Orientador: Áureo Tatsumi Yamada / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T04:02:31Z (GMT). No. of bitstreams: 1 Lima_PatriciaDanieleAzevedo_D.pdf: 2840118 bytes, checksum: 470a2cf0b7d63e81fd8f48aba5af44fc (MD5) Previous issue date: 2012 / Resumo: Células Natural Killer uterina (uNK) produzem moléculas angiogênicas e citocinas críticas ao sucesso da gestação , assim como proteínas citolíticas relacionadas à resposta imune inata. Contudo, se as capacidades angiogênicas e citolíticas são provenientes de diferentes subpopulações de células uNK não é conhecido; da mesma forma, estes fenótipos ainda não são estabelecidos. Assim, a proposta inicial deste trabalho foi avaliar...Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: Angiogenic and cytokine molecules produced by uterine natural killer (uNK) cells are critical for successful pregnancy. Cytolytic proteins are also express by uNK cells. However, it is unknown whether the angiogenic and cytolytic capacities are from different uNK subsets, or the same cells. Thus, we initially proposed to evaluate...Note: The complete abstract is available with the full electronic document / Doutorado / Histologia / Doutor em Biologia Celular e Estrutural

Orientador: Beatriz Appezzato da Glória, Vera Lúcia Garcia Rehder / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T10:55:10Z (GMT). No. of bitstreams: 1 Bombo_AlineBertolosi_M.pdf: 4141417 bytes, checksum: 23d44ff8ee2ca0a59a65cebea23db4a2 (MD5) Previous issue date: 2012 / Resumo: A família Asteraceae é uma das maiores famílias de angiospermas, com ampla distribuição e hábitos muito variados. O gênero Viguiera pertence à subtribo Heliantheae e conta com aproximadamente 180 espécies. No Brasil ocorrem 35 espécies e 27 dessas são endêmicas. Por se tratar de um gênero com muitos representantes, as relações filogenéticas não estão bem estabelecidas e estudos morfológicos e moleculares têm sido realizados com o objetivo de auxiliar a circunscrição do gênero. As espécies de Viguiera possuem potencial resinífero e as contempladas neste estudo são aromáticas em seu ambiente natural. Diversos estudos fitoquímicos têm destacado o potencial farmacológico de espécies do gênero, atribuído principalmente, à atividade das lactonas sesquiterpênicas e dos diterpenos. No entanto, estudos sobre a composição química dos óleos essenciais são escassos. A ocorrência das espécies do gênero Viguiera no Brasil se dá, principalmente, em regiões com inverno seco, devido à presença de um sistema subterrâneo espessado. O sistema subterrâneo das espécies incluídas neste estudo, Viguiera filifolia, V. linearifolia e V. trichophylla, consiste em um xilopódio, que apesar da capacidade gemífera, não possui capacidade de propagação vegetativa. As três espécies são muito semelhantes morfologicamente em estado vegetativo sendo diferenciadas principalmente por caracteres reprodutivos. Há registros na literatura de confusões na delimitação das mesmas. Além disso, V. filifolia e V. trichophylla constam na lista vermelha das espécies brasileiras como criticamente em perigo. Assim, os estudos anatômicos visaram contribuir com o levantamento de caracteres diagnósticos entre as três espécies. Os dados referentes à composição dos óleos essenciais também podem ser úteis do ponto de vista taxonômico, auxiliando na delimitação das espécies e ainda, no levantamento de possíveis compostos bioativos. Por fim, o estudo da germinação das sementes visou fornecer dados para futuros estudos de cultivo dessas espécies / Abstract: Asteraceae is one of the largest families of angiosperms with wide distribution and habits varied. Viguiera genus is included in the Heliantheae subtribe and has approximately 180 species. In Brazil, there are 35 species and 27 are endemic. Given that this genus has many representatives, phylogenetic relationships are not well established and morphological and molecular studies have been conducted to help limit the genus. Viguiera species have resin potential and the species used in this study are aromatic in their natural environment. Several phytochemical studies have shown the pharmacological potential to Viguiera species, attributed mainly to the activity of sesquiterpene lactones and diterpenes. However, studies on the chemical composition of essential oils are rare. In Brazil, the Viguiera species occurs mainly in regions of dry winter, due the presence of a thickened underground system. The underground system of the species used in this study, Viguiera filifolia, V. linearifolia and V. trichophylla is represented by a xylopodium, incapable of vegetative propagation despite forming buds. The three species are morphologically very similar in vegetative state and could be differentiated only by reproductive features. In the literature, there is confusion in individuating them. In addition, V. filifolia and V. trichophylla are classified in the Brazilian Species lias critically endangered. Thus, anatomical studies aimed at contributing to obtain diagnostic features among the three species. The essential oil composition data could help in chemosystematics and in the identification of possible chemotherapeutic compounds. Finally, the study of seed germination aimed to provide information for future studies for the cultivation of these species / Mestrado / Biologia Vegetal / Mestre em Biologia Vegetal

La voie de sécrétion bactérienne de type V permet l’exportation à la surface cellulaire de protéines dont certaines ont été identifiées comme d’importants facteurs de la pathogénicité bactérienne. Le type V regroupe la sécrétion des autotransporteurs et la sécrétion à deux partenaires (TPS). Les autotransporteurs sont constitués d’un domaine en tonneau β; et d’un domaine passager. L’interaction de l’autotransporteur avec le complexe protéique Bam, dont la pièce centrale est le transporteur BamA, permet l’insertion dans la membrane externe du tonneau β; et la sécrétion du passager. En revanche, la sécrétion à deux partenaires fait intervenir deux protéines, l’une appelée TpsA correspondant à la protéine exportée et l’autre, TpsB, formant un tonneau β qui contrôle le transport à travers la membrane externe. Les protéines TpsB sont spécifiques à leur(s) TpsA associée(s), et font partie de la superfamille des transporteurs Omp85 qui effectuent l’insertion de protéines dans la membrane externe bactérienne comme BamA, et dans celles des organites eucaryotes dont les chloroplastes et les mitochondries. Au cours de mon doctorat, je me suis intéressé à la sécrétion de l’hémagglutinine filamenteuse (FHA), qui est l’adhésine majoritaire de Bordetella pertussis, l’agent étiologique de la coqueluche. Cette adhésine qui permet à la bactérie de coloniser le tractus respiratoire de l’hôte est une protéine TpsA de 220 kD. Elle est très efficacement sécrétée par la voie de sécrétion à deux partenaires grâce à son transporteur spécifique TpsB nommé FhaC. L’étude cristallographique de FhaC a révélé un tonneau β; à 16 brins qui forme un canal dans la membrane externe obstrué par l’hélice-α; amino-terminale, H1, partagée par la majorité des TpsB, et par une boucle de surface, L6, conservée dans la superfamille Omp85. Cette conformation suggère un état au repos dans lequel le canal bouché ne pourrait pas transporter son partenaire. Afin de comprendre comment la FHA transite à l’intérieur du pore, il est donc nécessaire de connaître les changements de conformations que subit FhaC. Durant mon travail de thèse, nous avons apporté une vision plus dynamique de la sécrétion à deux partenaires en utilisant le couple FHA/FhaC comme modèle d’étude. Pour cela nous avons utilisé principalement la Résonance Paramagnétique Electronique (RPE). Cette technique de biophysique permet d’étudier FhaC en solution ou réincorporée dans une bicouche lipidique et de rendre compte de la mobilité à un site donné par l’utilisation de sondes paramagnétiques. Ainsi nous avons pu montrer que FhaC est en équilibre entre plusieurs conformations, avec H1 dans le pore ou du côté périplasmique de FhaC. La présence de la FHA déplace cet équilibre, favorisant ainsi la sortie de l’hélice hors du pore. Nous avons, par ailleurs, pu démontrer expérimentalement que la FHA transitait bien à l’intérieur du pore formé par FhaC et que l’hélice H1 se trouvait alors dans le périplasme. L’étude de la boucle L6 nous a permis de montrer que la mobilité de cette boucle était fortement contrainte à l’intérieur du pore même lors de la reconnaissance avec la FHA. Ce ralentissement de mobilité est lié, en autre, à une interaction avec un résidu d’un motif conservé présent sur le brin β13 qui influence la taille du pore. De manière plus générale, cette étude de la dynamique de FhaC contribue à la compréhension des mécanismes moléculaires de la voie TPS et des transporteurs de la superfamille Omp85. / Type V secretion in bacteria mediates the export to the cell surface of proteins, some of which have been identified as important factors of pathogenicity. Type V includes the secretion of autotransporters and the ‘Two-partner Secretion’ (TPS) pathway. Autotransporters consist of a β barrel domain and a passenger domain. The interaction of autotransporters with the Bam complex, of which the BamA transporter is the central component, allows the insertion of the β; barrel in the outer membrane and the secretion of passenger domain. In contrast, the two-partner secretion involves two proteins, the exported ‘TpsA’ protein and its TpsB partner that controls its transport across the outer membrane. TpsB proteins are specific to their associated TpsA(s) and belong to the superfamily of the Omp85 transporters, which carry out the insertion of proteins into the bacterial outer membrane, like BamA, or in the outer membranes of eukaryotic organelles including chloroplasts and mitochondria. For my PhD work, I have been interested in the secretion of filamentous hemagglutinin (FHA), which is the major adhesin of Bordetella pertussis, the causative agent of whooping cough. This adhesin allows the colonization by this bacterium of its host’s respiratory tract. This protein corresponds to a 220kD TpsA protein efficiently secreted by its specific transporter TpsB named FhaC. Crystallographic studies have revealed that FhaC harbours a 16-stranded β;-barrel occluded by both the N-terminal α;-helix, H1, shared by the majority of TpsB proteins, and by a surface loop, L6, that carries a conserved, hallmark motif of the Omp85 superfamilly. This conformation suggests that FhaC is in a resting state in which the channel does not transport its partner. To understand how the FHA passes through the FhaC pore, it is necessary to address the conformational changes undergone by FhaC. During my thesis work, we provided a more dynamic view of the TPS pathway using the FHA/FhaC couple as study model. For this we used Electron Paramagnetic Resonance (EPR). This biophysical technique allows to study of FhaC in solution or reincorporated into a lipid bilayer and it reports the mobility at specific sites of the protein by using paramagnetic probes. Thus we have shown that FhaC is in equilibrium between multiple conformations, with H1 in the pore or at the periplasmic side of FhaC. The presence of FHA displaces the conformational equilibrium, promoting the exit of the helix going from the pore. We have also experimentally demonstrated that FHA does transit through the pore formed by FhaC while helix H1 is then in the periplasm. The study of the L6 loop enabled us to show that the mobility of this loop is highly constrained in the pore and remains so upon the recognition of FHA. Its slow mobility is linked to an interaction between an invariant L6 residue and a conserved motif present on the β; strand 13 of the barrel. This interaction affects the size of the FhaC pore.More generally, the study of the dynamics of FhaC contributes to the understanding the molecular mechanisms of the TPS pathway and of transporters of the Omp85 superfamily.

Parmi les cinq isotypes d’anticorps présents chez l’Homme, les immunoglobulines A (IgA) prédominent dans les muqueuses. Les IgA sont produites dans le chorion sous forme majoritairement polymérique (pIgA) et sécrétées dans la lumière des muqueuses. Leur transport trans-épithélial est assuré par le récepteur aux immunoglobulines polymériques (pIgR), exprimé au pôle basal des épithéliums. Les pIgA ainsi sécrétées conservent le domaine extracellulaire du pIgR et sont appelées IgA sécrétoires (SIgA). Une fonction essentielle des SIgA intestinales est de maintenir microorganismes et antigènes alimentaires dans la lumière des muqueuses afin d’empêcher leur pénétration dans l'organisme. Néanmoins, la transcytose inverse des SIgA couplées à des bactéries a été décrite dans les plaques de Peyer où elle participe à la génération de la réponse immune contre ces bactéries. En outre, les travaux passés du laboratoire ont suggéré que le récepteur de la transferrine (CD71), surexprimé au pôle apical des entérocytes chez les patients cœliaques, interagit avec les SIgA couplées à des peptides de gliadines et permet leur transcytose inverse à travers l’épithélium intestinal. Ce travail de thèse a eu pour objectif de mieux caractériser l’interaction SIgA-CD71 dans les entérocytes humains. Dans un premier temps, nous avons utilisé la technologie CRISPR-Cas9 pour générer une lignée cellulaire intestinale humaine (Caco-2 TC7) dépourvue du récepteur CD71 (CD71KO). De manière inattendue, nous n’avons observé aucune altération de la fixation des SIgA sur les cellules Caco-2 CD71KO. Ce résultat nous a amenés à réévaluer le rôle de CD71 comme récepteur aux SIgA. Dans un second temps, nous avons vérifié et confirmé que les SIgA interagissent avec CD71, mais nous avons montré que cette interaction est indirecte. Nous avons ensuite criblé les récepteurs aux IgA déjà décrits dans la littérature et montré qu'aucun n'est responsable de la fixation des SIgA à la surface des cellules Caco-2 TC7. Ces résultats nous ont amenés à chercher le(s) autre(s) partenaire(s) du complexe SIgA-CD71. Par immunoprécipitation couplée à la spectrométrie de masse, nous avons identifié les protéines Secretory Carrier Membrane Protein 3 (SCAMP3), B-Cell Receptor-Associated Protein 31 (BCAP31) et Histocompatibility minor 13 (HM13) comme membres du complexe SIgA-CD71. En nous appuyant à nouveau sur la technologie CRIPSR-Cas9, nous avons montré qu’aucun de ses partenaires n’interagit directement avec les SIgA. Néanmoins, nos résultats suggèrent que SCAMP3 est nécessaire à l’oligomérisation de surface des complexes de fixation des SIgA. Enfin, l’internalisation des SIgA n'est pas altérée par l’absence de chacun des partenaires du complexe, suggérant que leur rôle advient durant le transport trans-épithélial des SIgA. L’ensemble de nos travaux montre que les SIgA interagissent avec l'épithélium intestinal via un complexe protéique composé d'au moins cinq membres : CD71, SCAMP3, BCAP31, HM13 et le(s) récepteur(s) inconnu(s) aux SIgA. Ces résultats complètent les travaux antérieurs sur le rôle physiopathologique des SIgA dans la maladie cœliaque et contribuent à souligner la complexité des interactions entre IgA et entérocytes. Une question importante sera d’identifier le(s) récepteur(s) aux SIgA et de déterminer le rôle des partenaires identifiés dans la transcytose inverse des SIgA par l’entérocyte. / Among the five isotypes of antibodies found in Humans, immunoglobulins A (IgA) are the most abundant in the mucosae. In the lamina propria, IgA are produced mainly as polymeric IgA (pIgA) and then secreted in the lumen. In fact, pIgA are translocated across the epithelium via the polymeric immunoglobulin receptor (pIgR), which is expressed at the basolateral side of the epithelium. Once secreted in the lumen, pIgA retain the extracellular domain of the pIgR and are called secretory IgA (SIgA). One of the main functions of intestinal SIgA is to restrain microorganisms and dietary antigens in the lumen, therefore, preventing their uptake through the epithelial barrier. However, retrotranscytosis of SIgA conjugated to bacteria has been described in Peyer’s patches where it participates to immune response. Moreover, previous works from the laboratory have suggested that transferrin receptor (CD71), which is overexpressed at the apical side of enterocytes from coeliac patients, interacts with SIgA bound to gliadin peptides and allows their retrotranscytosis across the intestinal epithelium. This thesis work aimed to further characterize the interaction between SIgA and CD71 in human enterocytes. We, first, generated a human epithelial intestinal cell line (Caco-2 TC7) devoid of CD71 expression (CD71KO) using the CRISPR/Cas9 genome editing method. Unexpectedly, flow cytometry experiments did not reveal a significant reduction of SIgA binding at the cell surface of CD71KO cells. Overall, our results indicated that CD71-SIgA interaction is indirect and may occur via additional protein partners through the assembly of a multifactorial protein complex. Therefore, we screened IgA receptors already known in the literature and showed that all are dispensable for SIgA binding at the surface of Caco-2 TC7 cells. In the effort to identify partner(s) within SIgA-CD71 complex, we set out mass spectrometry-based immunoprecipitation proteomics experiments and identified secretory carrier membrane protein 3 (SCAMP3), B-cell receptor-associated protein 31 (BCAP31) and histocompatibility minor 13 (HM13) as members of SIgA-CD71 complex. By generating knockout cell lines with the CRISPR/Cas9 system, we showed that none of these partners directly interacts with SIgA. However, our results suggest that SCAMP3 is required for the oligomerization of SIgA complexes at cell surface. Finally, we did not find any role in SIgA internalization for the different members of the complex, suggesting that they may play a role later on during SIgA retrotranscytosis. In conclusion, our work shows that SIgA interact with the intestinal epithelium via a proteic complex composed of at least five members: CD71, SCAMP3, BCAP31, HM13 and one or more unknown SIgA receptor(s). These results complement the previous works on the pathophysiologic role of SIgA in coeliac disease and underline the highly complex interaction between IgA and enterocytes. An important point to address will be to identify SIgA receptor(s) and to determine the role of the four other identified partners in SIgA retrotranscytosis across the intestinal epithelium.

Secretory phospholipase A2 hydrolyzes phospholipids at a lipid-water interface, resulting in pro-inflammatory products being released from cell membranes. Healthy cells are resistant to cleavage by this enzyme, but apoptotic cells become susceptible to its activity. Only bilayers with certain characteristics are able to be hydrolyzed. Most recently, studies in this lab have emphasized the idea that the biophysical state of the bilayer (in terms of lipid order, spacing, and fluidity) is relevant in determining the probability of one phospholipid escaping the membrane to be hydrolyzed. Prior to this study, it had been shown that apoptotic cells undergo biophysical alterations that weaken inter-lipid interactions early in apoptosis. The purpose of this dissertation was to examine these changes in more detail, define them more clearly on the molecular level, and suggest possible mechanisms responsible for their occurrence. First, the role of increased membrane permeability in susceptibility to the phospholipase was investigated. S49 cells were treated with ionomycin or apoptotic agents and assayed for merocyanine 540 staining of the membrane and membrane permeability to a vital dye. Human group X and snake venom isoforms were active towards all treated cells, but human groups V and IIa only hydrolyzed cells that were moderately permeable to the vital dye. Different isoforms must then be sensitive to different membrane properties. Second, the role of membrane oxidation in cell membrane vulnerability to the phospholipase (specifically human group IIa) was tested. The temporal onset of lipid peroxidation was assayed during apoptosis. This correlated with the onset of susceptibility to the IIa isoform. Direct oxidizers were then used to verify this result in isolation from other apoptotic membrane changes. Third, biophysical alterations during thapsigargin-induced apoptosis were examined using TMA-DPH and Patman. Data from these probes in artificial bilayers undergoing phase transitions were used to quantify the decrease in interlipid interactions and predict a 50 -- 100-fold increase in the probability of phospholipid protrusions. Patman equilibration kinetics also revealed more molecular detail about the biophysical changes related to susceptibility. Finally, temperature- and ionomyin-induced alterations in membrane properties were compared. Both increased fluidity, but only ionomycin caused susceptibility. Patman equilibration kinetic analysis could distinguish responsible membrane properties. Actin fragmentation during apoptosis or calcium loading is proposed as the mechanism.

No description available.

No description available.

Cornichon (CNI) proteins are a conserved family of proteins among eukaryotes, from Erv14 in the yeast Saccharomyces cerevisiae to CNI homologs (CNIHs) in mammals and plants. Erv14 functions as a cargo receptor of coat protein complex II (COPII) for protein trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus, en route to their final destinations. By interacting with specific cargo proteins, CNI proteins regulate key steps of embryo polarity in Drosophila, budding in yeast, and synaptic transmission in the mammalian brain. However, we have very limited understanding of plant CNIHs. Positive-strand RNA viruses assemble their viral replication complexes (VRCs) at specific host organelle membranes. With a better understanding of host factors involved in targeting viral replication proteins to the preferred organelles, we expect to block trafficking of viral replication proteins and thus, viral infection, by manipulating the required host proteins. Brome mosaic virus (BMV) is a model of positive-strand RNA viruses and its replication can be recapitulated in yeast. Importantly, BMV replication protein 1a is the only required viral protein to form VRCs at the perinuclear ER membrane in yeast. I demonstrate that Erv14 and COPII coat proteins are required for targeting BMV 1a to the perinuclear ER in yeast, suggesting a novel function of COPII vesicles in protein trafficking to the perinuclear ER membrane and in the BMV VRC formation. As for cellular functions, I show that plant CNIHs complement the defective distribution of BMV 1a in yeast mutant lacking Erv14. Taking advantage of Arabidopsis thaliana knockout mutants and knockdown of gene expression in Nicotiana benthamina, I also discover that CNIHs unexpectedly play crucial roles in pollen development, infection of a bacterial pathogen, and maintenance of ER tubules. I further confirm that CNI proteins are also required for maintaining ER tubules in yeast, suggesting a novel and conserved role in shaping ER morphology. Therefore, these findings indicate the functional diversity and redundancy of CNI proteins in key cellular processes and suggest a novel strategy to control plant pathogenic viruses and bacteria by manipulating plant CNIHs. / Ph. D.

No description available.

Neuraminidase (NA) is one of two major antigens on the surface of influenza A viruses. It is comprised of a single N-terminal transmembrane domain (TMD), a stalk domain, and a C-terminal enzymatic head domain that cleaves sialic acid, most notably to release new particles from the host cell surface. NA is only enzymatically active as a homo-tetramer. However, it is not known which properties facilitate the oligomerization of NA during assembly. Our results show that, apart from anchoring the protein to the membrane, the NA TMD also contributes to the assembly process by keeping the stalk in a tetrameric conformation. The ability of the TMD to oligomerize is shown to be dependent on its amphiphilic characteristics that was largely conserved across the nine NA subtypes (N1-N9). Over time the NA TMDs in human H1N1 viruses were found to have become more amphiphilic, which correlated with stronger oligomerization. An old H1N1 virus with a more recent N1 TMD had impaired growth, but readily acquired compensatory mutations in the TMD to restore growth, by reverting the TMD oligomerization strength back to that of the old TMD, demonstrating a biological role of the TMD in folding and assembly. NA and the other viral proteins are spatially and temporally coordinated to achieve optimal viral production. By using a co-transfection analysis, the high AU-content in the NA and HA ER-targeting sequence coding regions (for NA TMD as well as the HA signal sequence) were found to inhibit their expression. The inhibition was alleviated by the early expressed influenza RNA-binding protein NS1, which promoted translation and showed enriched foci at the endoplasmic reticulum (ER). NS1, which expresses early during infection, is therefore likely the regulator of NA and HA to prevent premature expression. These results show that the NA TMD is under substantial selection pressure at both the nucleotide and amino acid level to accommodate its roles in ER-targeting, protein folding, and post-transcriptional regulation. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Accepted.</p>