Phenotypic heterogeneity and the biological significance of a pyruvate sensing network in Escherichia coli

When facing nutrient limitation, bacterial populations are forced to optimize uptake systems for available substrates in order to adapt and survive. However, the physiological diversity of each individual in a population leads to differential activation of the uptake systems in response to the same environmental condition. Hence, heterogeneous phenotypes can be generated in a clonal population. A way for each bacterial individual to sense nutrient availability and transmit this information from the extra- to the intracellular spaces is via two-component systems (TCSs). Escherichia coli contains 30 TCSs, each composed of one histidine kinase (HK) and at least one response regulator (RR). The HK is responsible for perceiving the extracellular stimulus and the RR consequently mediates the output, generally a regulation of gene expression. BtsS/BtsR and YpdA/YpdB are examples of two TCSs in E. coli. BtsS/BtsR activation leads to the induction of yjiY, whereas YpdA/YpdB activates yhjX expression. yjiY and yhjX code for putative transporters. Both systems are functionally interconnected and are though to form a large signalling network. This thesis focuses on the analysis of the transcriptional activation of yjiY and yhjX at the single-cell level and on the biological significance of the two TCSs. The activation of the target promoters at the single-cell level was found to be heterogeneous and strongly influenced by the available nutrients. To exploit the biological relevance of the two TCSs, wild-type cells were compared to cells of a btsSRypdAB mutant under two metabolically modulated conditions: protein overproduction and persister formation. BtsS/BtsR and YpdA/YpdB network was shown to contribute to a balancing of the physiological state of all cells within a population. YjiY, the putative transport protein resulting from BtsS/BtsR activation, was found to function as a high-affinity and specific pyruvate/H+ symporter. The protein was renamed for BtsT (Bts is an abbreviation of the German name for pyruvate, Brenztraubensäure, and T stands for Transporter). The correlation between the heterogeneous transcriptional activation of btsT and the number of persister cells was explored using microfluidic technology. It was showed that individuals with low btsT transcriptional activation were more prone to form persisters. A relevant biological application of a pyruvate transporter, the resuscitation from the viable but nonculturable (VBNC) state of E. coli was tested in bulk and single-cell assays and revealed that the concominant presence of pyruvate and BtsS/BtsR and YpdA/YpdB network is required for proper resuscitation from this state. For the first time, phenotypes associated to a mutant lacking the BtsS/BtsR and YpdA/YpdB systems were identified, which in general are related to metabolic challenges. This thesis contributes to a better understanding of the biological role of the two TCSs, BtsS/BtsR and YpdA/YpdB of E. coli.Eine Nährstofflimitation zwingt Bakterienpopulationen dazu, ihre Aufnahmesysteme für verfügbare Substrate zu optimieren, um Adaptation und Überleben zu gewährleisten. Die physiologische Diversität einer Population impliziert trotz gleicher Umweltbedingung eine je nach Individuum verschiedene Aktivierung der Aufnahmesysteme. Infolgedessen können sich in einer klonalen Population heterogene Phänotypen entwickeln. Zweikomponentensystem (TCSs) ermöglichen es jedem bakteriellen Individuum, die Nährstoffverfügbarkeit festzustellen und diese Information von dem extra- in den intrazellulären Raum zu übertragen. Escherichia coli besitzt 30 TCSs, jeweils bestehend aus einer Histidinkinase (HK) und mindestens einem Antwortregulator (RR). Die HK ist dafür verantwortlich, den extrazellulären Stimulus wahrzunehmen, während der RR anschließend den Ausgabewert vermittelt, üblicherweise in Form einer Regulation der Genexpression. BtsS/BtsR und YpdA/YpdB sind Beispiele für zwei TCSs in E. coli. Die Aktivierung von BtsS/BtsR führt zur Induktion von yjiY, während YpdA/YpdB die Expression von yhjX aktiviert. YjiY und yhjX kodieren mutmaßliche Transporter. Die beiden Systeme sind funktionell miteinander verbunden und bilden vermutlich ein großes Signalnetzwerk. Diese Arbeit konzentriert sich auf die Einzelzellanalyse der transkriptionellen Aktivierung von yjiY und yhjX, sowie auf die biologische Signifikanz der beiden TCSs. Auf Einzelzellebende konnte eine heterogene Aktivierung der Zielpromotoren nachgewiesen werden, die stark von den verfügbaren Nährstoffen beeinflussbar ist. Um die biologische Relevanz der beiden TCSs zu untersuchen, wurden Wildtypzellen mit einer btsSRypdAB Mutante unter zwei metabolisch modifizierten Bedingungen verglichen: Proteinüberproduktion und Persistenz. Es konnte gezeigt werden, dass das Netzwerk aus BtsS/BtsR, sowie YpdA/YpdB zum Ausbalancieren des physiologischen Zustandes aller Zellen einer Population beitragen. YjiY, das aus der BtsS/BtsR Aktivierung resultierende, mutmaßliche Transportprotein, funktioniert nachweislich als hochaffiner und spezifischer Pyruvat/H+ Symporter. Dementsprechend wurde das Protein umbenannt in BtsT (Bts: Abkürzung für Brenztraubensäure, T: Transporter). Die Korrelation zwischen heterogener transkriptioneller Aktivierung von btsT und der Anzahl an Persisterzellen wurde mittels Mikrofluidiktechnologie untersucht. Es konnte gezeigt werden, dass Individuen mit niedrigem btsT Expressionslevel tendenziell anfälliger für den Übergang in den Zustand der Persistenz sind. In Gesamt- und Einzelzelltests konnte belegt werden, dass die gleichzeitige Präsenz von Pyruvat und BtsS/BtsR, sowie YpdA/YpdB Netzwerk Voraussetzung ist für eine korrekte Umkehrung des VBNC Zustandes (viable but nonculturable) von E. coli. Damit wurden zum ersten Mal Phänotypen, die gewöhnlich mit metabolisch herausfordernden Bedingungen in Zusammenhang stehen, bei einer Mutante identifiziert, der die BtsS/BtsR und YpdA/YpdB Systeme fehlen. Diese Arbeit trägt zu einem besseren Verständnis der biologischen Rolle der beiden E. coli TCSs BtsS/BtsR und YpdA/YpdB bei

Dietary exposure to imidacloprid, and sub-lethal effects in springtails

Tese de mestrado, Biologia Humana e Ambiente, 2021, Universidade de Lisboa, Faculdade de CiênciasNeonicotinoids are the most widely used insecticides worldwide. A large proportion ends up in the soil, where they degrade slowly, and are likely to affect non-target species in the soil community, such as Collembola (springtails). Springtails play an important role in soil ecosystems by contributing to litter decomposition and nutrient cycling. Standard toxicity tests use soil exposure to toxicants, which does not allow for detailed observation of the individuals during the experiment. These tests are developed to expose organisms through soil to certain types of contaminants, but in nature springtails can also be exposed to them through their diet. We therefore aimed to, and successfully developed, a method for dietary exposure to toxicants, allowing us to observe springtails performance during exposure. The neonicotinoid imidacloprid was applied as a test substance, and we studied two springtail species, Folsomia quadrioculata and Hypogastrura viatica, with different life histories, habitat, and ecology, to assess potential difference in sensitivity. For each species, five replicates of 20 newly hatched animals were exposed through feed treated with one of two different applications: moistening the feed with a micropipette (estimated final concentration of 129 mg/kg imidacloprid) or soaking it in a solution with 129 ug/L imidacloprid, at room temperature. Moistening the Tilia cordata bark was most accurate, giving concentrations similar to the estimated nominal ones, while soaking resulted in 38 times higher concentrations than moistened measured method. With our established method of moistening the bark, we conducted a concentration-response experiment to study how imidacloprid affects different life history traits in juvenile H. viatica using 5 sub-lethal concentrations from 0.01 to 1.2 mg/kg dry bark. Due to time constraints as a result of the COVID-19 pandemic, which prevented access to the laboratory, it was difficult to analyze some endpoints for Folsomia quadrioculata, and since it was possible to register molting and measure body size in Hypogastrura viatica we conducted the second experiment only on that species. Exposure to the soil relevant concentrations of imidacloprid caused H. viatica to reach maturity late, reduced its molting frequency, and thus reduced its body size. Since these endpoints have negative impacts on Collembola egg production, the studied neonicotinoid shown negative effects on population growth and may therefore cause population-level effects. There is still little information about these two springtails species and especially on the life history traits for springtails in general. Therefore, in the future it would be important to use methods that allow the monitoring of effects on life history traits

Social inclusion of refugees and asylum seekers: The role of public libraries in the Lisbon Metropolitan Area

Social inclusion of refugees and asylum seekers includes addressing their information needs. This research seeks to examine the role of public libraries in this process through the analysis of public librarians’ perceptions. Based on the constructivist paradigm, the case study method, and the semi-directive interview survey technique, 16 public municipal libraries in the Lisbon Metropolitan Area (Portugal) are studied. The results highlight that these libraries do not identify forced migrants as their users. Still, they consider that the information needs and behaviors and the use of libraries by these communities are similar to those of economic migrants, with emphasis on the relevance of free access to the internet, foreign language resources, Portuguese language and digital literacy courses, and help to interact with online platforms. The gaps include the existence of few Portuguese courses for foreigners, a limited practice in assisting with job and housing searches, promoting health literacy, and inclusive collaboration with schools, as well as space, financial, and human resources constraints. To overcome these difficulties, librarians emphasize the motivation to fulfill the inclusive social mission of the public library, the universal accessibility and informal environment of the library, and its action to promote social cohesion and social capital. The respondents consider that the mission of public libraries is adjusted to the goal of social inclusion of forced migrants, but that there is room for improvement in the training of librarians. This research points to the need for more initiative-taking public library collaboration with existing inclusion networks. In conclusion, we seek to alert to the urgency of the involvement of Portuguese public libraries in the social inclusion of forced migrants, which will also contribute to their institutional legitimization.info:eu-repo/semantics/publishedVersio

A evolução da ameaça terrorista no discurso político transâtlantico

Este estudo analisa a evolução da narrativa política transatlântica sobre a ameaça terrorista radical islâmica. A interpretação do terrorismo islamista como uma das principais ameaças contemporâneas tem vindo a ser construída ao longo do tempo. Não só o próprio fenómeno se tem vindo a alterar como a também a narrativa que lhe está associada. A data de 11 de setembro de 2001 é considerada um ponto de viragem na abordagem elaborada pelo mundo ocidental, e marca o ponto de partida desta análise. A hipótese avançada considera que, nos 14 anos seguintes, o terrorismo islamista se tornou uma ameaça prioritária e existencial, o que equivale a afirmar que se tornou uma ameaça securitizada. O processo de securitização ilustra a deslocação de um fenómeno da esfera da gestão política para a gestão securitária, que supõe um estado de exceção e a implementação de medidas extraordinárias. Essa alteração tem início numa dimensão discursiva, nomeadamente, de âmbito político, por corresponder ao ator responsável pela garantia de segurança da população, e da opinião pública depender a sua legitimidade. O contexto transatlântico analisado compõe-se por quatro atores estatais, Estados Unidos, Espanha, Reino Unido e França, e dois institucionais, a União Europeia e a Organização do Tratado do Atlântico Norte. De forma complementar à análise, são expostos os antecedentes de cada um dos atores no que toca à sua experiência com a ameaça terrorista. É também apresentada uma síntese da evolução do conceito de terrorismo e do método islamista, com ênfase na trajetória do grupo al-Qaeda. O objetivo do estudo é alcançado pela análise crítica da narrativa transatlântica, reunindo comunicações de reação a atentados, com discursos anuais generalistas, e documentos de orientação estratégica sobre segurança nacional e terrorismo em particular. A análise discursiva evidencia o contexto como fator decisivo para o posicionamento da ameaça terrorista islamista numa perspetiva politizada ou securitizada. A circunstância dos atentados despoleta o discurso securitário, cuja influência no contexto transatlântico difere de Estado para Estado. Contudo, esse discurso não se mantém ao longo do tempo, revelando-se ritmos diferentes – europeu e norte-americano – no contexto transatlântico.This study analyses the evolution of the political transatlantic narrative on the radical Islamic terrorist threat. The interpretation of the islamist terrorism as one of the main contemporary threats has been built over time: not only the phenomenon itself has changed but also has the narrative associated with it. September 11th 2001 is considered a turning point of the western world approach and therefore marks the beginning of this analysis. The hypothesis put forward considers that, on the 14 years that followed, islamist terrorism became an existential and primary threat, which is to say is became a securitized threat. The process of securitization, which illustrates the shift of a phenomenon from the politics management to the security management, supposes a state of exception and the employment of extraordinary measures. This change arises on a discursive dimension, namely that of political scope, seeing as it belongs to the actor responsible for the population’s warranty of safety and its validity depends on the public opinion. The transatlantic context that was studied is made up of four state actors (the United States of America, Spain, the United Kingdom and France) and two institutional actors (the European Union and the North Atlantic Treaty Organization). Complementary to the analysis are exposed each actor’s antecedents in regards to their experience with the terrorist threat. A synthesis of the evolution of the concept of terrorism and its Islamic method is also presented, highlighting the al-Qaeda group. The purpose of this study is reached by the critical analysis of the transatlantic narrative, assembling together reaction declarations to attacks and general annual speeches, as well as strategic orientation documents regarding national security and, specifically, terrorism. This discursive analysis emphasizes context as the decisive factor for placing an islamist threat on a more politicized or securitized perspective. The circumstances in which the attacks take place trigger the security discourse, the influence of which differs from state to state in the transatlantic context. However, this type of discourse does not remain over time, exposing different rhythms – the European and the North American – in the transatlantic setting

The generalized identification of truly interfacial molecules (ITIM) algorithm for nonplanar interfaces

We present a generalized version of the ITIM algorithm for the identification of interfacial molecules, which is able to treat arbitrarily shaped interfaces. The algorithm exploits the similarities between the concept of probe sphere used in ITIM and the circumsphere criterion used in the α-shapes approach, and can be regarded either as a reference-frame independent version of the former, or as an extended version of the latter that includes the atomic excluded volume. The new algorithm is applied to compute the intrinsic orientational order parameters of water around a dodecylphosphocholine and a cholic acid micelle in aqueous environment, and to the identification of solvent-reachable sites in four model structures for soot. The additional algorithm introduced for the calculation of intrinsic density profiles in arbitrary geometries proved to be extremely useful also for planar interfaces, as it allows to solve the paradox of smeared intrinsic profiles far from the interface. © 2013 American Institute of Physics

The Experience of a Pioneer Research Program in Architecture in Évora

Three years ago the University of Évora implemented a research PhD program in architecture. Generally a doctorate of architecture has been an academic title awarded to architects who present a theoretical dissertation; however, for us as a young but promising school of architecture it was just natural that a project (as a methodology, a process of knowledge or simulation of a hypothesis) could be part of an advance research in architecture. Thus, we started with this doctoral program seeking to question the current model of PhD programs and established a new pioneering paradigm syllabus in the national context with the intention to reach the international arena During the course syllabus the project lab integrates the formulation of a theoretical hypothesis (a conjecture), that becomes an architectural design, which is unique, but simultaneously an universal knowledge. The program has already two editions were PhD students have been encouraged to develop advance research and fostered interaction between the theoretical and architecture production. No research is yet finished. Students, although much interested in this type of research, are divided in their approach to architecture as a theoretical, speculative and critical field, and architecture as a field of research. Students and teacher are interested in research that develops their architecture design skills, as a relevant process of advancement knowledge in architecture. We believe that PhD syllabus will contribute to demystify and implement the concept of advanced studies in architecture based on architectural research. This paper will share some of the ideas, doubts, and results of our PhD program

Fostering collaboration in kindergarten through an augmented reality game

Kindergarten children are a very special class of users, since they are in a primary stage of life, when they must learn how to live in society, e.g. to listen and respect the others’ opinions, share the same objects and also help each other. This study presents an Augmented Reality game, in which kinder garten children were able to collaborate in a spontaneous way supported by motivation, enjoyment and curiosity. This game allows children to explore concepts like the animals and the en vironments they live in by using Augmented Reality markers and a wooden board. These markers are the game pieces and through them children are able to manipulate 3D virtual models. Experiments were performed with several classes of students in different schools. Results suggest that the game is effective in maintaining high levels of motivation and collaboration among children, particularly when using immediate feedback.info:eu-repo/semantics/publishedVersio