Le paludisme constitue un problème majeur de santé publique en zone tropicale et subtropicale. La morbidité ainsi que la mortalité sont principalement dues au parasite Plasmodium falciparum transmis à l'homme par la piqûre de moustiques femelles du genre Anopheles. Dans le but d'orienter au mieux les stratégies d'élimination du paludisme et d'une meilleure évaluation de l'efficacité des méthodes de lutte, les indicateurs mesurant le risque de transmission doivent être plus sensibles. Il a été montré que la réponse anticorps humaine contre des protéines/peptides salivaires d'Anopheles représente un bio-marqueur d'exposition aux piqûres de moustiques et pouvait être un indicateur de la transmission du paludisme. Toutefois cet outil doit être optimisé. Ce travail a ainsi un double objectif : i) valider la protéine salivaire CE5 comme bio-marqueur d'exposition aux piqûres d'Anopheles et comme indicateur évaluant l'efficacité de stratégie de lutte anti-vectorielle, et 2) identifier de nouvelles protéines salivaires comme candidat bio-marqueur spécifique à l'exposition de l'homme aux seules piqûres infectantes d'Anopheles. Tout d'abord, nous avons démontré que la réponse anticorps IgG contre la protéine CE5 pourrait être un indicateur du contact homme-vecteur, complémentaire et très sensible, en mesurant l'exposition de l'homme aux piqûres d'Anopheles et un outil évaluant l'efficacité, à court terme, des moustiquaires imprégnées d'insecticide. Par la suite, les méthodes de protéomique 2D-DIGE et de spectrométrie de masse ont permis d'identifier cinq protéines salivaires (gSG6 , gSG1b , TRIO , SG5 et la forme longue D7) qui sont surexprimées dans les glandes salivaires d'An. gambiae infectées par P. falciparum. Des peptides de chaque protéine, définis in silico, apparaissent antigéniques chez des individus exposés aux piqûres d'Anopheles, après évaluation par la technique d'épitope mapping. L'ensemble de ces travaux est non seulement une première étape pour optimiser cet outil immuno-épidémiologique évaluant le contact homme-vecteur mais démontre également la possibilité de définir un nouveau bio-marqueur qui serait spécifique des piqûres infectantes d'Anopheles. / Malaria is a major public health problem in tropical and subtropical areas. Morbidity and mortality are mainly due to Plasmodium falciparum transmitted to human individuals by the bite of female Anopheles mosquitoes. In order to orientate appropriate strategies for malaria elimination and for a better evaluation of the efficacy of control methods, the indicators measuring the risk of transmission should be more sensitive. It has been shown that the human antibody response against Anopheles salivary proteins/peptides represents a biomarker of exposure to mosquito bites and could be an indicator of malaria transmission. However, this tool must be optimized. This work has thus two objectives: i) to validate the salivary protein cE5 as biomarker of exposure to Anopheles bites and as an indicator for evaluating the efficacy of vector control strategy, and 2) to identify new salivary proteins as a candidate biomarker only specific to human exposure to infective bites of Anopheles.First, we demonstrated that the IgG antibody response to cE5 protein could be an indicator of human-vector contact, complementary and very sensitive, measuring the human exposure to Anopheles bites and a tool evaluating the short-term efficacy of insecticide treated nets. Subsequently, the proteomic methods, 2D - DIGE and mass spectrometry, allowed to identify five salivary proteins (gSG6, gSG1b, TRIO, SG5 and the long form D7) which are overexpressed in the salivary glands of An . gambiae infected by wild P. falciparum. Peptides for each protein, identified in silico, appear antigenic in individuals exposed to Anopheles bites, after the evaluation by the epitope mapping technique.Altogether, this work is not only the first step to optimize this immuno-epidemiological tool assessing the human-vector contact, but also demonstrates the possibility to define a new biomarker specific to the infective bites of Anopheles.

Le paludisme demeure un problème de santé majeur en Afrique subsaharienne. Le nombre limité d'antipaludiques, l’apparition de résistances et l’absence d’un vaccin efficace, font de la lutte anti-vectorielle (LAV) la principale stratégie préventive de cette maladie. Les méthodes actuelles de LAV visant à limiter ou à interrompre le développement du parasite chez le moustique vecteur, il est donc nécessaire d’améliorer notre compréhension des interactions entre le vecteur Anopheles, son environnement et le parasite Plasmodium. A Madagascar, Anopheles gambiae s.l. et Anopheles funestus sont les vecteurs majeurs de Plasmodium falciparum et de Plasmodium vivax. Anopheles mascarensis, espèce endémique, peut également être un vecteur important. Dans ce contexte, l’objectif premier de ma thèse a été d’approfondir les connaissances sur An. mascarensis à travers une revue. Les données collectées plaident davantage qu’An. mascarensis est un complexe d'espèces et permettent de poser les bases pour une analyse moléculaire ciblée. En parallèle, j’ai contribué à la mise en place de la première plateforme expérimentale de Madagascar pour infecter des anophèles par P. falciparum et P. vivax, afin d’évaluer leur compétence vectorielle. Enfin, en associant entomologie et immuno-parasitologie, nous avons analysé la contribution des vecteurs à la transmission du paludisme dans deux villages adjacents. L’ensemble des travaux réalisés durant de ma thèse contribue à une meilleure connaissance de la diversité de la transmission du paludisme à Madagascar. De plus, la mise en place de la plateforme expérimentale d’infection permettra l’analyse de la compétence des populations d’anophèles vecteurs. / Malaria remains a major health concern in sub-Saharan Africa. The limited number of antimalarial drugs, the emergence of resistances and the lack of an effective vaccine, make vector control the main preventive strategy for this disease. Current methods of vector control aim at limiting or interrupting parasite development in the vector mosquito. It is therefore necessary to improve our understanding on interactions between the Anopheles vector, its environment and the parasite Plasmodium. In Madagascar, Anopheles gambiae s.l. and Anopheles funestus are the major vectors of Plasmodium falciparum and Plasmodium vivax. Anopheles mascarensis, an endemic species, may also be an important vector. In this context, the main objective of my PhD was to deepen the knowledge on An. mascarensis through a review. The data collected indicate that An. mascarensis is a complex of sibling species. I could thus provide the foundation for targeted molecular analysis. In parallel, in order to evaluate their vector competence, I contributed in a major way to the establishment of the first experimental platform of Madagascar to infect anopheline mosquitoes by P. falciparum and P. vivax. Finally, combining entomology and immuno-parasitology, we analysed the contribution of vectors to malaria transmission in two neighbouring villages. All the work done during my PhD contributes to a better knowledge of the diversity of malaria transmission in Madagascar, especially on the effective contribution of the different vector species. In addition, the establishment of the experimental platform for infections will further allow the analysis of the competence of vector Anopheles populations.

Malaria ist eine Krankheit, die durch den Protozoen Plasmodium verursacht und von Anopheles Moskitos durch infektiöse Stiche übertragen wird. Diese ̈Übertragung kann durch verschiedene Interventionsstrategien blockiert werden. Eine relativ neue Strategie, die bisher nur im Labor getestet wurde, ist der Einsatz genetisch veränderter Moskitos, die den Parasiten nicht auf einen neuen menschlichen Wirt übertragen können. Ein Ansatz ist die Entwicklung von Moskitos, die mit murinen Antikoepern ausgestattet sind, die gegen relevante Oberflächenproteine des Parasiten, dem Circumsporozoite Protein (CSP) gerichtet sind. Es ist jedoch nach wie vor unklar, welches Entwicklungsstadium angegriffen werden soll und welche Antikörper für diesen Ansatz effizient sind. Hier zeige ich, dass in Stechmücken, die mit einem humanen Anti-CSP-Antikörper ausgestattet sind, die Sporogonie der Oozysten in Abhängigkeit von der Parasiten-dichte blockiert wird und somit die Sporozoitenlast in den Mücken signifikant verringern. Insbesondere Antikörper, die sich an die ’Repeat Region’ des CSP binden, können die Sporozoitenlast in der Stechmücke verringern. Des Weitern, zeigen diese Stechmücken kaum Defekte in der Entwicklung und im Überleben. Diese Ergebnisse bestätigen die zuvor beschriebene Bedeutung von CSP während der Sporogonie und unterstreichen die Effizienz von humanen, ’Repeat Region’ bindenden Anti-CSP-Antikörpern bei der Beeinträchtigung der Parasitenentwicklung in dem Vektor. Darüber hinaus ist in Stechmücken, die mit humanen Anti-CSP Antikörpern ausgestattet sind, die Entwicklung von Sporozoiten teils limitiert und teils komplett verhindert. Dies macht sie zu einem vielversprechenden Instrument für Maßnahmen zum Malaria Kontrolle. In dieser Arbeit habe ich weitere Einblicke in den Mechanismus , durch den Anti-CSP-Antikörper die Parasitenentwicklung in der Mücke stören, und gezeigt, dass Oozysten ein effizientes Ziel für diesen Ansatz sind. / Malaria is a disease caused by the protozoan parasite Plasmodium and transmitted by Anopheles mosquitoes trough infectious bites, these transmission events can potentially be blocked by different intervention strategies. A relatively new strategy which has been only tested in the laboratory is the use of genetically modified mosquitoes unable to transmit the parasite to a new human host. In the past, murine derived antibodies directed against relevant parasite surface proteins were used with limited success. It remains unclear which developmental stage is targeted, and which antibodies are useful. Here, I show that in mosquitoes equipped with a human derived anti-CSP repeat binding antibody, oocyst sporogony is blocked in a parasite density dependent manner. Only repeat binding antibodies could decrease sporozoite loads in the mosquito. These results confirm the previously described importance of CSP during sporogony and highlight the efficiency of human derived repeat binding anti-CSP antibodies in interfering with parasite development even in a different host. Additionally, mosquitoes equipped with human derived anti-CSP antibodies show little (in high density infections) to no (in low density infections) sporogonic development, making them a promising tool for malaria transmission blocking interventions. I provided additional insights into the mechanism by which anti-CSP antibodies interfere with parasite development in the mosquito showing that oocysts are an efficient target for this approach. Therefore, the mosquitoes used here are potentially resistant in a more natural setting. Additionally, I provided a new tool allowing a faster screening of antibodies in a mosquito context by injection of single chain Fabs into the mosquito hemolymph. Taken together, this approach could one day give rise to alternative strategies in tackling malaria transmissions.

Einer der ausschlaggebenden Faktoren für das Übertragungspotenzial von Malaria ist die Häufigkeit ihres Überträgers, der Anopheles-Mücke. Da diese Insekten besonders empfindlich auf ihre Umgebung reagieren, wurde in vielen Laborexperimenten das Verhältnis zwischen Umweltfaktoren und Malaria untersucht. In natürlichen Umgebungen ist über diesen Zusammenhang wenig bekannt. Untersuchungsgegenstand dieser Arbeit mit multidisziplinärem Ansatz ist die Beziehung zwischen verschiedenen Umwelteinflüssen und der Häufigkeit von Anopheles-Mücken in natürlicher Umgebung sowie die möglichen diesem Prozess zugrundeliegenden Mechanismen. Zunächst lässt sich anhand von Feldstudien und empirischen dynamischen Modellen eine verzögerte Auswirkung der Umwelt auf die Mückenhäufigkeit feststellen, was wiederum einen Einfluss auf die Entwicklung der Larvenstadien bedeuten könnte. Im Anschluss werden mögliche Hintergründe untersucht mit Hilfe eines kontrollierten Versuchsaufbaus, in dem Anopheles-coluzzii-Larven und Erwachsenentiere auf Umweltbedingungen reagieren. Dabei lässt sich feststellen, dass Temperatur und Besatzdichte vielschichtige Auswirkungen auf Larvenentwicklung und -sterblichkeit haben. Darüber hinaus gibt es Übertragungseffekte auf die Alterung und Fruchtbarkeit der Erwachsenentiere. Abschließend zeigt sich durch die Entwicklung eines vollständigen Modells für den Lebenszyklus von Anopheles-Mücken, dass die ermittelten Umwelteinflüsse auf Larven und erwachsene Mücken einen komplexen Einfluss auf die Größe der Mückenpopulation haben. Anhand mehrere mathematischer Methoden werden in dieser Arbeit Informationen aus Feldsammlungen und Versuchsdaten erfolgreich kombiniert. Dies unterstreicht die Bedeutung multidisziplinärer Ansätze für das Verständnis der komplexen Dynamik zwischen Mücken und der Umwelt. So entsteht ein detailliertes Bild der Lebensmerkmale der Mücken, die von der Umwelt beeinflusst werden, und deren Auswirkungen auf die Populationsdynamik. / One of the main determinants of malaria transmission is the abundance of its vector, the Anopheles mosquito. As these insects are susceptible to the environment in which they live, many laboratory experiments have explored the connection between environmental factors and the abundance of mosquitoes. However, little is known about this relationship in natural settings. In this work, I used a multidisciplinary approach to determine the relationship between environmental drivers and Anopheles abundance and the possible mechanisms that characterise these relationships. First, using field study data and an empirical dynamic modelling approach, I identified a delayed effect of the environment on mosquito abundance, suggesting an impact on the development of larval stages. Second, I explored the possible mechanisms behind this process using a controlled experimental setup in which Anopheles coluzzii larvae and adults responded to the environmental conditions. I identified a complex response to temperature and rearing density of larval development and mortality, as well as carry-over effects on adult ageing and fecundity. Finally, by developing a full model for the life cycle of Anopheles mosquitoes, I determined that the identified environmental impacts on larvae and adult mosquitoes have a complex impact on the mosquito population size. Taken together, this work combined the information from field collections and experimental data successfully using multiple mathematical methods, highlighting the importance of multidisciplinary approaches in understanding the complex dynamics between mosquitoes and the environment. With this approach, we obtained a detailed picture of the mosquito life traits that are impacted by the environment and its effects on mosquito population dynamics.

Master of Science / Department of Biochemistry / Michael R. Kanost / Laccases are multicopper oxidases that catalyze the oxidation of a broad range of substrates, typically phenols and anilines. Research on laccases in fungi, plants, and bacteria has indicated that they have roles in detoxification, pigmentation, wound healing, morphogenesis and lignin synthesis and degradation. However, there has been relatively little investigation on laccases that exist in insects or other invertebrates. Insects have multiple laccase genes, but the function of just one type is known; laccase-2 (Lac2) orthologs are required for tanning of newly synthesized exoskeleton. In the mosquito Anopheles gambiae and other insect species whose genomes have been sequenced, alternative exon splicing may generate two isoforms of Lac2. The objective of this study was to characterize the two isoforms of AgLac2. They are identical in their first 500 residues, but the carboxyl-terminal 262 residues derived from alternative exons are 81% identical. Recombinant Lac2A and Lac2B were expressed and purified. They are both glycoproteins of ~81 kDa, and both can oxidize the laccase substrate ABTS as well as the catechols, N-β-alanyldopamine (NBAD) and N-acetyldopamine (NADA). Lac2A and Lac2B with ABTS have pH optima of 5.0-5.5 and 4.5-5.0, respectively. The pH optima with NBAD and NADA are 5.5-6.5. The Km values (mM) for Lac2A and Lac2B with NBAD are 5.4 ± 2.1 and 5.0 ± 2.6, respectively. The Km values (mM) for Lac2A and Lac2B with NADA are 0.7 ± 0.2 and 1.4 ± 0.5, respectively. Thus, there is little difference between the isoforms in K[subscript]m for these two substrates. The K[subscript]m values do indicate that both isoforms have a greater affinity for the substrate NADA. The kcat values (s[superscript]-1) for Lac2A and Lac2B with NBAD are 14.2 ± 3.5 and 6.0 ± 1.8, respectively. The k[subscript]cat values (s[superscript]-1) for Lac2A and Lac2B with NADA are 2.4 ± 0.2 and 0.5 ± 0.04, respectively. The most apparent difference between the two isoforms detected in the study is that Lac2A was four-fold more active than Lac2B when NADA was used as a substrate. Although the two isoforms are very similar in their amino acid sequences, the differences in catalytic properties may indicate different roles in insect physiology.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Although malaria remains a major public health concern, especially in sub-Saharan Africa, little information exists on the physiological tolerances of malaria vectors. Here, I aimed to provide a comprehensive set of physiological tolerances for Anopheles arabiensis and An. funestus, by investigating thermal tolerance traits of adults, larvae and pupae; desiccation resistance of adults and development rate-temperature relationships for both vectors. Critical thermal limit (CT) and desiccation data showed significant effects of increasing adult age on reducing tolerance to temperature or dry conditions. Females of both species were more tolerant of high or low temperatures in CT experiments and were more desiccation tolerant than males in desiccation trials. Anopheles funestus was more desiccation tolerant than An. arabiensis, despite the common misconception that An. arabiensis is the more arid-adapted of the two species. Comparisons between thermal tolerance traits of adult laboratory and wild strain progeny of both species indicated a high degree of similarity between critical thermal limits in wild and laboratory strains, suggesting that the use of laboratory populations of both mosquito strains can provide an accurate estimate of wild population responses to thermal change. Lethal temperature estimates for both vectors indicated a higher tolerance to high temperature in An. arabiensis larvae and pupae when compared with An. funestus, and a greater tolerance of high or low temperatures in adult females when compared with adult males. Species differences between the vectors were further highlighted in development rate-temperature experiments. Under fluctuating and constant temperatures, An. arabiensis developed significantly faster than An. funestus and had higher survival to the adult stage. Under fluctuating temperatures, An. arabiensis developed faster or no different to constant temperatures, while survival under fluctuating temperatures was also comparable to constant temperature estimates. This faster development rate of this species is likely a consequence of the puddle-breeding nature of An. arabiensis and the need to develop to adulthood before evaporation of breeding sites. Anopheles funestus on the other hand, showed reduced survival and development under fluctuating temperatures when compared with constant temperatures, probably as a result of the more thermally stable breeding sites usually used by this species. Distribution data of these species, combined with developmental parameters in a process-based distribution model, suggests that both species will show range changes in response to climate change. Areas where these species were previously only present on a seasonal basis might become more suitable for vector population establishment and persistence, while areas on the northern margins of current distributions will become less favourable, leading to an overall southerly shift in habitat suitability for both species. Increases in temperature and changes in rainfall patterns as predicted to occur with climate change are likely to impact the distribution of both malaria vectors. Combining the physiological tolerance data collected in this thesis in a future, planned mechanistic distribution model, will provide an accurate indication of potential range shifts of these vectors and hence, provide an indication of areas that may be at increased risk of malaria. / AFRIKAANSE OPSOMMING: Alhoewel malaria „n groot publieke gesondheidskwelling bly, veral in sub-Sahara Afrika, bestaan min inligting rakende die fisiologiese toleransies van malaria vektore. Hier het ek gepoog om 'n omvattende reeks van fisiologiese toleransies te voorsien vir Anopheles arabiensis en An. funestus, deur termiese verdraagsaamheidseienskappe, uitdrogingsweerstand en ontwikkelingstempo-temperatuur verhoudings vir beide vektore te ondersoek. Kritiese termiese limiet (CT) en uitdroging data het beduidende uitwerkings getoon van toenemende ouderdom op die vermindering van verdraagsaamheid teenoor temperatuur of droë toestande. Wyfies van beide spesies was meer verdraagsaam vir hoë of lae temperature in CT eksperimente en was meer verdraagsaam teenoor uitdroging as mannetjies in die uitdrogingsproewe. Anopheles funestus was meer verdraagsaam teenoor uitdroging as An. arabiensis, ten spyte van die algemene wanopvatting dat An. arabiensis die meer ariede aangepaste van die twee spesies is. Vergelykings tussen termiese verdraagsaamheidseienskappe van laboratorium-en wilde stamlyn nageslagte van beide spesies het 'n hoë mate van ooreenkoms tussen kritieke termiese limiete in wilde en laboratorium stamlyne aangedui, wat voorstel dat die gebruik van laboratorium bevolkings van beide muskiet stamlyne 'n akkurate skatting kan gee van wilde bevolkingsreaksies tot termiese verandering. Fatale temperatuur beramings vir beide vektore het „n hoër toleransie getoon by hoë temperature in An. arabiensis larwes en papies wanneer dit vergelyk word met An. funestus, en 'n groter verdraagsaamheid van hoë of lae temperature in wyfies, wanneer vergelyk word met mannetjies. Spesies verskille tussen die vektore is verder uitgelig in die ontwikkelingstempo-temperatuur eksperimente. Onder wisselende en konstante temperature ontwikkel An. arabiensis aansienlik vinniger as An. funestus en het hoër oorlewing tot die volwasse stadium getoon. Onder wisselende temperature ontwikkel An. arabiensis vinniger of met geen verskil van konstante temperature nie, terwyl oorlewing onder wisselende temperature ook vergelykbaar was met konstante temperatuur beramings. Die vinniger tempo van hierdie spesie is waarskynlik 'n gevolg van die poel-broeiende aard van An. arabiensis en die behoefte om tot volwassenheid te ontwikkel voor die verdamping van broeiplekke. Anopheles funestus aan die ander kant, het verminderde oorlewing en ontwikkeling onder wisselende temperature gewys wanneer dit vergelyk word met konstante temperature, waarskynlik as gevolg van die meer termies stabiele broeiplekke wat gewoonlik gebruik word deur hierdie spesie. Verspreidingsdata van hierdie spesies, gekombineer met ontwikkelings-parameters in 'n proses-gebaseerde verspreidingsmodel, dui daarop dat beide spesies reeks veranderinge sal wys in reaksie tot klimaatsverandering. Gebiede waar hierdie spesies voorheen slegs teenwoordig was op 'n seisoenale basis, mag dalk meer geskik word vir vektor bevolkingsvestiging en volharding, terwyl areas op die noordelike grense van die huidige verspreidings minder gunstig sal word, wat sal lei tot algehele suidelike verskuiwing in die habitat geskiktheid vir beide spesies. Toenames in temperatuur en veranderinge in reënvalpatrone, soos voorspel word om voor te kom met verandering van die klimaat, sal waarskynlik die verspreiding van malaria vektore beïnvloed. Deur die fisiologiese toleransie data, versamel in hierdie tesis, te kombineer met 'n toekoms, beplande meganistiese verspreidingsmodel, sal dit 'n akkurate aanduiding gee van die potensiële verspreidingsverskuiwings van hierdie vektore en dus 'n aanduiding gee van gebiede wat onder verhoogde risiko van malaria sal wees.

The development of behavioural resistance in Anopheles mosquitoes is an important threat to the sustainability of current vector interventions, which selectively target mosquitoes that are found indoors. Changes in the following behaviour have been reported in several Anopheles vectors: biting time, host preference, biting and resting behaviour. However, a critical review of the published literature reveals that behavioural resistance has not been demonstrated clearly in Anopheles, highlighting important limitations in using entomological data to investigate the evolution of behaviour. The availability of next generation sequencing and low-cost custom genotyping technologies makes it possible to investigate behavioural resistance using genetic information as an alternative approach. Using such technologies, this thesis aims to investigate whether Anopheles vectors are evolving towards outdoor-biting behaviour ('exophagy') by examining the genetic difference between the indoor- and -outdoor population of the main African malaria vectors, An. coluzzii and An. gambiae from The Gambia and Uganda, respectively. We also investigated the role of olfactory-related genes in driving the potential genetic difference between the two populations. We found limited divergence between the two populations overall, however close inspection identified SNPs potentially associated with outdoor-biting behaviour as well as genomic regions displaying elevated levels of differentiation between the two populations. Most notably, gustatory receptors, Gr11 and Gr12 and the odour receptor, OR1 were identified as well as, genes related to transcription, signal transduction and catalysis. We discuss the potential role of these genes in determining biting behaviour and suggest that further investigation of these genomic regions is required to confirm the early-stage divergence of indoor- and outdoor-biting Anopheles populations.

In der vorliegenden Arbeit haben wir unter Nutzung bioinformatischer Methoden eine innovative Strategie zur Eindämmung der Malaria entwickelt. Die genetische Modifikationsstrategie beinhaltet sowohl Manipulationen aufseiten des gefährlichsten Erregers, Plasmodium falciparum, als auch des Hauptvektors, Anopheles gambiae. In den Genomen beider Spezies wurden eine Reihe neuer konkreter targets identifiziert. Auch bereits beschriebene targets und Ansätze wurden in die Strategie einbezogen bzw. weiter ausgestaltet. Bezüglich der Vektormoskitos wird die Verbreitung eines gegenüber Plasmodien resistenten Genotyps angestrebt. Es werden einerseits effiziente natürliche und künstliche Resistenzgene diskutiert und andererseits eine bekannte Strategie zur Fixierung natürlicher Resistenzallele in natürlichen Populationen verbessert. Auf der Seite der Plasmodien erweiterten wir einen bereits von A. Burt (2003) beschriebenen Eradikationsansatz um weitere targets. Aus ethischen und evolutionsbiologischen Erwägungen bevorzugen wir jedoch eine alternative Strategie, welche die Etablierung von in ihrer Virulenz gemilderten Parasiten zum Ziel hat. Der attenuierte Genotyp wird unter anderem durch komplexe Pathway-Remodellierungen beschrieben (Löwe, Sauerborn, Schirmer, Dandekar, A refined genome engineering strategy against parasites and vectors, Manuskript beim Journal „Genome Biology“ eingereicht). Da sich Mutanten in der Natur gegen Wildtyp-Organismen kaum durchsetzen können, werden zwei drive-Systeme beschrieben, welche für die Implementierung der genetischen Manipulationsstrategie entwickelt wurden. Beide Konstrukte wurden zur Patentierung angemeldet (Patentanmeldung U30010 DPMA bzw. Aktenzeichen 102006029354.1). Zusätzlich zur deutschen wurde für eines der beiden Konstrukte eine PCT-Anmeldung eingereicht, welche in Zukunft einen internationalen Patentschutz ermöglichen soll. Es werden Kalkulationen vorgelegt, welche die Verbreitungstendenzen der Konstrukte in natürlichen Populationen vorhersagen. Die Beschreibung der entwickelten Konstrukte beschränkt sich nicht auf das primäre Anwendungsgebiet der Arbeit (Malaria), sondern beinhaltet auch andere Anwendungsgebiete, vor allem im Bereich der Medizin und Molekularbiologie. / Background: Gene drive strategies are an important alternative to control tropical diseases such as malaria. Results: Here we introduce a new gene drive strategy based on gene conversion constructs. We identify a gene drive strategy both for plasmodia and for anopheles including design of an inducible modification vector. Our constructs are based on group II introns or homing endonuclease genes. They include besides the intron to modify vector or parasite genome sites inducible promoters for gene activation. We thus separate gene modification from activation of the modified gene. Moreover, we provide a detailed list of suitable targets in vector and plasmodia for the modification strategy. Finally, we discuss the control effect of an eradication strategy versus a mild strategy of the gene construct for vector and parasite populations. Conclusions: A new eukaryotic vector and parasite control strategy using gene drive systems is presented and discussed.

Foi feito o estudo da Bromélia-Malária no Brasil, e de acordo com os dados disponíveis foi reconstituída a distribuição geográfica e evidenciada a incidência dessa enfermidade entre nós. Para o levantamento do estado atual dispusemos de dados inéditos referentes a São Paulo e a Santa catarina, nestes últimos, englobados também os referentes ao Rio Grande do Sul. Pudemos verificar que nessas áreas em diferentes estágios de erradicação, São Paulo, cujo plano de erradicação quase concluído, luta para não ser restabelecida a transmissão, e Santa Catarina, ainda com uma grande parte a ser concluída, luta não só para interromper a transmissão nas áreas ainda maláricas, como para não ser restabelecida, ou mesmo estabelecida a transmissão nas áreas não maláricas. / Actualized data about Bromelia-Malaria in Brazil, are presentedthrough a reconstituted geographic distribution and new records about the disease incidence. New data are related to São Paulo and Santa Catarina States, including the ones belonging to Rio Grande do Sul. There are several areas in different erradication stages. In São Paulo, it is almost concluded and struggle is against the menace of a transmission restablishment. At other places there are a quite different picture because the transmission in malaria areas but also for to avoid its introduction in disease - cleaned ones.

Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-02-11T16:04:49Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_IdentificacaoFontesAlimentares.pdf: 390524 bytes, checksum: 30da67281c86b34fe1809bf589257afc (MD5) / Approved for entry into archive by Ana Rosa Silva (arosa@ufpa.br) on 2014-04-10T14:24:46Z (GMT) No. of bitstreams: 2 Dissertacao_IdentificacaoFontesAlimentares.pdf: 390524 bytes, checksum: 30da67281c86b34fe1809bf589257afc (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Made available in DSpace on 2014-04-10T14:24:46Z (GMT). No. of bitstreams: 2 Dissertacao_IdentificacaoFontesAlimentares.pdf: 390524 bytes, checksum: 30da67281c86b34fe1809bf589257afc (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Previous issue date: 2006 / O ensaio imunoenzimático para identificação de repastos sangüíneos apresenta especificidade até nível de gênero, sensibilidade para identificação de repastos sangüíneos parciais e detecção de repastos múltiplos. Foram identificadas as fontes alimentares de 82% dos anofelinos coletados em campo. Foram testados seis anticorpos monoclonais (humano, suíno, cão, bovino, rato e galinha) e destes, apenas o anti- IgG bovino apresentou instabilidade. Obteve-se 55,7% (519/932) dos repastos positivos para sangue humano, o que demonstra, a preferência alimentar destes anofelinos por humanos. Dos 206 mosquitos que apresentaram repasto único, 27,6% foi em humanos. O Anopheles darlingi apresentou 41% dos repastos em humanos e o Índice de Sangue Humano (HBI) no intradomicílio foi de 0,71. An. marajoara apresentou 51,3% dos repastos em humanos, embora tenha sido encontrada em grande quantidade no ambiente extradomiciliar e apresentando HBI no intradomicílio de 0,76. O An. nuneztovari foi a espécie mais abundante, apresentando comportamento exofílico e antropofílico, com 53,8% de repastos em humano e HBI no intradomicílio de 0,65. O método ELISA Sanduíche está implantado, identificando a fonte alimentar das espécies de anofelinos coletadas em campo, exceto para bovino. É a primeira vez que esta técnica é utilizada para determinação de repastos sanguíneos em anofelinos na região Amazônica brasileira. É importante a determinação da fonte alimentar das espécies de anofelinos no sentido de caracterizar o comportamento antropofílico e assim associá-las ou não à transmissão de malária. / The immunoenzymatic assay for the bloodmeal identification has specificity till genus level, sensitivity for the identification of partial bloodmeal and detection of multi bloodmeal. It was detected the blood source of 82% of all mosquitoes specimens collected at the transmission area. We had testes six monoclonal antibodies for human, pig, dog, bovine, rat and chicken, and from them, only the bovine one had been unstable. It was obtained 55,7% (519/932) of the blood source for human, which demonstrated, these mosquitoes preference for human blood. From the 206 mosquitoes that had just a unique blood source, 27,6% were in human. The Anopheles darlingi had fed in human (41%) and his human bloodmeal index (HBI) indoor was 0,71. An. marajoara had presented 51,3% of the bloodmeal in humans, although had been collected in large amount outdoor and showed the HBI indoor of 0,76. The An. nuneztovari was the specie more abundant, demonstrating an exophilic and anthropophilic behavior, with 53,8% of blood source in humans and the HBI indoor of 0,65. The ELISA test is established, identifying the blood source of the anopheline mosquitoes collected in the malaria transmission area, except for bovine. This is the first time that this technique is being used for the determination of the blood source in anopheline mosquitoes in Brazilian Amazonia. It is very important the determination of the blood source of the anopheline species in order to characterize their anthropophilic behavior and to associate them or not with the malaria transmission.