Les dimères chromosomiques se produisant lors de la réparation de chromosomes circulaires peuvent être dommageables pour les bactéries en bloquant la ségrégation des chromosomes et le bon déroulement de la division cellulaire. Pour remédier à ce problème, les bactéries utilisent le système Xer de monomérisation des chromosomes. Celui-ci est composé de deux tyrosine recombinases, XerC et XerD, qui vont agir au niveau du site dif et procéder à une recombinaison qui aura pour effet de séparer les deux copies de l’ADN. Le site dif est une séquence d’ADN où deux répétitions inversées imparfaites séparées par six paires de bases permettent la liaison de chacune des recombinases. Cette recombinaison est régulée à l’aide de FtsK, une protéine essentielle de l’appareil de division. Ce système a été étudié en profondeur chez Escherichia coli et a aussi été caractérisée dans une multitude d’espèces variées, par exemple Bacillus subtilis. Mais dans certaines espèces du groupe des Streptococcus, des études ont été en mesure d’identifier une seule recombinase, XerS, agissant au niveau d’un site atypique nommée difSL. Peu de temps après, un second système utilisant une seule recombinase a été identifié chez un groupe des epsilon-protéobactéries. La recombinase fut nommée XerH et le site de recombinaison, plus similaire à difSL qu’au site dif classique, difH. Dans cette thèse, des résultats d’expériences in vitro sur les deux systèmes sont présentés, ainsi que certains résultats in vivo. Il est démontré que XerS est en mesure de se lier de façon coopérative à difSL et que cette liaison est asymétrique, puisque XerS est capable de se lier à la moitié gauche du site prise individuellement mais non à la moitié droite. Le clivage par XerS est aussi asymétrique, étant plus efficace au niveau du brin inférieur. Pour ce qui est de XerH, la liaison à difH est beaucoup moins coopérative et n’a pas la même asymétrie. Par contre, le clivage est asymétrique lui aussi. La comparaison de ces deux systèmes montrent qu’ils ne sont pas homologues et que les systèmes Xer à seule recombinase existent sous plusieurs versions. Ces résultats représentent la première découverte d’un espaceur de 11 paires de bases chez les tyrosine recombinases ainsi que la première étude in vitro sur XerH. / The chromosome dimers produced during the repair of circular chromosomes can be harmful to bacteria by blocking the segregation of the chromosome and cell division. To overcome this problem, bacteria use the Xer system for the monomerisation of chromosome dimers. It has two components, XerC and XerD, which act on the dif site and complete a recombination that will lead to the separation of the two copies of the DNA. The dif site is a DNA sequence where two imperfect inverted repeats separated by six base pairs allow the binding of each recombinase. This recombination is regulated by the protein FtsK, an essential member of the cell division machinery. The Xer system has been well studied in Escherichia coli and has also been characterized in a variety of species, for example Bacillus subtilis. Furthermore, in certain species of Streptococcus, studies have identified only a single recombinase, XerS, which acts on an atypical site named difSL in order to monomerize dimeric chromosomes. Not long after, a second system using a single recombinase was identified in a group of epsilon-proteobacteria. This recombinase was named XerH and the recombination site, difH, was found to more similar to difSL than to the classical dif sites. In this thesis, results from in vitro experiments on both systems are presented, as well as some results from in vivo experiments. We show that XerS is capable of binding cooperatively to difSL and that this binding is asymmetrical. This is because XerS is able to bind to the left half of the site but not to the right half when they are separated. The cleavage by XerS is also asymmetrical, as it is more efficient on the bottom strand. As for XerH, its binding to difH is much less cooperative and doesn’t have the same asymmetry. But the cleavage is also asymmetrical like the one seen in XerS. Comparing the two systems show that they are not homologuous and that more than one version of Xer systems using a single recombinase exists. These results represent the first discovery of an 11 bases pairs spacer for tyrosine recombinase. It is also the first in vitro studies of XerH.

No Brasil o bicudo da cana-de-açúcar, (Sphenophorus levis Vaurie) tem se tornado uma praga importante da cultura, podendo causar perdas expressivas na produtividade. Percebe-se que são raros os estudos sobre a distribuição espacial desta praga no campo, e tais estudos são imprescindíveis para o desenvolvimento de planos de amostragem, visando à aplicação em programas de manejo integrado de pragas. O objetivo deste trabalho foi caracterizar a distribuição espacial de S. levis em cana-deaçúcar, utilizando análise geoestatística, a fim de validar o procedimento de amostragem georreferenciada adotado pelo usuário ou propor um método confiável, prático e viável. Também foi objetivo deste trabalho gerar mapas de aplicação localizada para poder intervir numa área de produção, possibilitando comparar o consumo de insumo de uma aplicação convencional com uma aplicação localizada. O estudo se desenrolou em quatro áreas pertencentes à Usina Iracema, em Iracemápolis, SP. Na Fazenda Iracema foi realizada uma amostragem adensada para avaliar a melhor densidade amostral para caracterizar a dependência espacial da praga, e os resultados mostraram que o padrão utilizado pelo usuário foi suficiente para caracterizar a dependência espacial de S. levis. Além disso, a variável que melhor representou a ocorrência da praga foi \"toco atacado\". Na Fazenda Santo Elias foram criados mapas com diferentes níveis de controle e mesmo adotando o nível mais conservador (0,2 tocos atacados) houve uma economia considerável de produto. Nas Fazendas Santo Antônio e Santa Lúcia, foi realizada a amostragem seguindo o padrão proposto pelo usuário e foram gerados mapas com nível de controle de 0,2 TA a fim de realizar a aplicação localizada. Nestas áreas houve uma economia de produto de no mínimo 43% e no máximo 87%. O sistema de amostragem utilizado pelo usuário (17 pontos ha-1) foi adequado para caracterizar a variabilidade espacial da praga. Mesmo utilizando o nível de controle mais rigoroso observou-se uma economia considerável na quantidade de inseticida utilizado na aplicação localizada, confirmando o potencial da utilização de técnicas de agricultura de precisão em trabalhos relacionados ao manejo integrado de pragas. / In Brazil the Sphenophorus levis has caused serious damages in sugarcane plantations, with significant losses in productivity. Studies about spatial distribution of this insect in the field are rare, but crucial for the development of sampling plans seeking the application of programs for its integrated management. The objective of this study was to characterize the spatial distribution of S. levis in a sugarcane plantation, utilizing geostatistics to validate sampling procedures used in the field. Also, the aim of this study was to generate maps of localized application in order to interfere in the production area, allowing product amount comparisons between conventional application and a localized application.The study was developed in four areas in Iracemápolis, SP. At the Iracema farm it was developed a very density sampling to evaluate the best sample density to characterize the spatial distribution of the plague, and the result showed that the standard used by the user was sufficient to characterize the spatial dependence of S. levis. Moreover, the variable that best represented the occurrence of the pest was \"attacked stumps\". At the St. Elias Farm, maps were created with different control levels and even adopting the more conservative level (0.2 attacked stumps) there was a considerable product saving. In Santo Antônio and Santa Lúcia farms, sampling was conducted based in standard proposed by the user and maps were generated with 0.2 attacked Stumps level control, in order to achieve a localized application. A minimum product saving of 43% and a maximum 87% was achieved in these areas. The sampling system used by the user (17 points ha-1) was adequate to characterize the spatial variability of the pest. Even using the most rigorous level of control there was a considerable saving in the amount of insecticide used in localized application, endorsing the potential use of precision farming techniques in plagues studies.

Der maschinengestützte Pflanzenmassesensor "Pendulum-Meter" kann online die teilflächenspezifischen Differenzierung der Bestandesfrischmassen und -trockenmassen der meisten Wachstumsstadien von Winterweizen, Winterroggen und Naßreis bestimmen. Das Pendulum-Meter ist in Weizen und Roggen sehr gut geeignet für die Stadien BBCH 39 bis 69, und mit geringerer Genauigkeit auch für die Stadien BBCH 32 bis 34. In Naßreis wurde die Biomasse in allen getesteten Wachstumsstadien von BBCH 25 bis BBCH 65 gut erfaßt. In früheren Wachstumsstadien ist eine Messung nicht möglich. Die Kraft-Winkel-Beziehung des Sensors ist nicht linear. Der wichtigste Faktor für diese Kontaktmessung ist der Biegewiderstand der Getreidehalme. Zur Reduzierung der Rohdaten zu repräsentativen Werten für die Parzellen sind sowohl der Mittelwert des Auslenkungswinkels und der Mittelwert des Vektors, als auch der Median des Auslenkungswinkels geeignet. Die Ergebnisse der Optimierungsversuche in bezug auf die Wiederholgenauigkeit der Meßwerte und die Abbildungsgenauigkeit der Pflanzenmasse zeigten eine große Bandbreite von geeigneten Einstellungsparametern und keine einzelnen optimalen Parameter. Trotzdem sollte die Pendelmasse mP niedrig sein, um eine Zerstörung einiger Pflanzen zu vermeiden. Die Höhe des Zylinderkörpers hA0 sollte die Halme berühren, um den Biegekontakt sicherzustellen. Und die Drehpunkthöhe hP sollte in Bestandeshöhe sein, um eine größtmögliche Bandbreite an Auslenkwinkeln für eine gegebene Biomasse zu erhalten. Die optimalen Einstellungen für hP, hA0, und mP sind in jedem Wachstumsstadium anders, aber eine einzige Pendeleinstellung für alle Wachstumsstadien ist möglich, jedoch läßt sie in vielen Wachstumsstadien eine gute Genauigkeit der Biomassebestimmung vermissen. Ohne Kalibrierung ermittelt das "Pendelum-Meter" immer noch gut die relative Verteilung der teilflächenspezifischen Biomasse, also der Heterogenität des Feldes. Wenn nur die Bestandesheterogenität von Interesse ist, können alle Pendeleinstellungen ohne Kalibrierung benutzt werden. Die Meßwiederholungen mit derselben Parametereinstellung zeigte eine Standardabweichung der Parzellenmittelwerte von weniger als 1°, in den meisten Fällen geringer als 0.3° für die meisten Wachstumsstadien. Der Variationskoeffizient ist für die meisten Einstellungsparameter geringer als 5 %, oft kleiner als 2 %, und er ist größer je kleiner die Bandbreite des gemessenen Winkels ist. Die Genauigkeit der Pflanzenmassebestimmung durch das Pendulum-Meter ist durch Bestimmtheitsmaße von 0.90 oder höher gekennzeichnet. Die lineare Abbildung zeigte dabei geringfügig niedrigere R2 als die quadratische, außer in Roggen, wo in den späteren Wachstumsstadien die Pflanzenmasse wesentlich besser quadratisch darstellt wurde. Auch zeigten die geplotteten Residuen dieser Regression allein im Roggen eine Verfälschung durch einen quadratischen Einfluß. Die Standardfehler dieser Regressionen zur Abbildung der Biomasse waren in der Regel geringer als 2 in Naßreis, geringer als 3 in Winterweizen, und geringer als 4 in Winterroggen, und mit dem Bestandeswachstum zunehmend. Die multiple und einfache Regression der Abhängigkeit der Meßwerte von den Parametereinstellungen des Sensors wurde stark von der Pflanzenmasse der Parzellen beeinflußt, weshalb eine Kalibrierung notwendig ist, wenn die Einstellungsparameter geändert werden. Die Geländeneigung lenkt das Pendel aus, ohne einen Getreidebestand zu messen, und benötigt eine Korrektur durch einen Neigungssensor. Die Fahrgeschwindigkeit muß konstant gehalten werden, da der gemessene Auslenkwinkel eine starke Abhängigkeit von der Geschwindigkeit zeigt, aber gleichzeitig auch von der Menge der Pflanzenmasse. Der Biomassesensor kann die Bestandesdichte bestimmen, wenn die Bestandeshöhe homogen ist, und bestimmt die Bestandeshöhe, wenn die Bestandesdichte konstant ist. Wind mit niedriger Geschwindigkeit ist verfälscht die Biomassemeßwerte nicht meßbar, aber bei höheren Windgeschwindigkeiten wird der Fehler größer. Weitere verfälschende Faktoren sind Unkräuter, das tägliche Pflanzenwachstum, die Neigung des Halmes, Vertiefungen der Fahrgasse, verschiedene Sorten, die eine Eichung des Biomassesensors unter den meisten Umständen notwendig machen. Die Genauigkeit des Meßprinzips wurde mit einem Geräteträger ermittelt, der seine Ausrichtung gegenüber dem Erdboden nicht veränderte, aber ein Traktor kann die Messungen durch seine Eigenbewegung verfälschen. Die Messungen des Biomassesensors können als Entscheidungsbasis zur teilflächenspezifischen Applikation von Wachstumsregeln und Fungiziden dienen, wenn auch die Entscheidungskriterien noch erarbeitet werden müssen. Teilflächenspezifische Applikationen von Wachstumsreglern und Fungiziden nach den Messungen des Pendulum-Meters, also nach der Pflanzenmasse, sind erfolgreich durchgeführt worden. Die Messungen des Pendulum-Meters können dabei als Entscheidungsbasis für die teilflächenspezifische Applikation von Wachstumsreglern und Fungiziden benutzt werden, obwohl die Beziehung zwischen Lager und Pflanzenmasse, und zwischen den meisten Schadpilzen und der Biomasse weitere Untersuchungen erfordert, genauso wie die Ermittlung der notwendigen Aufwandmenge an Wachstumsreglern und Fungiziden je nach Pflanzenmasse. Der Biomassesensor Pendulum-Meter ist für Kontrolle der mittleren und späten Applikationen von Wachstumsreglern und Fungiziden geeignet, hier die Stadien BBCH 32 - 59, aber nicht für die frühen Applikationen, hier die Stadien BBCH 25 - 31, wegen der fehlenden Eignung den Sensor in niedrigem Pflanzenbestand zu benutzen. In der Fungizidapplikation kann der Sensor ähnlich den LAI-Meßgeräten benutzt werden, und in der Ausbringung von Wachstumsreglern hat der Sensor eine große Gemeinsamkeit mit dem Widerstand gegen das Lagern. / The machine-based biomass sensor pendulum-meter can determine on-line the site-specific differentiation of cereal crop fresh masses and dry masses for the most growth-stages in winter wheat, winter rye, and irrigated rice. The pendulum-meter is well suited in wheat and rye for the growth-stages BBCH 39 to BBCH 69, and to a lesser degree for BBCH 34 and 32. Irrigated rice crop biomass was well sensed by the pendulum-meter at all tested growth-stages BBCH 25 to BBCH 65. Earlier growth-stages were not possible to measure. The angle-force relation of the pendulum-meter is non-linear. The most important factor for this contact measurement was found in the bending moment of resistance of the stems. For the reduction of the original data to representative plot values, the average of the angle, the average of the vector, and the median of the angle were suitable. The results of the optimisation trials, in terms of repeatability of the measurement and the accuracy of biomass determination, showed a wide range of suitable parameter settings and not a single optimal parameter. Nevertheless, the mass of the pendulum mP should be low to avoid destruction of the plants, the height of the cylindrical body hA0 should touch the stems to ensure bending contact, and the height of the pivot point hP should be at crop height to get a wide range of angles of deviation for a given range of biomass. For every growth-stage, the optimum hP, hA0, and mP are different, and although a single pendulum-meter setting for all growth-stages is possible, but lacking good accuracy of biomass determination in many growth-stages. Without calibration the pendulum-meter still senses well the relative distribution of the site-specific biomass, hence the field heterogeneity. When only the crop's heterogeneity is of interest, all pendulum settings can be used without calibration. The replicates with the same pendulum parameter settings show a standard deviation of the plot average of less than 1°, mostly less than 0.3° for the most growth-stages. The coefficient of variation is mostly less than 5 %, often less than 2 %, and it is higher the smaller the range of measured angles is. The accuracy of biomass determination of the pendulum-meter showed mostly R2 of 0.90 or higher. The linear regression performed with slightly lower R2 than the square, except for rye, where the square regression was much better for the late growth-stages. Solely in rye the plotted residuals showed a square bias. The standard errors of the regressions were less than 2 in rice, less than 3 in wheat, and less than 4 in rye, increasing during crop growth. The multiple and simple regression for the dependency of the measured angle on the parameter settings of the pendulum was strongly influenced by the biomass thus causing a re-calibration when changing the pendulum parameters. The slope of the terrain is deviating the pendulum-meter without crop and needed a correction by a slope sensor. The carrier speed has to be constant, and the angle of deviation is highly dependent on the carrier speed, but simultaneously dependent on the amount of biomass. The biomass sensor senses the tiller density, when the plant height is homogeneous, and the plant height when the tiller density is constant. Wind at low speeds is not a biasing factor, but at high wind speeds the bias will increase. Further biasing factors can be weeds, daily plant growth, stem inclination, tramline depth, and variety, thus requiring a re-calibration of the biomass sensor under most conditions. The accuracy of the measurement principle was determined with a carrier that was not changing its orientation towards the soil surface, but a tractor might bias the measurements because of its own movement. Site-specific application of growth-regulators and fungicides according to the measurements of the pendulum-meter, and hence the biomass, has been successfully conducted. The measurements of the pendulum-meter can be used as a decision base for the site-specific application of growth-regulators and fungicides, although the relationship between lodging and biomass, and between most fungi and biomass needs further examination, as well as determining the necessary amount of growth-regulators and fungicides according to biomass. The biomass sensor pendulum-meter is suitable for controlling the intermediate and late applications of growth-regulators and fungicides, here BBCH 32 - 59, but not for the early applications, here BBCH 25 - 31, due to impossibility of using the sensor in low plants. In fungicide application, the sensor can be used similar to LAI, and in growth-regulators sprayings, the sensor has in principle a high similarity with lodging resistance.

This dissertation considers the impact mobile media technologies have on the production and consumption of memory narratives and cultural memory discourses in Canada. Although this analysis pays specific attention to concepts of memory, heritage, and public history in its exploration of site-specific digital narratives, it is set within a larger theoretical framework that considers the relationship between mobile technology and place, and how the mobile phone in particular can foster both a sense of place and placelessness. This larger framework also includes issues of co-presence, networked identity, play, affect, and the phenomenological relationship between the individual and the mobile device. This is then considered alongside memory narratives (both on the national and quotidian levels) at specifically sanctioned sites of national commemoration (monuments, historic sites) and also in everyday urban spaces. To this end, this dissertation covers a wide range of augmented reality apps and forms of digital storytelling including locative media narratives, site-specific digital performances, social media and crowdsourced heritage archives, and urban mobile gaming and playful mapping. Despite common criticism that mobile phones only serve to distract us from our surrounding environment, I argue that mobile technology can generate deeper, more affective attachments to places by reformulating ways of perceiving and moving through them. They do this by insisting that place is more than just its material properties, but rather is composed of a fluctuating relationship between materiality, time, and affect. Following this framework, I also emphasize how mobile technology shifts the traditional mission of the archive to preserve and protect the past to something more playful, more affective, and more preoccupied with the circulation of the past in the present. Included in this analysis are crowdsourced archives created on social media platforms which, I argue, are particularly well suited to capturing the dynamic qualities of memory and living heritage practices. A contributing factor in this is the mobile phone’s position as a site of intimacy and co-presence, which situates it in a long history of communication technologies that employ rhetorical and technological strategies of co-presence, immediacy, and intimacy. Chapter one examines the role that locative media narratives play at official sites of memory in Canada’s capital region from app-based historical tours to more playful narrative encounters, through the lens of the archive and the repertoire. Chapter two then considers the digital site-specific performance piece, LANDLINE, to unpack how mobile media foster everyday place memories in urban spaces through the mobile phone’s position as a site of intimacy for geographically distant, but virtually co-present, individuals. Chapter three analyzes my own experimental method, Maplibs, which follows a mobile game structure to encourage participants to engage in acts of playful placemaking and collaborative storytelling in order to highlight an alternative process of engaging with place that carries the past forward in meaningful ways. And finally, chapter four analyzes the social media group “Lost Ottawa” to explore how collaborative memory communities mobilize through social media platforms like Facebook and create new forms of participatory heritage. In all of this, place is understood as a dynamic assemblage of stories and memories that the mobile phone, through its ubiquitous impact on social practices, plays a key role in shaping.

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.

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

This study evaluated the applicability of remote sensing instrumentation for site- specific management of abiotic and biotic stress on cotton grown under a center pivot. Three different irrigation regimes (100%, 75%, and 50% ETc) were imposed on a cotton field to 1) monitor canopy temperatures of cotton with infrared thermometers (IRTs) in order to pinpoint areas of biotic and abiotic stress, 2) compare aerial infrared photography to IRTs mounted on center pivots to correlate areas of biotic and abiotic stress, and 3) relate yield to canopy temperatures. Pivot-mounted IRTs and IR camera were able to differentiate water stress between the irrigation regimes, however, only the IR camera was effectively able to distinguish between biotic (cotton root rot) and abiotic (drought) stress with the assistance of groundtruthing. The 50% ETc regime had significantly higher canopy temperatures, which were reflected in significantly lower lint yields when compared to the 75% and 100% ETc regimes. Deficit irrigation up to 75% ETc had no impact on yield, indicating that water savings were possible without yield depletion.

This study evaluated the applicability of remote sensing instrumentation for site- specific management of abiotic and biotic stress on cotton grown under a center pivot. Three different irrigation regimes (100%, 75%, and 50% ETc) were imposed on a cotton field to 1) monitor canopy temperatures of cotton with infrared thermometers (IRTs) in order to pinpoint areas of biotic and abiotic stress, 2) compare aerial infrared photography to IRTs mounted on center pivots to correlate areas of biotic and abiotic stress, and 3) relate yield to canopy temperatures. Pivot-mounted IRTs and IR camera were able to differentiate water stress between the irrigation regimes, however, only the IR camera was effectively able to distinguish between biotic (cotton root rot) and abiotic (drought) stress with the assistance of groundtruthing. The 50% ETc regime had significantly higher canopy temperatures, which were reflected in significantly lower lint yields when compared to the 75% and 100% ETc regimes. Deficit irrigation up to 75% ETc had no impact on yield, indicating that water savings were possible without yield depletion.

This project examines contemporary performances that disrupt normative understandings of time/history. I argue that the complimentary regimes of heterosexuality and capitalism produce the temporal logics that create the psychic and material conditions under which U.S. queer subjects experience everyday, national, and transnational trauma. These logics include the construction of time/history as linear, teleological, and progress-oriented, and the idealized citizen as similarly straight, productive, and amnesic. I then analyze the ways in which queer performance can resist and transform chrono-normativity by creating "time slips": worlds in which past and present are given permission to touch; history/memory to repeat; and the future to reside in the now. Case studies include Ann Carlson and Mary Ellen Strom's Geyser Land (2003); floodlines (2004-2010), which I conceived and directed; and Peggy Shaw and The Clod Ensemble's Must: The Inside Story (2011). I situate my analysis against the backdrop of a post-9/11 security state that makes these performative disruptions particularly vital at this historical moment. / text

As a class, protein-based therapeutics offer tremendous advantages over traditional small molecule drugs. Due to their sizes and folding energies, proteins are ideal for catalyzing chemical reactions, and can bind tightly and selectively to extended target surfaces. However, due to their large size, virtually all proteins are unable to spontaneously enter cells, and as a result protein therapeutics are restricted to extracellular targets. We developed a platform for delivery of proteins to intracellular target sites by engineering the surface chemistry of a model protein, green fluorescent protein (GFP). We found that 'supercharged' cationic GFP variants (scGFPs) bind to anionic cell surface molecules and initiate endocytosis, resulting in the efficient delivery of translationally fused cargo to intracellular targets. We discovered that scGFPs, and cationic delivery reagents in general, alter endosomal trafficking in a manner proportional to both their charge and their delivery efficiency, suggesting that avoidance of endosomal maturation is a key step in the endosomal escape of delivered protein cargos. We also developed a method for encapsulation of recombinant proteins by cationic lipid delivery reagents using negatively supercharged GFP. Genetic modification technologies have matured rapidly following the discovery of protein classes with programmable DNA-binding specificities. While site-directed genetic knockout technologies are highly effective, targeted integration and repair remain comparatively inefficient. Site-specific recombinases directly catalyze strand exchange and ligation between DNA molecules, offering an approach to efficient genomic integration. However, most site-specific recombinases are not easily reprogrammable. To address this problem, we developed a genetic selection technique based on the Phage-Assisted Continuous Evolution (PACE) system, to enable the rapid evolution of recombinase proteins towards targets of interest. Using Cre recombinase as a model, the PACE system was optimized, validated, and used to evolve Cre variants with higher activity on their native loxP target site, as well as altered specificity towards a human genomic sequence within the hROSA26 locus. Finally, we developed a method for enhancing the specificity of RNA-guided nucleases by restricting activity to sites of obligate dimeric nuclease assembly. We engineered a FokI nuclease fusion to a catalytically inactivated Cas9 protein that mediates efficient modification with significantly reduced off-target activity.