Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 6 uppsatser.

Les structures protéiques sont de nature hautement dynamique contrairement à leur représentation dans les structures cristallines. Une composante majeure de la dynamique structurelle est la flexibilité des protéines inhérentes. L'objectif principal de cette thèse est de comprendre le rôle de la dynamique inhérente dans les structures protéiques et leur propagation. La flexibilité des protéines est analysée à différents niveaux de complexité structurelle, du niveau d'organisation primaire au niveau quaternaire. Chacun des cinq premiers chapitres traite un niveau différent d'organisation structurelle locale avec le premier chapitre traitant des structures secondaires classiques tandis que le second analyse la même chose en utilisant un alphabet structurel - les blocs protéiques. Le troisième chapitre se concentre sur l'impact d'événements physiologiques spéciaux comme les modifications post-traductionnelles et le désordre sur les transitions d'ordre sur la flexibilité des protéines. Ces trois chapitres indiquent une mise en œuvre dépendante du contexte de la flexibilité structurelle dans leur environnement local. Dans les chapitres suivants, des structures plus complexes sont prises en compte. Le chapitre 4 traite de l'intégrine αIIbβ3 impliquée dans des troubles génétiques rares. L'impact des mutations pathologiques sur la flexibilité locale est étudié dans deux domaines rigides de l'intégrine αIIbβ3 ectodomaine. La flexibilité inhérente dans ces domaines est montrée pour moduler l'impact des mutations vers les boucles. Le chapitre 5 traite de la modélisation structurelle et de la dynamique d'une structure protéique plus complexe du récepteur des chimiokines des antigènes du groupe Duffy incorporé dans un système de membrane mimétique érythrocytaire. Le modèle est soutenu par l'analyse phylogénétique la plus complète sur les récepteurs de chimiokines jusqu'à ce jour, comme expliqué dans le dernier chapitre de la thèse. / Protein structures are highly dynamic in nature contrary to their depiction in crystal structures. A major component of structural dynamics is the inherent protein flexibility. The prime objective of this thesis is to understand the role of the inherent dynamics in protein structures and its propagation. Protein flexibility is analyzed at various levels of structural complexity, from primary to quaternary levels of organization. Each of the first five chapters’ deal with a different level of local structural organization with first chapter dealing with classical secondary structures while the second one analysis the same using a structural alphabet - Protein Blocks. The third chapter focuses on the impact of special physiological events like post-translational modifications and disorder to order transitions on protein flexibility. These three chapters indicate towards a context dependent implementation of structural flexibility in their local environment. In subsequent chapters, more complex structures are taken under investigation. Chapter 4 deals with integrin αIIbβ3 that is involved in rare genetic disorders. Impact of the pathological mutations on the local flexibility is studied in two rigid domains of integrin αIIbβ3 ectodomain. Inherent flexibility in these domains is shown to modulate the impact of mutations towards the loops. Chapter 5 deals with the structural modelling and dynamics of a more complex protein structure of Duffy Antigen Chemokine Receptor embedded in an erythrocyte mimic membrane system. The model is supported by the most comprehensive phylogenetic analysis on chemokine receptors till date as explained in the last chapter of the thesis.

Le Web est un environnement éphémère. Alors que de nouveaux sites Web émergent chaque jour, il arrive que certaines communautés disparaissent entièrement de la surface de la toile, ne laissant derrière elles que des traces incomplètes voire inexistantes. Face à la volatilité du Web vivant, plusieurs initiatives d’archivage cherchent malgré tout à préserver la mémoire du Web passé. Mais aujourd’hui, force est de constater qu’un mystère demeure : Pourquoi, alors qu’elles n’ont jamais été aussi vastes et aussi nombreuses, les archives Web ne font-elles pas déjà l’objet de multiples recherches historiques ? Initialement construites pour inscrire la mémoire de la toile sur un support durable, ces archives ne doivent pourtant pas être considérées comme une représentation fidèle du Web vivant. Elles sont les traces directes des outils de collecte qui les arrachent à leur temporalité d’origine. Partant de là, cette thèse ambitionne de redonner aux chercheurs les moyens théoriques et techniques d’une plus grande maniabilité du Web passé, en définissant une nouvelle unité d’exploration des archives Web : le fragment Web, un sous-ensemble cohérent et auto-suffisant d’une page Web. Pour ce faire, nous nous inscrirons dans l’héritage des travaux pionniers de l’Atlas e-Diasporas qui permit, dans les années 2000, de cartographier et d’archiver plusieurs milliers de sites Web migrants. Source principale de données à partir desquelles nous déploierons nos réflexions, c’est à travers l’angle particulier des représentations en ligne des diasporas que nous chercherons à explorer les archives Web de l’Atlas. / The Web is an unsteady environment. As Web sites emerge every days, whole communities may fade away over time by leaving too few or incomplete traces on the living Web. Facing this phenomenon, several archiving initiatives try to preserve the memory of the Web. But today, a mystery remains : While they have never been so vast and numerous, why are the Web archives not already the subject of many historical researches ? In reality, Web archives should not be considered as a faithful representation of the living Web. In fact, they are the direct traces of the archiving tools that tear them away from their original temporality. Thus, this thesis aims to give researchers the theoretical and technical means for a greater manageability of the Web archives, by defining a new unit of exploration : the Web fragment, a coherent and self-sufficient subset of a Web page. To that end, we will follow the pioneering work of the e-Diasporas Atlas which allowed, in the 2000s, to map and archive thousands of migrant Web sites. Main source of data from which we will unfold our reflections, it is through the particular angle of online representations of diasporas that we will explore the Web archives of the Atlas.

Afin de comprendre la production de fragments qui se déroule dans les collisions d'ions lourds aux énergies intermédiaires, nous avons fait une étude à la fois expérimentale et théorique du phénomène de multifragmentation. Les données recueillies avec le multidétecteur INDRA sur une large gamme en énergie incidente pour le système Xe+Sn ont permis de faire une étude des collisions centrales et de montrer que le maxim̀um de production de fragments se situe autour de 65 MeV/n d'énergie incidente. Un examen plus approfondi des collisions centrales entre 32 et 50 MeV/n d'énergie de bombardement, fondé sur les fonctions de corrélation fragment/particule, a aidé à déterminer les caractéristiques des fragments primaires produits au cours de la collision. Il a été montré que les énergies d'excitation de ces fragments saturent vers une valeur de 3 MeV/n à partir de 39 MeV/n d'énergie de faisceau, et que les particules évaporées représentent moins de 40% (23% à 50 MeV/n) de toutes les particules légères chargées, ce qui montre l'importance de la dynamique de la collision. Afin de mieux comprendre ces grandeurs, d'étudier le rôle de la dynamique et l'évolution en temps de la collision, des calculs avec le modèle de dynamique moléculaire antisymétrisée AMD ont été effectués. Les simulations donnent une bonne image des données expérimentales, notamment des collisions les plus centrales à 50 et 100 MeV/n d'énergie incidente pour le système Xe+Sn. Pour cela des développements du modèle ont été nécessaires afin de bien prendre en compte la diffusion des nucléons dans le milieu. Les calculs ont permis de situer le temps de formation des fragments entre 100 et 200 fm/c dans le cas des collisions centrales à 50 MeV/n d'énergie incidente, et de monter un effet de transparence, qui existe même dans les collisions les plus centrale. Les comparaisons avec les données expérimentales montrent que cet effet est légèrement surestimé dans les calculs.

In all kinds of disease models, many proteins involved in protein-protein interactions (PPIs) are mutated and do not function properly. The important role of PPIs in disease makes the design of small molecule inhibition an interesting proposition. This project looks at mouse double minute 2 (MDM2) and mouse double minute X (MDMX) which binds and inhibits the tumour suppressor protein p53. MDM2 and MDMX are therefore attractive therapeutic targets due to their role in tumour progression. The aim is to identify small molecule dual inhibitors that are able to disrupt MDM2 and MDMX from binding to p53. Both N-terminal MDM2 and MDMX were successfully expressed and purified with high purity and decent yield. These proteins were used to develop Fluoresence Polarization (FP) and Capillary Electrophoresis (CE) assays for small molecule inhibitors screening. This work has successfully developed FP and CE assays for detecting weakly interacting fragments. The CE assay is a novel method for detecting weak fragments for protein-protein interactions, which are a challenging target. Two approaches were employed to identify small molecule inhibitors for MDM2- N/p53 interaction. At first, small molecules were identified using in silico screening and these hits were verified using FP and CE assays. Second, analogue exploration was applied to identify fragments from the small molecule inhibitors discovered from the in silico screening. Diphenylamine and oxindole fragments were identified as the most potent. However, diphenylamine fragment was discovered to aggregate MDM2-N and was ranked as a false positive hit. No protein aggregation was found when incubated with the oxindole fragment. Therefore oxindole can provide a good starting point for the design of higher affinity analogues. Studying the interaction of MDMX has only recently been undertaken. MDMX contains a high homology binding site with MDM2. Hence, developing a dual MDM2/MDMX inhibitor has become an attractive target to focus on. FP and CE assays were developed to screen compounds against MDMX-N. In silico screening against MDM2-N and MDMX-N found several hits. One compound was discovered as a dual binder to MDM2-N and MDMX-N with low μM affinity. This novel hit is potentially a good starting point for the design of higher affinity analogues.

Multiple sclerosis is a neurodegenerative disorder affecting the central nervous system (CNS). Currently, the disease is incurable and immunomodulating drugs are the only option to control the disease. CD49d is an adhesion receptor expressed on most immune cells. Antibodies that bind to CD49d and block immune cells from trafficking toward the CNS are being pursued as one class of therapeutics. In this work, by combining recombinant antibody and phage display technologies we isolated 10 anti-CD49d single domain antibodies from a synthetic antibody light chain variable domain (VL) phage display library. Isolated VLs (~ 12 kDa) were expressed in Escherichia coli, purified and analysed for biophysical characteristics. The majority were expressed in good yields and were non-aggregating. All 10 VLs bound recombinant CD49d by ELISA, and 7 bound to CD49d-expressing cells in flow cytometry experiments. To empower the VLs for better therapeutic efficacy (thru increasing avidity and half-life), three of the lead VLs were re-engineered as fusions to fragment crystallisable (Fc) of human immunoglobulin gamma (IgG). The engineered hFc-VL fragments (~ 70 – 90 kDa) retained their specificity for CD49d by flow cytometry. With (i) being less immunogenic due to their human nature, (ii) their efficient access to cryptic epitopes (iii) having half-lives comparable to IgGs’ and (iv) being more cost effective compared to IgGs, these novel antibody fragments (monovalent VLs and bivalent hFc-VLs) provide a promising therapeutic platform against multiple sclerosis.

This dissertation examines the surviving remains of the two books of Pindar’s Prosodia. The introduction falls into four parts. The first is concerned with gathering the evidence for the books, through a review of their ancient testimonia (Chapter 1) and of the indirect and direct transmission of their fragments (Chapter 2); the second is concerned with the prosodion as a poetic genre, with some introductory remarks (Chapter 3) followed by an investigation of the collected evidence for the notion of prosodion in describing poetic texts (Chapter 4) and in later scholarship and generic theory (Chapter 5); the third combines the results of the first two into an analysis of the surviving fragments of Pindar’s Prosodia and an inquiry on the generic principles that shaped the collection (Chapter 6); the fourth consists of a descriptive catalogue of the papyrus manuscripts that contribute to the text of Pindar’s Prosodia (Chapter 7). The critical text of the eighteen main fragments and groups of fragments is followed by an introduction and line-by-line commentary to six of them, nos. 1, 2, 3, 5, *6, and *7 (= fr. 89 Snell-Maehler and ‘Paeans’ 14, 15, 6.123-183, 17, and 18).

O presente estudo descreve e caracteriza três dos gêneros poéticos que, segundo a tradição, teriam sido praticados por Píndaro: o hiporquema, o partênio e o prosódio. Essa descrição foi realizada, principalmente, a partir do estudo dos testemunhos dos autores antigos, da análise dos fragmentos remanescentes desses gêneros e da discussão do conhecimento acumulado pela crítica moderna com relação ao tema. Buscou-se identificar o que é cada um desses gêneros, suas origens, as diferenças e semelhanças com relação aos demais gêneros mélicos, sua funçao, finalidade, ocasião na qual eram executados e os principais autores que os praticaram. Além disso, foi realizado um comentário detalhado de cada um dos fragmentos pindáricos supérstites classificados nesses gêneros, destacando questões relacionadas com as dimensões poética, histórica, mítica, lexical, da tradição textual, gramatical e métrica, buscando, quando possível, uma interpretação ao mesmo tempo pormenorizada e completa de cada fragmento. Adicionalmente, discutiu-se a classificação, pelas fontes antigas, desses fragmentos nos gêneros estudados. Por fim, foi realizada uma tradução dessas odes para o português. / This study describes and characterizes three of poetic genres which, according to tradition, had been practiced by Pindar: hyporchemata, parthenia and prosodia. The description was mainly carried out from the study of the testimonies of ancient authors, from the analysis of the remaining fragments of these genres and from the discussion of knowledge accumulated by modern criticism on the issue. We sought to define what each of these genres, its origin, its basic characteristics, differences and similarities with respect to the other melic genres, purpose, occasion for which were made, the situation in which they were executed and the main authors who practiced them. In addition, we performed a detailed comment of pindaric fragments classified in these genres, highlighting issues related to the poetic, historical, mythical, lexical, textual and metrics dimensions, seeking to interpret each fragment. Additionally, it was discussed the classification, by ancient sources and the modern editors, of these fragments in the genres studied. Finally, we performed a translation of these odes to the Portuguese.

Atualmente, existe um grande número de ferramentas para montagem de fragmentos de DNA disponíveis. Neste trabalho apresentamos um estudo comparativo das ferramentas CAP2, FAKtory, TIGR e PHRAP. Para realizarmos este estudo, primeiramente executamos esses sistemas de montagem sobre 12 casos de testes distintos. Após isso, tomamos os resultados obtidos por cada um deles e os comparamos com as seqüências de onde os fragmentos foram originalmente obtidos. Os testes utilizados avaliam a eficiências dos programas com relação a três problemas associados ao processo de montagem (erros no sequenciamento, fragmentos quimeras e regiões repetidas) e pudemos ver que nenhum dos sistemas é claramente superior aos demais no tratamento de todos eles. Cada ferramenta de montagem parece tratar de melhor forma um problema em especial.Além de avaliarmos os resultados, realizamos também um estudo. / Noways, several peckages for DNA fragment assembly are aviable. In this wok we present a comparative study of the preformances of the programs CAP2, FAKtory, TIGR e PHrap. To get to our objetives, we firt ran each of these programs on twelve intances. After this,we compared the outputs with the sequences from wich the fragments were originally obtained. In this comparison,we took into consideration three problems related to fragments assembly (sequencing errors, chimeric fragments and repeats regions). We conclude that no one of the packages we tested is more efficient than the others when considering all the problems cited above. If we consider a particular problem, the we observed different performances among the programs. Even more, we compare the packages with respect to theirs to CPU times.

Esta tese apresenta uma tradução dos fragmentos de Aristófanes e três estudos relativos a eles. O capítulo I, mais geral, é dedicado às fontes. Trata do contexto em que esses fragmentos surgiram, no período helenístico, e da sua transmissão, sobretudo através de escoliastas e lexicógrafos. O segundo e terceiro capítulos tratam de duas comédias em particular, Geritades (Geritads) e Convivas (Daitals). Nesses dois capítulos, a análise dos fragmentos está conjugada a um paralelo com comédias preservadas: no caso de Geritades, que tem por tema a crítica literária, Rãs; e Nuvens, no caso de Convivas, cujo assunto é o embate entre a nova e a velha educação. A segunda parte da tese contém uma tradução acadêmica dos 589 fragmentos de Aristófanes, a partir do original grego para o português. Esse corpus corresponde a todos os fragmentos do comediógrafo, com exceção daqueles que não são atribuídos a uma comédia específica, as chamadas incertae fabulae. / This thesis presents a translation of Aristophanes fragments and three studies about them. The initial chapter, of a more general nature, deals with the sources where the fragments are to be found, the context in wich they were created and their transmission, mainly through scholiasts and lexicographers. The other two chapters focus on two specific comedies: Geritades (Geritads) and Banqueters (Daitals), and parallels are drawn with extant comedies. Geritades is the object of comparative analysis with Frogs, since both plays are concerned with literary criticism. As to Banqueters, the thesis looks into points of contact with Clouds: the contrast between the old education and the new education is a central theme in both comedies. The second part of the thesis consists of an academic translation of the five hundred eightynine fragments, from the Greek original into Portuguese. This corpus corresponds to the totality of the comedians fragments, with the exception of those fragments that are not attributed to any specific comedy, the so called incertae fabulae.