Hemocyanins (Hcs) and phenoloxidases (POs) are both members of the type-3 copper protein family, possessing di-cupric active sites which facilitate the binding of dioxygen. While Hcs and POs share a high degree of sequence homology, Hcs have been associated traditionally with oxygen transport whereas POs are catalytic proteins with a role in innate immunity. Evidence gathered in recent years details numerous immune functions for Hc, including an inducible PO activity. Unlike the pro-phenoloxidase activation cascade in arthropods, the endogenous mechanism(s) involved in the conversion of Hc into an immune enzyme is lacking in detail. The overall aim of this research was to characterise the physiological circumstances in which Hc is converted into a PO-like enzyme during immune challenge. A series of biochemical, biophysical and cellular techniques were used to assess the ability of phospholipid liposomes to mimic the well-characterised induction of PO activity in Hc by SDS micelles. Incubation of Hc purified from Limulus polyphemus, in the presence of phosphatidylserine (PS) liposomes, yielded ~ 90% of the PO activity observed upon incubation of Hc with the non-physiological activator, SDS. Phospholipid–induced PO activity in Hc was accompanied by secondary and tertiary structural changes similar to those observed in the presence of SDS. Subsequent analysis revealed that electrostatic interactions appear to be important in the PS-Hc activation complex. In vivo, PS-Hc interactions are assumed to be limited in quiescent cells. However, amebocytes undergoing apoptosis redistribute PS onto the outer leaflet of the plasma membrane, resulting in the potential for increased Hc-PS interactions. In the absence of a reliable culturing technique for L. polyphemus amebocytes, in vitro conditions were optimised for the short term maintenance of this labile cell type. Amebocytes retained viability and functionality in a medium that mimicked most-closely, the biochemical properties of L. polyphemus hemolymph. When presented with a fungal, bacterial or synthetic challenge, ~9% of amebocytes in vitro were found to be phagocytically active. Target internalisation was confirmed via the use of fluorescent quenchers and membrane probes. Within 4 hours of target internalisation, amebocytes underwent apoptosis, characterised by the loss of plasma and mitochondrial membrane potential, increased caspase-3 activity and extracellularisation of PS. Phagocytosis-induced cell death led to a proportional increase in the level of Hc-derived PO activity, suggesting that Hc may be interacting with PS present on terminal amebocyte membranes. The PO activity of Hc was investigated further in order to address an economically important issue; hyperpigmentation in commercial shellfish. While PO enzymes are thought to be the cause of hyperpigmentation in Nephrops norvegicus, evidence presented here suggests that cellular PO is inactivated after freeze-thawing, while extracellular Hc retains stability and displays a heightened level of inducible PO activity under similar treatments. Known PO inhibitors were used successfully to reduce Hc-derived PO activity, with inhibitors assumed to bind Hc in a manner similar to PO-inhibitor complexes. Structural and functional studies of hemocyanins and immune cells presented here provide new insights into the interactions of hemocyanin-activator complexes in invertebrates.

The cross sections for neutral current (NC) deep inelastic scattering (DIS) in e+p collisions with a longitudinally polarised positron beam are measured at high momentum transfer squared (Q2 > 185 GeV2) at the ZEUS detector at HERA. The HERA accelerator provides e+-p collisions at a centre-of-mass energy of 318 GeV, which allows the weak contribution to the NC process to be studied at high Q2. The measurements are based on a data sample with an integrated luminosity of 135.5 pb-1 collected with the ZEUS detector in 2006 and 2007. The single differential NC cross sections dsigma/dQ2, dsigma/dx and dsigma/dy and the reduced cross section are measured. The structure function xF3 is determined by combining the e+p NC reduced cross sections with the previously measured e-p measurements. The interference structure function xF 3^gamma,Z is extracted at Q2 = 1500 GeV2. The cross-section asymmetry between the positive and negative polarisation of the positron beam is measured and the parity violation effects of the electroweak interaction are observed. The predictions of the Standard Model of particle physics agree well with the measurements.

Reading non-alphabetic script can be a serious challenge to second language (L2) learners with alphabetic backgrounds. Many L2 learners of Japanese or Chinese who are fluent in speaking the language do not necessarily acquire an advanced-level reading ability. The aim of my thesis was to investigate the development of L2 word recognition ability, one of the most important abilities that learners need to develop for efficient reading, among English-speaking learners of Japanese. By analysing the results of behavioural tests and a verbal protocol administered to both L1 and L2 readers of Japanese, the study described the changes in developing L2 learners’ kanji recognition skills and their awareness of the structure and function of characters at the different stages of L2 exposure. / The overall findings suggest that the changes in processing patterns demonstrated by the participants in the present study may be fundamentally similar to those of L1 children, which have been found to be similar regardless of the types of script involved. The changes in L2 readers’ developing kanji recognition were accounted for by the transformation of the internal processing system; this transformation seems to occur by continuous link formation through learning corresponding information, and information processing based on the learned information. The process of transformation, which is affected by the frequency of exposure and the amount of practice, and therefore appears to be item-based, generally progresses on a stage-based developmental trajectory; the processing begins with local and incomplete information and progresses via intentional and analytical processing to develop into sophisticated attention-free processing. / Although the developmental trajectory may be universal, the findings of the present study suggest that, when L1 and L2 are orthographically distant, L2 readers repeat the developmental phases due to lack of their ability to process script-specific information. L2 readers with alphabetic backgrounds cannot simply transfer the recognition skills and awareness that they have acquired in their L1 in the new environment of character recognition. The findings of the study suggest that script-specific recognition skills and awareness develop over time as the L2 readers’ internal processing system undergoes successive transformations. By identifying several critical skills and awareness, the present study has discussed the possibility of enhancing character recognition ability with the use of explicit instruction at critical moments.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / Nesta tese são apresentadas a medida da taxa de produção de eventos difrativos e a extração da função de estrutura do antipróton para eventos de difração simples. A análise aqui apresentada é baseada em dados do detector DØ no Tevatron/Fermilab, colisor de prótons-antiprótons à energia de centro de massa de 1.96 TeV. A seleção de eventos difrativos é feita através de intervalos de rapidez - região do detector desprovida de partículas - determinados através da deposição de energia nas células do calorímetro DØ. A função de estrutura obtida nessa análise é comparada com resultados existentes dos experimentos H1 e CDF. / In this thesis we present a measurement of the diffractive to non-diffractive production rate and the extraction of the antiproton diffractive structure function. The analysis presented uses data from the DØ proton-antiproton collider at Tevatron/Fermilab with = 1.96 TeV. The data sample was selected using rapidity gap signatures determined through the sum of energy depositions in the DØ calorimeter cells. The measurements were performed on 0.19 pb-1 of Run IIa DØ data. The results are compared to H1 and CDF results."

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / Nesta tese são apresentadas a medida da taxa de produção de eventos difrativos e a extração da função de estrutura do antipróton para eventos de difração simples. A análise aqui apresentada é baseada em dados do detector DØ no Tevatron/Fermilab, colisor de prótons-antiprótons à energia de centro de massa de 1.96 TeV. A seleção de eventos difrativos é feita através de intervalos de rapidez - região do detector desprovida de partículas - determinados através da deposição de energia nas células do calorímetro DØ. A função de estrutura obtida nessa análise é comparada com resultados existentes dos experimentos H1 e CDF. / In this thesis we present a measurement of the diffractive to non-diffractive production rate and the extraction of the antiproton diffractive structure function. The analysis presented uses data from the DØ proton-antiproton collider at Tevatron/Fermilab with = 1.96 TeV. The data sample was selected using rapidity gap signatures determined through the sum of energy depositions in the DØ calorimeter cells. The measurements were performed on 0.19 pb-1 of Run IIa DØ data. The results are compared to H1 and CDF results."

As enzimas diidroorotato desidrogenases (DHODHs) são flavo-enzimas que catalisam a oxidação do diidroorotato em orotato na quarta etapa da biossíntese de novo de nucleotídeos de pirimidina. Durante a rápida proliferação celular em mamíferos, a via de salvação de pirimidinas é insuficiente para suprir deficiências na síntese de nucleotídeos. Além disso, certos parasitas não possuem a via de salvação e contam somente com a biossíntese de novo para a produção de nucleotídeos. Por esta razão, DHODH se tornou um excelente alvo na busca por inibidores que interrompam a síntese de nucleotídeos. As enzimas DHODHs de E. coli (EcDHODH) e de X. fastidiosa (XfDHODH) são membros da classe 2 das DHODHs e encontram-se associadas à membrana citoplasmática através de uma extensão em seu N-terminal, enquanto que DHODH de T. cruzi (TcDHODH), membro da classe 1 de DHODHs, é uma proteína citosólica. Neste trabalho, usamos uma combinação de metodologias de biologia molecular e bioquímica com técnicas espectroscópicas para obter informações estruturais e funcionais acerca da enzima DHODH. Assim, Ressonância Paramagnética Eletrônica (RPE) associada à marcação de spin sítio dirigida (SDSL) e simulação espectral foram empregadas para estudar a interação da EcDHODH com modelos de membrana. Mudanças na dinâmica estrutural das vesículas induzidas pela enzima foram monitoradas via marcadores de spin localizados em diferentes posições ao longo da cadeia acil de fosfolipídios. Além disso, técnicas de DNA recombinante e mutações sítio dirigidas foram utilizadas para produzir mutantes de EcDHODH no qual um sondas paramagnéticas foram seletivamente ligadas em resíduos localizados na extensão N-terminal da proteína para experimentos subseqüentes de RPE-SDSL. Esses são os primeiros experimentos de marcação de spin sítio dirigida realizados no Brasil e com os quais monitoramos a dinâmica experimentada na região do N-terminal. Além disso, várias tentativas foram feitas para se expressar e purificar a enzima XfDHODH e a estabilidade estrutural da enzima TcDHODH na presença de um de seus inibidores naturais, o orotato, foi monitorada através de experimentos de Dicroísmo Circular (CD). / Dihydroorotate dehydrogenases (DHODHs) are flavin-containing enzymes which catalyse the conversion of (S)-dihydroorotate to orotate, in the fourth step of the de novo biosynthesis of pyrimidine nucleotides. In rapidly proliferating mammalian cells, pyrimidine salvage pathway is insufficient to overcome deficiencies for nucleotide synthesis. Moreover certain parasites lack salvage enzymes, relying solely on the de novo pathway to produce nucleotides. Thus, DHODH has turned out an excellent target to the development of inhibitors that block nucleotide biosynthesis. E. coli DHODH (EcDHODH) and X. fastidiosa DHODH (XfDHODH) are class 2 DHODHs found associated to cytosolic membranes through an N-terminal extension, whereas T. cruzi DHODH (TcDHODH) is a class 1 DHODH localizated in the cytoplasm. In the present work, we used a combination of molecular biology and biochemical methodologies with spectroscopic techniques to obtain structural and functional information on DHODH. On one hand, Electronic Paramagnetic Resonance (EPR) associated with Site-directed Spin Labeling (SDSL) and spectral simulation were employed to study the interaction of EcDHODH with vesicles. Changes in vesicle dynamic structure induced by the enzyme were monitored via spin labels located at different positions along the phospholipid acyl chain and via spin labels located at enzyme specific positions. On the other hand, DNA techniques and site-directed mutagenesis were used to produce mutants of EcDHODH where a nitroxide spin probe was selectively attached to some residues located at the protein N-terminal extension for subsequent EPR-SDSL experiments. These are the first site-directed spin labeling experiments performed in Brazil and the spectra allowed us to monitor dynamics experienced by those residues at the EcDHODH N-terminal domain. Furthermore, molecular biology and biochemical assays were employed with the objective of expressing and purifying XfDHODH and Circular Dichroism (CD) was utilized to probe the structural stability of TcDHODH in the presence of its natural inhibitor (orotate).

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / As lectinas sÃo proteÃnas multiativas que apresentam pelo menos um sÃtio capaz de reconhecer de maneira reversÃvel carboidratos especÃficos sem modificÃ-los. A famÃlia das lectinas de leguminosas representa o grupo desta classe proteica mais bem estudada, em especial destaque a subtribo Diocleinae. As lectinas de Diocleinae apresentam um alto grau de similaridade estrutural, porÃm o mesmo nÃo se observa quanto Ãs atividades biolÃgicas. Esta variabilidade reside em detalhes que podem ser analisados em estudos baseados em estruturas. A sequÃncia primÃria da lectina de C. grandiflora (ConGF) apresenta grande similaridade com lectinas do mesmo gÃnero, porÃm concentra o maior nÃmero de mutaÃÃes representativas do gÃnero Dioclea, caracterizando-o como o subgÃnero de Canavalia mais prÃximo de Dioclea, e dentre as canavalias à a mais primitiva. A ConGF apresentou efeito relaxante em mÃsculo liso de aortas de ratos endotelizadas, no entanto, os efeitos mostram-se fracos frente a outras lectinas de Diocleinae. A justificativa para este fato nÃo reside em uma baixa similaridade estrutural, mas em pequenas mudanÃas na orientaÃÃo de aminoÃcidos-chave, que se tornam responsÃveis pela diversidade na aÃÃo biolÃgica apresentada aqui, como deve ocorrer em outros fenÃmenos elicitados por lectinas. Para a lectina de Cymbosema roseum (CRLI), alÃm de avaliado o efeito relaxante, foi observado o papel do cÃlcio extracelular nesta atividade. Surpreendentemente, o cÃlcio nÃo foi definitivo para determinar o mecanismo de CRLI como dependente ou independente deste Ãon. Nossa investigaÃÃo permitiu a formulaÃÃo de uma hipÃtese em que esta lectina apresenta um duplo mecanismo de ativaÃÃo da Ãxido nÃtrico sintetase endotelial (eNOS). A primeira via à baseada em um receptor especÃfico na membrana do endotÃlio capaz de ativar a eNOS atravÃs da calmodulina. A segunda via à baseada na habilidade de ligaÃÃo de CRLI ao heparano sulfato do glicocÃlice em um sÃtio diferente do CRD demonstrada por docking molecular, o que justifica a ativaÃÃo mecÃnica da eNOS. Este proteoglicano à o principal candidato a mecanoreceptor da tensÃo de cisalhamento, principal fenÃmeno da manutenÃÃo do tÃnus vascular pela produÃÃo de NO. Dentre as proteÃnas de Diocleinae, foi ainda avaliada uma lectina do gÃnero Dioclea. D. sclerocarpa apresentou, como as outras lectinas deste trabalho, a habilidade de relaxar mÃsculos lisos de aortas. FenÃmeno que ocorreu com a dependÃncia do endotÃlio via produÃÃo de Ãxido nÃtrico e com a participaÃÃo do CRD de DSL. Tanto seu efeito como sua estrutura apresentam alto grau de semelhanÃa com lectinas do mesmo gÃnero e um conjuntos de caracterÃsticas corrobora para seu baixo efeito relaxante. DSL apresenta um desenho de CRD pouco favorÃvel para esta atividade e, alÃm disso, a presenÃa de um glutamato na posiÃÃo 205 demonstrou ser um fator determinante na regulaÃÃo desta atividade. Este resÃduo modula negativamente a capacidade relaxante frente a lectinas que do mesmo gÃnero que possuem um resÃduo de aspartato nesta mesma posiÃÃo. / Lectins are multiactive proteins that have at least one domain capable of recognizing specific carbohydrates reversibly without changing them. The legume lectin family is a group of this protein class further studied, in particular highlighted the subtribe Diocleinae. These lectins have a high degree of structural similarity, but it does not follow the biological activities. This variability must reside in details, small differences that can be analyzed in studies based in structures. The primary sequence of C. grandiflora lectin (ConGF) shows great similarity with lectins of the same genus, but it has the largest number of mutations representative of the genus Dioclea, characterizing it as the Canavalia subgenus closest to Dioclea, and it is the most primitive among the canavalias. ConGF presented relaxing effect on smooth muscles of endothelial aortas of rats; however, the effects are weak against other Diocleinae lectins. The justification for this does not lie in a low structural similarity but in small changes in the orientation of key amino acids residues, which become responsible for biological diversity in action presented here, as required in other phenomena elicited by lectins. For Cymbosema roseum lectin (CRLI), the relaxing effect was also evaluated and the role of extracellular calcium was observed for this activity. Surprisingly, the calcium was not definitive for determining CRLI mechanism as dependent or independent of calcium ions. Our research has led to the construction of a theory which this lectin has dual mechanism of nitric oxide synthase (eNOS) activation. The first path is based on a specific membrane endothelial receptor able to activate eNOS by calmodulin. The second path relies on the ability of CRLI binding to the glycocalyx heparano sulfate at a domain different from the CRD demonstrated by molecular docking, which explain mechanical activation of eNOS. This proteoglycan is the main mechanoreceptor candidate of shear stress and this phenomenon is the major agent of maintenance of vascular tone by NO production. A lectin from gender Dioclea was also evaluated to increase the range of legume proteins tested. As other lectins of this work, D. sclerocarpa presented the ability to relax smooth muscle of the aorta dependent on the endothelium nitric oxide production. Both its effect and its structure have a high degree of similarity with lectins of the same genus. A feature set corroborates with its low relaxant effect. DSL has a CRD design less favorable for this activity. In addition, the presence of a glutamate at position 205 proved to be a decisive factor in the activity regulation and it negatively modulates Dioclea lectins relaxant effect.

L’intégrase (IN) du VIH-1 est une enzyme clé catalysant l’insertion de l’ADN proviral dans le génome cellulaire au sein d’un complexe nucléoprotéique d’intégration.Les nombreux efforts apportés à l’étude du mécanisme d’intégration ont permis d’accumuler de multiples données sur ce processus complexe. Cependant plusieurs questions essentielles demeurent sans réponses en particulier concernant l’architecture fonctionnelle des complexes d’intégration de VIH-1. En effet même si les complexes actifs pour l’intégration sont relativement bien définis leur chronologie précoce de formation demeure inconnue. De même les phases tardives de leur association au substrat naturel d’intégration que constitue la chromatine restent floues. Enfin le rôle des facteurs du complexe de préintégration (CPI) dans la régulation des ces mécanismes doit être déterminé.Mon travail de thèse s’est reposé sur trois axes s’articulant autour de ces questions:-Comment est régulée la dynamique des phases précoces d’attachement des oligomères d’intégrase sur les extrémités virales ?-Quel est l’impact de la structure de l’ADN cible et de la chromatine sur l’association active des complexes d’intégration ?-Quel(s) rôle(s) joue(nt) les facteurs du CPI dans ces phases ? / HIV-1 integrase (IN) is a key enzyme catalyzing the proviral DNA insertion into the cellular genome within a nucleoprotein integration complex.The numerous efforts made on the mechanism of integration have led to the accumulation of substantial data on this process. However many important questions are still open particularly on the functional architecture of the HIV-1 integration complexes. Indeed even if the active complexes involved in the catalysis of the integration are well described, the chronology of their early formation is still unknown. Moreover, the late association of these complexes with the host chromatin is also obscure. Finally the involvement of the IN cofactors inside the preintegration complex on these steps has to be elucidated.The project of my PhD relied on three axis articulated on these questions:-What is the dynamic of the IN oligomers association on the DNA viral ends?-What is the impact of the target DNA chromatin structure on the functional association of the integration complexes?-Involvement of the PIC factors on these steps?

Pentameric ligand-gated ion channels (pLGICs) are membrane-embedded receptors found extensively in pre- and post-synaptic membranes throughout the nervous system where they play an important role in neurotransmission. The function of the prototypic pLGIC, the nicotinic acetylcholine receptor (nAChR) is highly sensitive to changes in its lipid environment, while other pLGICs display varying lipid sensitivities. This thesis presents a multidisciplinary investigation into the features of the transmembrane domain (TMD) that determine the unique functional and physical traits of different pLGICs. Using two prokaryotic homologues of the nAChR, ELIC and GLIC, as models, I focus on the outermost, lipid-exposed α-helix, M4, which, despite being distant from the primary allosteric pathway coupling agonist binding to channel gating, exercises significant control over channel function. Here, I present evidence that M4 acts as a lipid sensor, detecting changes in the surrounding lipids and transmitting these changes to the channel pore via contacts with the adjacent TMD α-helices, M1 and M3, and/or with structures in the extracellular domain. Using ELIC and GLIC chimeras, I first show that the TMD is the main driver of pLGIC thermal stability. I then demonstrate that the M4 α-helices in each channel play different roles in channel maturation and function, which suggests a divergent evolutionary path. Following this, I show that the M4 C-terminus is essential to both maturation and function in GLIC, while in ELIC its role is less defined, again showcasing possible evolutionary differences. Building on these findings, I examined the role of aromatic residues at the M4 – M1/M3 interface, and found that they predictably determine the interactions between M4 and M1/M3. Notably, the addition of aromatic residues to enhance M4-M1/M3 interactions in ELIC promotes channel function, while the elimination of aromatic residues at the M4-M1/M3 interface in GLIC is detrimental to channel function. Furthermore, I show that these same aromatics alter the strength of pLGIC lipid sensing and the sensitivity to certain disease-causing mutations, both indicating that aromatic residues are key players in channel function, stability and modulation. Finally, I and my collaborators identified and characterized a novel desensitization-linked lipid binding site in ELIC. Extensive mutagenesis studies coupled with biophysical measurements allowed us to develop a model describing how lipid binding influences the rates of ELIC desensitization to shape the agonist-induced response.

Les mécanismes adhésifs jouent un rôle crucial en biologie. Les cadhérines classiques constituent une des principales familles d'adhésion cellulaire dépendante du calcium. Ces glycoprotéines transmembranaires sont impliquées dans des interactions principalement homophiles. Ces interactions régulent des voies de signalisation impliquées dans de nombreux phénomènes biologiques. Cette thèse porte sur l'étude comparative des dynamiques d'interactions des cadhérines E- et -11, prototypes respectivement des cadhérines classiques de type I et II. Le ciblage d'acides aminés particuliers de l'interface adhésive nous a permis de montrer que pour les cadhérines de type I, l'échange de brin avec le Trp2 ont un rôle clé ; pour les types II un mécanisme différent intervient. Nous avons aussi développé une chimie innovante pour contrôler l'immobilisation orientée et covalente de protéines. Enfin une revue décrit une étude de l'activation de voies de signalisation par engagement des cadhérines. / Cell adhesion receptors of the classical cadherin family are involved in Ca2+-dependent homophilic interactions. In order to dissect the molecular mechanisms of cadherin-based cellcell adhesion, this Ph.D. thesis describes a comparative dynamic study of interactions between cadherins E- & -11, chosen as classical type I and II cadherins prototypes respectively. Modifications of particular residues in the E-cadherin adhesive interface showed that the ?-strand exchange with its Trp2 had a prominent feature; for type II cadherins, a different mechanism was described involving a larger domain swapping. We then developed a new protocol for immobilizing proteins in an orientated and covalent manner on surfaces. These interactions regulate signalization pathways in various biological processes. Studies describing Stat3 activation through direct cadherin engagement are reviewed.