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Reducing disease prevalence through selection for host resistance offers a desirable alternative to chemical treatment which is a potential environmental concern due to run-off, and sometimes only offers limited protection due to pathogen resistance for example (Chen et al., 2010). Genetic analyses require large sample sizes and hence disease phenotypes often need to be obtained from field data. Disease data from field studies is often binary, indicating whether an individual became infected or not following exposure to infectious pathogens. In genetic analyses of binary disease data, however, exposure is often considered as an environmental constant and thus potential variation in host infectivity is ignored. Host infectivity is the propensity of an infected individual to infect others. The lack of attention to genetic variation in infectivity stands in contrast to its important role in epidemiology. The theory of indirect genetic effects (IGE), also known as associative or social genetic effects, provides a promising framework to account for genetic variation in infectivity as it investigates heritable effects of an individual on the trait value of another individual. Chapter 2 examines to what extent genetic variance in infectivity/susceptibility is captured by a conventional model versus an IGE model. The results show that, unlike a conventional model, which does not capture the variation in infectivity when it is present in the data, a model which takes IGEs into account captures some, though not all, of the inherent genetic variation in infectivity. The results also show that genetic evaluations that incorporate variation in infectivity can increase response to selection and reduce future disease risk. However, the results of this study also reveal severe shortcomings in using the standard IGE model to estimate genetic variance in infectivity caused by ignoring dynamic aspects of disease transmission. Chapter 3 explores to what extent the standard IGE model could be adapted for use with binary infectious disease data taking account of dynamic properties within the remit of a conventional quantitative genetics mixed model framework and software. The effect of including disease dynamics in this way was assessed by comparing the accuracy, bias and impact for estimates obtained for simulated binary disease data with two such adjusted IGE models, with the Standard IGE model. In the first adjusted model, the Case model, it was assumed that only infected individuals have an indirect effect on their group mates. In the second adjusted IGE model, the Case-ordered model, it was assumed that only infected individuals exert an indirect effect on susceptible group mates only. The results show that taking the disease status of individuals into account, by using the Case model, considerably improves the bias, accuracy and impact of genetic infectivity estimates from binary disease data compared to the Standard IGE model. However, although heuristically one would assume that the Case-ordered model would provide the best estimates, as it takes the disease dynamics into account, in fact it provides the worst. Moreover, the results suggest that further improvements would be necessary in order to achieve sufficiently reliable infectivity estimates, and point to inadequacy of the statistical model. In order to derive an appropriate relationship between the observed binary disease trait and underlying susceptibility and infectivity, epidemiological theory was combined with quantitative genetics theory to expand the existing framework in Chapter 4. This involved the derivation of a genetic-epidemiological function which takes dynamic expression of susceptibility and infectivity into account. When used to predict the outcome of simulated data it proved to be a good fit for the probability of an individual to become infected given its own susceptibility and the infectivity of its group mates. Using the derived function it was demonstrated that the use of a linear IGE model would result in biased estimates of susceptibility and infectivity as observed in Chapters 2 & 3. Following the results of Chapter 4, the derived expression was used to develop a Markov Chain Monte Carlo (MCMC) algorithm in order to estimate breeding values in susceptibility and infectivity in Chapter 5. The MCMC algorithm was evaluated with simulated disease data. Prior to implementing this algorithm with real disease data an adequate experimental design must be determined. The results suggest that there is a trade-off for the ability to estimate susceptibility and infectivity with regards to group size; this is in line with findings for IGE models. A possible compromise would be to place relatives in both larger and smaller groups. The general discussion addresses such questions regarding experimental design and possible areas for improvement of the algorithm. In conclusion, the thesis advances and develops a novel approach to the analysis of binary infectious disease data, which makes it possible to capture genetic variation in both host susceptibility and infectivity. This approach has been refined to make those estimates increasingly accurate. These breeding values will provide novel opportunities for genome wide association studies and may lead to novel genetic disease control strategies tackling not only host resistance but also the ability to transmit infectious agents.

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Made available in DSpace on 2014-10-09T12:28:36Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:57:31Z (GMT). No. of bitstreams: 0 / A doença de Chagas, zoonose causada pelo protozoário Trypanosoma cruzi, apresenta uma ampla distribuição na América Latina estendendo-se do Sul dos Estados Unidos até a Argentina. Estima-se existirem 10 milhões de pessoas infectadas e outras 25 milhões expostas ao risco. Apesar de descoberta há mais de um século, o mal de Chagas ainda é uma infecção grave que provoca grande impacto sócio-econômico, sem tratamento efetivo na fase crônica e que carece de conhecimentos científicos. O presente trabalho teve como objetivo a obtenção e o uso de linhagens consômicas de camundongos, na investigação da resistência. As linhagens consômicas foram produzidas por meio de acasalamentos programados e monitoração com marcadores polimórficos de DNA, onde um de seus cromossomos foi substituído pelo seu homólogo da outra linhagem. Como parentais foram utilizadas as linhagens isogênicas C57BL/6/J Unib, de fenótipo resistente (doadora) e A/JUnib, susceptível (receptora) empregadas na produção de cinco linhagens consômicas para os cromossomos 7 (CSs7), 11 (CSs11),14 (CSs14),17 (CSs17) e 19 (CSs19); descritos por Passos et al. (2003), como importantes no controle da infecção causada pela cepa Y do T.cruzi. Nos ensaios experimentais, os consômicos foram inoculados pela via i.p., com as doses de 101, 102, 103 e 104 empregando-se como controles animais de ambas as linhagens parentais. Em todos os consômicos, a resistência foi superior àquela observada no parental susceptível. Em um segundo protocolo, os consômicos foram acasalados com associações programadas e as progênies foram desafiadas empregando-se inóculos com doses crescentes de tripomastigotas. A resistência observada neste grupo foi também superior àquela observada no parental de fenótipo susceptível. Os resultados observados demonstram que o emprego das linhagens consômicas produzidas possibilitam avaliar a participação de cada cromossomo na resistência, bem como os efeitos da associação entre os cromossomos, que são uma estratégia eficiente no estudo de doenças multifatoriais de trato complexo, como a doença de Chagas. / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

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Microsporidia infect invertebrate and vertebrate animals. Human pathogenic microsporidia are associated with severe disease in immunocompromised individuals, and mostly asymptomatic infection in the immunocompetent. Treatment options for microsporidiosis are limited, incompletely effective, and associated with toxicity. Furthermore, microsporidia infection of healthy individuals is poorly understood, and the consequences of asymptomatic infection have not been determined. Little is known about the molecular mechanisms of microsporidia infection, but such information is essential for the development of new therapies. Spores adhere to host cell surfaces in vitro. Our laboratory has focused on determining specific host cell and microsporidia spore surface participants in spore adherence. Our previous studies have shown that host cell sulfated glycosaminoglycans and the spore surface protein EnP1 participate in spore adherence to host cells. Additionally, in vitro inhibition or augmentation of spore adherence decreased or increased host cell infection, respectively. These studies demonstrated the importance of spore adherence in host cell infection and began to characterize the host cell and spore determinants of adherence. The goal of this research was to further characterize host cell and spore participants in microsporidia adherence and infection of host cells in vitro. We characterized an intracellular microsporidia protein and related antibodies for analyses of microsporidia spore surface proteins; characterized a spore surface protein, MsADAM, involved in spore adherence to and infection of host cells in vitro; and suggested a role for host cell integrins in microsporidia adherence to and infection of host cells in vitro.

Die vorliegende Arbeit beschäftigt sich mit der Synthese und Charakterisierung potenzieller Inhibitoren des Oberflächenproteins Mip von Legionella pneumophila. Der gramnegative Mikroorganismus ist der ursächliche Erreger der Legionellose. Die Erkrankung kann in zwei verschiedenen Formen auftreten, dem Pontiac-Fieber, einer Grippe-ähnlichen Atemwegserkrankung, und der Legionärskrankheit, einer schweren Lungenentzündung mit einer Mortalitätsrate von bis zu 30 %. Natürliche und künstlich geschaffene Süßwassersysteme bilden den biologischen Lebensraum der Bakterien. Aus dieser Umgebung werden sie über technische Vektoren wie Duschen oder Klimaanlagen durch Inhalation kontaminierter Aerosole auf den Menschen übertragen, wo sie alveoläre Makrophagen besiedeln und eine pulmonale Infektion hervorrufen. Um die Zellen in der Lunge zu erreichen, müssen die Mikroorganismen jedoch zuerst die alveoläre Barriere, bestehend aus einer Epithelzellschicht und extrazellulärer Matrix, überwinden. Dafür ist das Oberflächenprotein Mip, der Hauptvirulenzfaktor, verantwortlich. Mip ist ein Homodimer, das sich aus einer C- und einer N-Domäne zusammensetzt. Während der N-Terminus für die Dimerisierung des Proteins verantwortlich ist, weist der C-Terminus die typische Faltung einer Peptidyl-Prolyl-cis/trans-Isomerase (PPIase) auf. Der Vergleich der Aminosäuresequenz der Mip-C-Domäne mit der Domäne verschiedener humaner PPIasen zeigte eine besonders große Homologie zu FKBP12, welches zur Familie der FK506-bindenden Proteine gehört und eine wichtige Rolle innerhalb des menschlichen Immunsystems spielt. Bemerkenswerterweise hemmen bekannte immunsuppressive FKBP12-Inhibitoren wie FK506 und Rapamycin neben der humanen PPIase ebenfalls das bakterielle Mip. Außerdem wurde beobachtet, dass die C-terminale Mip-PPIase an Kollagen IV, den Hauptbestandteil in der menschlichen Lunge, bindet und somit für die Transmigration der Legionellen in die Lunge verantwortlich ist. Mip-Inhibitoren sollten demnach eine Legionellen-Infektion verhindern können. Zur Verifizierung der Hypothese sollten daher im Rahmen dieser Arbeit neue Leitstrukturen für Mip-PPIase-Inhibitoren entwickelt werden. Mit Hilfe von Molecular-Modelling-Untersuchungen basierend auf der NMR-Struktur 2VCD wurde als eine mögliche Leitstruktur N,N-Dimethylphenylsulfonsäureamid identifiziert. Deshalb sollten diese Verbindung sowie Analoga hergestellt werden. Obwohl die Immunsuppressiva FK506 und Rapamycin Mip-Inhibitoren darstellen, können sie auf Grund ihrer immunsuppressiven Eigenschaften nicht in der Legionellosetherapie eingesetzt werden. Die makrozyklischen Immunsuppressiva setzen sich im Gegensatz zu N,N-Dimethylphenylsulfonsäureamid allerdings aus zwei strukturellen Einheiten, einer Binde- sowie einer Effektordomäne, zusammen. Die Bindedomäne mit dem Pipecolinsäure-Grundgerüst ist für die Wechselwirkungen mit Mip und FKBP12 verantwortlich. Die Effektordomäne hingegen ist der aliphatische Teil der Makrozyklen, der erst durch die Bildung eines ternären Komplexes eine Immunsuppression hervorruft. Somit können Verbindungen vom Pipecolinsäure-Typ keine immunsuppressive Wirkung haben und stellen demnach optimale, neue Leitstrukturen für Mip-Inhibitoren dar. Aus diesem Grund wurden die zwei literaturbekannten, nicht-immunsuppressiven FKBP12-Inhibitoren A und B ausgewählt und in verschiedenen Docking-Studien untersucht. Das Molecular-Modelling zeigte, dass nur Verbindung B reproduzierbare Interaktionen mit Mip eingehen kann und demnach ein potenzieller Inhibitor ist. Um dies zu überprüfen, sollten beide Verbindungen A und B sowie eine Mischform hergestellt werden. Neben diesen Verbindungen wurden weiterhin Variationen an der Struktur vorgenommen. Alle Verbindungen wurden in einem In-vitro-Enzymassay gezielt auf ihre Mip-Interaktion untersucht. Die In-vitro-Untersuchungen zeigten, dass nur Pipecolinsäure-Derivate vom Sulfonsäureamid-Typ B die Mip-PPIase inhibieren, die besten Verbindungen sind 22b, 23a und 24a. Neben den enzymatischen In-vitro-Testungen wurden exemplarisch für die Verbindungen 1a, 12a, 22a und 22b HSQC-NMR-Experimente zur Bestimmung der Inhibitor-Proteinbindung durchgeführt. Für Verbindung 22b wurde zusätzlich die In-vivo-Wachstumshemmung der Legionellen mittels Gentamicin-Infektionsstudien ermittelt. / The present work focused on the synthesis and characterization of potential inhibitors of the surface protein Mip of Legionella pneumophila. The gram-negative microorganism is the causative agent of Legionellosis, which may occur in two different progressive forms, the Pontiac fever, a flu-like respiratory disease, and the Legionnaires disease, a severe pneumonia with mortality rate of up to 30 %. Natural and man-made fresh water systems provide a biological habitat to the bacteria. From this environment they were transmitted into humans via technical vectors such as showers or air conditioners by inhaling the contaminated aerosols. Within the human lung, they affect alveolar macrophages and cause a pulmonary infection. Though, to affect the alveolar macrophages the microorganisms have first to cross the alveolar barrier, which consists of epithelial cells and an extracellular matrix. For this, the surface protein Mip, which represents the main virulence factor of the bacterium, is responsible. Mip forms a stable homodimer, which consists of two domains, a C- and an N-domain. While the N-terminus is responsible for the dimerization of the protein, the C-terminus exhibits the typical folding of a peptidyl-prolyl-cis/trans-isomerase (PPIase). The comparison of the amino acid sequence of the Mip-C-domain and the domain of different human PPIases shows a particularly high homology to FKBP12. This human PPIase belongs to the family of the FK506 binding proteins and plays an important role within the human immune system. Remarkably, known immunosuppressive FKBP12-inhibitors such as FK506 and Rapamycin are also able to inhibit the bacterial Mip. Furthermore, it was observed that the C-terminal Mip-PPIase binds to collagen IV, the main component of the human lung, and therefore, Mip is responsible for the transmigration of Legionella. Due to this fact, Mip-inhibitors should prevent a Legionellosis. To verify this hypothesis, the intention of this work was to develop novel lead structures for Mip-PPIase-inhibitors. By means of molecular modeling studies based on the NMR structure 2VCD N,N-dimethylbenzenesulfonamide was identified as a potential lead structure. Therefore, this compound as well as analogues were aimed to prepare. Although the macrolides FK506 and Rapamycin inhibit Mip, they cannot be used for the treatment of Legionellosis due to their immunosuppressive effects. Compared to N,N-dimethylbenzenesulfonamide the macro cyclic, immunosuppressive drugs consist of two structural domains, a binding domain and an effector domain. The binding domain with the pipecolic acid feature is responsible for the interactions with Mip and FKBP12, respectively, whereas the effector domain, the aliphatic part of the molecule, causes the immunosuppression by forming a ternary complex. Therefore, compounds of the pipecolic acid type cannot affect an immunosuppression and thus, represent optimal, novel lead structures. Due to this fact, the two common, non-immunosuppressive FKBP12-inhibitors A and B were analyzed by means of different docking studies with the result that only compound B is able to interact with Mip. Therefore, it was assumed that B must be a potential Mip-inhibitor. To verify this hypothesis, both compounds A and B as well as a hybrid of them should be prepared. In addition to these compounds, further structural variations were prepared. To determine the interaction with Mip, all synthesized compounds were analyzed by means of an in vitro enzyme assay. During the in vitro studies, it was observed that only pipecolic acid compounds of the sulfonamide type B inhibit the Mip-PPIase, the most promising compounds are 22b, 23a und 24a. To determine the inhibitor-protein binding, compounds 1a, 12a, 22a and 22b were analyzed by means of HSQC-NMR experiments. Furthermore, for compound 22b an in vivo determination of growth inhibition of Legionella by means of a Gentamicin infection assay was carried out.

Selon le dernier rapport de l’EFSA-ECDC (EFSA Journal 2014), les parasites se classent en 8ème position des agents étiologiques impliqués dans les épidémies d’origine alimentaire reportées en 2012 en Europe. Par ailleurs, un récent rapport de l’OMS et la FAO (2014) classe Toxoplasma gondii en première position des parasites protozoaires à considérer dans le domaine alimentaire, suivi de Cryptosporidium spp et Giardia duodenalis. Les oocystes de T. gondii et Cryptosporidium spp et les kystes de G. duodenalis sont des formes très résistantes excrétées en très grande quantité dans les selles des individus malades. Lorsqu’ils se retrouvent dans l’environnement, ils peuvent y persister longtemps et contaminer certaines matrices alimentaires (végétaux et mollusques) lors de leur production primaire. A l’heure actuelle, en l’absence de méthodes d’analyse standardisées dans les aliments pour ces 3 parasites, peu de données de prévalence sont disponibles dans la littérature et les épidémies d’origine alimentaire restent négligées. Pour combler ce manque, une norme pour la détection/quantification des kystes de G. duodenalis et des oocystes de Cryptosporidium spp. dans les végétaux à feuilles vertes et fruits rouges à baies par microscopie à fluorescence est en cours de rédaction (ISO 18744). Des approches moléculaires, plus compatibles avec l’analyse de routine ont été développées par ACTALIA et l’équipe PROTAL pour détecter les 3 parasites simultanément sur des matrices végétales (Protofood, ANR-09-ALIA-009). Cependant, quelque soit la méthode de détection utilisée, elle met en évidence les parasites vivants et morts. Or, seul un parasite viable pourra être infectieux et donc potentiellement provoquer une maladie. A l’heure actuelle, les modèles in vivo constituent la méthode de choix pour évaluer l’infectiosité de manière précise, sensible et quantitative. Ils sont en revanche coûteux, lourds à mettre en œuvre et présentent un délai de réponse de plusieurs jours voire semaines qui n’est pas compatible avec les attentes des professionnels de l’agroalimentaire. L’objectif de la thèse est de développer des méthodes moléculaires pour caractériser la viabilité des 3 protozoaires dans des matrices alimentaires et disposer d’un outil permettant l’évaluation du risque lié à la détection de ces dangers dans les aliments. Ces méthodes seront comparées à celles qui permettent de mesurer l’infectiosité. Elles seront ensuite mises en œuvre pour évaluer leur potentiel pour déterminer l’efficacité d’inactivation de traitements technologiques sur des matrices alimentaires. / In the latest report from EFSA-ECDC (EFSA Journal 2014), parasites are ranked in the 8th position of the etiological agents involved in foodborne outbreaks reported in Europe in 2012. Moreover, in a recent report from the WHO and FAO (2014), Toxoplasma gondii is designated as the first protozoan parasite to be considered in the food domain, followed by Cryptosporidium spp. and Giardia duodenalis. Oocysts of T. gondii and Cryptosporidium spp., and cysts of G. duodenalis are excreted in big quantity by infected hosts and are particularly resistant. Consequently they can be found in the environment during long period and contaminate food matrices (vegetables and molluscs) during primary production. For now, since there are no standard methods to detect these 3 parasites in food samples, only few occurrence data are available and foodborne outbreaks remain neglected. To fill this gap, an ISO standard which describes a method for the detection and quantification of Cryptosporidium and Giardia in green leafy vegetables and red berries fruit by fluorescence microscopy is being draft (ISO 18744). Molecular approaches which are more suitable for routine analyses were developed by ACTALIA and PROTAL to simultaneously detect the 3 parasites in vegetable matrices (Protofood, ANR-09-ALIA-009). Nevertheless, whatever the used detection method, it highlights alive and dead parasites. But solely a living parasite can be infectious and induce pathology. For the moment, animal models are the favorite method to quantitatively evaluate infectivity with accuracy and sensitivity. However they are costly, heavy to implement and display a long time-to-result (from days to weeks) which does not fit with the agro-industrial needs. The objective of the thesis is to develop molecular methods to characterize the viability of the three protozoa in food matrices in order to have a tool allowing risk assessment in food safety. These methods will be compared to infectivity measurement methods. Then they will be implemented to evaluate their potential to determine the efficiency of technological treatments to inactivate protozoa in food matrices.

Die Melioidose und die Legionärskrankheit werden von den beiden Erregern Burkholderia pseudomallei bzw. Legionella pneumophila verursacht. Eine hohe Mortalitätsrate trotz langwieriger Behandlungen sowie die zunehmende Resistenz vieler Bakterien gegenüber den eingesetzten Antibiotika verdeutlichen die Notwendigkeit alternativer Behandlungsmethoden. Als neues Angriffsziel gilt das bereits in vielen Pathogenen gefundene „macrophage infectivity potentiator“-Protein, kurz Mip, das als Virulenzfaktor die Infektion forciert. Bei Legionella pneumophilia ist LpMip dafür verantwortlich, dass das Bakterium in die Lunge eindringen kann. Dabei überwindet der Erreger mit Hilfe des Mips die Epithelzellschicht und die extrazelluläre Matrix. Für BpMip ist der Sachverhalt der Invasion noch Gegenstand aktueller Forschung. Beide Mips zeigen eine hohe Sequenzhomologie zu humanem FKBP12 (FK506-bindende Proteine) und gehören deshalb zur Superfamilie der Peptidyl-Prolyl-cis/trans-Isomerasen (PPIasen), die die Fähigkeit besitzen, die cis/trans-Isomerisierung von Peptidbindungen der Aminosäure Prolin zu katalysieren. Die bereits bekannten FKBP12- und Mip-Inhibitoren Rapamycin und FK506 sind aufgrund ihrer immunsuppressiven Wirkung nicht zur Behandlung der beiden Krankheiten einsetzbar. Im Vorfeld dieser Arbeit konnte durch Synthese des literaturbekannten nicht-immunsuppressiven FKBP12-Inhibitors eine Leitstruktur gewonnen werden, die sowohl die PPIase-Aktivität von LpMip als auch von BpMip inhibiert. Zunächst konnten in dieser Arbeit durch Optimierung des Synthesewegs die Inhibitoren enantiomerenrein hergestellt werden. Ebenso wurde verifiziert, dass das S-Enantiomer das aktivere Konfigurationsisomer ist. Daneben wurde durch Synthese der Verbindung 8a/S-8a die anti-PPIase-Aktivität und die Löslichkeit im PBS-Puffer verbessert sowie die Zytotoxizität im Vergleich zu S-1a gesenkt Diese Verbindung zeigte jedoch eine schlechte Aktivität im Infektionsassay. In weiteren Kooperationen mit dem Biozentrum Würzburg und dem Dstl wurden die Inhibitoren ebenfalls erfolgreich an den Mips von Chlamydia trachomatis, Neisseria gonorrhoeae, Francisella tularensis undYersinia pestis getestet. In dieser Arbeit wurden erstmals Mip-Inhibitoren an Burkholderien in einer In-vivo-Studie untersucht. Die Wirksamkeit der Inhibitoren im Tiermodell soll in Folgestudien bewiesen werden. Damit ist eine aussichtsreiche Basis für zukünftige alternative Behandlungsmethoden der gram-negativer Bakterien gelegt. / Development of macrophage infectivity potentiator inhibitors