Pathogen infection dynamics and the evolution of host resistance and tolerance

Since the dawn of time, multicellular life has been exposed to a wide range of microbes. A share of them will not interfere with the host development or daily homeostasis, some might even contribute to it and potentially become essential, such as gut microbiota. However, a smaller, but significant, fraction of them will threaten the correct livelihood of their host, often defined as parasites or pathogens, depending on their properties or the scientific school that is addressing them. Hosts face this kind of organisms on a daily basis. Most parasites we face every day present low virulence and frequently their menace happens by chance, commonly referred as opportunistic pathogens, as is the case of microbiota that following a given stress might grow uncontrollably. On the other side of the spectrum, we have parasites that under normal conditions hosts might be exposed much less often but exhibit a higher level of virulence and pathogenicity, the so-called pathogens. Therefore, just by focusing on a parasite virulence we observe a wide range of threats to the host. Nevertheless, there are an immense range of properties intrinsic to the parasite that might influence the outcome of a host-parasite, or better a host-pathogen, interaction. These properties can include infection site, transmission rate and which resources they hitchhike. In face of these threats, hosts were selected to develop an equivalent wide set of immune responses with different costs and benefits to themselves. Immunity can be further divided into innate and adaptive. The latter is a complex branch only found in vertebrates, and therefore, I will focus on the most universal branch of the immune system throughout this thesis, the innate immune responses. Hosts evolved resistance and tolerance strategies, two disparate sets of mechanisms that in a stricto sensu aim to clear or cope with the infection. Naturally, these strategies will have very distinct consequences for the ecology and evolution of a host-parasite interaction and each of the populations' evolutionary history. Moreover, in the current medicine state and health crisis, the study of these strategies offers new insights and solutions for immunology and epidemiology. Up to now, when we address an infection, fighting and hopefully clearing the parasite burden is the first solution that comes to mind. We define the latter as resistance, particularly quantitative resistance as it reduces the parasite number. Nevertheless, resistance comes with a number of cons. Resistance mechanisms are usually very costly, either by the effectors induction or the collateral damage caused by these (e.g. oxidative stress and consequent inflammation). Furthermore, resistance acts on the parasite number and therefore selects for increased virulence alleles on the parasite side that will in turn select for further host increased resistance and so forth. This antagonistic coevolution between the host and parasite is often referred as Red Queen dynamics and it is one of the major selective forces in nature. On the other hand, tolerance acts by reducing or controlling the damage employed by either the parasite itself or by the immune response. Hence, this strategy does not affect the parasite burden and consequently, it is not expected to select for higher virulence. Experimentally, we quantify tolerance as the reduction of a given infection cost in fitness measures, such as fecundity-tolerance for reproductive fitness or mortality tolerance for survival. Unfortunately, tolerance studies are scarce and often reflect theoretical predictions from modelling or studies from plant biology, where most of our understanding comes from. In this thesis, we started by investigating how different pathogens shape infection dynamics outcome in an outbred population of Drosophila melanogaster and second, how does a host population respond to the exposure to different pathogens in the short and long-term. In Chapter 3, we exposed a D. melanogaster population to a variety of parasites from low to high virulence and low to high inoculum size. This allowed us to characterize bacterial infections during the host lifetime. From the host side, we focused on common points such as clearance ability, while on the parasite side we turned to its aptitude to persist within the host. Furthermore, we decompose parasitic virulence into smaller factors, such as host exploitation or per-parasite pathogenicity, i.e. how well the bacterial species survive and replicate within the host, and the amount of damage they inflict. In Chapter 4, we turn our focus to the host and addressed how the variation in parasite burden at given time-points during infection reflects variation in the immune strategies. Here we showed that throughout a bacterial infection with Lactococcus lactis, individuals become less tolerant and less resistant across the two time-points measured, potentially indicating immunity costs. Curiously, we also observed that individuals unable to control parasite burden in the acute phase are particularly less tolerant than their counterparts. Lastly, in Chapter 5 we examined what are the evolutionary implications of selection for host resistance and fecundity-tolerance in a specific host-parasite interaction, D. melanogaster and L. lactis. Interestingly, our results show that after eight generations of selection, tolerance seems to be the favoured immune strategy to evolve in both resistance and tolerance selection regimes. This result suggests tolerance mechanisms might offer less costs or be more promptly available under the settings of this study. Altogether, our results help to clarify some of the implications and properties of these strategies on the dynamics of host-parasite interactions, as well as bring to light a new set of questions regarding tolerance and all its underpinnings.Tiere sind täglich einer Vielzahl von Mikroorganismen ausgesetzt. Die Wechselwirkung zwischen ihnen kann von positiv bis negativ reichen. Letztere Art von Mikroorganismen werden je nach ihren Eigenschaften als Parasiten oder Krankheitserreger bezeichnet. Wirte reagieren auf eine Infektion mit verschiedenen Strategien, nämlich mit Resistenz oder Toleranz. Resistenz zielt darauf ab, die Anzahl der Parasiten zu reduzieren oder eine Infektion vollständig zu beseitigen. Auf der anderen Seite erlaubt Toleranz dem Wirt, mit den Auswirkungen einer Infektion und der Immunantwort zurechtzukommen. Natürlich führen diese beiden Strategien zu sehr unterschiedlichen Ergebnissen sowohl in der Evolution, als auch in der Ökologie der Wirt-Parasit-Interaktion, aber auch zu unterschiedlichen Implikationen für Medizin, Immunologie und Epidemiologie. Resistenz wirkt sich auf die Parasitenzahl aus und selektiert daher auf erhöhte Virulenz. Auf der anderen Seite beeinflusst Toleranz nicht die Parasitenlast und sollte daher laut Studien einen neutralen bis positiven Effekt auf die Parasitenprävalenz und die Evolution haben. In dieser Arbeit untersuchten wir: i) wie verschiedene Parasiten die Infektionsdynamik und deren Ergebnis in einer Population von Drosophila melanogaster beeinflussen; ii) wie ein Wirt kurzfristig auf eine Infektion mit einem bestimmten Parasiten reagiert; iii) ob der Wirt langfristig eine Resistenz oder Toleranz entwickeln kann. In Kapitel 3 wurde unsere Population von D. melanogaster mit verschiedenen Krankheitserregern in unterschiedlichen Inokulationsdosen infiziert. Die Infektionsdynamik wurde über die gesamte Lebensdauer des Wirts verfolgt. Auf der Seite des Wirts konzentrierten wir uns auf die Clearance-Fähigkeit. Auf der Seite der Parasiten analysierten wir deren Persistenz im Wirt. Wir zerlegten die Virulenz der Parasiten weiter in kleinere Faktoren. In Kapitel 4 konzentrierten wir uns auf die Dynamik der Wirtsresistenz und -toleranz während verschiedener Phasen der Infektion. Unsere Ergebnisse zeigen, dass bei einer Infektion mit Lactococcus lactis der Wirt mit der Zeit weniger tolerant und weniger resistent wird. Darüber hinaus sind Individuen, die in der akuten Phase nicht in der Lage sind, die Parasitenzahl zu kontrollieren, weniger tolerant als ihre Gegenspieler. In Kapitel 5 haben wir bei D. melanogaster, die mit L. lactis infiziert wurde, auf Wirtsresistenz und Fruchtbarkeitstoleranz selektiert. Unsere Ergebnisse zeigen, dass sich nach acht Generationen der Selektion die Toleranz sowohl bei der Resistenz-, als auch bei der Toleranzselektion entwickelt. Dieses Ergebnis deutet darauf hin, dass Toleranz unter den Bedingungen dieser Studie weniger Energie verbraucht und für den Wirt schneller verfügbar ist. Insgesamt zeigen unsere Ergebnisse die Auswirkungen und Eigenschaften dieser Immunstrategien auf die Dynamik von Wirt-Parasit-Interaktionen und bringen eine Reihe neuer Fragen zur Toleranz und ihren Grundlagen ans Licht

The role of Down syndrome cell adhesion molecule 1 for the bacterial microbiota of Tribolium castaneum

Tese de mestrado em Biologia Evolutiva e do Desenvolvimento, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2017Organismos multicelulares estabelecem diversos tipos de interações com microrganismos. Estas interações podem ser positivas, neutras ou negativas para o hospedeiro. As diferentes interações moldaram a evolução do sistema imunitário. Interações negativas levam a uma co-evolução parasita-hospedeiro. Os hospedeiros necessitam de apresentar mecanismos e respostas imunes mais eficientes para sobreviverem e reproduzirem-se, enquanto que os parasitas requerem novas formas de escapar ou resistir ao sistema imune do hospedeiro e infectá-lo. Contudo, grande parte dos organismos estabelecem interações neutras ou benéficas para o hospedeiro. Num cenário de mutualismo, a interação pode ser necessária ao desenvolvimento de estruturas ou crucial para que o hospedeiro apresente uma imunidade eficiente. No caso das interações neutras, pensa-se que sejam dinâmicas e facilmente possam evoluir para uma interação positiva/negativa. Desta forma, os diversos tipos de interação levaram à evolução do sistema imunitário e à necessidade de uma forma de diferenciação entre microrganismos benéficos e prejudiciais por parte do hospedeiro. Uma possível explicação para a diferenciação entre diferentes tipos de interação foi sugerida recentemente por Lazzaro and Rolff. Esta hipótese sugere que o sistema imunitário do hospedeiro é capaz de ajustar o tipo de resposta conforme o rácio de sinais a que é sujeito. Enquanto que uma ferida levaria à expressão de sinais de danos, um simbionte apenas apresentaria os seus padrões moleculares associados a micróbios a receptores nas células do hospedeiro. Contudo, um parasita apresentaria não só os padrões moleculares associados a micróbios, como também causaria danos no tecido infetado, levando à expressão de sinais de danos pelas células. Assim, o estabelecimento de interações positivas e respostas imunes apropriadas dependem do reconhecimento por parte do hospedeiro e possivelmente de um balanço entre diferentes sinais. A diferenciação entre diferentes microrganismos deverá depender de receptores que permitam o reconhecimento e levem à produção uma resposta adequada. Dscam1 tem sido sugerido como possível receptor de reconhecimento dada a grande quantidade de isoformas que é capaz de produzir por alternative splicing. Down syndrome cell adhesion molecule 1 (Dscam1) é um gene expresso em neurónios e em alguns invertebrados em células e tecidos do sistema imunitário. Por alternative splicing é capaz de originar cerca de 18.000 isoformas em Drosophila melanogaster e 15.000 em Tribolium castaneum. Esta proteína é essencial para o desenvolvimento de circuitos neuronais A grande variabilidade de isoformas permite a identificação e consequente repulsão de dendrites-irmãs. Isto assegura que as dendrites cubram a maior área possível e assim estabeleçam um correto circuito neuronal. A análise da estrutura proteica de Dscam1 permitiu identificar dois epítopos (I e II). O epítopo I está envolvido em interações homofílicas no sistema nervoso e é bastante conservado, coerente com a sua função no desenvolvimento de circuitos neuronais e elevada pressão seletiva para um grupo limitado de isoformas. Por outro lado, o epítopo II apresenta grande variabilidade na sua sequência. Dada esta variabilidade, foi proposto que este epítopo poderá estar envolvido no reconhecimento de microrganismos. Recentemente, vários estudos têm proposto não só uma função no reconhecimento de microrganismos, mas também como possível opsonina e/ou regulador de bactérias no hospedeiro. Sabe-se que diferentes bactérias induzem a expressão de diferentes isoformas de Dscam1. Contudo, ainda permanece um mistério como é que é produzida esta expressão diferencial e qual a sua função na imunidade de invertebrados. No mosquito Anopheles gambiae, um knock-down de Dscam1 levou a uma redução no index de fagocitose, bem como uma proliferação das bactérias na hemolinfa. Seis anos mais tarde, ao sobre-expressarem uma isoforma de Dscam1 induzida na presença de bactérias, observaram uma redução nas bactérias no mosquito. Porém, há resultados contraditórios. Em Laodelphax striatellus após o knock-down de Dscam1, observa-se uma redução na infeção por parasitas intracelulares como Wolbachia e nenhuma alteração no número de bactérias extracelulares. Deste modo, o objetivo desta tese foi clarificar o papel de Dscam1 na microbiota bacteriana de T. castaneum. Para tal, procedeu-se ao knock-down de Dscam1 e posteriores análises à quantidade total de microbiota bacteriana em três pontos de desenvolvimento: estádio de larva (15 dias pós-oviposição); estádio de pupa (23 dias pós-oviposição) e estádio de adulto (30 dias pós-oviposição). Através de RTqPCR foi possível quantificar quer o knock-down de Dscam1, quer a quantidade de bactérias em cada tratamento utilizando como proxy o gene bacteriano 16S rRNA. Os resultados deste projeto mostram que em Tribolium castaneum, um knock-down de Dscam1 parece não afetar a quantidade total de bactérias. Isto foi observado para os três pontos de desenvolvimento analisados. Estes resultados sugerem que neste modelo, Dscam1 poderá não regular a flora bacteriana como observado em mosquito. No entanto, foi observada uma forte correlação entre a expressão relativa do 16S rRNA bacteriano e a expressão relativa do Dscam1 para dois dos três pontos de desenvolvimento. A correlação é de caráter negativo: uma maior expressão de Dscam1 está correlacionada com uma menor expressão de 16S rRNA e por consequente, menor quantidade de bactérias no sistema. Esta tendência foi observada por Dong e colegas em mosquitos adultos. Assim, é possível que Dscam1 faça parte de um sistema robusto que reconhece/regula bactérias e não é afetado por baixos níveis de Dscam1. É ainda possível que caso Dscam1 tenha uma função no reconhecimento/regulação de bactérias, haja outros receptores com função redundante que possam compensar a falta deste em particular. De forma a responder a estas questões, um estudo mais exaustivo onde se analisasse também a composição bacteriana após knock-down de Dscam1 seria necessário. É possível que não se observe alterações na quantidade total de bactérias, mas sim diferentes proporções de cada classe após redução de Dscam1. Seria expectável que classes reguladas por Dscam1 teriam as suas proporções aumentadas face a outras classes. Dada a evidência de forte competição bacteriana dentro da microbiota, é possível que não se observe diferenças significativas no número total de bactérias, mas sim diferenças na sua identidade.Hosts establish different types of interactions with the elements of their microbiota. These interactions can be broadly classified as positive, neutral or negative and shaped the evolution of the immune system. Negative interactions may lead to host-parasite coevolution. While the host may only survive and reproduce by exhibiting more efficient immune responses, the parasite requires novel ways of dodging the host's immune system. On the other hand, positive interactions can evolve to the point where the lack of specific microorganisms may interfere with tissue development, immunity or resistance to pathogens. Neutral interactions are not only difficult to define but are also thought to be dynamic and could evolve to either positive or negative interactions. Consequently, the host needs to be able to differentiate between harmful and beneficial microbes. This identification is thought to be performed through pattern recognition receptors that recognise microbe-associated molecular patterns and other environmental cues, enabling an appropriate response from the host. Dscam1 is a gene expressed in neurons and essential to the correct development of neuronal circuits. Through alternative splicing, it is able to originate around 18.000 isoforms in Drosophila melanogaster and 15.000 in Tribolium castaneum. More recently, its expression was observed in immune cells and tissues of some invertebrates. Recent studies have shown differential isoforms expression after infection by different classes of bacteria. Hence, it was suggested that Dscam1 may work as a pattern recognition receptor, opsonin and/or bacteria regulator. Concerning bacteria regulation, there are studies in Anopheles gambiae that show a proliferation of gut bacteria after a Dscam1 knock-down. In the same model, overexpression of bacteria-induced Dscam1 isoforms resulted in a reduction of bacteria in the haemolymph. The above-mentioned results suggest that Dscam1 is involved in negative regulation of bacteria. Nonetheless, there is also evidence suggesting a decrease in the intracellular parasites and no effect on extracellular bacteria after Dscam1 knock-down in Laodelphax striatellus. Hence, I aimed to clarify the role of Dscam1 in the dynamics of Tribolium castaneum bacterial microbiota. This was achieved through Dscam1 knock-down and bacterial load analysis through RTqPCR for the bacterial 16S rRNA at three time-points of the beetle development: 15-days old larvae, 23-days old pupae and 30-days old adults. Our results show that a Dscam1 knock-down does not affect the total amount of bacteria in the beetle in any of the three developmental time-points analysed. This is in agreement with previous studies. Nevertheless, a correlation between the relative expression of the bacterial 16S rRNA and Dscam1 was observed. The correlation had a negative tendency: a higher expression of Dscam1 was correlated with a lower expression of 16S rRNA and therefore less bacteria in the system. Such a tendency was also observed in Dong and colleagues’ studies on adult mosquitoes. A knock-down may not be sufficient to disturb a robust system capable of bacterial regulation in this model. Further analyses of the composition of bacterial microbiota are required to clarify the role of Dscam1 in bacterial regulation in T. castaneum. It is for example possible that changes occur in the proportion of different bacterial taxa

Astrophysical signatures of boson stars: quasinormal modes and inspiral resonances

Compact bosonic field configurations, or boson stars, are promising dark matter candidates which have been invoked as an alternative description for the supermassive compact objects in active galactic nuclei. Boson stars can be comparable in size and mass to supermassive black holes and they are hard to distinguish by electromagnetic observations. However, boson stars do not possess an event horizon and their global spacetime structure is different from that of a black hole. This leaves a characteristic imprint in the gravitational-wave emission, which can be used as a discriminant between black holes and other horizonless compact objects. Here we perform a detailed study of boson stars and their gravitational-wave signatures in a fully relativistic setting, a study which was lacking in the existing literature in many respects. We construct several fully relativistic boson star configurations, and we analyze their geodesic structure and free oscillation spectra, or quasinormal modes. We explore the gravitational and scalar response of boson star spacetimes to an inspiralling stellar-mass object and compare it to its black hole counterpart. We find that a generic signature of compact boson stars is the resonant-mode excitation by a small compact object on stable quasi-circular geodesic motion.Comment: 20 pages, 8 figures. v2: minor corrections, version to be published in Phys. Rev. D. v3: final versio

Slowly Rotating Anisotropic Neutron Stars in General Relativity and Scalar-Tensor Theory

Some models (such as the Skyrme model, a low-energy effective field theory for QCD) suggest that the high-density matter prevailing in neutron star interiors may be significantly anisotropic. Anisotropy is known to affect the bulk properties of nonrotating neutron stars in General Relativity. In this paper we study the effects of anisotropy on slowly rotating stars in General Relativity. We also consider one of the most popular extensions of Einstein's theory, namely scalar-tensor theories allowing for spontaneous scalarization (a phase transition similar to spontaneous magnetization in ferromagnetic materials). Anisotropy affects the moment of inertia of neutron stars (a quantity that could potentially be measured in binary pulsar systems) in both theories. We find that the effects of scalarization increase (decrease) when the tangential pressure is bigger (smaller) than the radial pressure, and we present a simple criterion to determine the onset of scalarization by linearizing the scalar-field equation. Our calculations suggest that binary pulsar observations may constrain the degree of anisotropy or even, more optimistically, provide evidence for anisotropy in neutron star cores.Comment: 19 pages, 7 figures, 1 table. Matches version in press in CQG. Fixed small typo

Prediction of dam concrete structural properties based on wet-screened test results and mesoscale modelling

Dam or full-mixed concrete is produced with large aggregates which implies the use of large specimens, heavy laboratory equipment and non-standard embedding monitoring devices. The wet-screened concrete, obtained from the full-mixed concrete by sieving the larger aggregates while the concrete is still fresh, is used to cast smaller specimens and embed standard monitoring devices. This thesis focuses on the prediction of the structural properties of dam concrete based on wet-screened concrete experimental results, analytical models and detailed particle models in which the mesostructure is represented. In order to study the effect of wet-screening, an in situ experimental setup and a series of laboratory tests were carried out for both dam and wet-screened concretes. Three sets of creep cells were installed in a dam which allowed for the improvement of the experimental setup and the testing procedures, aiming to reduce costs, to facilitate the installation in situ and to increase the reliability of the results. The test results revealed the differences between the two types of concrete and the influence of the coarse aggregate on the deformability and strength properties. Different analytical models based on composite models, on the equivalent age method, on size effect and on the Abrams law were developed to describe the instantaneous and delayed behaviour of concrete using the composition data and to establish a practical relationship between dam and wet-screened concrete behaviour. Similarly, a numerical solution based on the discrete element method applied to particle models was developed to predict the behaviour of dam concrete. A new fast numerical procedure for long-term analysis taking into account the aging viscoelastic behaviour of cementitious material is proposed. Particle models are especially suited for modelling dam concrete since the coarse aggregate structure can be explicitly represented, allowing the study of stress distribution inside the specimen and the study of complex failure patterns both in tension and compression. For the two types of approaches, the obtained experimental results were used to calibrate the main parameters and to validate the analytical and numerical prediction models. The experimental results and the development of physically-based models highlighted the particular properties of dam concrete, allowed for a significant contribution to the analysis of concrete instantaneous and delayed behaviour and presented new approaches for the prediction of dam concrete behaviour based on the wet-screened properties