Добірка наукової літератури з теми "Lhc protein"

A collection of 17 monoclonal antibodies elicited against the light-harvesting chlorophyll a/b protein complex which serves photosystem II (LHC-II) of Pisum sativum shows six classes of binding specificity. Antibodies of two of the classes recognize a single polypeptide (the 28- or the 26- kD polypeptides), thereby suggesting that the two proteins are not derived from a common precursor. Other classes of antibodies cross-react with several polypeptides of LHC-II or with polypeptides of both LHC-II and the light-harvesting chlorophyll a/b polypeptides of photosystem I (LHC-I), indicating that there are structural similarities among the polypeptides of LHC-II and LHC-I. The evidence for protein processing by which the 26-, 25.5-, and 24.5-kD polypeptides are derived from a common precursor polypeptide is discussed. Binding studies using antibodies specific for individual LHC-II polypeptides were used to quantify the number of antigenic polypeptides in the thylakoid membrane. 27 copies of the 26-kD polypeptide and two copies of the 28-kD polypeptide were found per 400 chlorophylls. In the chlorina f2 mutant of barley, and in intermittent light-treated barley seedlings, the amount of the 26-kD polypeptide in the thylakoid membranes was greatly reduced, while the amount of 28-kD polypeptide was apparently not affected. We propose that stable insertion and assembly of the 28-kD polypeptide, unlike the 26-kD polypeptide, is not regulated by the presence of chlorophyll b.

AbstractLHC (light-harvesting complex) proteins of plants and algae are known to be involved both in collecting light energy for driving the primary photochemical reactions of photosynthesis and in photoprotection when the absorbed light energy exceeds the capacity of the photosynthetic apparatus. These proteins usually contain three transmembrane (TM) helices which span the thylakoid membranes and bind several chlorophyll, carotenoid and lipid molecules. In addition, the LHC protein family includes LHC-like proteins containing one, two, three or even four TM domains. One-helix proteins are not only present in eukaryotic photosynthetic organisms but also in cyanobacteria where they have been named high light-inducible proteins. These small proteins are probably the ancestors of the members of the extant LHC protein family which arouse through gene duplications, deletions and fusions. During evolution, some of these proteins have diverged and acquired novel functions. In most cases, LHC-like proteins are induced in response to various stress conditions including high light, high salinity, elevated temperature and nutrient limitation. Many of these proteins play key roles in photoprotection, notably in non-photochemical quenching of absorbed light energy. Moreover, some of these proteins appear to be involved in the regulation of chlorophyll synthesis and in the assembly and repair of Photosystem II and also of Photosystem I possibly by mediating the insertion of newly synthesized pigments into the photosynthetic reaction centers.

Abstract The light-harvesting chlorophyll a/b-protein complex of photosystem II (LHC II) was isolated from carnation (Dianthus caryophyllus L.) leaves by K+-induced aggregation of n-hep-tylthioglucoside-treated photosystem II particles. When solubilized with a mixture of lithium docedyl sulphate, octyl-β-D-glucopyranoside and dodecyl-β-D-maltoside the LHC II was re­ solved by mild sodium dodecyl sulphate-polyacrylamide gel electrophoresis into four oligo­meric forms and a monomeric one. LHC II contained five major polypeptides only two of which (27 and 26 kDa) were found to be its authentic components. The oligomeric forms of LHC II were found to differ in the stoichiometric ratios of the polypeptides present. The 26 kD a polypeptide was enriched in the largest oligomeric forms while the 27 kDa polypep­tide tended to form a monomer or to assemble as lower oligomeric states of LHC II.

This paper addresses the question of whether the PsbS protein of photosystem two (PS II) is located within the LHC II–PS II supercomplex for which a three–dimensional structure has been obtained by cryoelectron microscopy and single particle analysis. The PsbS protein has recently been implicated as the site for non–photochemical quenching. Based both on immunoblotting analyses and structural considerations of an improved model of the spinach LHC II–PS II supercomplex, we conclude that the PsbS protein is not located within the supercomplex. Analyses of other fractions resulting from the solubilization of PS II–enriched membranes derived from spinach suggest that the PsbS protein is located in the LHC II–rich regions that interconnect the supercomplex within the membrane.

Chlorophyll a/b light-harvesting complexes (chl a/b LHC) and photosystem II (PSII) cores were isolated from an octyl glucoside-containing sucrose gradient after solubilization of barley thylakoid membranes with Triton X-100 and octyl glucoside. No cation precipitation step was necessary to collect the chl a/b LHC. PAGE under mildly denaturing and fully denaturing conditions showed that the chl a/b LHC fraction contained chlorophyll-protein complexes CP27, CP29, and CP64. The PSII core material contained CP43 and CP47, and little contamination by other nonpigmented polypeptides. Freeze-fracture electron microscopy of the chl a/b LHC after reconstitution into digalactosyldiglyceride (DG) or phosphatidylcholine (PC) vesicles showed that the protein particles (approximately 7.5 +/- 1.6 nm) were approximately 99 and 90% randomly dispersed, respectively, in the liposomes. Addition of Mg++ produced particle aggregation and membrane adhesion in chl a/b LHC-DG liposomes in a manner analogous to that described for LHC-PC liposomes. Reconstitution of PSII cores into DG vesicles also produced proteoliposomes with randomly dispersed particles (approximately 7.5 +/- 1.6 nm). In contrast, PSII-PC mixtures formed convoluted networks of tubular membranes that exhibited very few fracture faces. Most of the protein particles (approximately 7.0 +/- 1.5 nm) were seen trapped between, rather than embedded in, the membranes. The interaction between the zwitterionic head group of the phosphatidyl choline and the negatively charged PSII core may be responsible for the unusual membrane structures observed.

Biochemical and spectroscopic studies on the effects of high temperatures (45-47� C) over a 1 h period on the protein composition, fluorescence and photochemical activities of the barley thylakoid membrane were made. Photosystem II (PS II) activity decreased as expected, and photosystem I (PS I) activity also unexpectedly decreased. Our data support previous conclusions that the decrease in PS I activity is largely due to inactivation (or loss) of a component between the two photosystems. A two-dimensional electrophoretic system permitted first the separation of the thylakoid pigment-protein complexes of unstressed and stressed plants, followed by a determination of their subunit composition. The changes in the protein composition of each pigment-protein complex in response to elevated temperatures were monitored. Heat changed the quaternary structure of PS II and resulted in removal of the oxygen-evolving enhancer proteins from the thylakoid, but did essentially no damage to the PS I complex. The PS II core complex dissociated from a dimeric form to a monomeric one, and the major LHC II component (LHC IIb) changed from a trimeric to a monomeric form. The pigments that are lost from thylakoids during heat stress are mainly removed from the PS II pigment-proteins.

Light-dependent reduction of the plastoquinone pool regulates the activity of the thylakoid-bound protein kinase which phosphorylates the light harvesting chlorophyll a,b-protein complex (LHC II) and regulates energy distribution between photosystems II (PS II) and I (Staehelin, L. A., and C. J. Arntzen, 1983, J. Cell Biol., 97:1327-1337). Since reduction of plastoquinone by PS II is abolished in photoinhibited thylakoids due to loss of the secondary electron acceptor QB protein (Kyle, D. J., I. Ohad, and C. J. Arntzen, 1984, Proc. Natl. Acad. Sci. USA, 81:4070-4074), it was of interest to examine the activity of the LHC II protein kinase system during photoinhibition and recovery of PS II activity. The kinase activity was assessed both in vivo and in vitro in Chlamydomonas cells exposed to high light intensity (photoinhibition) and recovery at low light intensity. The kinase activity was progressively reduced during photoinhibition and became undetectable after 90 min. The inactive LHC II-kinase system could not be reactivated in vitro either by light or by reduction of the plastoquinone pool following addition of reduced duroquinone (TMQH2). The LHC II polypeptides were dephosphorylated in vivo when cells, prelabeled with [32P]orthophosphate before exposure to high light intensity, were transferred to photoinhibiting light in the presence of [32P]orthophosphate. In vivo recovery of the LHC II-kinase activity, elicited by the addition of TMQH2 to the assay system, did not require restoration of QB-dependent electron flow or de novo protein synthesis, either in the cytoplasm or in the chloroplast. Mild sonication of thylakoids isolated from photoinhibited cells restored the ability of the LHC II protein kinase system to be activated in vitro by addition to TMQH2. Restoration of the light-activated LHC-II kinase required recovery of QB-dependent electron flow. At the structural level, photoinhibition did not affect the ratio of grana/stroma thylakoids. A reduction of approximately 20% of the 11-17-nm intramembrane particles and an equivalent increase in the number of 6-10.5-nm particles was observed on the E-fracture faces of stacked thylakoid membranes. Similar but smaller changes were observed also on the E-fracture faces of unstacked thylakoid membranes (more 10-14-nm and less 6-9-nm particles) and P-fracture faces of stacked thylakoid membranes (more 6-8- and less 9.5-13-nm particles). All these structural changes were reversed to normal values during recovery of PS II activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Three characteristics of shade plants are reviewed. Firstly, they have relatively more chlorophyll b and the associated light-harvesting chlorophyll a/b-protein complex (LHC). Two currently accepted reasons for this are not supported by quantitative analysis. Instead, the reduced protein cost of complexing chlorophyll in LHC and the turnover of the 32 kDa herbicide binding protein are considered. Secondly, shade plants have low electron transport capacities per unit of chlorophyll. This is primarily related to a reduction in the amount of electron transport components such as the cytochrome f complex and the ATPase. The nitrogen cost of the thylakoid membranes per unit of light absorbed is thereby reduced, but the irradiance range over which light is used with high efficiency is also reduced. Thirdly, shade plants have less RuP2 carboxylase and other soluble proteins for a given amount of chlorophyll. However, while the ratio of RuP2 carboxylase protein to thylakoid protein declined, the ratio of the RuP2 carboxylase activity to electron transport activity increased. For several species, the relationship between the rate of CO2 assimilation and leaf nitrogen content depends on the irradiance during growth.

We have found that treatment of the photosynthetic membranes of green plants, or thylakoids, with the nonionic detergent Triton X-114 at a 10:1 ratio has three effects: (a) photosystem I and coupling factor are solubilized, so that the membranes retain only photosystem II (PS II) and its associated light-harvesting apparatus (LHC-II); (b) LHC-II is crystallized, and so is removed from its normal association with PS II; and (c) LHC-II crystallization causes a characteristic red shift in the 77 degrees K fluorescence from LHC-II. Treatment of thylakoids with the same detergent at a 20:1 ratio results in an equivalent loss of photosystem I and coupling factor, with LHC-II and PS II being retained by the membranes. However, no LHC-II crystals are formed, nor is there a shift in fluorescence. Thus, isolation of a membrane protein is not required for its crystallization, but the conditions of detergent treatment are critical. Membranes with crystallized LHC-II retain tetrameric particles on their surface but have no recognizable stromal fracture face. We have proposed a model to explain these results: LHC-II is normally found within the stromal half of the membrane bilayer and is reoriented during the crystallization process. This reorientation causes the specific fluorescence changes associated with crystallization. Tetrameric particles, which are not changed in any way by the crystallization process, do not consist of LHC-II complexes. PS II appears to be the only other major complex retained by these membranes, which suggests that the tetramers consist of PS II.

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

This chapter focuses on the March 18, 1970, sit-in at Ladies' Home Journal (LHJ), a crucial episode in feminist media activism that had dramatic internal and external consequences for women's liberation. Conceived as a radical action by a small group of women incensed at the demeaning portrayal of women in a publication that touted itself as “the magazine women believe in,” the LHJ protest was an unpredictable success, precipitating significant changes in editorial and employment practices at women's magazines. That outcome was the product of several factors, including the emphases of the print and broadcast coverage of the LHJ events as well as the action's timing among a wave of protests and discrimination complaints launched in 1970 by women employees of major media institutions. Equally important was the recognition of the magazine's editors—and those of their sister publications—that incorporating and commodifying women's liberation was more profitable than resisting it, processes that would soon escalate across all forms of mass media.

Focusing specifically on Jean-Luc Godard’s experimental 3-D outing, Adieu au langage (2014), this chapter equates becoming-animal (manifested through Godard’s dog, Roxy) with the time of the intolerable, a crisis in the action-image which precludes any absorption of affect within the sensory-motor schema. In this sense, Roxy becomes an immanent, animistic force that remakes the world (and by extension, as in all of Godard’s films, cinema itself) into a protean force of becoming, where even 3-D bifurcates and deterritorialises as a new form of machinic fabulation. In the case of the intolerable, where our sensory-motor schema is allowed to jam or break, a different type of image appears, what Deleuze calls a pure optical-sound image: ‘opsigns’ and ‘sonsigns’, images where the seen and heard no longer extend into action. In Adieu au langage, Godard’s use of 3D also offers a new kind of time-image, a lectosign (a visual image which must be ‘read’ as much as seen) that requires the eye’s negotiation of conflicting points of attention within the shot, thereby inducing crisis and the intolerable within the image itself.

The light-harvesting chlorophyll a/b protein complex (LHC) has been shown to form crystalline arrays in vitro. These arrays have P321 symmetry. The unit cell is composed of trimeric protein oligomers arranged with a 3-fold symmetry axis running centrally through each complex[1]. Each polypeptide has an apparent molecular weight of 25–27kd within which are bound 6-11 chlorophyll molecules.

The intriguing finding that functions of t-PA coincide with structural domains and that these domains occur in related proteins, has been the basis to construct hybrid proteins by artificial exon shuffling to prove the conservation of functions in the shuffled domains. The heavy chain (Hch) of t-PA mediates both binding to fibrin and stimulation of plasminogen activator activity via its Finger- and Kringle-2 domain, whereas the light chain (Lch) contains the serine protease moiety of the protein. The Hch of u-PA is very homologous to the Lch of t-PA, but exhibits a higher plasminogen activator activity. This activity of u-PA is not stimulated by fibrin. We employed the ‘M13 in vitro outlooping’ technique to fuse the Hch of t-PA cDNA and the Hch of u-PA cDNA, to create two different hybrid cDNAs. On one hybrid cDNA, the t-PA and the u-PA sequences are coupled precisely at the exon-intron boundaries of the corresponding genes, while the other hybrid cDNA lacks a u-PA segment at the junction, encoding 13 amino acids of u-PA. The hybrid cDNAs were transiently expressed in mouse Ltk- cells and the recombinant proteins were characterized. The plasminogen activator activity of these proteins was determined in an indirect amidolytic assay, using plasminogen and the chromogenic substrate S2251. As anticipated, the activity of both t-PA/u-PA hybrid proteins is stimulated by fibrin, however, not to the same extent as t-PA. Remarkably, we found a decreased inhibition of the hybrid proteins by the endothelial plasminogen activator inhibitor (PAI-1) as compared to t-PA and u-PA, although stable complexes between the hybrid proteins and the inhibitor are formed. We conclude that functions of structural domains are maintained during exon shuffling

Introduction The light-harvesting (LH) pigment-proteins of photosynthetic purple bacteria have been extensively studied with picosecond [1] and now recently with femtosecond [2] pulses. These investigations have helped to make these pigments to the probably best studied and characterized LH systems. Several bacteria, including the presently studied species Rhodobacter sphaeroides, contain two types of LH pigments, a core LH1 antenna surrounding the photochemical reaction centers and a more peripheral LH2 antenna. Both these LH pigments are believed to be organized in small minimum units of 2-4 polypeptides holding about the same number of chromophores, which aggregate to form the network of the intact antenna. Energy transfer between minimum units is believed to occur in a non-coherent hopping fashion. Interaction within a unit is probably quite strong and spectral evidence suggest that it should be characterized as strong exciton interaction leading to the formation of delocalized exciton states. In this report we will mainly be concerned with the ultrafast dynamics within such a minimum unit.

Over the past twenty-five years, genetic methods have generated a wealth of information on the regulation and the structure-function relationship of bacterial genes.These methods are based on the introduction of random mutations in a gene to alter its function. Subsequently, genetic techniques cure applied to localize the mutation, while the nature of the impairedfunction could be determined using biochemical methods. Classic examples of this approach is now considered to be the elucidation of the structure and function of genes, constituting the Escherichia coli lactose (lac) and tryptophan (trp) operons,and the detailed establishment of the structure and function of the repressor (lacl) of the lac operon. Recombinant DNA techniques and the development of appropriate expression systems have provided the means both to study structure and functionof eukaryotic (glyco-) proteins and to create defined mutations with a predestinedposition. The rationale for the construction of mutant genes should preferentiallyrely on detailed knowledge of the three-dimensional structure of the gene product.Elegant examples are the application of in vitro mutagenesis techniques to substitute amino-acid residues near the catalytic centre of subtilisin, a serine proteasefrom Bacillus species and to substituteanamino acid in the reactive site (i.e. Pi residue; methionine) of α-antitrypsin, a serine protease inhibitor. Such substitutions have resulted into mutant proteins which are less susceptible to oxidation and, in some cases, into mutant proteins with a higher specific activity than the wild-type protein.If no data are available on the ternary structure of a protein, other strategies have to be developed to construct intelligent mutants to study the relation between the structure and the function of a eukaryotic protein. At least for a number of gene families, the gene structure is thought to be created by "exon shuffling", an evolutionary recombinational process to insert an exon or a set of exons which specify an additional structural and/or functional domain into a pre-existing gene. Both the structure of the tissue-type plasminogen activator protein(t-PA) and the t-PA gene suggest that this gene has evolved as a result of exon shuffling. As put forward by Gilbert (Science 228 (1985) 823), the "acid test"to prove the validity of the exon shuffling theory is either to delete, insert or to substitute exon(s) (i.e. in the corresponding cDNA) and toassay the properties of the mutant proteins to demonstrate that an exon or a set of adjacent exons encode (s) an autonomousfunction. Indeed, by the construction of specific deletions in full-length t-PA cDNA and expression of mutant proteins intissue-culture cells, we have shown by this approach that exon 2 of thet-PA gene encodes the function required forsecretion, exon 4 encodes the "finger" domain involved in fibrin binding(presumably on undegraded fibrin) and the set of exons 8 and 9 specifies kringle 2, containing a lysine-binding sit(LBS) which interacts with carboxy-terminal lysines, generated in fibrin after plasmic digestion. Exons 10 through 14 encode the carboxy-ter-minal light chain of t-PA and harbor the catalytic centre of the molecule and represents the predominant "target site" for the fast-acting endothelial plasminogen activator inhibitor (PAI-1).As a follow-up of this genetic approach to construct deletion mutants of t-PA, we also created substitution mutants of t-PA. Different mutants were constructed to substitute cDNA encoding thelight chain of t-PA by cDNA encoding the B-chain of urokinase (u-PA), in order to demonstrate that autonomous structural and functional domains of eitherone of the separate molecules are able toexert their intrinsic properties in a different context (C.J.M. de Vries et al., this volume). The possibilities and the limitations of this approach to study the structure and the function of t-PA and of other components of the fibrinolytic process will be outlined.

The well-recognized Lac repressor protein (LacI) regulates transcription by bending DNA into a loop. In addition to the known role of DNA flexibility, there is accumulating evidence suggesting that the flexibility of LacI also plays a role in this gene regulation. Here we extend our elastic rod model for DNA (previously used to model DNA only) to represent LacI. Specifically, we represent sites of concentrated flexibility in the protein with flexible elastic rod domains; and we represent relatively rigid domains of the protein with stiff elastic rod domains. Our analysis shows the sensitivity of looping energetics to the degree of flexibility within the protein over a large range of DNA lengths. In addition, we show that the predicted energetically dominant binding topology (A) remains upon introducing protein flexibility.

This study is focused on testing “immuno-sensors” of micro-cantilever beams for the purpose of future design of high-throughout bioassays. We currently study the aberrant expression, deletion and mutation of hDMP1 (Human DMP1) in human lung cancer. Lung cancer is the leading cause of cancer deaths among men and women in North America. There are four major histological subtypes of human lung cancer: small-cell carcinoma (SCC), adenocarcinoma (AC), squamous cell carcinoma (SCC), and large-cell carcinoma (LCC). The hDMP1 locus is localized on chromosome 7q21, a region frequently deleted as part of the 7q-minus and monosomy 7 abnormalities of acute myeloid leukemia and myelodysplastic syndrome. Recent data demonstrate the critical role of Dmp1 in Ras-Raf-Arf signaling and cellular senescence. In order to study the interactions of hDMP1 gene product and selected tumor markers with BioMEMS devices, protein coating (Antibody) conduct on cantilevers with silicon nitride (SiNx) surfaces. Silicon nitride surface has the potential to provide a good interface for BioMEMS devices, due to enhanced adherence of substances on this surface. The cantilever biosensors coated with hDMP1 antibody were used for the detection of hDMP1 antigen, which is known to be a tumor suppressor protein. Results revealed that the changes in nano-mechanical forces provided sufficient differential torque to bend the cantilever beam upon injection of the antigen. Theoretical models have been developed for the prediction of the vibrational responses of atomic force microscope (AFM) cantilevers before and after antigen/antibody interaction. Exposure of the proteins to micro-cantilever (MC) resulted in un-reversible large stress. Static deflection of micro-cantilever appeared as a result of the surface stresses that are induced by changes upon antibody-antigen interaction. This indicated that the micro-cantilever approach is useful for detecting proteins and tumor markers, and this system is applicable as a model to design miniaturized biosensor systems. We also applied gene micro-array to identify unknown targets for hDMP1 and extend our observation of the complicated process of carcinogenesis.

Lab-on-a-chip (LOC) devices integrate multiple laboratory functions and processes to a miniaturized chip format. Many LOC devices are used in a wide range of biomedical and other analytical applications including rapid pathogen detection, clinical diagnosis, electrophoresis, flow cytometry, and protein and DNA analysis. LOC devices can be fabricated from many types of material including various polymers, glass/silicon, or combinations of these materials. A broad variety of fabrication technologies are used for LOC device fabrication. LOC systems have several common features including microfluidics (e.g. channels, micro-pumps, mixers and valves) and sensing capabilities. This paper describes a technique for the fabrication of a micro-pump that can be used in implantable biocompatible devices. This paper will also discuss the synthesis and properties of biocompatible iron oxide nanoparticles (NPs) for AC electrokinetic micro-pumping. Iron oxide NP will be incorporated with PDMS (polydimithylsiloxane) to obtain biocompatible nanocomposite. The use of photoluminescence, hydrophobic, and reflective optical response of these materials for in-vitro and in-vivo sensing and micro-pumping will be highlighted.

Introduction: Long RNAs are non-coding RNAs with more than 200 nucleotides in length, with essential regulatory roles in several biological processes, including in breast cancer (BC). The human genome contains 481 ultraconserved regions, which are genomic stretches of over 200 base pairs conserved among humans, rats, and mice. Most of these regions are transcriptionally active (T-UCRs), and several are differentially expressed in tumors. Some T-UCRs have been functionally characterized, but few have been associated with BC. Objectives: In this study, we aimed to expand the knowledge of T-UCRs in BC and characterize the lnc-uc.147, a long RNA transcribed from an ultraconserved region. Methods: We evaluated the expression level of 481 T-UCRs and their association with clinical parameters from TCGA data. For confirmation, 102 Brazilian BC samples were analyzed by RT-qPCR. Cytosolic and nuclear cell fractions and RT-qPCRs were done to determine the cell compartment of the transcript. Northern blotting and RACE were performed to determine the sequence and precise size of lnc-uc.147. Using two luminal cell lines (CAMA and BT474), a siRNA-based approach was applied to investigate the effects of lnc-uc.147 knockdown in cell viability, colony formation, and apoptosis level. To understand the interactions of lnc-uc.147 and proteins, we performed a pull-down assay. Results: Using TCGA (The Cancer Genome Atlas) data, we found 302 T-UCRs related to clinical features in BC: 43% were associated with molecular subtypes, 36% with estrogen-receptor positivity, 17% with HER2 expression, 12% with stage, and 10% with overall survival. We found that uc.147 is highly expressed in luminal A and B patients, which was also confirmed in Brazilian samples. For luminal A, a subtype usually associated with better prognosis, high uc.147 expression was associated with a poor prognosis and suggested as an independent prognostic factor. The lncRNA from uc.147 (lnc-uc.147) is in the nucleus. Northern blotting results show that uc.147 is a 2,8 kb monoexonic transcript. The silencing of uc.147 increases apoptosis, arrests the cell cycle and reduces cell viability and colony formation in luminal BC cell lines. Additionally, we identified 19 proteins that interact with uc.147 through mass spectrometry. These proteins are mainly involved in cytoskeletal and centrosome organization as well as in epithelial-mesenchymal transition. Conclusions: We show herein evidence that neoplastic BC cells exhibit a unique expression profile of T-UCRs. This study characterized the lnc-uc.147, a transcript that has never been described before. Indeed, lnc-uc.147 has an oncogenic effect in the luminal BC cell line and can interact with proteins. Furthermore, uc.147 has the potential as a BC prognostic marker in luminal patients.

The major goal of the proposed research was to study the role of a 70-kDa heat shock cognate protein from chloroplasts (ct-HSP70) in the assembly of chlorophyll-protein complexes. The latters are mostly important in allowing photosynthesis to occur. Photosynthesis is at the heart of crop productivity and the knowledge of the biogenesis of the photosynthetic apparatus is essential to manipulate the efficiency of photosynthesis. The characterization of the function of the ct-HSP70 was planned to be studied in vitro by assaying its capability to physically interact with the thylakoid proteins and to assist their assembly into thylakoid membranes. We planned to identify regions in the light-harvesting complex protein (LHCP) that interact with the ct-HSP70 and characterize the interaction between them. We also intended to isolate cDNA clones encoding ct-HSP70, sequence them, express one of them in E. coli and use the purified protein for functional assays. The research in this BARD proposal aimed at providing insights and aid in understanding the mechanism by which plants may respond to the heat stress. Since plants often experience increased temperatures.

Innate immune responses in animals and plants involve receptors that recognize microbe-associated molecules. In plants, one set of this defense system is characterized by large families of TIR–nucleotide binding site–leucine-rich repeat (TIR-NBS-LRR) resistance genes. The direct interaction between plant proteins harboring the TIR domain with proteins that transmit and facilitate a signaling pathway has yet to be shown. The Arabidopsis genome encodes TIR-domain containing genes that lack NBS and LRR whose functions are unknown. Here we investigated the functional role of such protein, TLW1 (TIR LECTIN WOUNDRESPONSIVE1). The TLW1 gene encodes a protein with two domains: a TIR domain linked to a lectin-containing domain. Our specific aim in this proposal was to examine the ramifications of the TL1-glycan interaction by; A) The functional characterization of TL1 activity in the context of plant wound response and B) Examine the hypothesis that wounding induced specific polysaccharides and examine them as candidates for TL-1 interactive glycan compounds. The Weizmann group showed TLW1 transcripts are rapidly induced by wounding in a JA-independent pathway and T-DNA-tagged tlw1 mutants that lack TLW1 transcripts, fail to initiate the full systemic wound response. Transcriptome methodology analysis was set up and transcriptome analyses indicates a two-fold reduced level of JA-responsive but not JA-independent transcripts. The TIR domain of TLW1 was found to interact directly with the KAT2/PED1 gene product responsible for the final b-oxidation steps in peroxisomal-basedJA biosynthesis. To identify potential binding target(s) of TL1 in plant wound response, the CCRC group first expressed recombinant TL1 in bacterial cells and optimized conditions for the protein expression. TL1 was most highly expressed in ArcticExpress cell line. Different types of extraction buffers and extraction methods were used to prepare plant extracts for TL1 binding assay. Optimized condition for glycan labeling was determined, and 2-aminobenzamide was used to label plant extracts. Sensitivity of MALDI and LC-MS using standard glycans. THAP (2,4,6- Trihydroxyacetophenone) showed minimal background peaks at positive mode of MALDI, however, it was insensitive with a minimum detection level of 100 ng. Using LC-MS, sensitivity was highly increased enough to detect 30 pmol concentration. However, patterns of total glycans displayed no significant difference between different extraction conditions when samples were separated with Dionex ICS-2000 ion chromatography system. Transgenic plants over-expressing lectin domains were generated to obtain active lectin domain in plant cells. Insertion of the overexpression construct into the plant genome was confirmed by antibiotic selection and genomic DNA PCR. However, RT-PCR analysis was not able to detect increased level of the transcripts. Binding ability of azelaic acid to recombinant TL1. Azelaic acid was detected in GST-TL1 elution fraction, however, DHB matrix has the same mass in background signals, which needs to be further tested on other matrices. The major findings showed the importance of TLW1 in regulating wound response. The findings demonstrate completely novel and unexpected TIR domain interactions and reveal a control nexus and mechanism that contributes to the propagation of wound responses in Arabidopsis. The implications are to our understanding of the function of TIR domains and to the notion that early molecular events occur systemically within minutes of a plant sustaining a wound. A WEB site (http://genome.weizmann.ac.il/hormonometer/) was set up that enables scientists to interact with a collated plant hormone database.

The Problem: Abscission is a highly regulated process, occurring as a natural terminal stage of development, in which various organs are separated from the parent plant. In most plant species, the process is initiated by a decrease in active auxin in the abscission zone (AZ) and an increase in ethylene, and may be accelerated by postharvest or environmental stresses. Another potential key regulator in abscission is IDA (Inflorescence Deficient in Abscission), which was identified as an essential peptide signal for floral organ abscission in Arabidopsis. However, information is still lacking regarding the molecular mechanisms integrating all these regulators. In our previous BARD funded research we made substantial progress towards understanding these molecular events in tomato, and the study is still in progress. We established a powerful platform for analysis of genes for regulatory proteins expressed in AZ. We identified changes in gene expression for several transcription factors (TFs) directly linked to ethylene and auxin signaling and several additional regulatory proteins not so obviously linked to these hormones. Moreover, we demonstrated using a virus-induced gene silencing (VIGS) assay that several play a functional role in the onset of abscission. Based on these results we have selected 14 genes for further analysis in stably transformed tomato plants. All 14 genes were suppressed by RNA interference (RNAi) using a constitutive promoter, and 5 of them were also suppressed using an abscission-specific promoter. Transformations are currently at different stages of progress including some lines that already display an abscission phenotype. Objectives: We propose here to (1) complete the functional analysis of the stably transformed tomato plants with T2 lines and perform transcriptome analysis using custom abscission-specific microarrays; (2) conduct an indepth analysis of the role of IDA signaling in tomato leaf and flower abscission; (3) perform transcriptome and proteome analyses to extend the earlier gene expression studies to identify transcripts and proteins that are highly specific to the separation layer (i.e., target cells for cell separation) prior to the onset of abscission; (4) extend and compliment the work in tomato using a winnowed set of genes in soybean. Methodology: Next Generation Sequencing (NGS) of mRNA will be used to further increase the list of abscission-associated genes, and for preparation of a custom tomato abscission microarray to test altered gene expression in transgenic plants. Tandem mass spectrometry (LC-MS/MS) of protein extracts from leaf petiole, flower pedicel and their AZ tissues will be used to identify the proteome of the AZ before and during abscission. AZ-specific gene promoters will be used in stably transformed tomato plants to reduce non-target phenotypes. The bean pod mottle virus (BPMV) plasmid vectors will be used for VIGS analysis in soybean. Expected Contribution: Our study will provide new insights into the regulation of ethylene-induced abscission by further revealing the role of key regulators in the process. This will permit development of novel techniques for manipulating leaf and flower abscission, thereby improving the postharvest performance of agriculturally important crops.

The main goal of this research plan was to elucidate regulatory mechanisms controlling the development, function of the bovine corpus luteum (CL). The CL contains two different sterodigenic cell types and therefore it was necessary to obtain pure cell population. A system was developed in which granulosa and theca interna cells, isolated from a preovulatory follicle, acquired characteristics typical of large (LL) and small (SL) luteal cells, respectively, as judged by several biochemical and morphological criteria. Experiments were conducted to determine the effects of granulosa cells removal on subsequent CL function, the results obtained support the concept that granulosa cells make a substaintial contribution to the output of progesterone by the cyclic CL but may have a limited role in determining the functional lifespan of the CL. This experimental model was also used to better understand the contribution of follicular granulosa cells to subsequent luteal SCC mRNA expression. The mitochondrial cytochrome side-chain cleavage enzyme (SCC), which converts cholesterol to pregnenolone, is the first and rate-limiting enzyme of the steroidogenic pathway. Experiments were conducted to characterize the gene expression of P450scc in bovine CL. Levels of P450scc mRNA were higher during mid-luteal phase than in either the early or late luteal phases. PGF 2a injection decreased luteal P450scc mRNA in a time-dependent manner; levels were significantly reduced by 2h after treatment. CLs obtained from heifers on day 8 of the estrous cycle which had granulosa cells removed had a 45% reduction in the levels of mRNA for SCC enzymes as well as a 78% reduction in the numbers of LL cells. To characterize SCC expression in each steroidogenic cell type we utilized pure cell populations. Upon luteinization, LL expressed 2-3 fold higher amounts of both SCC enzymes mRNAs than SL. Moreover, eight days after stimulant removal, LL retained their P4 production capacity, expressed P450scc mRNA and contained this protein. In our attempts to establish the in vitro luteinization model, we had to select the prevulatory and pre-gonadotropin surge follicles. The ratio of estradiol:P4 which is often used was unreliable since P4 levels are high in atretic follicles and also in preovulatory post-gonadotropin follicles. We have therefore examined whether oxytocin (OT) levels in follicular fluids could enhance our ability to correctly and easily define follicular status. Based on E2 and OT concentrations in follicular fluids we could more accurately identify follicles that are preovulatory and post gonadotropin surge. Next we studied OT biosynthesis in granulosa cells, cells which were incubated with forskolin contained stores of the precursor indicating that forskolin (which mimics gonadotropin action) is an effective stimulator of OT biosynthesis and release. While studying in vitro luteinization, we noticed that IGF-I induced effects were not identical to those induced by insulin despite the fact that megadoses of insulin were used. This was the first indication that the cells may secrete IGF binding protein(s) which regonize IGFs and not insulin. In a detailed study involving several techniques, we characterized the species of IGF binding proteins secreted by luteal cells. The effects of exogenous polyunsaturated fatty acids and arachidonic acid on the production of P4 and prostanoids by dispersed bovine luteal cells was examined. The addition of eicosapentaenoic acid and arachidonic acid resulted in a dose-dependent reduction in basal and LH-stimulated biosynthesis of P4 and PGI2 and an increase in production of PGF 2a and 5-HETE production. Indomethacin, an inhibitor of arachidonic acid metabolism via the production of 5-HETE was unaffected. Results of these experiments suggest that the inhibitory effect of arachidonic acid on the biosynthesis of luteal P4 is due to either a direct action of arachidonic acid, or its conversion to 5-HETE via the lipoxgenase pathway of metabolism. The detailed and important information gained by the two labs elucidated the mode of action of factors crucially important to the function of the bovine CL. The data indicate that follicular granulosa cells make a major contribution to numbers of large luteal cells, OT and basal P4 production, as well as the content of cytochrome P450 scc. Granulosa-derived large luteal cells have distinct features: when luteinized, the cell no longer possesses LH receptors, its cAMP response is diminished yet P4 synthesis is sustained. This may imply that maintenance of P4 (even in the absence of a Luteotropic signal) during critical periods such as pregnancy recognition, is dependent on the proper luteinization and function of the large luteal cell.