Plasmodium falciparum est un parasite protozoaire responsable de la forme la plus sévère de la malaria. Depuis quelques années, les cas de résistance aux antipaludiques sont devenus de plus en plus fréquents et de plus en plus répandus. En plus de sa résistance aux drogues actuellement disponibles, ce parasite reste jusqu' à aujourd'hui réfractaire aux vaccinations. L’identification de nouvelles approches basées sur l`inhibition spécifique de certaines de ses cibles moléculaires vitales est devenue une nécessité. La recombinase à site spécifique de P. falciparum (Pf-Int) est un enzyme qui a été récemment identifié dans le laboratoire à partir de PlasmoDB. Cette recombinase à site spécifique joue potentiellement un rôle clé dans le système de recombinaison nécessaire à la viabilité du parasite. Cette protéine de 490 acides aminés, soit ~57 kDa, contient une région C-terminale qui porte les résidus conservés du site catalytique des recombinases à tyrosine R-H-K-R-(H/W)-Y. La prédiction montre une région N-terminale qui ressemble à celle de l’intégrase du phage lambda avec un mélange de structures secondaires α et β.Lors de ces travaux, nous avons d’abord montré par RT-PCR que le gène (MAL13P1.42) qui code pour PF-Int est transcrit pendant le cycle intra-érythrocytaire avec un maximum pendant la phase schizont. Nous avons ensuite essayé de montrer l`implication de Pf-Int dans le cycle parasitaire. Ceci a été réalisé grâce à un parasite (KO: knock-out) dont le gène Pf-Int a été invalidé. Ces analyses montrent que Pf-Int n'a aucun impact apparent sur le cycle de développement intra-érythrocytaire du parasite, en particulier sur la durée du cycle et le taux de croissance. Au niveau moléculaire, nous avons également procédé à la production d'anticorps anti-Pf-Int en utilisant le fragment C-162 (Résidus 162-490). La comparaison des profils de marquage, par cet anticorps, des extraits protéiques du KO et du parasite sauvage par la technique de Western blot n'a pas permis d'identifier la protéine endogène dans le parasite sauvage. Dans le but de déterminer la localisation sub-cellulaire de Pf-Int, nous avons réalisé des essais de sur-expression de différentes protéines de fusion dans le parasite. Nous avons essayé de déterminer l’impact de trois codons d’initiation différents ainsi que l’impact de la présence de la région N-terminale (1-190aa) de Pf-Int sur sa localisation subcellulaire en utilisant une chimère entre la partie N-terminale et la protéine GFP. Lors de ces travaux, nous avons réussi à sur-exprimer différentes régions de Pf-Int sous forme recombinante dans E. coli. Nous l’avons d’abord caractérisé par des études biophysiques. Ainsi nous avons pu déterminer, par dichroïsme circulaire (CD), le contenu en structures secondaires de Pf-Int, qui est proche de celui des autres membres de la même famille. Nous avons également démontré sa stabilité par CD couplé à la dénaturation thermique. Le spectre RMN-1D a aussi pu être enregistré. La troisième partie de nos travaux a concerné l’identification des cibles ADN de Pf-Int. Deux stratégies de recherche de cibles par affinité ont été utilisées au laboratoire en utilisant une première bibliothèque de séquences synthétisées chimiquement et une deuxième bibliothèque formée de fragments d’ADN génomique de P. falciparum. Ces deux approches ont permis l’identification de deux séries de cibles ADN. Grace aux cibles ADN identifiées, nous avons pu démontrer l’interaction de différents fragments de Pf-Int avec ces cibles par des expériences de retard sur gel natif (EMSA). Nous avons aussi pu démontrer que les protéines recombinantes sont actives in vitro. En effet, ces dernières sont capables de former des complexes covalents en présence de l’ADN cible. La conservation de la protéine, ainsi que son expression différentielle nous laisse à penser que son rôle est certes loin d’être élucidé, mais que Pf-Int reste une cible potentielle pour P. falciparum. / Plasmodium falciparum is a protozoan parasite responsible for the most severe form of malaria. In recent years, cases of resistance to antimalarial drugs have become increasingly frequent and common. In addition to its resistance to drugs currently available, there is no vaccine available against this parasite till now. The identification of new approaches based on the specific inhibition of some of its molecular targets has become vital.The identification of the Pf-Int site specific recombinase in Plasmodium falciparum by analysis of PlasmoDB is a new opportunity to study the role of genetic variation in this parasite as it needs to adapt to its hosts. This ~ 57 kDa protein contains a C-terminal domain carrying the putative tyrosine recombinase conserved active site residues R-H-K-R-(H/W)-Y, an N-terminus with a predicted alpha-helical bundle and a mixed alpha-beta domain resembling Lambda-Int. Here, we show that the sequence is highly conserved among members of the Plasmodia. It is expressed differentially during distinct life stages as estimated by RT-PCR, namely with a peak in the schizont phase. We then tried to show the involvement of Pf-Int in the parasitic cycle. We were able to create a parasite where the Pf-Int gene was knocked-out. The comparison test showed that Pf-Int has apparently no impact on the intraerythrocytic developmental cycle of the parasite, particularly in the cycle length and the growth rate.At the molecular level, we produced two sets of anti-Pf-Int antibodies using the purified recombinant fragment C-162 (residues 162-490). Comparison of protein extracts from KO and wild parasite by Western blot technique using our antibody has failed to identify the endogenous protein in the wild type parasite.We also tried to determine the subcellular localization of Pf-Int and the role of possible alternate initiation codons by over-expressing different constructs in the parasite Plasmodium falciparum. In order to determine the impact of the N-terminal region (1-190aa) of Pf-Int on its subcellular localization, we also created a chimeric protein using a fusion of Pf-Int(1-190aa) with the GFP. We successfully expressed a variety of the recombinant form of Pf-Int in E. coli. We have first determined its secondary structure content by circular dichroism (CD) and its solution stability by thermal denaturation-CD. An 1-D NMR spectrum was also recorded. The third part of our work has involved the identification of the DNA targets of Pf-Int. Two search strategies conducted in the laboratory using a library of chemically synthesized sequences and a second library made of fragments of genomic DNA of P. falciparum. Both approaches have allowed the identification of two sets of target DNA. Secondly, electrophoretic mobility shift assays (EMSA) were used to show its affinity and specificity for DNA. The recombinant proteins were shown to be functional as they form a covalent complex with DNA. Thus Pf-Int could be a potential agent that binds to and alters DNA, either in a specific or in random fashion. Its conservation and differential expression leads us to conclude that although its role is far from being understood, Pf-Int remains a key target for P. falciparum.

Le mercure (Hg) est un polluant global et ses effets écotoxicologiques sont fortement dépendants de sa forme chimique principalement sous forme de méthylmercure (MeHg). En raison de la forte bioaccumulation et bioamplification du MeHg dans les réseaux trophiques, l'exposition humaine est associée à la consommation de poissons, mais aussi du riz produit à proximité des régions minières riches en Hg (Chine). La composition isotopique du Hg est un outil potentiellement puissant qui a été récemment utilisé pour le traçage de la contamination par mercure et les processus biogéochimiques dans l'environnement et les organismes vivants. Dans ce travail, la spéciation et la composition isotopique du mercure d'échantillons bio-environnementaux ont été étudiées afin d'élucider les voies de contamination par Hg entre les milieux contaminés et certains organismes vivants cibles, tels que le système sol-riz dans une région minière (Hg) et le biote aquatique dans une rivière polluée par une usine de production chlore/soude. Une étude écotoxicologique a également démontré que la composition isotopique de Hg dans les différents organes des poissons permet de mieux comprendre les voies métaboliques du Hg et la réponse toxicologique induite. / Mercury (Hg) is a global pollutant and its ecotoxicological effects are strongly dependent on its chemical form mainly as methylmercury (MeHg). Due to the high bioaccumulation and biomagnification of MeHg in food webs, human exposure occurs through the consumption of fish, but also rice nearby Hg mining area (China). Hg isotopes composition is a potentials powerful tool and has been recently used to trace Hg contamination and biogeochemical processes in environment and living organisms. In this work, Hg speciation and isotopic composition of bio-environmental samples has been investigated in order to elucidate Hg contamination pathways between contaminated sites and target living organisms such as soil-rice plant system in a Hg mining region and aquatic biota in a river impacted by a chlor-alkali plant. An ecotoxicological study has also demonstrated how Hg isotopic composition in different fish organs allows to better understand Hg metabolic pathways and induced toxicological response.

Es ADCs (Antibody-Drug Conjugates) correspondent à une nouvelle stratégie thérapeutique anti-tumorale particulièrement prometteuse. Néanmoins, les ADCs actuellement utilisés en clinique sont obtenus par conjugaisons chimiques, resultant en des mixtures hétérogènes impactant négativement leurs pharmacocinétiques et leurs performances in vivo.Récemment, différentes strategies de couplage site-spécifique ont été développées afin de réduire cette hétérogénéité. Dans cette thèse, nous rapportons le développement d’une nouvelle technologie CovIsoLink™ (Covalently Isopeptide Crosslinking) permettant la génération d’ADCs par utilisation de nouveaux peptides glutamine Q-Tag présentant des affinités optimisées par rapport à des peptides disponibles (ZQG, LLQG) pour une enzyme bactérienne la transglutaminase (mTG).La preuve de concept de cette technologie a été réalisée par insertion de ces peptides Q-Tag en C-ter de la région codant pour la chaine lourde des anticorps anti-HER2 (Trastuzumab). Nous avons ainsi pu démontrer la conjugaison homogène et reproductible de différentes drogues sans contamination par des chaines d’anticorps non conjuguées. Nous avons pu montrer que l’immunoréactivité et la capacité d’internalisation de ces ADCs n’étaient pas altérées par la conjugaison et qu’ils présentaient in vitro et in vivo, des propriétés de lyse de cellules tumorales similaires au Trastuzumab emtansine (Kadcyla®), actuellement en clinique. Par ailleurs, afin de généraliser notre technologie à différents formats d’anticorps nous avons générés des fragments Fab et scFv et évalué leur fonctionnalité. Ainsi, nous avons pu prouver que l’utilisation de nouveaux peptides optimisés Q-Tag substrat de la transglutaminase permettait une stratégie de couplage alternative plus homogène par couplage de différentes molécules sans espèce contaminante non couplée / Antibody-drug conjugates (ADCs) are a powerful class of therapeutic agents, demonstrating success in the treatment of several malignancies. The currently approved ADCs are produced by chemical conjugations and exist as heterogeneous mixtures that negatively influence the pharmacokinetics and in vivo performance. Recently many of site-specific conjugation technologies have been developed to reduce heterogeneity and batch-to batch variability. Microbial transglutaminase (mTG) has been demonstrated as efficient tool for site-specific conjugation. In this thesis we report the development CovIsoLink™ (Covalently Isopeptide Crosslinking) technology for the generation of homogenous immunoconjugates using a novel glutamine donor peptides (Q-tag) with improved affinity compared to the known peptides (ZQG, LLQG). As a proof of concept, the peptides sequences were engineered into the heavy chain C-terminal of Trastuzumab antibody. We demonstrated the reproducible and homogeneous conjugation of Q-tagged Trastuzumab with different payloads, without any unconjugated species. The ADCs were evaluated in series of in vitro and in vivo assays. We confirmed that the immunoreactivity and internalisation are not altered by the conjugation. Furthermore similar in vitro and in vivo tumor cell killing potency was demonstrated than Trastuzumab emtansine (Kadcyla®), which is already used in the clinic. Morover we extend our site-specific conjugation technology to antibody fragments (Fab and scFv), evaluating their functionality by conjugation with AlexaFluor488-cadaverine and in antigen binding assays. Thus, using novel glutamine donor peptides, our technology provides an alternative enzymatic conjugation strategy for the engrafment of different payloads resulting in homogeneous batches, without unconjugated species

A partir de um olhar e de uma escuta direcionadas à cidade, objetivou-se a experiência do corpo no espaço urbano por meio do caminhar como ação poética capaz de suscitar um corpo-crítico que percebesse na paisagem e na arquitetura dos mobiliários urbanos instalados os ideais de cerceamento que fomentam os desequilíbrios sociais e a segregação urbana. A metodologia utilizada inspirou-se na flanerie na cidade, com um mapeamento do uso de espaços públicos, seguido de levantamento das arquiteturas dos mobiliários urbanos, para que as mesmas fossem Re-projetadas, além da coleta feita da paisagem sonora do lugar. Buscou-se assim revelar as segregações, brechas e vazios produzidos por uma anti-arquitetura que cauteriza a experiência sensorial de imersão do corpo na cidade quando estas revelam, na escala fílmica aqui proposta, a solidão e angústia, frutos da leitura de uma cidade fragmentada pelo desenho urbano de onde os vazios, as ausências e o caos engolfam a vida, anulando a experiência do corpo na cidade. / From a direct observation and an attentive listening to the city, this research emphasizes the experience of the body in the urban space taking the act of walking as poetical action as a means to excite a critical-body that could perceive in the landscape and in the architecture of the urban furniture installed the method of clipping ideals that foment social unbalance and urban segregation. The methodology used was inspired in flanerie in the city, with a mapping of the use of public spaces, followed by a survey of the architectures of the urban furniture, so that the same ones were re-projected, beyond the collection of the sonorous landscape of the place. One thus searched to disclose to the segregations, breaches and emptinesses produced by an anti-architecture that cauterizes the sensorial experience of immersion of the body in the city when these polarize on the filmic scale disclosing to the solitude and anguish, fruits of the reading of a city broken up for the urban drawing from where the emptinesses, the absences and the chaos engulf life, annulling the experience of the body in the city.

A partir de um olhar e de uma escuta direcionadas à cidade, objetivou-se a experiência do corpo no espaço urbano por meio do caminhar como ação poética capaz de suscitar um corpo-crítico que percebesse na paisagem e na arquitetura dos mobiliários urbanos instalados os ideais de cerceamento que fomentam os desequilíbrios sociais e a segregação urbana. A metodologia utilizada inspirou-se na flanerie na cidade, com um mapeamento do uso de espaços públicos, seguido de levantamento das arquiteturas dos mobiliários urbanos, para que as mesmas fossem Re-projetadas, além da coleta feita da paisagem sonora do lugar. Buscou-se assim revelar as segregações, brechas e vazios produzidos por uma anti-arquitetura que cauteriza a experiência sensorial de imersão do corpo na cidade quando estas revelam, na escala fílmica aqui proposta, a solidão e angústia, frutos da leitura de uma cidade fragmentada pelo desenho urbano de onde os vazios, as ausências e o caos engolfam a vida, anulando a experiência do corpo na cidade. / From a direct observation and an attentive listening to the city, this research emphasizes the experience of the body in the urban space taking the act of walking as poetical action as a means to excite a critical-body that could perceive in the landscape and in the architecture of the urban furniture installed the method of clipping ideals that foment social unbalance and urban segregation. The methodology used was inspired in flanerie in the city, with a mapping of the use of public spaces, followed by a survey of the architectures of the urban furniture, so that the same ones were re-projected, beyond the collection of the sonorous landscape of the place. One thus searched to disclose to the segregations, breaches and emptinesses produced by an anti-architecture that cauterizes the sensorial experience of immersion of the body in the city when these polarize on the filmic scale disclosing to the solitude and anguish, fruits of the reading of a city broken up for the urban drawing from where the emptinesses, the absences and the chaos engulf life, annulling the experience of the body in the city.

Esta dissertação tem por objetivo analisar o trabalho artís tico Projeto para a Ocupação de uma Casa, realizado dentro do programa de mestrado em Poéticas Visuais entre 2004 e 2005. Através desse trabalho pretendeu-se compreender e aprofundar o interesse no ambiente doméstico presente na produção que o antecedeu. Para isso, foi feita uma revisão crítica dessa produção, onde foram identificados os principais assuntos, recursos e problemas que formaram o interesse no ambiente doméstico. Essa revisão constitui a primeira parte desta dissertação. Na segunda parte, é analisado o Projeto. Partindo de um conjunto de referências prioritariamente literárias, uma série de objetos foi elaborada para ocupar cada um dos cômodos de uma casa escolhida, de modo que, ao percorrê-la, uma narrativa de caráter ficcional se formava. No texto, verificam-se as intenções e resultados de cada peça e ambiente; se elas cumpriram a meta de aprofundamento e entendimento da questão doméstica, as suas soluções formais, os sentidos que engendraram e o papel que desempenharam na constituição da narrativa. / The objective of this thesis is to analyze the art work Project for the Occupation of a House, which was developed within the masters program in Visual Poetics, between 2004 and 2005. The thesis has aimed at understanding and deepening the interest in the domestic environment that was present in the production that preceded it. In order to achieve this, a critical review of the production was carried out, in which the main subjects, resources and problems involved in the interest in the domestic environment were identified. This review constitutes the first part of the thesis. In the second part, the Project is analyzed. With the background of a number of primarily literary references, a series of objects was elaborated to occupy each of the rooms of a chosen house, in a way that as the house was visited, a narrative of fictional character was formed. In the text, the intentions and results of each piece and surrounding are checked: whether or not they have achieved the result of deepening the understanding of the domestic question, their formal solutions, the meanings that were generated, and the role they played in the cons titution of the narrative.

Importance of web services increases as the technology improves and the need for the challenging e-commerce strategies increases. This thesis focuses on web market analysis of web sites by evaluating from the perspectives of static, dynamic and content. Firstly, web site evaluation methods and web analytic tools are introduced. Then evaluation methodology is described from three perspectives. Finally, results obtained from the evaluation of 113 web sites are presented as well as their correlations.

This article is an attempt to put forward the use of new digital techniques and data for understanding prehistoric landscapes. The starting point is that the specific characteristics of the landscape and of the sites included affect the interpretation. One character is the contemporary landscape and its topographies. Ancient landscapes can be successfully recreated digitally using GIS. By applying GIS methodology, a regression equation and new data, we reinvestigated an hypothesis proposed by Welinder in 1978 concerning the acculturation of the Pitted Ware Culture. The results indicate that a reconstruction of the landscape may alter the understanding of the Neolithic land use and the question of the relocation and termination of the Pitted Ware Culture at the end of Middle Neolithic B.

Abstract This report describes our ten week long thesis work which is the last task in our education in mechanical engineering at Linköpings University. The project has been performed at Siemens Industrial Turbomachinery AB’s service department. The company which is located in Finspång produces, sells and provides service for steam and gas turbines around the world. The purpose of this project is to help Siemens develop their maintenance programs for two of the auxiliary systems which are a part of the complete steam turbine system. These are lube and control oil systems. The project is restricted to two of Siemens maintenance products, No Stop Check (NSC) and the annual Safety Inspection (SI). Some parts of the major and limited overhauls have also been investigated. During the work different maintenance concepts have been investigated and evaluated. Focus has been centered on finding possible solutions that allow maintenance work while the steam turbine system is running. The economical consequences are enormous if the turbine has to be shut down. This due to today’s expanded capacity of the plants. Many discussions and interviews have taken place during the project. Information has also been obtained from documents, literature and the Internet. A Failure Mode and Effect Criticality Analysis (FMECA) has also been input for the task, especially while maintenance measures and concepts were discussed. The present maintenance that Siemens supplies are mostly based on time and experience. We have tried to implement a more condition based maintenance. A lot of time has therefore been spent finding methods for reliable measuring of the machinery condition. The work has generated a lot of different recommendations for the future maintenance. These are more or less condition based and tailor-made for Siemens steam turbine systems. We hope that these propositions will be used in the future maintenance work and also to give some new ideas on how to develop the condition based maintenance at Siemens. / Sammanfattning Denna rapport beskriver vårt tio veckor långa examensarbete som är den avslutande delen i vår maskiningenjörsutbildning. Arbetet har utförts vid serviceavdelning på Siemens Industrial Turbomachinery AB (Siemens). Siemens som ligger i Finspång tillverkar, säljer och servar ång - och gasturbinanläggningar över hela världen. Syftet med arbetet är att hjälpa Siemens att utveckla sina serviceprogram för två av de kringsystem som ingår i en ångturbinanläggning, dessa är smörj- och hydrauloljesystem. Arbetet är avgränsat till två av Siemens serviceprodukter, No Stop Check (NSC) och Säkerhetsinspektion (SI). Även vissa delar av de större revisionerna har undersökts. I arbetet har olika underhållsfilosofier undersökts och utvärderats. Fokus har riktats mot att undersöka möjligheter att utföra underhållsåtgärder med turbinanläggningen i drift. I och med den höga kapacitet dagens ångturbinanläggningar har blir de ekonomiska förlusterna enormt stora vid ett eventuellt produktionsbortfall. Under arbetets gång har många diskussioner och intervjuer ägt rum. Information har också inhämtats genom dokument, böcker och Internet. En så kallad Failure Mode and Effect Criticality Analysis (FMECA) har också legat till grund för arbetet när metoder och åtgärder för underhållet diskuterats. Siemens nuvarande underhåll är till stor del erfarenhets- och tidsbaserat. Vi har försökt att applicera ett mer tillståndsbaserat underhåll och därför har mycket tid ägnats åt att hitta metoder att tillståndskontrollera utrustningen med. Arbetet har genererat ett flertal olika rekommendationer på underhållsaktiviteter som är tillståndsbaserade och anpassade för Siemens turbinanläggningar. Tanken är att dessa förslag ska användas i det fortsatta underhållsarbetet och dessutom väcka idéer till fortsatt utveckling av tillståndsbaserat underhåll.

Molecular chaperones help many proteins in the cell reach their native conformation. The mechanism with which they do this has been studied extensively, but has not been entirely elucidated. This work is a continuation of the study done by Laila Villebeck et al. (2007) on the conformational rearrangements in the eukaryotic protein actin in interaction with the eukaryotic chaperone TRiC. In this study the intentions were to analyze the protein MreB, a prokaryotic homologue to actin, when interacting with the prokaryotic chaperone GroEL. The purpose was to investigate if the mechanisms of GroEL and TRiC are similar. The analysis of the conformation of MreB was to be made through calculations of fluorescence resonance energy transfer (FRET) between two positions in MreB labeled with fluorescein. A MreB mutant was made through site-specific mutagenesis to enable labeling at a specific position. Another single mutant and a corresponding double mutant needed for these measurements were avaliable from earlier studies. The results from fluorescence measurements on these mutants indicated that the degree of labeling was insufficient for accurate determination of FRET. Suggestions are made on improvements of the experimental approach for future studies.