHP1/2) bilden ausschließlich Heterodimere und binden Chlorophyll sowie Carotinoide im Verhältnis 3:1. Die Pigmentbindung basiert auf den konservierten Aminosäuren im Chlorophyllbindemotiv. An das OHP1-OHP2-Dimer bindet der PSII-Assemblierungsfaktor HCF244 und wird dadurch an der Membran verankert. HCF244 stabilisiert das OHP-Heterodimer und beide OHPs stabilisieren sich gegenseitig. Der heterotrimere OHP1-OHP2-HCF244-Komplex ist für die D1-Synthese wesentlich. Es wird vermutet, dass die OHPs an der co-translationalen Beladung von (p)D1 mit Pigmenten beteiligt sind sowie frühe Assemblierungsintermediate von PSII vor überschüssiger Anregungsenergie schützen.
The plant light-harvesting complex protein family comprises different members with a variety of functions. However, the function of the light-harvesting-like 3 proteins (LIL3) as well as the one-helix proteins (OHPs) is largely unknown. In this thesis, an interaction of LIL3 not only with geranylgeranyl-reductase (CHLP), but also with protochlorophyllide-oxidoreductase (POR) could be established. LIL3 tethers CHLP and POR to the thylakoid membrane, thereby conferring stability to both enzymes. Both CHLP and POR are synthesizing the direct chlorophyll precursors which are combined to chlorophyll by the subsequent chlorophyll synthase (CHLG). In addition to the chlorophyll binding ability of LIL3 reported earlier, an affinity of LIL3 towards the chlorophyll biosynthesis intermediates Proto IX, MgP, MgPMME, and Pchlide could be shown. Interestingly, the highest affinity of LIL3 was exerted towards Pchlide which is the substrate of POR. Therefore, LIL3 is postulated to shuffle the intermediates between enzymes and brings CHLP and POR in close proximity, which may help to supply CHLG with its substrates. Regarding the function of the OHPs an exclusive heterodimer formation of both the OHP1 and OHP2 variants could be shown. The OHP1-OHP2-heterodimer is able to bind chlorophyll and carotenoids in an approximate 3:1 ratio and pigment binding depends on dimer formation as well as the presence of the conserved amino acids in the chlorophyll binding motif. The PSII-assembly factor HCF244 is anchored to the thylakoid membrane by binding to both OHPs, thereby stabilizing the OHP-heterodimer. The heterotrimeric OHP1-OHP2-HCF244-complex is essential for D1 biosynthesis, although the exact molecular function of HCF244 is still unknown. It is suggested that the OHP-dimer is responsible for co-translational loading of (p)D1 with pigments as well as photoprotection of early PSII assembly intermediates.

The Large Hadron collider (LHC) is a 7\,TeV per beam proton-proton collider. The high stored beam energy of the LHC (361.6\,MJ) requires unprecedented machine protection systems. As demonstrated in September 2008, failure of any one of these systems can lead to considerable damage, and hence delays in the physics schedule. In addition to various quench protection systems and interlocks, a beam collimation system is installed to protect against regular and irregular beam loss, with the aim of protecting against superconducting magnet quenches. The high 8.33T magnetic field of the main arc bending magnets leads to a quench when exposed to beam energy leakage of $5\,\textrm{mW cm}^{-3}$. In order to ensure accurate running of this system, computer simulations must be created. A tracking code, Merlin, has been adapted to simulate the features of the collimation system. New physics models for the simulation of protons interacting in a collimator jaw have been created, and these have been fitted to all experimental scattering data for the first time. Merlin has been used to simulate beam loss in the LHC, and a comparison of the effect of these different physics models have been made. Finally, a luminosity upgrade, the High Luminosity Large Hadron Collider is to be installed in the 2020s. Merlin has also been used to simulate the collimation system for this upgrade in order to ensure reliable future operation of this upgrade.

Negli organismi eucaritici fotosintetici il sistema antenna è composto da subunità codificate dalla famiglia multigenica Light harvesting complex (Lhc). Queste proteine sono coinvolte sia nella raccolta della luce che nella fotoprotezione. In particolare, le proteine antenna del PSII, le subunità Lhcb, sembrano essere implicate nel meccanismo di dissipazione termica dell’energia di eccitazione in eccesso (NPQ, Non Photochemical Quenching). Chiarire i dettagli molecolari dell’induzione dell’NPQ nelle piante superiori si è dimostrata essere una grande sfida. Durante il mio dottorato di ricerca, ho deciso di indagare il ruolo delle subunità Lhcb nel quenching dell’energia di eccitazione utilizzando un approccio di genetica inversa: ho ottenuto mutanti privi di ciascuna delle subunità per capire il loro coinvolgimento nel meccanismo. Qui di seguito sono riassunti i principali risultati ottenuti. Sezione A. Mutanti per le subunità monomeriche Lhc e fotoprotezione È stata studiata la funzione delle proteine antenna CP26, CP24 e CP29 nella raccolta della luce e nella regolazione della fotosintesi, mediante l’isolamento di mutanti knockout (ko) di Arabidopsis thaliana che mancano completamente di una o due di queste subunità. In particolare nella sezione A.1 sono trattati i singoli mutanti koCP24, koCP26 e il doppio mutante koCP24/26. Tutte queste tre linee mostrano una ridotta efficienza di trasferimento di energia dai complessi trimerici di raccolta della luce (LHCII) al centro di reazione del fotosistema II (PSII) a causa della disconnessione fisica degli LHCII dal PSII. Abbiamo osservato che il trasporto di elettroni è diminuito nel genotipo koCP24, ma non nelle piante che mancano di CP26: koCP24 ha una diminuita velocità di trasporto elettronico, un più basso gradiente di pH transmembrana, una ridotta capacità di NPQ, e una crescita limitata. Inoltre, i complessi PSII di queste piante sono organizzati in array bidimensionali nelle membrane granali. Sorprendentemente, il doppio koCP24/26 mutante, mancante sia di CP24 che CP26, recupera la capacità di trasporto elettronico, di NPQ e il tasso di crescita ai livelli del WT. Abbiamo quindi approfondito lo studio del mutante koCP24 per comprendere le ragioni di tali alterazioni fenotipiche. L’analisi della cinetica di induzione di fluorescenza e di misure di trasporto di elettroni nei vari passaggi all’interno della catena fotosintetica hanno suggerito che la limitazione nel trasporto degli elettroni in koCP24 è dovuta alla restrizione del trasporto degli elettroni tra i siti QA e QB del PSII, ritardando la diffusione del plastoquinone. Abbiamo concluso che la mancanza di CP24 altera l’organizzazione dei PSII e limita, di conseguenza, la diffusione del plastoquinone. Tale limitazione è ripristinata in koCP24/26. Nella sezione A.2, è descritta la caratterizzazione della funzione della subunità CP29, estendendo l'analisi alle diverse isoforme CP29. A questo scopo, ho ottenuto mutanti knock-out privi di una o più isoforme CP29 ed analizzato la loro capacità fotosintetica e di fotoprotezione. La mancanza di CP29 non comporta alcuna variazione significativa del trasporto elettronico lineare/ciclico e della capacità di transizione di stato, mentre l’efficienza quantica del PSII e la capacità di NPQ risultano alterati. L’efficienza di fotoprotezione è inferiore in koCP29 rispetto sia al WT che ai mutanti che conservano una singola isoforma. È interessante notare che, mentre l’espressione di una delle isoforme CP29.1 o CP29.2 ripristina la capacità di fotoprotezione, l’espressione di solo CP29.3 non porta all’accumulo della proteina né al recupero del fenotipo fotoprotettivo. Sezione B. Riorganizzazione dinamica delle membrane: dissociazione della B4 e identificazione di due siti di quenching. Le subunità antenna sembrano essere il sito del quenching, mentre l'innesco del meccanismo è mediato da PsbS, una subunità del PSII coinvolta nella rilevazione dell’acidificazione lumenale. Abbiamo indagato il meccanismo molecolare attraverso il quale PsbS è in grado di regolare l’efficienza di raccolta della luce, studiando mutanti di Arabidopsis che mancano dei singoli Lhcbs monomerici. Nella Sezione B.1 è mostrato come PsbS è in grado di regolare l'associazione/dissociazione di un complesso membrana di cinque subunità, composto dalle due proteine monomeriche CP29 e CP24 e dal complesso trimerico LHCII-M (Band 4 Complex - B4C). Abbiamo dimostrato che la dissociazione di questo supercomplesso è indispensabile per l'attivazione dell’NPQ in luce alta. Coerentemente, abbiamo scoperto che mutanti knock-out mancanti delle due subunità componenti la B4, koCP24 e koCP29, sono fortemente influenzati nella dissipazione dell’energia. L'osservazione diretta mediante microscopia elettronica ha mostrato che la dissociazione della B4C porta alla ridistribuzione dei PSII all'interno delle membrane granali. Proponiamo che la dissociazione della B4C renda i due siti di quenching, possibilmente CP29 e CP24, disponibili per lo switch a una conformazione quenchiata. Questi cambiamenti sono reversibili e non richiedono la sintesi/degradazione proteica, consentendo in tal modo cambiamenti di dimensione dell'antenna PSII e l'adattamento a rapide variazioni delle condizioni ambientali. Nella sezione B.2 abbiamo studiato questo meccanismo di quenching mediante analisi di fluorescenza ultra-rapida. Recenti risultati sui tempi di vita di fluorescenza in vivo propongono l’attivazione di due siti indipendenti di quenching durante l’NPQ: Q1 si localizza nei complessi LHCII, funzionalmente staccati dal PSII/RC (centro di reazione) con un meccanismo che richiede PsbS ma non Zea; Q2 si trova ed è collegato al complesso PSII, e dipende dalla formazione di Zea. Questi due eventi di quenching potrebbero originarsi in ciascuno dei due domini fisici granali rivelati dall’analisi di microscopia elettronica come precedentemente riportato. Abbiamo quindi studiato la modulazione del quenching in mutanti knock out confrontando i tempi di vita di fluorescenza in condizioni di quenching e non quenching in foglie intatte: abbiamo ottenuto risultati coerenti con il modello di due siti di quenching situati, rispettivamente, nel dominio C2S2 e nel dominio arricchito in LHCII. I dati indicano che il sito Q1 manca nel koCP24 mentre il Q2 è attenuato nel koCP29. Sulla base dei risultati di questa sezione, possiamo concludere che durante l’induzione dell’NPQ in vivo il supercomplex del PSII si dissocia in due frazioni, separate in domini distinti della membrana granale e protetti ciascuno dalla sovra-eccitazione grazie all’attività di siti di quenching localizzati in CP24 e CP29. Sezione C. Trasferimento di energia di eccitazione e organizzazione della membrana: ruolo delle subunità antenna del PSII. In questa sezione è riportato lo studio del ruolo dei singoli complessi antenna fotosintetici di PSII sia nell’organizzazione di membrana che nel trasferimento dell’energia di eccitazione, utilizzando i mutanti knock out precedentemente isolati. Membrane tilacoidali wild-type e dei tre mutanti mancanti dei complessi CP24, CP26 o CP29, sono stati studiati con spettroscopia di fluorescenza rapida, utilizzando combinazioni differenti di lunghezze d'onda di eccitazione e di detection, al fine di separare le cinetiche del PSI e PSII. Tali misurazioni spettroscopiche hanno rivelato che la mancanza di CP26 non ha modificato l'organizzazione del PSII. Al contrario, l'assenza di CP29 e soprattutto di CP24 porta a cambiamenti sostanziali dell'organizzazione del PSII come evidenziato da un aumento significativo del tempo di migrazione apparente, dimostrando una cattiva connessione tra una parte significativa dell’antenna periferica e i RC. Sezione D.
In eukaryotes the photosynthetic antenna system is composed by subunits encoded by the light harvesting complex (Lhc) multigene family. These proteins play a key role in photosynthesis and are involved in both light harvesting and photoprotection. In particular, antenna protein of PSII, the Lhcb subunits, have been proposed to be involved in the mechanism of thermal dissipation of excitation energy in excess (NPQ, non-photochemical quenching). Elucidating the molecular details of NPQ induction in higher plants has proven to be a major challenge. In my phD work, I decided to investigate the role of Lhcbs in energy quenching by using a reverse genetic approach: I knocked out each subunit in order to understand their involvement in the mechanism. Here below the major results obtained are summarized. Section A. Mutants of monomeric Lhc and photoprotection: insights on the role of minor subunits in thermal energy dissipation. In this section I investigate the function of chlorophyll a/b binding antenna proteins, CP26, CP24 and CP29 in light harvesting and regulation of photosynthesis by isolating Arabidopsis thaliana knockout (ko) lines that completely lacked one or two of these proteins. In particular in Section A.1 I focused on single mutant koCP24, koCP26 and double mutant koCP24/26. All these three mutant lines have a decreased efficiency of energy transfer from trimeric light-harvesting complex II (LHCII) to the reaction center of photosystem II (PSII) due to the physical disconnection of LHCII from PSII. We observed that photosynthetic electron transport is affected in koCP24 plants but not in plants lacking CP26: the former mutant has decreased electron transport rates, a lower pH gradient across the grana membranes, a reduced capacity for non-photochemical quenching, and a limited growth. Furthermore, the PSII particles of these plants are organized in unusual two-dimensional arrays in the grana membranes. Surprisingly, the double mutant koCP24/26, lacking both CP24 and CP26 subunits, restores overall electron transport, non-photochemical quenching, and growth rate to wild type levels. We further analysed the koCP24 phenotype to understand the reasons for the photosynthetic defection. Fluorescence induction kinetics and electron transport measurements at selected steps of the photosynthetic chain suggested that koCP24 limitation in electron transport was due to restricted electron transport between QA and QB, which retards plastoquinone diffusion. We conclude that CP24 absence alters PSII organization and consequently limits plastoquinone diffusion. The limitation in plastoquinone diffusion is restore in koCP24/26. In Section A.2 I characterized the function of CP29 subunits, extending the analyses to the different CP29 isoforms. To this aim, I have constructed knock-out mutants lacking one or more Lhcb4 isoforms and analyzed their performance in photosynthesis and photoprotection. We found that lacks of CP29 did not result in any significant alteration in linear/cyclic electron transport rate and maximal extent of state transition, while PSII quantum efficiency and capacity for NPQ were affected. Photoprotection efficiency was lower in koCP29 plants with respect to either WT or mutants retaining a single Lhcb4 isoform. Interestingly, while deletion of either isoforms Lhcb4.1 or Lhcb4.2 get into a compensatory accumulation of the remaining subunit, photoprotection capacity in the double mutant Lhcb4.1/4.2 was not restored by Lhcb4.3 accumulation. Section B. Membrane dynamics and re-organization for the quenching events: B4 dissociation and identification of two distinct quenching sites. Antenna subunits are hypothesized to be the site of energy quenching, while the trigger of the mechanism is mediated by PsbS, a PSII subunit that is involved in detection of luminal acidification. In this section we investigate the molecular mechanism by which PsbS regulates light harvesting efficiency by studying Arabidopsis mutants specifically devoid of individual monomeric Lhcbs. In Section B.1 we showed that PsbS controls the association/dissociation of a five-subunit membrane complex, composed of two monomeric Lhcb proteins, CP29 and CP24 and the trimeric LHCII-M (namely Band 4 Complex - B4C). We demonstrated that the dissociation of this supercomplex is indispensable for the onset of non-photochemical fluorescence quenching in high light. Consistently, we found that knock-out mutants lacking the two subunits participating to the B4C, namely CP24 and CP29, are strongly affected in heat dissipation. Direct observation by electron microscopy showed that B4C dissociation leads to the redistribution of PSII within grana membranes. We interpret these results proposing that the dissociation of B4C makes quenching sites, possibly CP29 and CP24, available for the switch to an energy-quenching conformation. These changes are reversible and do not require protein synthesis/degradation, thus allowing for changes in PSII antenna size and adaptation to rapidly changing environmental conditions. In Section B.2 we studied this quenching mechanism by ultra-fast Chl fluorescence analysis. Recent results based on fluorescence lifetime analysis in vivo proposed that two independent quenching sites are activated during NPQ: Q1 is located in the major LHCII complexes, which are functionally detached from the PSII/RC (reaction centre) supercomplex with a mechanism that strictly requires PsbS but not Zea; Q2 is located in and connected to the PSII complex and is dependent on the Zea formation. These two quenching events could well originate in each of the two physical domains of grana revealed by electron microscopy analysis previously reported. We thus proceeded to investigate the modulation of energy quenching in knock out mutants by comparing the fluorescence lifetimes under quenched and unquenched conditions in intact leaves: we obtained results that are consistent with the model of two quenching sites located, respectively, in the C2S2 domain and in the LHCII-enriched domain. Data reported suggest that Q1 site is released in the koCP24 mutant while Q2 is attenuated in the koCP29 mutant. On the bases of the results of this section, we conclude that during the establishment of NPQ in vivo the PSII supercomplex dissociates into two moieties, which segregates into distinct domain of the grana membrane and are each protected from over-excitation by the activity of quenching sites probably located in CP24 and CP29. Section C. Excitation energy transfer and membrane organization: role of PSII antenna subunits. In this section we investigated the role of individual photosynthetic antenna complexes of PSII both in membrane organization and excitation energy transfer, by using the knock out mutants previously isolated. Thylakoid membranes from wild-type and three mutants lacking light harvesting complexes CP24, CP26 or CP29 respectively, were studied by ps-fluorescence spectroscopy on thylakoids, using different combination of excitation and detection wavelengths in order to separate PSI and PSII kinetics. Spectroscopic measurements revealed that absence of CP26 did not alter PSII organization. In contrast, the absence of CP29 and especially CP24 lead to substantial changes in the PSII organization as evidenced by a significant increase of the apparent migration time, demonstrating a bad connection between a significant part of the peripheral antenna and the RCs. Section D.

The purpose of this study was to establish whether curcumin protects renal proximal tubule cells against ischemic injury, determine whether this postulated cytoprotective effect is mediated through the upregulation of HSP70, and investigate whether the mechanism by which curcumin induces HSP70 expression and confers its protective effect is through activation of the Unfolded Protein Response. LLC-PK1 cells were cultured on collagen-coated filters to mimic conditions of in vivo renal proximal tubule cells and induce cell polarization. Injury with and without curcumin treatment was studied by using chemically-induced ATP-depletion which mimics renal ischemic injury. Cell injury was assessed using a TUNEL assay in order to evaluate DNA cleavage associated with ischemia-induced apoptosis and actin staining used to assess cytoskeletal disruption. Renal ischemic damage was further investigated by determining detachment of the Na-K ATPase from the basolateral membrane, which represents loss of cell polarity. Cells were incubated with curcumin in a dose- and time-response fashion and subsequent levels of HSP70 expression were assessed. Cells were then incubated with AEBSF, an inhibitor of the Unfolded Protein Response (UPR) and HSP70 and BiP/GRP78 (an ER resident chaperone that is upregulated by the UPR) expression levels were evaluated. Results demonstrated that treatment with curcumin during two hours of injury results in significantly less injury-related apoptosis and cytoskeletal disruption compared to control injured cells. It was demonstrated that curcumin induces HSP70 in both a dose- and time-response fashion. Moreover, curcumin treatment resulted in profound stabilization of Na-K ATPase on the basolateral membranes as there was significantly less Na-K ATPase detachment in cells treated with curcumin during two hours of injury compared to control injured cells. Finally, treatment with AEBSF inhibited HSP70 upregulation in curcumin-treated cells as well as inhibiting the GRP78 over-expression otherwise demonstrated in curcumin-treated cells. Protection of proximal tubule cells against renal ischemic injury by curcumin was therefore indicated to be mediated by the activation of the UPR through which HSP70 is upregulated. Curcumins activation of the UPR and induction of HSP70 explains the stabilization of Na-K ATPase on the cytoskeleton and also provides a potential mechanism explaining many of curcumins therapeutic and protective qualities.

Evolución dirigida de proteínas: La evolución dirigida es una técnica que nos permite explorar funciones enzimáticas que no son requeridas en el ambiente natural. Esta técnica, simula procesos genéticos naturales y de selección. Esta estrategia se utiliza cuando un diseño racional es muy complicado. Consiste en una repetición de ciclos de diversificación y selección que llevan a la acumulación de mutaciones benéficas. Aquí se presenta dos ejemplos de evolución dirigida con los cuales se ha trabajado directamente: la ADN polimerasa del organismo  Thermus aquaticus usada comúnmente en PCR, y la proteína LacI que regula la expresión de genes usados para el metabolismo de lactosa en E. Coli.
Directed evolution allows us to explore protein functionalities not required in the natural environment. It mimics natural genetic processes and selective pressures. This approach is used when the molecular basis is not completely understood and rational design is a difficult task. This approach consists of serial cycles of consecutive diversification and selection which eventually lead to the accumulation of beneficial mutations. Here are presented two cases where directed evolution is used to modify two different proteins: Taq polymerase, enzyme used for DNA extension in PCR, and the LacI repressor protein which regulates gene expression on E.coli.

Les diatomées constituent le principal groupe du phytoplancton dans les océans, contribuant à près de 20% de la production primaire globale. Dans leur environnement très variable, les diatomées sont particulièrement efficaces dans leur capacité à ajuster leur activité photosynthétique en dissipant sous forme de chaleur l’énergie lumineuse absorbée en excès, par un processus appelé le « Non-Photochemical Quenching of chlorophyll fluorescence », (NPQ). Chez la diatomée modèle, Phaeodactylum tricornutum, il a été montré que LHCX1, une protéine proche des antennes photosynthétiques, est impliquée dans le NPQ. Par des approches intrégrées de génétique, biologie moléculaire, biochimie, imagerie des cinétiques de fluorescence et spectroscopie ultrarapide, j’ai étudié le rôle de la famille des LHCX chez P. tricornutum. J’ai tout d’abord pu corréler une expression différentielle des 4 gènes LHCX de P. tricornutum avec différentes dynamiques de NPQ et activités photosynthétiques, dans différentes conditions de lumiére et nutriments. En localisant les LHCX dans les differents complexes photosynthétiques et les différents sites de dissipation d’énergie, j’ai pu proposer un modèle de régulation dynamique du NPQ impliquant à court terme principalement LHCX1 au niveau des centres réactionnels, et une autre isoforme, possiblement LHCX3, au niveau des antennes lors d’un stress lumineux prolongé. Enfin, par le criblage d’une série de mutants potentiellement dérégulés dans leur contenu en LHCXs, j’ai pu identifier des lignées avec un NPQ altéré qui pourront constituer des nouveaux outils de recherche. Dans l’ensemble ce travail de thèse a permis de mettre en évidence la diversification fonctionnelle et l’importance de la famille des LHCX dans la fine modulation des capacités de collecte de lumière et de photoprotection, expliquant sans doute en partie le succès des diatomées dans leur environnement très fluctuant
Diatoms dominate phytoplanktonic communities in contemporary oceans, contributing to 20% of global primary productivity. In their extremely variable environment, diatoms are especially efficient in adjusting their photosynthetic activity by dissipating as heat the light energy absorbed in excess, through a process called “Non-Photochemical Quenching of chlorophyll fluorescence”, (NPQ). In the model diatom Phaeodactylum tricornutum, it has been shown that LHCX1, a photosynthetic antenna-related gene, is involved in the NPQ process. Through integrated approaches of genetics, molecular biology, biochemistry, study of the kinetics of chlorophyll fluorescence yields and ultrafast spectroscopy, I studied the role of the LHCX family in the photoprotection activity of P. tricornutum. I first correlated a differential regulation of the 4 P. tricornutum LHCX genes with different dynamics of NPQ and photosynthetic activity, in different light and nutrient conditions. By localizing the LHCXs in fractioned photosynthetic complexes and the different sites of energy dissipation, I was able to propose a model of dynamic regulation of NPQ capacity involving mainly the LHCX1 in the reaction centers, during short-term high light responses. During prolonged high light stress, the quenching occurs mainly in the antennas, potentially mediated by the LHCX3 isoform. Finally, using photosynthetic parameters, I screened a series of transgenic lines putatively deregulated in their LHCX amount, and I identified lines with altered NPQ, which could represent novel investigation tools. Altogether, this work highlighted the functional diversification and the importance of the LHCX protein family in the fine-tuning of light harvesting and photoprotection capacity, possibly contributing to explain diatoms success in their highly fluctuating environment

In eukaryotischen Zellen sind die Lipidspezies häufig asymmetrisch zwischen den Hälften der Plasmamembran verteilt. Insbesondere Phosphatidylserin (PS) weist oft eine ausgeprägte transversale Asymmetrie auf, da es fast ausschliesslich auf die innere Hälfte der Plasmamembran beschränkt ist. In den letzten Jahren wurden mehrere Proteine diskutiert, die Lipide zwischen den Membranhälften transportieren und möglicherweise die transversale Lipidasymmetrie sowie damit verbundene Zelleigenschaften beeinflussen. Im Mittelpunkt der vorliegenden Promotion steht der Auswärtstransport fluoreszierender (C6-NBD-) Lipid-Analoga und endogener Lipide durch das Multidrug Resistance 1 P-Glycoprotein (MDR1 Pgp), das der ATP Binding Cassette (ABC) Transporter Superfamilie angehört. Interessanter Weise wird für MDR1 Pgp eine ungewöhnlich breite Substratspezifität angenommen. Das anionische Lipid PS war hier von besonderem Interesse, obgleich es in vorhergehenden Arbeiten nicht als MDR1 Pgp Substrat betrachtet wurde. Der Auswärtstransport von Phosphatidylcholin-, Phosphatidylethanolamin-, Glucosylceramid- und Sphingomyelin-Analoga durch MDR1 Pgp konnte in einer humanen Magenkarzinomlinie (EPG85-257), die MDR1 überexprimiert, mittels Fluoreszenzspektroskopie bestätigt werden. Zudem legt die verringerte Akkumulation von Diacylglycerol- und Ceramid-Analoga den Transport dieser Lipidspezies durch MDR1 Pgp nahe. Im Anschluß an die intrazelluläre Markierung mit C6-NBD-PS mittels eines neuen Verfahrens konnte der signifikant erhöhte Auswärtstransport dieses Analogons in MDR1 überexprimierenden Zellen durch Verwendung spezifischer Inhibitoren MDR1 Pgp zugeschrieben werden. In flusscytometrischen Versuchen war die Exponierung von endogenem PS auf der äusseren Membranhälfte von MDR1 überexprimierenden Zellen signifikant höher als in Kontrollzellen. Verringerung der PS-Exponierung durch einen Inhibitor von MDR1 Pgp deutet auf den Transport von endogenem PS durch MDR1 Pgp hin. Zusätzlich wurde hier der Transport von C6-NBD-PS in vier weiteren Zellinien mit verschiedener Spezies- und Gewebezugehörigkeit charakterisiert, die unterschiedliche Mengen an MDR1 Pgp synthetisieren. Wie Experimente in einer BCRP überexprimierenden EPG85-257-Sublinie nahelegen, ist ausser MDR1 Pgp möglicherweise ebenfalls der ABC Halb-Transporter Breast Cancer Resistance Protein (BCRP) am Transport von C6-NBD-PS und an der verstärkten Exponierung von endogenem PS beteiligt.
In eukaryotic cells, the lipid species are frequently distributed asymmetrically between the plasma membrane leaflets. Phosphatidylserine (PS), in particular, often exhibits a distinct transverse asymmetry, being restricted almost exclusively to the inner leaflet. In the past years, several proteins were suggested to transport lipids between the leaflets of a membrane, and to potentially influence transverse lipid asymmetry and related cell properties. This thesis focuses on outward transport of fluorescent (C6-NBD-) lipid analogs and endogenous lipids by the Multidrug Resistance 1 P-Glycoprotein (MDR1 Pgp), a member of the ATP binding cassette (ABC) transporter superfamily. Interestingly, MDR1 Pgp has been suggested to exhibit an unusually broad substrate specificity. Here, the anionic PS was of particular concern, although previously reported not to be an MDR1 Pgp substrate. In a human gastric carcinoma cell line (EPG85-257) overexpressing MDR1, outward transport of phosphatidylcholine, phosphatidylethanolamine, glucosylceramide and sphingomyelin analogs via MDR1 Pgp was confirmed using fluorescence spectroscopy. In addition, decreased accumulation of analogs of diacylglycerol and ceramide suggest MDR1 Pgp mediated transport of these lipid species. Upon intracellular labelling with C6-NBD-PS using a novel approach, significantly increased outward transport of this analog in MDR1 overexpressing cells could be attributed to MDR1 Pgp by employing specific inhibitors. In a flow cytometry setup, the exposure of endogenous PS on the outer plasma membrane leaflet was significantly elevated in MDR1 overexpressing cells compared to controls. Reduction of PS exposure by an MDR1 Pgp inhibitor suggests transport of endogenous PS by MDR1 Pgp. Transport of C6-NBD-PS was furthermore characterized here in four additional cell lines of different species and tissue origin with varying synthesis levels of MDR1 Pgp. Besides MDR1 Pgp, the ABC half-size transporter Breast Cancer Resistance Protein (BCRP) is possibly also involved in transport of C6-NBD-PS and in increased exposure of endogenous PS, as found in a BCRP overexpressing EPG85-257 subline.

Five target genes were selected in Toxoplasma gondii tachyzoites for localization studies. These five genes, detected through proteomics studies, included TgME49_227450, TgME49_223080, TgME49_262390, TgME49_230940, and TgME49_269620. Localization of these five target proteins is a first step to confirm their interaction with TgCrk2 and understand their function and role in TgCrk2 regulation of the tachyzoite cell cycle. Gene models for the targets were analyzed using ToxoDB and ApE analysis tools. Endogenous tagging constructs were created for each target. Transgenic parasites were created. Finally, localization analysis of the target proteins in tachyzoites was completed using immunofluorescent microscopy following. One protein was found to be nuclear, two were located in the cytoplasm, and two were unable to be analyzed. Future research should be completed in order to prove these putative interactors are really correlated with TgCrk2 through Co-immunoprecipitation.

During my PhD program I worked on two Nuclear Mitotic Apparatus (NuMA)-related projects. The first one centred on the biochemical and structural characterization of the NuMA-dynein mitotic interaction and was published last year on Structure. The second project focused on the study of the largely unknown role of NuMA in the nucleus during interphase. Regarding this part, I got interesting details on the NuMA-53BP1 (p53-binding protein 1) interaction in the context of liquid-liquid phase separation (LLPS). In multicellular organisms, the proper organization of the mitotic spindle is essential for accurate cell division, tissue development and homeostasis. In vertebrate cells, the protein NuMA is a master regulator of mitotic spindle functions, implicated in spindle assembly and orientation, working together with the high molecular weight dynein-dynactin microtubule-motor complex. The domain structure of NuMA consists of an N-terminal globular domain, a central extended coiled-coil, and an unstructured C-terminal cargo-binding region. Whether NuMA is a dynein-dynactin activating adaptor is still not known. On these premises, the first part of my PhD project focused on the characterization of the NuMA-dynein binding interface, which I performed in collaboration with other members of the group. The crystal structure of the N-terminal head of NuMA (NuMA_1-153) revealed that it folds into a hook domain, a conserved feature of the Hook-family dynein-dynactin adaptors interacting directly with the Light Intermediate Chain (LIC) subunit of dynein. Pulldown assays performed with purified proteins indicated a direct interaction between NuMA_1-705 and LIC and identified four conserved residues in the NuMA hook domain that are crucial for LIC binding. Interestingly, sequence alignment between NuMA and known CC1-box containing dynein-dynactin adaptors revealed the existence of a CC1-box-like motif in the NuMA N-terminal coiled-coil domain (NuMA_365-376) that we demonstrated to be also implicated in contacting LIC. Thus, our studies identified two sites on NuMA’ N-terminus required for the interaction with a conserved hydrophobic helix in LIC1 C-terminus. Spindle positioning assays in human HeLa cells showed that these newly identified dynein-binding interfaces of NuMA are essential for correct mitotic progression. Collectively, these results support the notion that NuMA acts as a mitotic dynein-dynactin adaptor, forming complexes with similar topology to what observed for other known hook and CC1-box containing adaptors. In vertebrate cells, NuMA accumulates in the nucleus during interphase and contributes to the DNA damage response (DDR), negatively regulating the 53BP1 double strand break (DSB) repair function. The second part of my PhD project focused on the characterization of the NuMA-53BP1 binding interface. By co-immunoprecipitation (co-IP) experiments in human HEK293T nuclear extracts with anti-NuMA antibodies, I confirmed that endogenous NuMA interacts with 53BP1, and that this interaction is decreased upon DNA damage induction. Interestingly, analytical size-exclusion chromatography (SEC) experiments with purified fragments revealed that the C-terminus of 53BP1 (53BP1_1484-1972) interacts directly with the C-terminus of NuMA (NuMA_1821-2115). These are two intrinsically disordered domains, common to proteins that undergo LLPS, a mechanism conferring spatial and temporal regulation to biological processes. Since 53BP1 forms DNA damage foci, which are LLPS condensates promoted by its C-terminal disordered region, I tested whether also NuMA is involved in this mechanism. Interestingly, I found that NuMA_1821-2115 forms liquid droplets in vitro at 20 uM and physiological salt concentrations, promoted by electrostatic and polar interactions. By co-IP experiments in HEK293T nuclear extracts, I also detected an interaction of NuMA with the MT nucleator TPX2. Since TPX2 counteracts the 53BP1 DSB repair function during replication stress and undergoes LLPS, I hypothesized that NuMA could work with TPX2 in regulating the DDR by forming dynamic LLPS condensates. Surprisingly, by co-IP experiments, an interaction between NuMA and 53BP1 was also scored during mitosis, where 53BP1 is known to be part of the centrosome surveillance pathway, another condensate-associated regulatory process. Further studies are required to uncover the molecular basis and the functional role of the NuMA interaction with 53BP1 both in the DDR and in the centrosome surveillance pathway.

RASSF1A is a tumor suppressor gene which loses its function due to methylation of CpG islands on its promoter region. Detection of methylation leads to early diagnosis of cancer. Zinc finger proteins are capable of detecting a specific DNA sequence and Methyl binding domain can bind to the methyl group on the CpG, using this idea mCpG SEER- Lac system makes use of a split protein, β-lactamase. Lac A attached to the ZFP and Lac B attached to the MBD protein. On binding to the DNA, the Lac A and Lac B come in close proximity with each other causing a reassembly and activation of the enzyme. In the presence of a substrate, the activated β-lactamse enzyme hydrolyzes the β-lactam bond in the substrate and shows a color change from yellow to red in the presence of a methylated cognate DNA. The study suggests that a solution based assay was not as specific in differentiating signal intensities between methylated and non-methylated DNA. It was also not sensitive in measuring dose dependent signals. Zinc finger array could successfully show relatively low signals for non-methylated DNA. The findings of the study show that MBD2 shows higher preference for mCpG than MBD1 in the mCpG SEER-Lac system and oligonucleotides with a 2 bp spacing between methylation and ZF target site shows higher signals than the 3 bp spacing. Due to it’s specificity and sensitivity, it serves as a potential diagnostic tool to detect cancer.

Ce projet vise à étudier, par une approche pluridisciplinaire, l'influence des la cristallisation des protéines membranaires (PM) en prenant pour protéine modèle le complexe photosynthétique RC-LH1-pufX de Rhodobacter blasticus. Des cristaux de ce complexe avaient été obtenus en présence de dodécyl-!-maltoside (DDM) et avaient diffractés à 8 Å de résolution. L'objectif final est de pouvoir améliorer, de façon rationnelle, la qualité des cristaux du complexe RC-LH1-pufX grâce à une meilleure compréhension des mécanismes mis en jeu. Dans un premier temps, trois tensioactifs dérivés du DDM ont été conçus et synthétisés. L'intérêt est d'augmenter la rigidité et le caractère lipophobe des parties hydrophobes des tensioactifs par rapport au DDM, pour les rendre moins déstabilisants envers la protéine: soit par l'incorporation d'un groupement bicyclohexyle (PCC-maltoside), soit par l'ajout d'un segment fluoré de longueur modulable (F4H5- et F2H9-maltoside). Nous avons inclus également le F8TAC, tensioactif fluoré utilisé depuis une vingtaine d'années pour le maintien en solution des PM, et les "tripodes", amphiphiles faciaux dont la géométrie particulière n'avaient jamais été testée. Nous avons ensuite réalisé la caractérisation physico-chimique, en solution, de ces tensioactifs et du DDM en terme de CMC (concentration micellaire critique), nombre d'agrégation, taille (par diffusion de la lumière dynamique, DLS), facteur de forme (par diffusion des rayons X aux petits angles, SAXS) et facteur de structure (par mesure du second coefficient du viriel, indicateur du potentiel des tensioactifs à initier la cristallisation)afin de déterminer les caractéristiques importantes au maintien en solution et à la cristallisation des PM. Le PCC-malt présentant le même comportement que le DDM,nous l'avons sélectionné pour réaliser une étude en présence de la protéine.Après avoir mis au point une méthode de dosage des tensioactifs par HPTLC (HighPerformance Thin Layer Chromatography) et identifier les lipides présents dans les de Rhodobacter blasticus, nous avons pu quantifier les quantités de lipides et de tensioactifs associés à la protéine en présence de DDM et de PCC-malt.Enfin, dans une dernière partie, nous avons réalisé des essais de cristallisation du complexe RC-LH1-pufX en présence des tensioactifs sélectionnés pour faire le lien entre les conditions de cristallisation et l'étude physico-chimique des micelles en solution.

Chronic Lymphocytic Leukemia is the primary lymphoid neoplasm in adults and and it is especially manifested in the elderly. Because it is a heterogeneous disease it awakens great interest regarding its prognosis. Rai and Binet developed staging systems to predict the evolution of the disease and currently, the analysis of expression of CD38 and Zap-70 has been investigated as a prognostic factor for indicating presence or absence of the mutation in the gene IgVH, so, the objective of this study was to analyze the clinical and laboratory profiles of patients with Chronic Lymphocytic Leukemia taking as reference the clinical staging of Rai and Binet and quantification of CD38 and Zap-70 expression as prognosis factors. We searched the medical records of 64 patients treated at University Hospital of Santa Maria and the variables considered were swollen lymph nodes, presence or absence of hepatomegaly and / or splenomegaly, hematological evaluation of peripheral blood and immunophenotype. The data obtained were correlated with the staging of Rai (1975) and Binet (1981), the expression of CD38 and Zap-70 and clinical stage. The results showed no association between ataging Rai and Binet and the expression of CD38, Zap-70 and Binet clinical staging.
A Leucemia Linfocítica Crônica é a principal neoplasia linfóide em adultos e se manifeta principalmente em indivíduos idosos. Por ser uma doença heterogênea, desperta grande interesse quanto ao seu prognóstico. Rai e Binet desenvolveram sistemas de estadiamento capazes de prever a evolução da doença e atualmente, a análise da expressão de CD38 e Zap- 70 tem sido investigada como fator prognóstico por indicar presença ou ausência da mutação no gene IgVH, assim, o objetivo deste estudo foi analisar o perfil clínico-laboratorial dos pacientes com Leucema Linfocítica Crônica, tomando como referência os estadiamentos clínicos de Rai e Binet e a quantificação da expressão de CD38 e Zap-70 como fatores prognóstico. Foram pesquisados 64 prontuários médicos de pacientes atendidos no Hospital Universitário de Santa Maria e as variáveis consideradas foram aumento de linfonodos, presença ou ausência de hepatomegalia e/ou esplenomegalia, avaliação hematológica de sangue periférico e imunofenótipo. Os dados obtidos foram correlacionados com o estadiamento de Rai (1975) e Binet (1981), a expressão de CD38 e Zap-70 com o estádio clínico de Binet. Os resultados demonstraram que não há associação entre o estadiamento de Raí e Binet e a expressão de CD38, Zap-70 com o estadiamento clínico de Binet.

Deux sondes moléculaires, DNA complémentaire de la petite sous-unité de ribulose-1,5. Bisphosphate carboxylase (PSU) et de la "light harvesting chlorophyll a/b binding proteins" (LHCP) ont été clonées. Leur utilisation a permis de mettre en évidence des modifications du génome reliées à l'état de différenciation et/ou de l'irradiation donnée. La transcription de ces deux polypeptides (PSU) et (LHCP) est régulée par le phytochrome chez le radis (raphanus sativus)

La luce è essenziale per la fotosintesi e la vita sulla terra e tuttavia può diventare dannosa per le piante. Quando i fotoni vengono assorbiti in eccesso, rispetto alla capacità del trasporto elettronico fotosintetico, si producono specie reattive dell'ossigeno che provocano fotoinibizione, limitando la crescita delle piante e la loro produttività. Gli organismi fotosintetici hanno sviluppato meccanismi di fotoprotezione per prevenire/evitare i danni foto-ossidativi. Tra questi, il Non-Photochemical Quencing o NPQ è di particolare interesse. Il meccanismo di NPQ smorza gli stai eccitati della clorofilla catalizzando la dissipazione, sottoforma di calore, dell’energia assorbita in eccesso. Nel corso degli ultimi decenni, molti sforzi sono stati fatti per chiarire i meccanismi alla base di questi processi. Oltre alla curiosità accademica, la manipolazione della dissipazione termica e la sua regolazione in risposta agli stimoli ambientali sembra essere la chiave per aumentare sia la resistenza allo stress sia la produttività di cibo e combustibili. Durante mio dottorato ho usato un approccio di genetica inversa, sull’organismo modello Arabidopsis thaliana, per dissezionare e caratterizzare il ruolo dei diversi componenti dei meccanismi di fotoprotezione, nonché il loro contributo all’acclimatazione agli stress abiotici. Di particolare interesse è stata la generazione e l'analisi di mutanti di diversi enzimi della via di biosintesi dei carotenoidi, di proteine antenna che legano i carotenoidi e di mutanti privi dellla capacità di muovere i cloroplasti all’interno della cellula per ridurre l’assorbimento luminoso.
Light is essential for photosynthesis and life on earth and yet it is harmful for plants. When photons are absorbed in excess with respect to the capacity of photosynthetic electron transport, reactive oxygen species are produced that causes photoinhibition, limiting plant growth and productivity. Oxygenic photosynthetic organisms have evolved photoprotective mechanisms to prevent/avoid photodamage. Among these, the Non-Photochemical Quenching (of chlorophyll fluorescence) or NPQ is of particular interest. NPQ has been reported to quench the chlorophyll excited states thus catalyzing the thermal dissipation of energy absorbed in excess. Over the past decades many efforts have been made to elucidate the mechanisms underlying these processes. Besides academic curiosity, manipulation of thermal dissipation rate and its regulation in response to environmental cues appears to be the key for both enhancing stress resistance and productivity for food and fuels. In my PhD I used a reverse genetic approach on the model organism Arabidopsis thaliana to disentangle and characterize the role of different components of photoprotective mechanisms as well as their contribution to acclimation to abiotic stresses. Of particular interest have been the generation and analysis of mutants defective in carotenoids biosynthesis, specific xanthophyll binding proteins and in the chloroplast light avoidance mechanism.

Lfc is a guanine nucleotide exchange factor (GEF) for RhoA and is negatively regulated by its association with the microtubule array. Tctex-1, a light chain subunit of the dynein motor complex, was identified as an Lfc-interacting protein in a yeast two-hybrid screen. In mouse embryonic fibroblast (MEF) cells, over-expression of Tctex-1 represses Lfc-induced actin stress fiber and focal adhesion complex formation. Here, we present biochemical evidence obtained from a real-time, nuclear magnetic resonance (NMR)-based assay indicating that the microtubule exerts its inhibitory effect on Lfc through a mechanism that is dependent on the presence of Tctex-1. We also present NMR structure data showing that Lfc and the dynein intermediate chain (DIC) bind to different surfaces of Tctex-1. The biochemical and structural data together support a model in which Lfc is recruited to the microtubules through the dynein cargo adaptor function of Tctex-1, resulting in inhibition of Lfc function.

In eukaryotes the photosynthetic antenna system is composed by subunits encoded by the light harvesting complex (Lhc) multigene family. These proteins play a key role in photosynthesis and are involved in both light harvesting and photoprotection. In particular, antenna protein of PSII, the Lhcb subunits, have been proposed to be involved in the mechanism of thermal dissipation of excitation energy in excess (NPQ, non-photochemical quenching). Elucidating the molecular details of NPQ induction in higher plants has proven to be a major challenge. In my phD work, I decided to investigate the role of Lhcbs in energy quenching by using a reverse genetic approach: I knocked out each subunit in order to understand their involvement in the mechanism. Here below the major results obtained are summarized. Section A. Mutants of monomeric Lhc and photoprotection: in- sights on the role of minor subunits in thermal energy dissipation. In this section I investigate the function of chlorophyll a/b binding antenna proteins, CP26, CP24 and CP29 in light harvesting and regulation of photosynthesis by isolating Arabidopsis thaliana knockout (ko) lines that completely lacked one or two of these proteins. In particular in Section A.1 I focused on single mutant koCP24, koCP26 and double mutant koCP24/26. All these three mutant lines have a decreased eciency of energy transfer from trimeric light-harvesting complex II (LHCII) to the reaction center of photosystem II (PSII) due to the physical disconnection of LHCII from PSII and formation of PSII reaction center depleted domains in grana partitions. We observed that photosynthetic electron transport is aected in koCP24 plants but not in plants lacking CP26: the former mutant has decreased electron transport rates, a lower pH gradient across the grana membranes, a reduced capacity for non-photochemical quenching, and a limited growth. Furthermore, the PSII particles of these plants are organized in unusual two-dimensional arrays in the grana membranes. Surprisingly, the double mutant koCP24/26, lacking both CP24 and CP26 subunits, restores overall electron transport, non-photochemical quenching, and growth rate to wild type levels. We further analysed the koCP24 phenotype to understand the reasons for the photosynthetic defection. Fluorescence induction kinetics and electron transport measurements at selected steps of the photosynthetic chain suggested that koCP24 limitation in electron transport was due to restricted electron transport between QA and QB, which retards plastoquinone diusion. We conclude that CP24 absence alters PSII organization and consequently limits plastoquinone diffusion. The limitation in plastoquinone diusion is restore in koCP24/26. In Section A.2 I characterized the function of CP29 subunits, extending the analyses to the dierent CP29 isoforms. To this aim, I have constructed knock-out mutants lacking one or more Lhcb4 isoforms and analyzed their performance in photosynthesis and photoprotection. We found that lacks of CP29 did not result in any signicant alteration in linear/cyclic electron transport rate and maximal extent of state transition, while PSII quantum eciency and capacity for NPQ were aected. Photoprotection eciency was lower in koCP29 plants with respect to either WT or mutants retaining a single Lhcb4 isoform. Interestingly, while deletion of either isoforms Lhcb4.1 or Lhcb4.2 get into a compensatory accumulation of the remaining subunit, photoprotection capacity in the double mutant Lhcb4.1/4.2 was not restored by Lhcb4.3 accumulation. Section B. Membrane dynamics and re-organization for the quench- ing events: B4 dissociation and identication of two distinct quenching sites. Antenna subunits are hypothesized to be the site of energy quenching, while the trigger of the mechanism is mediated by PsbS, a PSII subunit that is involved in detection of lumenal acidication. In this section we investigate the molecular mechanism by which PsbS regulates light harvesting eciency by studying Arabidopsis mutants specically devoid of individual monomeric Lhcbs. In Section B.1 we showed that PsbS controls the association/dissociation of a ve-subunit membrane complex, composed of two monomeric Lhcb proteins, CP29 and CP24 and the trimeric LHCII-M (namely Band 4 Complex - B4C). We demonstrated that the dissociation of this supercomplex is indispensable for the onset of non-photochemical uorescence quenching in high light. Consistently, we found that knock-out mutants lacking the two subunits participating to the B4C, namely CP24 and CP29, are strongly aected in heat dissipation. Direct observation by electron microscopy and image analysis showed that B4C dissociation leads to the redistribution of PSII within grana membranes. We interpret these results proposing that the dissociation of B4C makes quenching sites, possibly CP29 and CP24, available for the switch to an energy-quenching conformation. These changes are reversible and do not require protein synthesis/degradation, thus allowing for changes in PSII antenna size and adaptation to rapidly changing environmental conditions. In Section B.2 we studied this quenching mechanism by ultra-fast Chl uorescence analysis. Recent results based on uorescence lifetime analysis \in vivo" (Holzwarth et al., 2009) proposed that two independent quenching sites are activated during NPQ: Q1 is located in the major LHCII complexes, which are functionally detached from the PSII/RC (reaction centre) supercomplex with a mechanism that strictly requires PsbS but not Zea; Q2 is located in and connected to the PSII complex and is dependent on the Zea formation (Miloslavina et al., 2008). These two quenching events could well originate in each of the two physical domains of grana revealed by electron microscopy analysis previously reported (see Section B.1). We thus proceeded to investigate the modulation of energy quenching in knock out mutants by comparing the uorescence lifetimes under quenched and unquenched conditions in intact leaves: we obtained results that are consistent with the model of two quenching sites located, respectively, in the C2S2 domain and in the LHCII-enriched domain. Data reported suggest that Q1 site is released in the koCP24 mutant while Q2 is attenuated in the koCP29 mutant. On the bases of the results of this section, we conclude that during the establishment of NPQ in vivo the PSII supercomplex dissociates into two moieties, which segregates into distinct domain of the grana membrane and are each protected from over-excitation by the activity of quenching sites probably located in CP24 and CP29. Section C. Excitation energy transfer and membrane organiza- tion: role of PSII antenna subunit. In this section we investigated the role of individual photosynthetic antenna complexes of PSII both in membrane organization and excitation energy transfer, by using the knock out mutants previously isolated (Section A). Thylakoid membranes from wild-type and three mutants lacking lightharvesting complexes CP24, CP26 or CP29 respectively, were studied by picosecond- uorescence spectroscopy on thylakoids, using dierent combination of excitation and detection wavelengths in order to separate PSI and PSII kinetics. Spectroscopic measurements revealed that absence of CP26 did not alter PSII organization, as evidenced by very similar excitons migration times in both wild-type and koCP26 samples. In contrast, the absence of CP29 and especially CP24 lead to substantial changes in the PSII organization as evidenced by a signicant increase of the apparent migration time, demonstrating a bad connection between a signicant part of the peripheral antenna and the RCs. Section D. A mutant without minor antenna proteins: towards a solution of the dierential role of monomeric Lhcb proteins vs major LHCII in Non Photochemical Quenching. In this section we attempt to answer to the question on the localization of the quenching site(s) in the major LHCII or in monomeric Lhcb proteins. In order to verify the implication of monomeric antenna CP24, CP26 and CP29 in quenching we tried to isolate a mutant knocked out for all the three subunit. Lhcb5 (CP26) and Lhcb6 (CP24) are coded by a single gene while three isoforms of Lhcb4 (CP29) are present in the Arabidopsis genome, we thus looked for a ve-gene mutant. We tried to isolate such a mutant from the screening of two dierent populations but without success. In particular, we found that is not possible to obtain a mutant lacking both CP26 and CP29 at the same time. We veried that is not a problem of seed germination but instead a defect of the embryo development. This unexpected genetic evidence shows that, for a correct and functional organization of the PSII supercomplexes, either Lhcb5 or Lhcb4.1 must be present: probably these two subunits can replace themselves each other when one lacks, while none of the other monomeric antenna (Lhcb6, Lhcb4.2, Lhcb4.3) are able to takes their place in PSII. Two mutants obtained in these screenings allowed us to make some considerations about the role of monomeric antenna proteins in NPQ: mutants koCP29/24 which dier for the condition at the Lhcb5 locus, either homozygous WT or heterozygous. We observed a dose eect for the expression of Lhcb5, that is lower in the heterozygous since one allele only is functional. Interestingly, the amount of CP26 on thylakoid membrane strictly correlated with the quenching amplitude measured at 1200 mol photons m��2s��1 of actinic light. These results suggest that the residual quenching in koCP29/24 plants is largely due to the capacity of CP26 subunit to mediate energy quenching. This is a further indication of the primary role of monomeric Lhcb in NPQ, as recently stated by CT (charge transfer) quenching models (Avenson et al., 2008; Ahn et al., 2008).
In eukaryotes the photosynthetic antenna system is composed by subunits encoded by the light harvesting complex (Lhc) multigene family. These proteins play a key role in photosynthesis and are involved in both light harvesting and photoprotection. In particular, antenna protein of PSII, the Lhcb subunits, have been proposed to be involved in the mechanism of thermal dissipation of excitation energy in excess (NPQ, non-photochemical quenching). Elucidating the molecular details of NPQ induction in higher plants has proven to be a major challenge. In my phD work, I decided to investigate the role of Lhcbs in energy quenching by using a reverse genetic approach: I knocked out each subunit in order to understand their involvement in the mechanism. Here below the major results obtained are summarized. Section A. Mutants of monomeric Lhc and photoprotection: in- sights on the role of minor subunits in thermal energy dissipation. In this section I investigate the function of chlorophyll a/b binding antenna proteins, CP26, CP24 and CP29 in light harvesting and regulation of photosynthesis by isolating Arabidopsis thaliana knockout (ko) lines that completely lacked one or two of these proteins. In particular in Section A.1 I focused on single mutant koCP24, koCP26 and double mutant koCP24/26. All these three mutant lines have a decreased eciency of energy transfer from trimeric light-harvesting complex II (LHCII) to the reaction center of photosystem II (PSII) due to the physical disconnection of LHCII from PSII and formation of PSII reaction center depleted domains in grana partitions. We observed that photosynthetic electron transport is aected in koCP24 plants but not in plants lacking CP26: the former mutant has decreased electron transport rates, a lower pH gradient across the grana membranes, a reduced capacity for non-photochemical quenching, and a limited growth. Furthermore, the PSII particles of these plants are organized in unusual two-dimensional arrays in the grana membranes. Surprisingly, the double mutant koCP24/26, lacking both CP24 and CP26 subunits, restores overall electron transport, non-photochemical quenching, and growth rate to wild type levels. We further analysed the koCP24 phenotype to understand the reasons for the photosynthetic defection. Fluorescence induction kinetics and electron transport measurements at selected steps of the photosynthetic chain suggested that koCP24 limitation in electron transport was due to restricted electron transport between QA and QB, which retards plastoquinone diusion. We conclude that CP24 absence alters PSII organization and consequently limits plastoquinone diffusion. The limitation in plastoquinone diusion is restore in koCP24/26. In Section A.2 I characterized the function of CP29 subunits, extending the analyses to the dierent CP29 isoforms. To this aim, I have constructed knock-out mutants lacking one or more Lhcb4 isoforms and analyzed their performance in photosynthesis and photoprotection. We found that lacks of CP29 did not result in any signicant alteration in linear/cyclic electron transport rate and maximal extent of state transition, while PSII quantum eciency and capacity for NPQ were aected. Photoprotection eciency was lower in koCP29 plants with respect to either WT or mutants retaining a single Lhcb4 isoform. Interestingly, while deletion of either isoforms Lhcb4.1 or Lhcb4.2 get into a compensatory accumulation of the remaining subunit, photoprotection capacity in the double mutant Lhcb4.1/4.2 was not restored by Lhcb4.3 accumulation. Section B. Membrane dynamics and re-organization for the quench- ing events: B4 dissociation and identication of two distinct quenching sites. Antenna subunits are hypothesized to be the site of energy quenching, while the trigger of the mechanism is mediated by PsbS, a PSII subunit that is involved in detection of lumenal acidication. In this section we investigate the molecular mechanism by which PsbS regulates light harvesting eciency by studying Arabidopsis mutants specically devoid of individual monomeric Lhcbs. In Section B.1 we showed that PsbS controls the association/dissociation of a ve-subunit membrane complex, composed of two monomeric Lhcb proteins, CP29 and CP24 and the trimeric LHCII-M (namely Band 4 Complex - B4C). We demonstrated that the dissociation of this supercomplex is indispensable for the onset of non-photochemical uorescence quenching in high light. Consistently, we found that knock-out mutants lacking the two subunits participating to the B4C, namely CP24 and CP29, are strongly aected in heat dissipation. Direct observation by electron microscopy and image analysis showed that B4C dissociation leads to the redistribution of PSII within grana membranes. We interpret these results proposing that the dissociation of B4C makes quenching sites, possibly CP29 and CP24, available for the switch to an energy-quenching conformation. These changes are reversible and do not require protein synthesis/degradation, thus allowing for changes in PSII antenna size and adaptation to rapidly changing environmental conditions. In Section B.2 we studied this quenching mechanism by ultra-fast Chl uorescence analysis. Recent results based on uorescence lifetime analysis \in vivo" (Holzwarth et al., 2009) proposed that two independent quenching sites are activated during NPQ: Q1 is located in the major LHCII complexes, which are functionally detached from the PSII/RC (reaction centre) supercomplex with a mechanism that strictly requires PsbS but not Zea; Q2 is located in and connected to the PSII complex and is dependent on the Zea formation (Miloslavina et al., 2008). These two quenching events could well originate in each of the two physical domains of grana revealed by electron microscopy analysis previously reported (see Section B.1). We thus proceeded to investigate the modulation of energy quenching in knock out mutants by comparing the uorescence lifetimes under quenched and unquenched conditions in intact leaves: we obtained results that are consistent with the model of two quenching sites located, respectively, in the C2S2 domain and in the LHCII-enriched domain. Data reported suggest that Q1 site is released in the koCP24 mutant while Q2 is attenuated in the koCP29 mutant. On the bases of the results of this section, we conclude that during the establishment of NPQ in vivo the PSII supercomplex dissociates into two moieties, which segregates into distinct domain of the grana membrane and are each protected from over-excitation by the activity of quenching sites probably located in CP24 and CP29. Section C. Excitation energy transfer and membrane organiza- tion: role of PSII antenna subunit. In this section we investigated the role of individual photosynthetic antenna complexes of PSII both in membrane organization and excitation energy transfer, by using the knock out mutants previously isolated (Section A). Thylakoid membranes from wild-type and three mutants lacking lightharvesting complexes CP24, CP26 or CP29 respectively, were studied by picosecond- uorescence spectroscopy on thylakoids, using dierent combination of excitation and detection wavelengths in order to separate PSI and PSII kinetics. Spectroscopic measurements revealed that absence of CP26 did not alter PSII organization, as evidenced by very similar excitons migration times in both wild-type and koCP26 samples. In contrast, the absence of CP29 and especially CP24 lead to substantial changes in the PSII organization as evidenced by a signicant increase of the apparent migration time, demonstrating a bad connection between a signicant part of the peripheral antenna and the RCs. Section D. A mutant without minor antenna proteins: towards a solution of the dierential role of monomeric Lhcb proteins vs major LHCII in Non Photochemical Quenching. In this section we attempt to answer to the question on the localization of the quenching site(s) in the major LHCII or in monomeric Lhcb proteins. In order to verify the implication of monomeric antenna CP24, CP26 and CP29 in quenching we tried to isolate a mutant knocked out for all the three subunit. Lhcb5 (CP26) and Lhcb6 (CP24) are coded by a single gene while three isoforms of Lhcb4 (CP29) are present in the Arabidopsis genome, we thus looked for a ve-gene mutant. We tried to isolate such a mutant from the screening of two dierent populations but without success. In particular, we found that is not possible to obtain a mutant lacking both CP26 and CP29 at the same time. We veried that is not a problem of seed germination but instead a defect of the embryo development. This unexpected genetic evidence shows that, for a correct and functional organization of the PSII supercomplexes, either Lhcb5 or Lhcb4.1 must be present: probably these two subunits can replace themselves each other when one lacks, while none of the other monomeric antenna (Lhcb6, Lhcb4.2, Lhcb4.3) are able to takes their place in PSII. Two mutants obtained in these screenings allowed us to make some considerations about the role of monomeric antenna proteins in NPQ: mutants koCP29/24 which dier for the condition at the Lhcb5 locus, either homozygous WT or heterozygous. We observed a dose eect for the expression of Lhcb5, that is lower in the heterozygous since one allele only is functional. Interestingly, the amount of CP26 on thylakoid membrane strictly correlated with the quenching amplitude measured at 1200 mol photons m��2s��1 of actinic light. These results suggest that the residual quenching in koCP29/24 plants is largely due to the capacity of CP26 subunit to mediate energy quenching. This is a further indication of the primary role of monomeric Lhcb in NPQ, as recently stated by CT (charge transfer) quenching models (Avenson et al., 2008; Ahn et al., 2008).

碩士
國立臺灣海洋大學
食品科學系
107
This study examined the possibility of Trametes versicolor LH1 to reduce the crude fiber content of sorghum distillery residue (SDR) and the feasibility of replacing 20% fish meal in carnivorous fish diet by SDR fermented with T. versicolor LH1 (f-SDR). Submerged fermentation (SmF) and solid-state fermentation (SSF) of T. versicolor LH1 on SDR at 25oC showed a logarithmic growth phase during the first 7 days, followed by slow growth. The mycelium biomass and phytosterol content were the highest after 20 days of SmF fermentation. T. versicolor LH1 had high cellulolytic enzymes activities in SmF being higher than in SSF. SDR cultivated by SmF for 7 days, the crude fiber content reduced from 11.95% to 5.11%, which was lower than that by SSF for 28 days (6.17%). The optimal fermentation to obtain low-fiber f-SDR was obtained by SmF for 7 days. The bioactive compounds including total polyphenols (3093.22 g GAE/g), total flavonoids (122.22 g QE/g dw) and triterpenoids (31.18 g UAE/g dw) were obtained. The antinutritional factors of f-SDR including, phytic acid (130.09 mg /g dw), tannin (0.53 mg CAE/g dw) and trypsin inhibitor (2.27 mg/g dw) still remained. Five dietary feeds were separated into, 20% soybean meal fermented products (f-SBM) as control, 10% f-SDR, 15% f-SDR, 20 % f-SDR and 20% SDR fed grouper (Epinephelus coioides) for 8 weeks. At the end of feeding trail, there was no significant difference in palatability indicated by consuming time, feed utilization rate and specific growth rate between those groupers fed 20% f-SBM and 15% f-SDR groups. Therefore, 15% f-SDR was the optimal ratio of f-SDR to substitute for fish meal. Cold stress was performed at 15oC for 4 h on grouper previously fed diet containing 20% f-SDR showed higher hematocrit, red blood cell counts, hemoglobin concentrations, oxyhemoglobin and higher dissolved oxygen in blood than those fed 20% SDR or 20% f-SBM. The concentration of cortisol and glucose in plasma of the 20% f-SDR group was lower than those fed f-SBM group or SDR. Overall, the crude fiber content of SDR was significantly reduced by SmF for 7 days. Moreover, the reduction of crude fiber content improved the feed palatability and the growth rate, enhance anti-cold stress tolerance of grouper. These findings indicated that low-fiber f-SDR can be developed into functional feed for grouper or other carnivorous fish species.

abstract: Molecular dynamics (MD) simulations provide a particularly useful approach to understanding conformational change in biomolecular systems. MD simulations provide an atomistic, physics-based description of the motions accessible to biomolecular systems on the pico- to micro-second timescale, yielding important insight into the free energy of the system, the dynamical stability of contacts and the role of correlated motions in directing the motions of the system. In this thesis, I use molecular dynamics simulations to provide molecular mechanisms that rationalize structural, thermodynamic, and mutation data on the interactions between the lac repressor headpiece and its O1 operator DNA as well as the ERK2 protein kinase. I performed molecular dynamics simulations of the lac repressor headpiece - O1 operator complex at the natural angle as well as at under- and overbent angles to assess the factors that determine the natural DNA bending angle. I find both energetic and entropic factors contribute to recognition of the natural angle. At the natural angle the energy of the system is minimized by optimization of protein-DNA contacts and the entropy of the system is maximized by release of water from the protein-DNA interface and decorrelation of protein motions. To identify the mechanism by which mutations lead to auto-activation of ERK2, I performed a series of molecular dynamics simulations of ERK1/2 in various stages of activation as well as the constitutively active Q103A, I84A, L73P and R65S ERK2 mutants. My simulations indicate the importance of domain closure for auto-activation and activity regulation. My results enable me to predict two loss-of-function mutants of ERK2, G83A and Q64C, that have been confirmed in experiments by collaborators. One of the powerful capabilities of MD simulations in biochemistry is the ability to find low free energy pathways that connect and explain disparate structural data on biomolecular systems. An extention of the targeted molecular dynamics technique using constraints on internal coordinates will be presented and evaluated. The method gives good results for the alanine dipeptide, but breaks down when applied to study conformational changes in GroEL and adenylate kinase.
Dissertation/Thesis
Ph.D. Chemistry 2011

碩士
國立臺灣大學
解剖學暨細胞生物學研究所
102
Previous studies have shown that Galanin modulated peripheral pain sensation via galanin receptor type 2 (GalR2). Following nerve injury, inflammation, spontaneous discharge and upregulation of pain related factors would involve in neuropathic pain development. To our knowledge, the correlation between median nerve demyelination and GalR2 and its substrate expression levels has not been documented; and yet the effect of GalR2 on medain neuropathic pain is not valid. Thus, using LPC treated median nerve injury model, we investigate the role of GalR2 and its pain corelated factors in the upper limb neuropathic pain. One week after LPC treatment of median nerve induced mechnical allodynia and thermal hyperalgesia. Immunohistochemistry analysis showed that GalR2-like immunoreactive (-LI) neurons were predominately in small-size DRG neurons of normal rats. However, one week after LPC treatment, GalR2-LI neurons not only increased in its percentage but also distributed in medium- and large-sized neurons. Moreover, to characterize GalR2-LI neurons in the DRG was using immunofluorescence double labeling for NF200, peripherin, pain-related factors including vanilloid receptor subtype 1 (VR1), P2X3, NPY, nNOS, Galanin, or MMP9. We found that the number and percentage of GalR2-LI neurons colocalized with NF200, P2X3, NPY, nNOS, Galanin and MMP9 were increased in the LPC-treated DRG. Furthermore, lidocaine pretreatment attenuated the number of upregulated GalR2-LI neurons in the LPC-treated DRG. Our study also found that one week afterLPC treatment, the number of GalR2-LI neurons in the cuneate nucleus of LPC treated rats was higer than that in the control group. The present results suggest that lidocaine pretreatment relieved the development of neuropathic pain partially pass through reducing GalR2 expression.

碩士
輔英科技大學
醫事技術系碩士班
94
Nickel (Ni), cadmium (Cd) and arsenic (As) are important environmental and industrial contaminants, which will produce varieties of effects on the renal system. These metals are recognized as carcinogen, however, the mechanisms remain unclear. In this study, we investigated the effects of Ni, Cd and As on oxidative stress, expression of apoptic proteins and Ca2+ influx in LLC-PK1 cells. LLC-PK1 cells were treated with nickel (II) acetate (Ni(CH3COO)2) (0-500μM), cadmium chloride (CdCl2 ) (0-8μM) and sodium arsenic (NaAsO2 ) (0-8μM) and then observed their later varcation. The oxidative stress was evaluated by lipid peroxidation and intracellular oxidants. After Ni, Cd or As treatment resulted in the decreasing of the viability, and the increasing of intracellular oxidants and lipid peroxidation. The expression of Bax, Bad, Bcl-x were increased, while Bcl-2 were decreased in Ni-treated LLC-Pk1 cells. In Cd or As treated, the expression of NF-κB, Bcl2 and Bad were increased, while it makes have not significant difference on Fas-L and Bax.As we treat LLP-PK1 cells by ways of Cd or As, we could saw the varcation of NF-κB by comparison of the expression of nuclear for 6hr. The result demonstrates significant expression with the increasing of time treated by any of the three elements. On the other hand, the intracellular Ca2+ levels ([Ca2+]i) in Ni-, Cd- and As-treated LLC-PK1 cells were explored. [Ca2+]i were measured by using the Ca2+-sensitive dye fura-2. However,in the treatment of Ni or As the Ca2+-contaning buffer of which wrer increased in [Ca2+]i , on the contrary, it was found the inhibit of [Ca2+]i in Ca2+-free buffer. With the treatment of Cd, it increased either in Ca2+-contaning buffer or Ca2+-free buffer. The results may indicate that Ni or As resulted inhibitor, while Cd play as enhancer of calcium entry pathway. The oxidative stress and intracellular Ca2+ may play important role in Ni-, Cd- or As-induced renal molecule cytotoxicity.

Thesis (Ph. D.)--University of Georgia, 2002.
Directed by Raghubir P. Sharma. Includes articles submitted to Journal of biological chemistry, Biochemistry, Toxicological Sciences, and Toxicology. Includes bibliographical references.

As the classical model for negative transcriptional control in prokaryotes and the subject of concentrated experimental attention, the lactose operon of Escherichia coli presents a well-defined system for studying genetic control through protein-DNA binding interactions. Binding of repressor at its cognate operator sequence within the regulatory region of the operon, while responsive to environmental conditions, efficiently inhibits transcription initiation by RNA polymerase. The high binding affinity and degree of specificity exhibited by this protein-DNA complex has encouraged investigation of the nature of the contacts formed. We have explored specific contacts between the lac repressor and operator using 5-bromodeoxyuridine-substituted DNA. Substitution of BrdU for single thymidine positions in a synthetic 40 bp operator provides an indirect means of probing the major groove of operator DNA for critical contacts between the repressors and the 5-methyl of individual thymidines. As a photoreactive species, BrdU provides substrate for ultraviolet irradiation. Upon irradiation, strand scission occurs at the BrdU residues. When bound, lac repressor protein provides protection against UV-induced breakage depending on the nature of the sites and type of interaction. We have confirmed thirteen unique sites of inducer-sensitive protection along the operator sequence (+1, 2, 3, 4, 6, 8, 13, 15, 16, 18, 19, 20, 21) using this method compared to per-substitution with BrdU (Ogata and Gilbert, 1977). The ability of these photosensitive DNAs to form short-range cross-links to bound protein has been used to determine the efficiency with which cross-linked protein-DNA complexes are generated at each individual site of BrdU substitution. Five sites of high efficiency cross-linking to the repressor protein have been identified (+3, 4, 14, 18, 19). Comparison of the UV protection results and the cross-linking data shows that these processes provide complementary tools for identifying and analyzing individual protein-DNA contacts. Using these same BrdU-substituted operator DNAs, we attempted to define individual protein-DNA interactions with respect to the specific amino acid(s) making contact at a selected site within the operator sequence. With the selection of the T$\sb{+3}$ site for our initial investigation, the cross-linked complex was formed and isolated. These polypeptide-DNA species were prepared for final analysis through a series of steps including proteolysis and anion-exchange HPLC. Protein sequence analysis on the purified peptide-operator complex identified a peptide spanning Val23 through Lys33. The data suggest His29 as the specifically crosslinked amino acid.