Effect of different host genetic backgrounds in the response against oral bacterial infection

Tese de mestrado, Biologia Evolutiva e do Desenvolvimento, Universidade de Lisboa, Faculdade de Ciências, 2021Os organismos vivos interagem naturalmente no seu habitat de formas que se estendem sobre uma vasta panóplia de interações bióticas, e de entre as quais o parasitismo parece ser a mais comum e bem-sucedida. O seu sucesso deve-se maioritariamente a um delicado e dinâmico equilíbrio entre a transmissão do parasita e a sua virulência, propriedades que são usualmente atribuídas exclusivamente ao patogénio. Contudo, estas características podem variar quer natural quer experimentalmente, dependendo de condições ambientais, da genética do hospedeiro e das comunidades microbianas que constituem a microbiota. Naturalmente, para sobreviver a uma infeção parasítica o hospedeiro tem de ser apto para montar uma resposta imune competente e eficaz. Em traços gerais, a resposta imune é constituída por mecanismos de resistência, que visam limitar a infeção bloqueando ou eliminado o patogénio e reduzindo a carga parasitária, e por mecanismos de tolerância à doença, que resultam numa melhoria no vigor ou estado de saúde do hospedeiro sem implicar uma redução da carga parasitária. A mosca da fruta, Drosophila melanogaster, passa grande parte do seu ciclo de vida em matéria orgânica em decomposição contactando intimamente com microrganismos, muitos deles patogénicos. Como tal, este organismo possui um vasto conjunto de respostas imunes que o protegem de bactérias, fungos, parasitas e vírus. Embora não possua sistema imunitário adaptativo, presente em vertebrados, a Drosophila tem sido amplamente usada como organismo modelo para estudar imunidade inata, devido a conservação evolutiva destes mecanismos. As defesas imunes da mosca da fruta consistem em: 1) Imunidade comportamental, que tem como propósito evitar contacto com microrganismos patogénicos para prevenir uma possível infeção; 2) Imunidade epitelial, que consiste em barreiras físicas que protegem contra o estabelecimento de microrganismos no interior do corpo; 3) Imunidade celular que depende da ação de células móveis e sésseis (hemócitos) presentes na cavidade corporal; 4) Imunidade humoral que tem por base a produção e secreção de péptidos antimicrobianos para a hemolinfa. Um dos múltiplos patogénios naturais capazes de infetar Drosophila é Pseudomonas entomophila, cujo genoma se encontra totalmente sequenciado e muitos dos seus fatores de virulência identificados, tornando este microrganismo num modelo ideal para estudos de imunidade em D. melanogaster. P. entomophila causa ativação da imunidade sistémica mesmo quando ingerida, e a sua patogenicidade neste cenário deve-se à capacidade que esta bactéria tem de persistir no sistema digestivo da mosca, excretando múltiplas substâncias tóxicas que causam danos no intestino. Muitos dos seus genes associados à sua entomopatogenicidade foram identificados como, por exemplo, genes que codificam toxinas, explicando o efeito altamente nefasto que esta bactéria tem quando ingerida pela mosca da fruta. Dada a importância do background genético do hospedeiro em face de uma infeção a que responde através de mecanismos de resistência e tolerância à doença, este trabalho tem como ambição compreender qual o contributo relativo de cada um destes mecanismos neste processo. Para este fim, infetámos um conjunto de 75 linhas do Drosophila Genetic Reference Panel (DGRP) com P. entomophila. O DGRP é um painel que consiste de cerca de 200 linhas isogénicas, sequenciadas na sua totalidade, que permite decompor a variabilidade genética presente numa população natural de Drosophila, constituindo uma poderosa ferramenta para análises de associação genética. Usando este sistema previamente estabelecido no laboratório o nosso objetivo é caracterizar dinâmicas de infeção em linhas do DGRP por P. entomophila. Com a recolha de dados relativos à sobrevivência após infeção, caracterizámos diferentes dinâmicas de sobrevivência dependentes no background genético do hospedeiro. Através da análise combinada de sobrevivência e dados referentes a carga parasitária recolhidos, inferimos fenótipos relativos a resistência e tolerância à doença neste conjunto de linhas do DGRP. No futuro, a extensão dos protocolos à totalidade do DGRP, juntamente com alguns ajustes que visam melhorar a qualidade dos dados recolhidos, poderá levar à identificação através de análises de associação genética (GWAS) de genes envolvidos em mecanismos quer de resistência quer de tolerância à doença.Organisms coexist naturally in their environment, interacting through a vast array of biotic interactions of which parasitism seems to be the most common and successful. This success comes from a balance between parasite transmission and virulence, traits which are often seen as properties of the pathogen. However, these traits can vary both experimentally or spontaneously, depending on environment, host genetics and the microbial communities forming the microbiota. Therefore, to survive infection it is particularly important that hosts are able to mount strong and competent immune responses that comprise resistance and tolerance mechanisms. The first is responsible for limiting infection by blocking or eliminating the pathogen, decreasing the parasitic load, while the second reveals itself in the increase in health status or fitness of the host without decreasing the parasitic load. The fruit fly, Drosophila melanogaster spends its whole life cycle in decaying organic matter in close contact with pathogenic microorganisms. Therefore, it possesses an array of immune mechanisms to protect itself against infecting bacteria, fungi, parasites or viruses. Although lacking the presence of an adaptive immune system, present in vertebrates, Drosophila has been widely used as model system for the study of innate immune defences, due to their evolutionary conservation. Fruit fly immune defences consist of: 1) Behavioural immunity, which involves avoiding the pathogen in order to prevent infection or reduce pathogen exposure; 2) Physical or epithelial immunity, which is composed by physical barriers against the establishment of microorganisms in the body; 3) Cellular component, which consists in the action of free-floating and sessile blood cells (haemocytes), that circulate through the body cavity; 4) Humoral immunity, that relies on the production and secretion of antimicrobial peptides (AMPs) to the haemolymph. One of the several natural Drosophila pathogens is Pseudomonas entomophila which has its genome fully sequenced and its virulence factors identified, making it a prime bacterial model for infection in D. melanogaster. P. entomophila is able to activate a systemic immune response in the fruit fly even through ingestion. Under oral infection, pathogenicity is strictly dependent on its ability to persist in the gut and the excretion of toxic substances that disrupt host physiology. A lot of its genes have also been associated with its entomopathogenicity. For example, genes that encode toxins explain why infection with this bacterium is so deleterious to the gut, causing irreversible damage to flies. Given that the host genetic background influences infection outcome through defence mechanisms of resistance and disease tolerance, this work aimed at shedding light on the contributions of each mechanism upon oral infection with a pathogen. As such, we infected a subset of 75 Drosophila Genetic Reference Panel (DGRP) lines with a natural pathogen of D. melanogaster, P. entomophila. The DGRP is a panel that consists of around 200 fully sequenced isogenic lines that allows for the breakdown and analysis of the genetic variability of a natural population, constituting a powerful tool for Genome Wide Association Studies (GWAS). Using this lab established system our objective was to characterize infection dynamics of oral infection by P. entomophila in DGRP lines. By gathering survival data upon infection, we were able to characterize the differences in infection outcome caused by differences in host genetic background. We also created several categories of survival profile based on mortality dynamics over the course of infection. We also measured bacterial loads such as initial inoculum, bacterial loads upon death and set point bacterial loads, in order to characterize how the host influences pathogen dynamics during infection. By combining all the data, we were able to infer phenotypes of resistance and disease tolerance on a set of DGRP lines. In the future, amplification of the dataset to the full DGRP, together with the identified necessary adjustments to improve data quality, may lead to Genome Wide Association Studies (GWAS) and the identification of genes that are involved in the mechanisms of resistance and disease tolerance

Participatory cartography, as a support for decision-making in public health, case: Metropolitan Area of Toluca

The work aims to revalidate environments participatory mapping, know the origin this practice, and the epistemological basis. Since this type of research can create links for reflection and action, between social perception and stakeholders focused on promoting social development and investigative actions. It articulate collective knowledge by the researcher becomes a challenge, involving a group of people in research and methodological schemes without losing objectivity cohesive give opinions, criticisms. From this idea analysis methodologies of the IAP, Geography of perception and participatory mapping was performed, and how they can help uncover the major public health problems currently facing the metropolitan area of Toluca because diagnoses by the Institute of health of the State of Mexico that identifies two focuses: environmental pollution, and chronic degenerative diseases (ISEM, 2015)

Occurrence and behavior of illicit drugs and metabolites in sewagewater from the Spanish Mediterranean coast (Valencia region)

In this work, a study on the occurrence and behavior of illicit drugs and metabolites in sewage water systems has been made. A comprehensive dataset was obtained by analyzing illicit drugs daily in influent and effluent waters from three sewage treatment plants (STPs), over three different weeks. To complete this dataset, monitoring was conducted during an international pop/rock festival, an interesting facet within this study. The STPs selected were sited along the Spanish Mediterranean coast (Castellón province, Valencia region) and represent towns of different sizes, with appreciable variations in the population in the summer period. Illicit drug concentrations in the influents were low, except during the celebration of the music festival, when the levels of cocaine, benzoylecgonine, amphetamine, MDA and MDMA increased. Comparing the influent and effluent concentration data allowed the rough estimation of the removal of illicit drugs and metabolites by each STP. Removal efficiencies were estimated between 75 and 100% for most of the analytes under investigation. The loads discharged into the aquatic ecosystem were also calculated from effluent data. Weekly discharges of drugs and metabolites via effluent sewage waters presented values commonly below 10 g for each individual drug, with the exception of benzoylecgonine, which usually exceeded this level. The increase in population and drug consumption during the music event led to a notable increase in the weekly discharges, reaching values up to 406 g of MDMA and 122 g of benzoylecgonine

Occurrence and removal of pharmaceuticals in wastewater treatment plants at the Spanish Mediterranean area of Valencia

A survey on the presence of pharmaceuticals in urban wastewater of a Spanish Mediterranean area (Castellon province) was carried out. The scope of the study included a wide variety of pharmaceuticals belonging to different therapeutical classes. For this purpose, 112 samples, including influent and effluent wastewater, from different conventional wastewater treatment plants were collected. Two monitoring programmes were carried out along several seasons. The first was in June 2008 and January 2009, and the second in April and October 2009. During the first monitoring, the occurrence of 20 analytes in 84 urban wastewater samples (influent and effluent) was studied. The selection of these pharmaceuticals was mainly based on consumption. From these, 17 compounds were detected in the samples, with analgesics and anti-inflammatories, cholesterol lowering statin drugs and lipid regulators being the most frequently detected groups. 4-Aminoantipyrine, bezafibrate, diclofenac, gemfibrozil, ketoprofen, naproxen and venlafaxine were the compounds most frequently found. In the highlight of these results, the number of analytes was increased up to around 50. A lot of antibiotic compounds were added to the target list as they were considered “priority pharmaceuticals” due to their more potential hazardous effects in the aquatic environment. Data obtained during the second monitoring programme (spring and autumn) corroborated the results from the first one (summer and winter). Analgesics and anti-inflammatories, lipid regulators together with quinolone and macrolide antibiotics were the most abundant pharmaceuticals. Similar median concentrations were found over the year and seasonal variation was not clearly observed. The removal efficiency of pharmaceuticals in the wastewater treatment plants was roughly evaluated. Our results indicated that elimination of most of the selected compounds occurred during the treatment process of influent wastewater, although it was incomplete

Fronteras subjetivas entre lucha y violencia desde la experiencia de luchadores profesionales de MMA

[ES] El objetivo de este estudio es comprender las experiencias de los luchadores profesionales de artes marciales mixtas (MMA) en la transición psicológica entre la lucha y la violencia, a partir de sus propios informes. Se realizaron entrevistas semiestructuradas con una muestra intencional de cinco participantes y su análisis se realizó desde una perspectiva fenomenológica. El cruce intencional de experiencias permitió la identificación de cinco categorías: mantener la concentración vs. perder la concentración; mantenimiento de la técnica vs. pérdida de la técnica; sentirse bien vs. vergüenza; deseo de probarse a sí mismo vs. agresión; represalia deseable vs. represalia desproporcionada. Los resultados de este estudio indican que la intensificación del intercambio combativo que conduce a la transición del primero al segundo ítem de cada categoría es el elemento clave en la transición subjetiva entre lucha y violencia.[EN] This study aimed to understand the experiences of professional mixed martial arts (MMA) fighters in the psychological transition between fight and violence, based on their own reports. Semi-structured interviews were conducted with an intentional sample of five participants and their analysis was carried out from a phenomenological perspective. The intentional crossing of experiences allowed the identification of five categories: maintaining focus vs. losing your head; maintenance of the technique vs. loss of the technique; feeling good vs. embarrassment; desire to test yourself vs. aggression; desirable retaliation vs. disproportionate retaliation. The results of this study indicate that the intensification of the combative exchange that leads to the transition from the first to the second item of each category is the key element in the subjective transition between fight and violence.[PT] O objetivo deste estudo é compreender as experiências de lutadores profissionais de artes marciais mistas (MMA) no trânsito psicológico entre a luta e a violência, a partir de seus próprios relatos. Entrevistas semiestruturadas foram conduzidas com uma amostra intencional de cinco participantes e sua análise foi realizada através de uma perspectiva fenomenológica. O cruzamento intencional das experiências permitiu a identificação de cinco categorias: manter o foco vs. perder a cabeça; manutenção da técnica vs. perda da técnica; sentir-se bem vs. constrangimento; desejo de se testar vs. agressão; revide desejável vs. revide desproporcional. Os resultados deste estudo indicam que a intensificação na troca combativa que conduz a passagem do primeiro ao segundo item de cada categoria é o elemento chave na transição subjetiva entre luta e violência

Exploring matrix effects in liquid chromatography–tandem mass spectrometry determination of pesticide residues in tropical fruits

Tropical fruits are being increasingly consumed around the world because of their appreciated characteristics, particularly their high nutritional value and distinctive taste, which are different from those of traditional fruits. Owing to their introduction into international markets it is necessary to have a reliable analytical methodology available for the sensitive determination of pesticide residues in order to monitor the compliance of maximum residue limits (MRLs). From an analytical point of view, tropical fruits have generally been far less studied than other fruits frequently consumed in the European Union or USA, which are among the most important markets. In this work, LC–MS/MS-based methodology using a triple quadrupole analyzer was developed for the multi-residue determination of selected pesticides and metabolites in tropical fruits, which were selected among the most popular in Colombia, one of the most important suppliers of tropical fruits around the world. After selection of a QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe)-based sample treatment, the study focused on the evaluation of matrix effects, in order to find a simple way for their correction. Twelve different food matrices were selected to perform this study: the seven Colombian tropical fruits of highest value for domestic and international markets (uchuva, tamarillo, granadilla, gulupa, maracuya, papaya, and pithaya), and five more matrices highly consumed in Colombia (lulo, carambolo, feijoa, mangostan, and guayaba). Twenty compounds, including pesticides widely applied in tropical fruits pest control and several metabolites considered in residue definition, were used as model compounds in this work. Correction factors were used on the basis of calibration graphs obtained with standards in solvent and in matrix, and their usefulness was supported by validation of the method in all the matrices tested at 0.01 and 0.1 mg/kg. The analysis of real-world samples revealed the presence of several target compounds that were identified by the acquisition of two MS/MS transitions, and by ion intensity ratio and retention time agreement.Generalitat Valenciana (Research Group of Excellence Prometeo 2009/ 054 and Prometeo II 2014/023; Collaborative Research on Environment and Food-Safety, ISIC/2012/016

Screening of Pesticides and Polycyclic Aromatic Hydrocarbons in Feeds and Fish Tissues by Gas Chromatography Coupled to High-Resolution Mass Spectrometry Using Atmospheric Pressure Chemical Ionization

This paper reports a wide-scope screening for detection and identification of pesticides and polycyclic aromatic hydrocarbons (PAHs) in feeds and fish tissues. QuEChERS sample treatment was applied, using freezing as an additional cleanup. Analysis was carried out by gas chromatography coupled to hybrid quadrupole time-of-flight mass spectrometry with atmospheric pressure chemical ionization (GC-(APCI) QTOF MS). The qualitative validation was performed for over 133 representative pesticides and 24 PAHs at 0.01 and 0.05 mg/kg. Subsequent application of the screening method to aquaculture samples made it possible to detect several compounds from the target list, such as chlorpyrifos-methyl, pirimiphos-methyl, and ethoxyquin, among others. Light PAHs (≤4 rings) were found in both animal and vegetable samples. The reliable identification of the compounds was supported by accurate mass measurements and the presence of at least two representative m/z ions in the spectrum together with the retention time of the peak, in agreement with the reference standard. Additionally, the search was widened to include other pesticides for which standards were not available, thanks to the expected presence of the protonated molecule and/or molecular ion in the APCI spectra. This could allow the detection and tentative identification of other pesticides different from those included in the validated target list

Aquatic pollution may favor the success of the invasive species A. franciscana

The genus Artemia consists of several bisexual and parthenogenetic sibling species. One of them, A. franciscana, originally restricted to the New World, becomes invasive when introduced into ecosystems out of its natural range of distribution. Invasiveness is anthropically favored by the use of cryptobiotic eggs in the aquaculture and pet trade. The mechanisms of out-competition of the autochthonous Artemia by the invader are still poorly understood. Ecological fitness may play a pivotal role, but other underlying biotic and abiotic factors may contribute. Since the presence of toxicants in hypersaline aquatic ecosystems has been documented, our aim here is to study the potential role of an organophosphate pesticide, chlorpyrifos, in a congeneric mechanism of competition between the bisexual A. franciscana (AF), and one of the Old World parthenogenetic siblings, A. parthenogenetica (PD). For this purpose we carried out life table experiments with both species, under different concentrations of the toxicant (0.1, 1 and 5. μg/l), and analyzed the cholinesterase inhibition at different developmental stages. The results evidence that both, AF and PD, showed an elevated tolerance to high ranges of chlorpyrifos, but AF survived better and its fecundity was less affected by the exposure to the pesticide than that of PD. The higher fecundity of AF is a selective advantage in colonization processes leading to its establishment as NIS. Besides, under the potential selective pressure of abiotic factors, such as the presence of toxicants, its higher resistance in terms of survival and biological fitness also indicates out-competitive advantages. © 2015This research was supported by the Spanish Ministry for Science and Innovation projects (CGL2005-02306 and CGL2008-04737-E) “Biodiversidad de Artemia (Branchiopoda, Anostraca) en el Mediterráneo Occidental, archipiélagos Balear y Canario. Efectos de A. franciscana como especie invasora. Implicaciones ecológicas y de interés en acuicultura” y “Biodiversidad amenazada en salinas mediterráneas”. S. Redón was supported by a Ph.D grant (FPI) from the Spanish Ministry of Science and Innovation. D. Guinot was supported by BANCAJA contract.Peer Reviewe

Aquatic pollution may favor the success of the invasive species A. franciscana

The genus Artemia consists of several bisexual and parthenogenetic sibling species. One of them, A. franciscana, originally restricted to the New World, becomes invasive when introduced into ecosystems out of its natural range of distribution. Invasiveness is anthropically favored by the use of cryptobiotic eggs in the aquaculture and pet trade. The mechanisms of out-competition of the autochthonous Artemia by the invader are still poorly understood. Ecological fitness may play a pivotal role, but other underlying biotic and abiotic factors may contribute. Since the presence of toxicants in hypersaline aquatic ecosystems has been documented, our aim here is to study the potential role of an organophosphate pesticide, chlorpyrifos, in a congeneric mechanism of competition between the bisexual A. franciscana (AF), and one of the Old World parthenogenetic siblings, A. parthenogenetica (PD). For this purpose we carried out life table experiments with both species, under different concentrations of the toxicant (0.1, 1 and 5 μg/l), and analyzed the cholinesterase inhibition at different developmental stages. The results evidence that both, AF and PD, showed an elevated tolerance to high ranges of chlorpyrifos, but AF survived better and its fecundity was less affected by the exposure to the pesticide than that of PD. The higher fecundity of AF is a selective advantage in colonization processes leading to its establishment as NIS. Besides, under the potential selective pressure of abiotic factors, such as the presence of toxicants, its higher resistance in terms of survival and biological fitness also indicates out-competitive advantages.Spanish Ministry for Science and Innovation project, CGL2005-02306, CGL2008-04737-E

Identification of new, very long-chain polyunsaturated fatty acids in fish by gas chromatography coupled to quadrupole/time-of-flight mass spectrometry with atmospheric pressure chemical ionization

This is a pre-print of an article published in Analytical and Bioanalytical Chemistry. The final authenticated version is available online at: https://doi.org/10.1007/s00216-020-03062-0The characterization of very long-chain (>C24) polyunsaturated fatty acids (VLC-PUFAs), which are essential in the vision, neural function, and reproduction of vertebrates, is challenging because of the lack of reference standards and their very low concentrations in certain lipid classes. In this research, we have developed a new methodology for VLC-PUFA identification based on gas chromatography coupled to quadrupole/time-of-flight mass spectrometry with an atmospheric pressure chemical ionization source (GC-APCI-QTOF MS). The mass accuracy attainable with the innovative QTOF instrument, together with the soft ionization of the APCI source, provides valuable information on the intact molecule, traditionally lost with electron ionization sources due to the extensive fragmentation suffered. We have identified, for the first time, VLC-PUFAs with chains up to 44 carbons in eyes, brain, and gonads of gilthead sea bream, a commercially important fish in the Mediterranean. The added value of ion mobility-mass spectrometry (IMS), recently developed in combination with GC-QTOF MS, and the contribution of the collisional cross section (CCS) parameter in the characterization of novel VLC-PUFAs (for which reference standards are not available) have been also evaluated. The methodology developed has allowed assessing qualitative differences between farmed and wild fish, and opens new perspectives in a still scarcely known field of research