Les études sur le patrimoine architectural bénéficient aujourd'hui de nombreuses méthodes numériques qui ouvrent de nouvelles perspectives pour la conservation, l'analyse et le partage des connaissances, en particulier sur les sites complexes. Ces nouveaux outils se banalisent aujourd'hui, mais à quel point sont-ils désormais nécessaires voire indispensables pour les études architecturales et archéologiques ? Comment s'inscrivent-ils dans les différentes étapes du travail ?Nous aborderons ces questions principalement à propos de deux terrains d'expérimentation : le site de Saint-Syméon-le-stylite en Syrie et l'abbaye de Saint-Michel de Cuxa en France. La méthodologie mise en œuvre pour l'analyse et la restitution des élévations se fonde sur les outils et techniques numériques d'acquisition et de gestion d'information tridimensionnelle et sur leur mise en relation avec les problématiques archéologiques pour construire un dialogue entre ces deux domaines. A partir de cette réflexion, nous avons élaboré un protocole de travail à l'échelle de l'étude monographique d'un édifice spécifique du site de Saint-Syméon « La Résidence ».Cette recherche s'appuie sur une combinaison de plusieurs outils numériques appliquées à l'édifice en vue de son analyse et en les utilisant comme instrument privilégié d'aide à la réflexion archéologique, et non comme une fin en soi.La principale contribution de cette thèse porte sur la méthodologie de l'intégration de ces outils à l'approche archéologique. Cette approche met en valeur plusieurs axes, le dynamisme et l'interactivité du travail, le contexte interdisciplinaire et collectif, l'objectif de la documentation et la diffusion. / Nowadays, the heritage architectural studies benefit of the contributions of diverse digital methods and open new perspectives for the conservation, the analyses and the knowledge sharing especially on complex sites. Those new tools and techniques have become common and very useful presently, but how much they become common or indispensable for architectural and archeological studies? How do they fit into the different phases of the research work?We will discuss these principal questions by working mainly on two experimented sites, the site of Saint-Simeon the stylite in Syria and the abbey of Saint-Michel de Cuxa in France. The methodology used for the analysis and the elevation restoration is based on the digital tools and acquisition techniques and of three-dimensional information management and also on their setting relation with archaeological issues to build a dialogue between these two domains. Based on this reflection, we have developed a work protocol in scale of monographic study of a specific building in Saint Simeon site “the Residence”. This research is based on a combination of digital tools applied on this building for its analysis and by using them as a privileged instrument to support archaeological reflection, not an end in itself.The main contribution of this thesis focuses on the methodology and the ethics of the use of 3D digital service in archaeological approach, from the filed acquisition till the restitution representations. This approach highlights several axes, dynamism and interactivity of the work, the multidisciplinary and the collectivity context, the objective of documentation and diffusion.

A lagarta da espécie Lonomia obliqua é medicamente importante, cujo veneno, presente nas espículas, causa uma síndrome hemorrágica caracterizada por equimoses, alterações da coagulação, dentre outros sintomas. Isto sugere a presença de peptídeos bioativos com potencial farmacêutico, devido à capacidade de modular o comportamento das células endoteliais. O objetivo deste estudo é analisar os potenciais efeitos do veneno de Lonomia obliqua na angiogênese. Uma linhagem celular endotelial (HUVEC) foi exposta a diferentes concentrações do extrato de espículas da Lonomia obliqua (Lonomia obliqua Bristle extract - LOBE) 5 μg/mL, 10 μg/mL, 20 μg/mL e 50 μg/mL. Empregando citometria de fluxo, observou-se que nenhuma das doses afetou o ciclo celular, viabilidade ou apoptose das células endoteliais após 24h de exposição. Os esferóides das células HUVEC foram plaqueados numa matriz 3D de colágeno e observou-se que LOBE (10 μg/mL, 20 μg/mL e 50 μg/mL) induz um aumento na migração celular, consistente com o processo de angiogênese. A análise da dinâmica da VE-caderina indica que a exposição imediata a LOBE (10 μg/mL) induz um desprendimento da junção célula-célula, o que corrobora com a hemorragia observada nas vítimas de envenenamento. Através de espectrometria de massa, observou-se que LOBE possui vários potenciais peptídeos bioativos. Grupos destes peptídeos foram isolados por fracionamento com metanol a partir do veneno bruto. Os peptídeos presentes, em cada uma das 10 frações, foram caracterizados por espectrometria de massa e foram analisados os efeitos de cada fração sobre a angiogênese. Os resultados sugerem que alguns dos efeitos do envenenamento por Lonomia obliqua são devidos à presença de peptídeos bioativos que modulam o comportamento das células endoteliais. / The caterpillar of the species Lonomia obliqua is medically important, whose venom present in the bristles leads to an hemorrhagic syndrome characterized by ecchymosis, coagulation disorders and others symptoms. This suggests the presence of bioactive peptides with pharmaceutical potencial due to the ability to modulate the behavior of endothelial cells. The aim of this study is to analyze the potential effects of Lonomia obliqua venom on angiogenesis. An endothelial cell line (HUVEC) was exposed to different concentrations (5 μg/mL, 10 μg/mL, 20 μg/mL and 50 μg/mL) of Lonomia obliqua bristle extract (LOBE). Using flow cytometry, it was observed that none of the doses affected endothelial cell cycle, cell viability or apoptosis after 24h of exposition. Spheroids of HUVEC cells were plated in a 3D-collagen matrix and it was observed that LOBE (10 μg/mL, 20 μg/mL and 50 μg/mL) induced an increase on cell migration consistent with the angiogenesis process. Analysis of VE-cadherin dynamics indicates that the immediate exposition to LOBE (10 μg/mL) induced a loosening of cell-cell junction, which corroborates with the hemorrhage observed in the victims. By mass spectroscopy, it was observed that LOBE possesses several potentially bioactive peptides. Groups of these peptides were isolated by a methanol-based fractioning of the crude venom. The peptides present in each of the 10 fractions were characterized by mass spectroscopy and it was analyzed the effects of each fraction on angiogenesis. The results suggest that some of the effects of Lonomia obliqua envenomation are due to the presence of bioactive peptides that modulate the behavior of endothelial cells.

Submitted by Luanna Matias (lua_matias@yahoo.com.br) on 2015-02-06T18:00:08Z No. of bitstreams: 2 Dissertação - Paula Francinete Faustino da Silva - 2014..pdf: 2636030 bytes, checksum: 541cdc7c62049af013a9e3343fd197d1 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-02-19T14:26:49Z (GMT) No. of bitstreams: 2 Dissertação - Paula Francinete Faustino da Silva - 2014..pdf: 2636030 bytes, checksum: 541cdc7c62049af013a9e3343fd197d1 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-02-19T14:26:50Z (GMT). No. of bitstreams: 2 Dissertação - Paula Francinete Faustino da Silva - 2014..pdf: 2636030 bytes, checksum: 541cdc7c62049af013a9e3343fd197d1 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-04-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Nearly 60% of the Paracoccidioides genes encode proteins annotated as hypothetical proteins or predicted proteins (HPs). Transcriptomes studies revealed 2364 HPs expressed in mycelium and yeast form; during the transition from mycelium to yeast; and under conditions that mimic the infection pathway. In this study, we describe a global detection and functional inference for the HPs in Paracoccidioides. For these analysis we used computational methods based on sequence similarity, search for targeting signals, presence of known protein domains and also a functional classification based on Gene Ontology. Our analysis allowed the HPs to be classified into different functional categories such as metabolism, organelle organization, cell communication, protein localization, cell cycle, signaling and cell differentiation. We also performed a functional enrichment analysis of the transcripts expressed under different growth conditions. The best represented functional domains are those involved in the regulation of gene expression, suggesting that these HPs may be involved in regulation of the gene response and adaptation. The transcriptional profiling of six HPs confirms that they are highly expressed in the presence of human blood and plasma and also during phase transition, a crucial event for establishment of the infection process. Additionally, we have cloned and expressed the gene encoding the hypothetical protein PAAG_08614 from Pb01. The transcript and protein expression were evaluated by qPCR e western-blot, respectively. PAAG_08614 is preferentially expressed in mycelium and during mycelium to yeast transition. This work constitutes the first large-scale characterization of the proteins of unknown functional from Paracoccidioides spp. and helps the functional assignment of hypothetical proteins, as well as the understanding of the data generated from structural and functional genome. / Aproximadamente 60% dos genes de Paracoccidioides ssp. codificam proteínas anotadas como proteínas hipotéticas ou proteínas preditas (HPs). Estudos Transcricionais revelaram 2.364 HPs foram expressas nas fases de micélio e levedura; durante a transição de micélio para levedura; e em condições que mimetizam as vias de infecção. Neste estudo, descrevemos uma detecção global e inferência funcional para as HPs de Paracoccidioides. Para essas análises foram utilizados métodos computacionais baseados na similaridade de sequência, procurar de peptídeos de sinais, presença de domínios de proteínas conhecidas e também uma classificação funcional baseada no Gene Ontology. Nossa análise permitiu a classificação das HPs em diferentes categorias funcionais, tais como o metabolismo, organização de organela, comunicação celular, localização de proteínas, ciclo celular, sinalização e diferenciação celular. Também realizamos uma análise de enriquecimento funcional dos transcritos expressos em diferentes condições de cultivo. Os domínios funcionais mais bem representados são os envolvidos na regulação da expressão gênica, o que sugere que estas proteínas estão envolvidas na regulação da resposta e adaptação do fungo às diferentes condições impostas pelo ambiente. A análise do perfil transcricional de seis HPs confirmou que elas são altamente expressas na presença de sangue e de plasma humano e também durante a fase de transição, um evento crucial para o estabelecimento do processo de infecção. Adicionalmente, o gene que codifica a proteína hipotética de PAAG_08614 de Pb01 foi clonado e expresso. A expressão dos transcritos e proteínas foram avaliadas por qPCR e western-blot, respectivamente. A PAAG_08614 é preferencialmente expressa na fase de micélio e durante a transição micélio para levedura. Este trabalho representa a primeira caracterização em grande escala das proteínas de função desconhecida de Paracoccidioides spp. e ajuda na atribuição funcional de proteínas hipotéticas, assim como uma melhor compreensão dos dados gerados a partir do genoma estrutural e funcional.

Ο ρόλος του χαλκού είναι πολύ σημαντικός για τη σωστή λειτουργία της κυτοχρωμικής c οξειδάσης (CcO), και συνεπώς για την κυτταρική αναπνοή στους ευκαρυωτικούς και προκαρυωτικούς οργανισμούς. Η συγκρότηση της CcO στον ενδομεμβρανικό μιτοχονδριακό χώρο είναι μια πολύπλοκη διαδικασία, εξαρτώμενη από πλήθος συνεργών πρωτεϊνών, υπεύθυνων για τη λειτουργική αναδίπλωση των υπομονάδων του ενζύμου και τη μεταφορά αίμης και ιόντων Cu σε αυτές. Ενώ οι πρωτεΐνες που ενέχονται στη διαδικασία είναι μάλλον γνωστές, οι μηχανισμοί μεταφοράς και ενσωμάτωσης των μεταλλικών ιόντων στα δυο ενεργά κέντρα της CcO, CuA και CuB, παραμένουν ανεξερεύνητοι. Το CuA κέντρο είναι ένα διπυρηνικό κέντρο χαλκού, του οποίου ο ρόλος εντοπίζεται στη μεταφορά e- από το κυτόχρωμα c στο καταλυτικό κέντρο CuB της CcO. Η σωστή συγκρότηση του CuA κέντρου είναι κρίσιμης σημασίας για την καταλυτική δράση του ενζύμου. Αρκετές πρωτεΐνες έχουν χαρακτηριστεί σαν ενεργοί παράγοντες στη μεταφορά ιόντων Cu στο CuA κέντρο, ο ακριβής, ωστόσο, μοριακός μηχανισμός και ρόλος της κάθε πρωτεΐνης είναι άγνωστος. Στους προκαρυωτικούς οργανισμούς, δυο οικογένειες πρωτεϊνών έχουν προταθεί για την εμπλοκή τους στη συγκρότηση του CuA. Η πρώτη περιλαμβάνει πρωτεΐνες που δεσμεύουν ιόντα Cu1+ με ένα συντηρημένο μοτίβο δέσμευσης H(M)x10Mx21HxM (υποθετικές πρωτεΐνες Hyp1) ενώ η δεύτερη περιλαμβάνει τις πρωτεΐνες της οικογένειας Sco, των οποίων ο ρόλος στον μηχανισμό του CuA κέντρου σαν θειορεδοξίνες ή χαλκομεταφορείς, παραμένει ασαφής. Στην παρούσα εργασία αποδείχθηκε ότι μια νέα περιπλασματική πρωτεΐνη (TtHyp1 ή PCuAC) εισάγει επιλεκτικά ιόντα Cu1+ στην Cox2 υπομονάδα της ba3-CcO του Thermus thermophilus προς σχηματισμό του φυσιολογικού διπυρηνικού TtCuA κέντρου, καθώς και ότι η Sco πρωτεΐνη του συγκεκριμένου οργανισμού (TtSco1) δεν μεταφέρει μεταλλικά ιόντα στο CuA, αλλά δρα σαν θειο-δισουλφιδική αναγωγάση ρυθμίζοντας τη σωστή οξειδωτική κατάσταση των κυστεϊνικών καταλοίπων του CuA. Οι πρωτεΐνες PCuAC, TtSco1 και TtCuA εκφράστηκαν, απομονώθηκαν και μελετήθηκαν τα βιοχημικά χαρακτηριστικά τους, η ικανότητα δέσμευσης μεταλλικών ιόντων και οι μεταξύ τους αλληλεπιδράσεις. Επιπλέον η PCuAC χαρακτηρίστηκε δομικά με φασματοσκοπία NMR στην απο και Cu(I) μορφή της. Ο ρόλος των προκαρυωτικών Sco διερευνήθηκε περαιτέρω με μελέτες γονιδιακής ανάλυσης και την έκφραση και τον προκαταρκτικό χαρακτηρισμό μιας νέας πρωτεΐνης, PpSco1/cytc της Pseudomonas putida, πρωτεΐνης αποτελούμενης από δυο επικράτειες, Sco1 και cytc, συνδέοντας το ρόλο των Sco πρωτεϊνών με τη θεωρια περί θειρεδοξινικής τους δράσης. Καινοτομία στην παρούσα εργασία αποτέλεσε η μεθοδολογική προσέγγιση της πολλαπλής κλωνοποίησης των γονιδίων-στόχων με μια νέα τεχνολογία κλωνοποίησης (Gateway) συνδυασμένης με τοποειδική ένθεση σε πολλαπλούς πλασμιδιακούς φορείς και η ανάπτυξη high throughput τεχνικών για πολλαπλές δοκιμές έκφρασης – απομόνωσης. Η συγκεκριμένη μελέτη παρέχει νέα δεδομένα για το μηχανισμό τη συγκρότησης του CuA κέντρου της προκαρυωτικής CcO, υποστηρίζοντας ένα νέο μοντέλο για τη συγκεκριμένη διαδικασία και παράλληλα συνδράμει στην αποκρυπτογράφηση του πολύπλοκου ρόλου των Sco πρωτεϊνών. / Copper is essential for the correct assembly and function of the cytochrome c oxidase (CcO), thus for the efficient cellular respiration in both eukaryotes and prokaryotes. CcO assembly in the inner mitochondrial membrane space is a multi complicated procedure, depended on a number of co-factors and their synergic action. These co-factors are proteins commissioned with the correct folding of the enzyme subunits and the transport/incorporation of heme moieties and Cu ions to them. While the proteins involved in this multistep procedure are rather known, the mechanisms of metal ion delivery and incorporation within the two active centers of CcO, CuA and CuB, still remain uncharted. The CuA center is a binuclear copper center, whose part in the respiratory chain is spoted in electron transport from the active cytochrome c to the catalytic CuB center of CcO. Efficient CuA assembly is crucial for the catalytic action of the entire enzyme. Several proteins have been characterized as essential factors for the transport of Cu ions to the CuA center; however their exact molecular mechanism of action still remains obscure. In prokaryotes, two protein families have been suggested to be involved in the CuA assembly. The first includes proteins that bind Cu1+ ions through a potential conserved motif H(M)x10Mx21HxM (hypothetical proteins, Hyp1), while the second includes proteins of the Sco family, whose exact role in CuA assembly as thioredoxins or copper chaperones is widely debated. In this work, it is propesed that a new periplasmic protein (TtHyp1 or PCuAC) selectively inserts Cu1+ ions in the Cox2 subunit of the ba3-CcO of Thermus thermophilus resulting the formation of the physiological binuclear TtCuA center, as well as that the Sco protein of the organism (TtSco1) is not able to transfer metal ions to the CuA center; instead it acts rather like a thio-disulfide reductase adjusting the proper redox state of the CuA cysteine residues. Proteins PCuAC, TtSco1 and TtCuA were over-expressed, purified and subjected to biochemical characterization, while their Cu binding capability and their inter se interactions were studied through NMR and UV spectroscopy. In addition, PCuAC was structurally characterized through NMR in its apo and Cu(I) form. The role of Sco proteins was further investigated through genome based analysis and the expression and biochemical characterization of a new protein, PpSco1/cytc from Pseudomonas putida, a unique bacterial protein consisted on two domains, a Sco1 and a cytc domain, presumptively connecting the role of Sco proteins with the suggested theory of thioredoxin action. A novelty in this work was the methodological aspect of the multiple cloning of the target genes with a new cloning technology (Gateway) combined with site specific recombination into multiple expression plasmid vectors and the development of a high throughput technique for parallel expression/purification tests. The infra work provides new insights to the CuA center assembly molecular mechanism of the prokaryotic CcO, supporting a new model for the particular procedure and also subscripts for the decipherment of the complicated role of Sco proteins.

Submitted by Andrea Hartke (andreamh@fiocruz.br) on 2014-11-24T13:25:54Z No. of bitstreams: 1 Dissertação Bruno Dias Ramos.pdf: 4916835 bytes, checksum: fa6000cbfecb89598eef24d308186fc7 (MD5) / Approved for entry into archive by Andrea Hartke (andreamh@fiocruz.br) on 2014-11-24T17:25:03Z (GMT) No. of bitstreams: 1 Dissertação Bruno Dias Ramos.pdf: 4916835 bytes, checksum: fa6000cbfecb89598eef24d308186fc7 (MD5) / Made available in DSpace on 2014-11-24T17:25:03Z (GMT). No. of bitstreams: 1 Dissertação Bruno Dias Ramos.pdf: 4916835 bytes, checksum: fa6000cbfecb89598eef24d308186fc7 (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Pós-Graduação em Biociências e Biotecnologia. Curitiba, PR, Brasil. / Desde sua descoberta, no inicio do século 20, o protozoário parasita causador da Doença de Chagas, Trypanosoma cruzi, se tornou alvo de estudo em diversas instituições de pesquisa ao redor do mundo. Portador de características peculiares interessantes, T. cruzi é um excelente modelo de estudo de processo de regulação pós-transcricional. O parasita teve o seu genoma sequênciado em 2005, e foi observado que muitos dos genes contidas em seu genoma não apresentaram uma descrição funcional confiável, sendo anotados como codificadores de "proteínas hipotéticas". Tendo em vista essa ausência de caracterização funcional de uma parcela considerável do genoma de T. cruzi, o presente trabalho se propôs a realizar a caracterização molecular de um grupo de 10 proteínas de T. cruzi anotadas como "hipotéticas", e que se apresentavam diferencialmente expressas na forma celular tripomastigota metacíclica do parasita, a forma infectiva não-replicativa que inicialmente infecta o hospedeiro mamífero na via usual de transmissão da doença via o inseto vetor. Após amplificação, todos os dez genes selecionados no estudo foram clonados em vetor de entrada (pDONRTM221, Invitrogen), a partir do qual foi possível a transferência do fragmento gênico para outros vetores, visando diferentes abordagens de análise. Todos os genes foram transferidos com sucesso para o vetor de expressão pDESTTM17, de expressão em Escherichia coli, e nove para o vetor pTcGFP, de expressão no próprio T. cruzi e que fusiona à proteína uma etiqueta fluorescente. Dos genes inseridos no pDESTTM17, sete deles foram com sucesso expressos em E. coli, sendo obtidos anticorpos policlonais contra seis das proteínas expressas. Os ensaios de western blot realizados com os soros obtidos contra extratos das quatro formas celulares de T. cruzi corroboram os dados previamente obtidos de sequenciamento de mRNA. Foram realizados ensaios de imunolocalização com os soros obtidos, no entanto, os padrões encontrados nas imunolocalizações foram diferentes dos observados pelas proteínas fusionadas à etiqueta fluorescente GFP. Ensaios de nocaute gênico das proteínas estudadas foram iniciados, e em breve virão a contribuir para o trabalho. Espera-se que os dados obtidos com esse estudo venham melhorar a anotação funcional e a compreensão das proteínas selecionadas. / Since it discovery in early 20th century, the Chagas Disease causative protozoan parasite, Trypanosoma cruzi, became the subject of study in several research institutions around de world. Carrier of interesting peculiar characteristics, T. cruzi is an excellent study model of post-transcriptional regulation process. The parasite had its genome sequenced in 2005, and it was observed that several of it genes did not show a reliable functional description, been annotated as coding for "hypothetical proteins". Given this lack of functional characterization of a considerable portion of T. cruzi genome, the present study proposed to perform a molecular characterization of a group of ten T. cruzi proteins annotated as "hypothetical" that in mRNA studies had showed to be differentially expressed in the parasite metacyclic trypomastigotes cell form, the infective non-replicative that initially infect the mammalian host in the usual route of transmission of the disease via the insect vector. After been amplified all the ten genes selected in the study were cloned into entry vector (pDONRTM221, Invitrogen) from which it was possible to transfer the gene fragment to other vectors, targeting different approaches to analysis. All genes were successfully transferred to pDESTTM17 expression vector, for expression in Escherichia coli, and nine to the pTcGFP vector, for expression of the protein fused to a fluorescent tag in T. cruzi. From all genes inserted into pDESTTM17, seven were successfully expressed in E. coli, which six were purified and inoculated in mice for obtaining polyclonal antibody. Western blot assays performed against protein extracts of the four T. cruzi cell forms using this sera corroborate the date previously obtained by mRNA sequencing. Immunolocalization assays were performed, however, the localization patterns observed in the immunolocalizations were different from those observed by the proteins fused to the GFP fluorescent tag. Gene knockout assays of the studied proteins were started and soon will come to contribute to this characterization work. It is hoped that the data obtained from this study will improve the functional annotation and understanding of the selected proteins.

A lagarta da espécie Lonomia obliqua é medicamente importante, cujo veneno, presente nas espículas, causa uma síndrome hemorrágica caracterizada por equimoses, alterações da coagulação, dentre outros sintomas. Isto sugere a presença de peptídeos bioativos com potencial farmacêutico, devido à capacidade de modular o comportamento das células endoteliais. O objetivo deste estudo é analisar os potenciais efeitos do veneno de Lonomia obliqua na angiogênese. Uma linhagem celular endotelial (HUVEC) foi exposta a diferentes concentrações do extrato de espículas da Lonomia obliqua (Lonomia obliqua Bristle extract - LOBE) 5 μg/mL, 10 μg/mL, 20 μg/mL e 50 μg/mL. Empregando citometria de fluxo, observou-se que nenhuma das doses afetou o ciclo celular, viabilidade ou apoptose das células endoteliais após 24h de exposição. Os esferóides das células HUVEC foram plaqueados numa matriz 3D de colágeno e observou-se que LOBE (10 μg/mL, 20 μg/mL e 50 μg/mL) induz um aumento na migração celular, consistente com o processo de angiogênese. A análise da dinâmica da VE-caderina indica que a exposição imediata a LOBE (10 μg/mL) induz um desprendimento da junção célula-célula, o que corrobora com a hemorragia observada nas vítimas de envenenamento. Através de espectrometria de massa, observou-se que LOBE possui vários potenciais peptídeos bioativos. Grupos destes peptídeos foram isolados por fracionamento com metanol a partir do veneno bruto. Os peptídeos presentes, em cada uma das 10 frações, foram caracterizados por espectrometria de massa e foram analisados os efeitos de cada fração sobre a angiogênese. Os resultados sugerem que alguns dos efeitos do envenenamento por Lonomia obliqua são devidos à presença de peptídeos bioativos que modulam o comportamento das células endoteliais. / The caterpillar of the species Lonomia obliqua is medically important, whose venom present in the bristles leads to an hemorrhagic syndrome characterized by ecchymosis, coagulation disorders and others symptoms. This suggests the presence of bioactive peptides with pharmaceutical potencial due to the ability to modulate the behavior of endothelial cells. The aim of this study is to analyze the potential effects of Lonomia obliqua venom on angiogenesis. An endothelial cell line (HUVEC) was exposed to different concentrations (5 μg/mL, 10 μg/mL, 20 μg/mL and 50 μg/mL) of Lonomia obliqua bristle extract (LOBE). Using flow cytometry, it was observed that none of the doses affected endothelial cell cycle, cell viability or apoptosis after 24h of exposition. Spheroids of HUVEC cells were plated in a 3D-collagen matrix and it was observed that LOBE (10 μg/mL, 20 μg/mL and 50 μg/mL) induced an increase on cell migration consistent with the angiogenesis process. Analysis of VE-cadherin dynamics indicates that the immediate exposition to LOBE (10 μg/mL) induced a loosening of cell-cell junction, which corroborates with the hemorrhage observed in the victims. By mass spectroscopy, it was observed that LOBE possesses several potentially bioactive peptides. Groups of these peptides were isolated by a methanol-based fractioning of the crude venom. The peptides present in each of the 10 fractions were characterized by mass spectroscopy and it was analyzed the effects of each fraction on angiogenesis. The results suggest that some of the effects of Lonomia obliqua envenomation are due to the presence of bioactive peptides that modulate the behavior of endothelial cells.

A lagarta da espécie Lonomia obliqua é medicamente importante, cujo veneno, presente nas espículas, causa uma síndrome hemorrágica caracterizada por equimoses, alterações da coagulação, dentre outros sintomas. Isto sugere a presença de peptídeos bioativos com potencial farmacêutico, devido à capacidade de modular o comportamento das células endoteliais. O objetivo deste estudo é analisar os potenciais efeitos do veneno de Lonomia obliqua na angiogênese. Uma linhagem celular endotelial (HUVEC) foi exposta a diferentes concentrações do extrato de espículas da Lonomia obliqua (Lonomia obliqua Bristle extract - LOBE) 5 μg/mL, 10 μg/mL, 20 μg/mL e 50 μg/mL. Empregando citometria de fluxo, observou-se que nenhuma das doses afetou o ciclo celular, viabilidade ou apoptose das células endoteliais após 24h de exposição. Os esferóides das células HUVEC foram plaqueados numa matriz 3D de colágeno e observou-se que LOBE (10 μg/mL, 20 μg/mL e 50 μg/mL) induz um aumento na migração celular, consistente com o processo de angiogênese. A análise da dinâmica da VE-caderina indica que a exposição imediata a LOBE (10 μg/mL) induz um desprendimento da junção célula-célula, o que corrobora com a hemorragia observada nas vítimas de envenenamento. Através de espectrometria de massa, observou-se que LOBE possui vários potenciais peptídeos bioativos. Grupos destes peptídeos foram isolados por fracionamento com metanol a partir do veneno bruto. Os peptídeos presentes, em cada uma das 10 frações, foram caracterizados por espectrometria de massa e foram analisados os efeitos de cada fração sobre a angiogênese. Os resultados sugerem que alguns dos efeitos do envenenamento por Lonomia obliqua são devidos à presença de peptídeos bioativos que modulam o comportamento das células endoteliais. / The caterpillar of the species Lonomia obliqua is medically important, whose venom present in the bristles leads to an hemorrhagic syndrome characterized by ecchymosis, coagulation disorders and others symptoms. This suggests the presence of bioactive peptides with pharmaceutical potencial due to the ability to modulate the behavior of endothelial cells. The aim of this study is to analyze the potential effects of Lonomia obliqua venom on angiogenesis. An endothelial cell line (HUVEC) was exposed to different concentrations (5 μg/mL, 10 μg/mL, 20 μg/mL and 50 μg/mL) of Lonomia obliqua bristle extract (LOBE). Using flow cytometry, it was observed that none of the doses affected endothelial cell cycle, cell viability or apoptosis after 24h of exposition. Spheroids of HUVEC cells were plated in a 3D-collagen matrix and it was observed that LOBE (10 μg/mL, 20 μg/mL and 50 μg/mL) induced an increase on cell migration consistent with the angiogenesis process. Analysis of VE-cadherin dynamics indicates that the immediate exposition to LOBE (10 μg/mL) induced a loosening of cell-cell junction, which corroborates with the hemorrhage observed in the victims. By mass spectroscopy, it was observed that LOBE possesses several potentially bioactive peptides. Groups of these peptides were isolated by a methanol-based fractioning of the crude venom. The peptides present in each of the 10 fractions were characterized by mass spectroscopy and it was analyzed the effects of each fraction on angiogenesis. The results suggest that some of the effects of Lonomia obliqua envenomation are due to the presence of bioactive peptides that modulate the behavior of endothelial cells.

This paper provides an exploration of major traded derivative instruments in Chile, the interest rate swaps, and most commonly used valuation methods, addressing financial risk management in the context of the International Financial Reporting Standards (IFRS). We analyze the requirements for adopting hedge accounting, particularly with regard to evidence of effectiveness, and discuss particular aspects of the Chilean case that companies must face the time to prove the validity of contracts and contract coverage. The study’s findings suggest that the local market still has problems of efficiency and availability of information to (i) properly manage financial risks and (ii) demonstrate the effectiveness of hedges under IFRS. / El presente trabajo realiza una revisión, análisis y exploración de uno de los principales instrumentos derivados transados en Chile, los swaps de tasas de interés, y sus métodos de valorización más utilizados, además de abordar la gestión de riesgos financieros en el contexto de las International Financial Reporting Standards (IFRS). Se analizan los requisitos exigidos para adoptar contabilidad de cobertura, especialmente las pruebas de efectividad, y se discuten otros aspectos particulares del caso chileno que las compañías deben enfrentar al momento de demostrar la validez de las coberturas contratadas y por contratar. Las conclusiones del estudio apuntan a que el mercado local aún presenta problemas de eficiencia y disponibilidad de información para (i) gestionar correctamente los riesgos financieros y (ii) demostrar la efectividad de las coberturas bajo IFRS.

Toxoplasma gondii, comme Plasmodium falciparum appartiennent au phylum des Apicomplexes. Ce groupe de parasites ont comme dénominateurs communs, trois organites apicaux : rhoptries, micronèmes et granules denses contenant des facteurs indispensables pour la reconnaissance, l’entrée et la survie du parasite au sein de la cellule hôte. Le récepteur transmembranaire de type 1 appelé TgSORTLR ("Toxoplasma gondii Sortilin-Like Receptor") est nécessaire à la biogenèse des organelles de sécrétion rhoptrie et micronème (Sloves et al., 2012). Le domaine C-terminale de la TgSORTLR, lie TgVps26 et TgVps35 deux protéines appartement au complexe Rétromère essentiel au recyclage protéique chez les mammifères et S. cerevisiae. Nous avons construit le premier interactome du CRC de T. gondii et des autres Apicomplexes. Contrairement aux mammifères, le Rétromère de T. gondii est composé du CRC (Complexe de Reconnaissance du Cargo) TgVps35-TgVps26-TgVsp29 et l’absence du dimère de Sorting Nexin (SNX). Nous avons identifié plusieurs protéines connues de l’ELC (Endosomal-like compartment) ainsi que des protéines parasitaires spécifiques. La déplétion conditionnelle de TgVps35 démontre que le complexe Rétromère n’est pas seulement crucial pour la biogenèse des rhoptries, micronèmes et granules denses, mais aussi pour l’architecture et l’intégrité du parasite. Nous avons montré que le recyclage de la TgSORTLR entre l’ELC et le TGN (Tans-Golgi-Network) est essentiel au trafic des protéines de sécrétion rhoptries et micronèmes. Par ailleurs nous avons décrit deux nouvelles protéines hypothétiques TgHP12 et TgHP03 pouvant être impliquées respectivement dans le trafic vers l’ELC et vers la membrane plasmique. Afin nous avons identifié et caractérisé une protéine chimérique TgHP25 avec les domaines BAR et SBF2, pouvant être impliquée dans la biogenèse de l’organite rhoptrie. En somme notre travail souligne l’importance du recyclage protéique et l’implication de protéines spécifiques dans la maturation des organites et l’intégrité du parasite. / Toxoplasma gondii, like Plasmodium falciparum are belong to the Apicomplexan phylum. This group of parasites have as a common denominator, three apical organelles: rhoptries, micronemes and dense granules containing the essential factors for recognition, entry and survival into the host cell. The Toxoplasma gondii Sortilin-Like Receptor (TgSORTLR), is essential for the biogenesis of apical secretory organelles rhoptries and micronemes (Sloves et al., 2012). The C-terminal tail of TgSORTLR specifically binds to TgVps26 and TgVps35 proteins, two components of a pentameric complex called retromer (RC), and known to play an essential role in retrograde transport in yeast and mammals. We now report the first retromer-trafficking interactome in T. gondii and other apicomplexan parasites. In contrast to yeast and mammals, T. gondii RC harbors a singular architecture typified by a Vps35-Vps26-Vps29 trimer complex and the absence of the dimer of sorting nexins. Rather, we identified several known endosomal-like compartment (ELC) proteins and unrelated parasite-specific proteins. The conditional ablation of TgVps35 demonstrates that the Retromer complex is not only crucial for the biogenesis of rhoptries, micronemes and dense granules but also for maintaining a proper parasite architecture and integrity. We showed that the recycling of TgSORTLR between ELC and Trans-Golgi Network (TGN), is essential for proper protein trafficking to secretory organelles rhoptries and micronemes. Furthermore, we will describe two novel parasite-specific proteins TgHP12 and TgHP03, whose functions are likely related to ELC and plasma membrane. So we identified and characterized a chimeric protein TgHP25 with the BAR and SBF2 domains, may be involved in the biogenesis of the organelle rhoptrie. In short our work emphasizes the importance of protein recycling and involvement of specific proteins in the maturation of organelles and integrity of the parasite.

The function concept is one of the most important concepts in the learning of mathematics (Dubinsky & Harel, 1992), yet it is considered by many researchers to be one of the least understood and most difficult concepts to master in the learning of high school mathematics (Eisenberg, 1992, Sfard, 1992). To this end, problems concerning its teaching and learning are often confronted (Mann, 2000) and few teachers know how learners come to understand functions (Yoon, 2007). As a result, most teachers teach functions using the conventional approach which starts by stating definitions followed by examples and then a few applications. The nature of this approach has not encouraged teachers to engage learners and their ways of reasoning in knowledge construction and adequately addressing their difficulties. The purpose of this study was to use design research to improve the teaching and learning of functions at grade 11 level. This was achieved by adapting design cycles of Wademan’s (2005) Generic Design Research model in which each cycle comprised different iterative APOS (Action, Process, Object, Schema) analysis, design, development and implementation of hypothetical learning trajectories (HLTs). I started by interrogating twelve grade 11 learners of a particular rural high school on the June 2011 mathematics paper 1 examination they had written to determine the APOS theory conception level each learner was operating at, and their difficulties. Learners’ difficulties from initial interviews and literature were grouped under the function definition and representation. I then designed instruction based on HLTs embedded with Realistic Mathematics Education (RME) activities and two separate tasks on the definition and representation as a form of intervention to help learners move up from their initial conception levels to the next and to overcome their difficulties. After each design cycle I interviewed learners based on the task for a particular concept and learners’ responses were analysed using APOS theory and used to design further instruction to help learners approximate the schema level of understanding concepts related to functions. The major findings of this study were that the use of learners’ conceptions and RME activities in designing instruction helped learners to progress smoothly through APOS theory conception levels though they did not fully reach the intended schema level. In addition, design research cycles and their HLTs implemented in a constructivist environment enabled learners to collectively derive working definitions of the function concept and to improve their conceptual understanding of the process of switching from a graph to an equation. Another contribution of this study has been a deeper understanding of the extent to which design research can be used to improve learners’ understanding of functions and an addition of some insights to the teaching and learning of functions. / Thesis (PhD)--University of Pretoria, 2013. / gm2014 / Science, Mathematics and Technology Education / unrestricted