CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / This work reports the biochemical characterization, cytotoxic and allergenic effects of a lipid transfer protein isolated from M. citrifolia seeds (McLTP1), with trypsin and alpha-amylase inhibition properties. McLTP1 was purified with a procedure involving trichloroacetic acid precipitation and gel filtration chromatography. This protein showed significant inhibitory activities against trypsin (767,10  8,36 TIU/mgP), chymotrypsin (25,36  0,86 IU/mgP), papain (65,419  0,152 IU/mgP) and alpha-amylase (24,40%). Atomic force microscopy displayed that McLTP1 oligomerized in tetramers showing a central channel. Fluorescence and CD assays revealed that the McLTP1 structure is highly stable, regardless of pH and temperature levels. In vitro, McLTP1 presented a selective cytotoxic effect to human ovarian cancer cells (OVCAR-8; IC50 of 16,6 μg/mL) and demonstrated hemolytic effect against fresh rabbit red blood cels. Similarly to other non-specific lipid transfer protein reported, McLTP1 showed allergenic properties in mice, being considered as a true food allergen since it was able to sensitize the animals via the gastrointestinal tract. / Morinda citrifolia L. à uma espÃcie nativa do Sudeste da Ãsia intensamente investigada em funÃÃo de suas propriedades terapÃuticas reportadas hà mais de 2.000 anos. Recentemente, uma proteÃna transferidora de lipÃdeos denominada McLTP1 (UniProt Accession Number: C0HJH5) foi isolada de sementes de noni pelo nosso grupo de pesquisa. McLTP1 à uma proteÃna termoestÃvel de massa molecular 9,4 kDa, resistente à proteÃlise e dotada de atividades moduladoras da inflamaÃÃo e da dor pela via oral, promissoras e inÃditas para esse grupo de molÃculas. Este trabalho objetivou caracterizar bioquimicamente McLTP1, bem como avaliar o seu potencial alergÃnico em camundongos, como etapas bÃsicas para o seu uso racional e seguro do ponto de vista farmacolÃgico. Em adiÃÃo, as propriedades terapÃuticas de McLTP1 foram tambÃm ampliadas, atravÃs da investigaÃÃo de seu efeito citotÃxico em diferentes linhagens de cÃlulas tumorais. A proteÃna em estudo foi isolada utilizando o protocolo jà estabelecido, envolvendo as etapas de precipitaÃÃo seletiva de proteÃnas do extrato total das sementes de noni com Ãcido tricloroacÃtico 2,5% e cromatografia de exclusÃo molecular. O ensaio de alergenicidade in vivo foi conduzido apÃs prÃvia aprovaÃÃo pelo Comità de Ãtica para Uso de Animais da Universidade Federal do Cearà e utilizou fÃmeas nulÃparas com massa corporal entre 25 e 30 g. McLTP1 apresentou in vitro atividades inibitÃrias de tripsina (767,10  8,36 UIT/mgP), quimotripsina (25,36  0,86 UI/mgP), papaÃna (65,419  0,152 UI/mgP) e alfa-amilase (24,40%). A atividade inibitÃria de tripsina de McLTP1 foi reduzida significativamente em temperaturas superiores a 37 ÂC, apresentando atividade residual de apenas 5,91% quando aquecida a 100 ÂC por 30 min. Essa atividade foi tambÃm influenciada pelo pH, sendo de apenas 30,13% e 39,05% quando a proteÃna foi incubada em tampÃes de pH 3,0 e 12,0. O padrÃo de oligomerizaÃÃo de McLTP1 demonstrou a formaÃÃo de agregados dimÃricos/tetramÃricos delimitando um canal central de diÃmetro de 4,4 nm. As anÃlises espectroscÃpicas mostraram que McLTP1 apresenta espectro de CD similar Ãquele apresentado por outras proteÃnas transferidoras de lipÃdeos e caracterÃstico de proteÃnas ricas em alfa-hÃlice. Espectro de CD de McLTP1 nÃo mostrou alteraÃÃes significativas em diferentes temperaturas e pHs, corroborando com os dados de estabilidade obtidos anteriormente. Diferentemente, em condiÃÃes redutoras (DTT 1 mM) o espectro de CD mostrou alteraÃÃo na estrutura secundÃria da proteÃna e os mÃnimos e mÃximos de elipticidade molar foram tambÃm alterados na presenÃa de micelas iÃnicas de SDS (10 mM). McLTP1 apresentou atividade citotÃxica seletiva contra cÃlulas de cÃncer de ovÃrio (Ovcar-8; CI50: 16,6 μg/mL), nÃo sendo citotÃxica para as cÃlulas tumorais de cÃlon humano (HCT-116), leucemia humano (HL-60) e glioblastoma humano (SF-295) testadas. McLTP1 foi capaz de promover hemÃlise significativa em hemÃcias de coelho a partir da concentraÃÃo de 0,005 mgP/mL. McLTP1 apresentou potencial efeito alergÃnico in silico e em camundongos imunizados pela via oral, induzindo a sÃntese de anticorpos IgG e IgG1. Tal como descrito na literatura para outras LTPs, anticorpos anti-McLTP1 produzidos em coelho foram tambÃm capazes de reconhecer proteÃnas presentes em extratos de Rosaceae, Cucurbitaceae e na polpa do fruto de noni. Os dados obtidos permitiram caracterizar parcialmente a proteÃna em estudo, bem como avaliar o seu potencial imunogÃnico apÃs administraÃÃo oral. Novos testes serÃo conduzidos objetivando avaliar a importÃncia clÃnica dessas respostas, uma vez que testes de toxicidade demonstraram que McLTP1 nÃo foi capaz de promover reaÃÃes adversas em camundongos, mesmo apÃs administraÃÃo da dose de 8 mg/kg por 28 dias.

Submitted by Maria Beatriz Vieira (mbeatriz.vieira@gmail.com) on 2018-06-11T17:00:10Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) resumo_tese_gabriela_peres_moraes_de_freitas.pdf: 25733 bytes, checksum: db07dd8031202634a6a7a48362f489ff (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2018-06-11T18:28:39Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) resumo_tese_gabriela_peres_moraes_de_freitas.pdf: 25733 bytes, checksum: db07dd8031202634a6a7a48362f489ff (MD5) / Made available in DSpace on 2018-06-11T18:28:39Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) resumo_tese_gabriela_peres_moraes_de_freitas.pdf: 25733 bytes, checksum: db07dd8031202634a6a7a48362f489ff (MD5) Previous issue date: 2017-06-23 / Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul - FAPERGS / Devido a sua origem tropical, o arroz (Oryza sativa L.) é muito impactado pelo estresse causado pelas baixas temperaturas e consequentemente apresenta grandes perdas de crescimento e produtividade. É o segundo cereal mais consumido no mundo e o decremento na produção causado por eventos de temperatura extrema, pode ocasionar impactos significativos na economia mundial. A sensibilidade e os sintomas das respostas das plantas ao estresse por baixas temperaturas variam com o estádio de desenvolvimento. Observa-se que no estádio inicial ocorre um retardo e diminuição da germinação das sementes e no vegetativo, clorose foliar, baixa estatura e diminuição do perfilhamento. Já no reprodutivo ocorre o aborto e esterilidade das espiguetas, exerção incompleta da panícula, anormalidades no desenvolvimento das anteras, mancha dos grãos e maturação tardia e incompleta dos mesmos. Este trabalho teve como objetivo avaliar nos estádios vegetativo e reprodutivo, os mecanismos adaptativos de resposta ao estresse por baixas temperaturas, a nível molecular, bioquímico e fisiológico. Foram analisados 22 genótipos de arroz, da coleção “mini-core” do USDA para tolerância ao frio, in vitro, através de parâmetros morfológicos. Destes 22, foram selecionados quatro genótipos (dois tolerantes e dois sensíveis), para avaliação, no estádio vegetativo, de trocas gasosas, análises bioquímicas, expressão gênica e de proteínas. As análises de trocas gasosas e expressão gênica foram repetidas durante o estádio reprodutivo. Os primeiros efeitos do estresse por baixa temperatura foram identificados na fotossíntese em todos os genótipos e em ambos os estádios. O perfil bioquímico e dos genes de proteínas de transferência de lipídeos (LTPs), OsGH3-2, OsSRO1a, OsZFP245, OsTPP1 e as proteínas LRR-RLKs, BHLH, GLYI e LTP1, no estádio vegetativo, demonstraram que, apesar dos impactos da baixa temperatura nos genótipos tolerantes, houve um ajuste rápido para que a homeostase celular fosse mantida, mostrando uma clara diferença na expressão gênica entre os genótipos. Foi visualizado também que a expressão dos genes OsLTP7, OsLTP10, fatores de transcrição e genes induzidos por baixa temperatura foram maiores nos genótipos tolerantes, em ambos os tecidos. O estresse por baixa temperatura afetou severamente os parâmetros de produção, tendo uma perda no rendimento dos grãos de 40 e 90%, nos genótipos tolerantes e sensíveis, respectivamente. Assim, este estudo identificou o genótipo Nipponbare como tolerante a baixas temperaturas em ambos os estádios. Os genes LTP, OsGH3-2, OsSRO1a, OsZFP245 e OsTPP1 e as proteínas LRR-RLKs, bHLH, GLYI e LTP1, são bons candidatos para o “screening” de tolerância a baixas temperaturas, no vegetativo, enquanto que as OsLTP7, OsLTP10, OsNAC9, OsNAC10 e OsNAP podem ser usados, com o mesmo fim, no reprodutivo. / Due to its tropical origin, rice (Oryza sativa) is very impacted by cold stress and consequently presents great losses of growth and yield. It is the second most consumed cereal in the world and the decrease in production caused by extreme temperature events can have significant impacts in the world economy. Sensitivity and symptoms of plant responses to stress due to low temperatures vary with the stage of developmental. It is observed that in the first developmental stage there is a delay and decrease of seed germination and on vegetative stage, foliar chlorosis, short stature and decrease of the profile. It is observed that in the first development stage there is a delay and decrease of the germination of the seeds and in the vegetative stage, leaf chlorosis, short stature and reduction of tillering. At reproductive stage, the abortion and sterility of the spikelets, incomplete panicle excretion, abnormalities in the development of the anthers, grain spots and late and incomplete maturation of the grain. The purposed of this study was to evaluate the adaptive mechanisms of response to stress induced by low temperatures at the molecular, biochemical and physiological levels at the vegetative and reproductive stages. Twenty-two rice genotypes from the USDA mini-core collection for cold tolerance, in vitro, were analyzed through morphological parameters. Of these 22, four genotypes (two tolerant and two sensitive) were selected for vegetative stage evaluation of photosynthetic parameters, biochemical and gene and protein expression. Analysis of photosynthetic parameters and gene expression were repeated during the reproductive stage. The first effects of low temperature stress were identified in photosynthesis in all genotypes and in both stages. Biochemical profile and genes of lipid transfer proteins (LTPs), OsGH3-2, OsSRO1a, OsZFP245, OsTPP1 and the LRR-RLKs, BHLH, GLYI and LTP1 proteins at the vegetative stage have demonstrated that, despite impacts of low temperature in the tolerant genotypes, had a fast adjustment for cellular homeostasis to be maintained, showing a clear difference in gene expression between the genotypes. It was also visualized that the expression of OsLTP7, OsLTP10, transcription factors and low temperature genes were higher in the tolerant genotypes in both tissues. Low temperature stress severely affected production parameters, with a yield loss of 40 and 90% in tolerant and sensitive genotypes, respectively. Therefore, this study identified the genotype Nipponbare as possible tolerant to low temperatures in both stages. LTP genes, OsGH3-2, OsSRO1a, OsZFP245 and OsTPP1, and the porteins LRR-RLKs, bHLH, GLYI and LTP1 are good candidates at the screening for cold tolerance at the vegetative stage, whereas OsLTP7, OsLTP10, OsNAC9, OsNAC10 and OsNAP can be used, for the same purpose, at reproductive stage.

La cuticule est une couche hydrophobe déposée à la surface des organes aériens des plantes terrestres. Elle joue de nombreux rôles allant de la résistance face à divers stress biotiques et abiotiques à son implication dans divers processus de développement. Bien que la compréhension de la biosynthèse des composés cuticulaires a considérablement augmenté ces dernières années, les mécanismes de transport de ces lipides cuticulaires à travers la paroi et d'assemblage au sein de la cuticule, sont encore peu caractérisés. Les nsLTPs (non-specific Lipid Transfer Protein), sont codées par une famille multigénique impliquée dans de nombreuses fonctions biologiques. Parmi les rôles proposés pour les nsLTPs, il est supposé depuis longtemps qu'elles pourraient transporter les précurseurs cuticulaires à travers la paroi et ainsi contribuer à la formation de la cuticule. Dans ce travail de thèse, nous nous sommes servis du modèle des hypocotyles étiolés d'A. thaliana afin de caractériser le fonction biologique d'AtLTP2. En effet, AtLTP2 est une protéine de transfert de lipides présente de façon abondante et est la seule représentante des nsLTPs dans le protéome pariétal des hypocotyles étiolés. Nous avons tout d'abord confirmé que l'expression d'AtLTP2 est forte dans les très jeunes stades de développement de la plantule étiolée et est restreinte aux cellules de l'épiderme des organes aériens, sur lesquelles se dépose la cuticule. Conformément aux résultats de protéomique obtenus au préalable, AtLTP2 fusionnée à un marqueur fluorescent est localisée au niveau de la paroi mais également et de façon surprenante au niveau des plastes. Cette remarquable double localisation d'une nsLTP dans la paroi et dans les plastes n'a à ce jour, jamais été décrite. De plus, le mode de transport d'AtLTP2 vers les plastes est particulièrement original puisque la protéine emprunte d'abord la voie de sécrétion avant d'être finalement adressée aux plastes. En analysant l'adressage de différentes versions d'AtLTP2 tronquée, nous avons pu montrer que c'est certainement sa conformation tertiaire qui est cruciale pour sa localisation plastidiale. Par des approches de génétique inverse, nous avons pu montrer que les mutants atltp2 présentaient une augmentation importante de la perméabilité de sa cuticule fortement corrélée à une ultrastructure de l'interface cuticule-paroi très perturbée alors qu'aucun changement dans la composition biochimique de la cuticule n'a été détecté. Ces résultats nous ont permis de proposer un nouveau rôle structural pour AtLTP2, elle interviendrait pour maintenir l'adhésion des deux couches que sont la cuticule hydrophobe et la paroi hydrophile. Ainsi, en maintenant l'intégrité de l'interface entre la cuticule et la paroi, AtLTP2 participerait au maintien de la fonction de barrière cuticulaire limitant les pertes d'eau. De façon intéressante, la double localisation d'AtLTP2 dans la paroi et les plastes nous laisse supposer que d'autres fonctions pourraient être assignées à AtLTP2. L'identification des mécanismes moléculaires mis en jeu dans le maintien de l'homéostasie cuticule-paroi et dans la double localisation d'AtLTP2 constituera un challenge pour de futures recherches visant à toujours mieux identifier les acteurs de la formation de cette barrière protectrice, la cuticule. / The cuticle is a hydrophobic layer that covers the surface of the aerial organs of land plants. The cuticle plays numerous roles in plants from resistance against biotic and abiotic stresses to several developmental processes. Although the understanding of the biosynthesis of cuticle has considerably increased last years, the mechanisms underlying the transport of cuticular lipids through the cell wall and their assembly within the cuticle have been poorly characterized. nsLTPs (non-specific Lipid Transfer Proteins) are encoded by a multigenic family in A. thaliana and are involved in several biological processes. Among the different roles proposed for nsLTPs, it has long been suggested that they could transport cuticular precursor across the cell wall and then could contribute to the cuticle formation, despite the absence of formal evidence for individual members. Here we took advantage of the A. thaliana etiolated hypocotyls model to characterize the biological function of AtLTP2. Indeed, AtLTP2 was found to be abundant and the unique nsLTP member in the cell wall proteome of etiolated hypocotyls. We have first confirmed the high level of AtLTP2 expression during the young developmental stages of etiolated seedlings that was restricted to the epidermal cells of aerial organs, that are covered by the cuticle. In agreement with the cell wall localization determined by previous proteomic studies, we localized AtLTP2 fused to a fluorescent marker to the cell wall, but also and surprisingly to the plastids. This remarkable dual localization in the cell wall and plastids was never described before for a nsLTP. Furthermore, the mechanism of AtLTP2 transport to the plastids was particularly original because AtLTP2 can first undergo import into the ER/ secretory pathway and then sorting to the cell wall and the plastids. By studying the sub-cellular localization of truncated version of AtLTP2, we have shown that its tertiary conformation was crucial for the plastidial localization. By using reverse genetic approaches, we have shown that atltp2 mutants displayed a high increase in cuticle permeability strongly correlated with a deep modification of the ultra-structure at the cuticle-cell wall interface, while no changes in biochemical composition of the cuticle were detected. These results prompt us to suggest a novel structural role for AtLTP2. AtLTP2 could be involved in maintaining the accurate sealing between the hydrophobic cuticle and the hydrophilic underlying cell wall. Then, by preserving the integrity of the cuticle-cell wall interface, AtLTP2 could act on the barrier function of the cuticle limiting water loss. Interestingly, the dual localization to the cell wall and plastids suggested that other functions could be assigned to AtLTP2. The elucidation of the molecular mechanisms by which AtLTP2 establish cell wall-cuticle homeostasis and the exact function of the dual targeting will be challenging tasks in the future to better identify the main actors of the formation of the cuticle.

La protéine OSBP est un transporteur de lipides qui régule la distribution cellulaire du cholestérol. OSBP comprend un domaine PH, deux séquences « coiled coil », un motif FFAT (deux phénylalanines dans un environement acide), et un domaine de liaison de lipides (ORD) à son extrémité C-terminale. Le domaine PH interagit avec le PI(4)P et la petite protéine G Arf1-GTP au niveau du Golgi, alors que le motif FFAT interagit avec la protéine VAP-A, résidente du réticulum endoplasmique (RE). En liant simultanément tous ces déterminants, OSBP stabilise des sites de contact membranaire entre RE et Golgi, permettant ainsi un contre-échange cholestérol / PI(4)P par l'ORD. OSBP contient également une longue séquence N-terminale d’environ 80 aa, intrinsèquement désordonnée, composée principalement de glycine, proline et d'alanine. Nous démontrons que la présence de ce N-terminus désordonné augmente le rayon de Stoke de OSBP tronquée du domaine ORD, et limite sa densité d’association sur la membrane portant le PI(4)P. La protéine dépourvue du N terminus favorise l'agrégation symétrique des liposomes PI(4)P (mimant la membrane du Golgi) par les deux domaines PH du dimère OSBP, alors que la présence de la séquence désordonnée empêche cette association symétrique. De même, nous observons que la distribution d’OSBP sur la membrane de vésicules unilamellaires géantes (GUV) varie selon la présence ou l'absence du N-terminus. En présence de la séquence désordonnée, la protéine est répartie de manière homogène sur toute la surface du GUV, alors que la protéine sans N-terminal a tendance à s'accumuler à l'interface entre deux GUV de type Golgi. Cette accumulation locale ralentit fortement la mobilité de la protéine à l’interface. Un effet similaire du N-terminal sur la dynamique des protéines est observé lorsque l’association de membranes de type ER et Golgi est assuré par des protéines monomériques (dépourvue du coiled coil) en présence de Vap-A. Les résultats de nos expériences in vitro ont été confirmés en cellules vivantes, où la séquence intrinsèquement désordonnée contrôle le recrutement d’OSBP sur les membranes Golgiennes, sa mobilité et sa dynamique d’activité au cours des cycles de transfert de lipides. La plupart des protéines de la famille d’OSBP contiennent des séquences N-terminales de faible complexité, suggérant un mécanisme général de régulation. / Oxysterol binding protein (OSBP) is a lipid transfer protein that regulates cholesterol distribution in cell membranes. OSBP consists of a pleckstrin homology (PH) domain, two coiled-coils, a “two phenylalanines in acidic tract” (FFAT) motif and a C-terminal lipid binding OSBP-Related Domain (ORD). The PH domain recognizes PI(4)P and small G protein Arf1-GTP at the Golgi, whereas the FFAT motif interacts with the ER-resident protein VAP-A. By binding all these determinants simultaneously, OSBP creates membrane contact sites between ER and Golgi, allowing the counter-transport of cholesterol and PI(4)P by the ORD. OSBP also contains an intrinsically disordered ~80 aa long N-terminal sequence, composed mostly of glycine, proline and alanine. We demonstrate that the presence of disordered N-terminus increases the Stoke’s radius of OSBP truncated proteins and limits their density and saturation level on PI(4)P-containing membrane. The N-terminus also prevents the two PH domains of OSBP dimer to symmetrically tether two PI(4)P-containing (Golgi-like) liposomes, whereas protein lacking the disordered sequence promotes symmetrical liposome aggregation. Similarly, we observe a difference in OSBP membrane distribution on tethered giant unilamellar vesicles (GUVs), based on the presence/absence of N-terminus. Protein with disordered sequence is homogeneously distributed all over the GUV surface, whereas protein without N-terminus tends to accumulate at the interface between two PI(4)P-containing GUVs. This protein accumulation leads to local overcrowding, which is reflected by slow in-plane diffusion. The effect of N-terminus is also manifested in monomeric OSBPderived proteins that tether ER-like and Golgi-like membranes in the presence of VAP-A. Findings from our in vitro experiments are confirmed in living cells, where N-terminus controls the recruitment of OSBP on Golgi membranes, its motility and the on-and-off dynamics during lipid transfer cycles. Most OSBP-related proteins contain low complexity N-terminal sequences, suggesting a general effect.