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

Seroprevalence of human cysticercosis in Maputo, Mozambique.

We carried out a serosurvey for cysticercosis among people visiting the Central Hospital of Maputo, the capital of Mozambique, between January and June 1993. A standardized questionnaire was designed to obtain information on demographic, socioeconomic, environmental, and behavioral characteristics related to the transmission of the infection. Four hundred eighty-nine individuals were tested for anti-cysticercosis antibodies: 222 blood donors and patients from the Department of Orthopedics, 148 patients from the Department of Neurology, and 119 patients from the Department of Psychiatry. The overall positivity rate was 12.1% (59 of 489). Anti-cysticercus antibodies was detected in 14.9% of the blood donors and patients from the Department of Orthopedics, 11.5% of the patients from the Department of Neurology, and 7.6% of the patients from the Department of Psychiatry. Living in poor sanitary conditions seems to be an important factor related to human cysticercosis in Maputo, Mozambique

Seroprevalence of human cysticercosis in Maputo, Mozambique

We carried out a serosurvey for cysticercosis among people visiting the Central Hospital of Maputo, the capital of Mozambique, between January and June 1993. A standardized questionnaire was designed to obtain information on demographic, socioeconomic, environmental, and behavioral characteristics related to the transmission of the infection. Four hundred eighty-nine individuals were tested for anti-cysticercosis antibodies: 222 blood donors and patients from the Department of Orthopedics, 148 patients from the Department of Neurology, and 119 patients from the Department of Psychiatry. The overall positivity rate was 12.1% (59 of 489). Anti-cysticercus antibodies was detected in 14.9% of the blood donors and patients from the Department of Orthopedics, 11.5% of the patients from the Department of Neurology, and 7.6% of the patients from the Department of Psychiatry. Living in poor sanitary conditions seems to be an important factor related to human cysticercosis in Maputo, Mozambique.publishersversionpublishe

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

Contributo para o estabelecimento de reserva ex situ de medronheiro (Arbutus unedo L.). Condições de germinação, instalação e crescimento em viveiro de plantas de diferentes proveniências

Mestrado em Engenharia Agronómica / Instituto Superior de Agronomia. Universidade de LisboaA variabilidade genética do medronheiro existente em Portugal levou à necessidade da criação de uma reserva de germoplasma, para utilizar em futuros projetos de melhoramento partindo de plantas silvestres. Este trabalho visa contribuir para a constituição dessa reserva pela identificação de locais de colheita de sementes que confiram elevadas taxas de germinação e originem plantas com maior potencial de crescimento. Para tal, colheram-se frutos de, aproximadamente, 25 plantas de cinco zonas de Portugal: serra de São Mamede, serra de Monchique, serra do Caldeirão, Barrocal e Penamacor. Extraíram-se, caracterizaram-se as sementes e selecionaram-se três lotes de semente de cada zona para realizar de ensaios de viabilidade (TTC) e germinação, segundo diferentes tratamentos: sem estratificação, 15, 29, 46, 60, 75 e 90 dias de estratificação a frio (4 ºC). Em viveiro monitorizou-se quinzenalmente o crescimento de aproximadamente cinco plântulas de cada lote de sementes, durante três meses e a taxa de mortalidade das plântulas. As sementes colhidas em São Mamede não se encontravam viáveis, talvez pelo elevado tempo de amolecimento da polpa do fruto em água (54 dias). 12% das sementes de Monchique, Caldeirão e Barrocal, que estiveram 39 dias em água, encontravam-se viáveis, em média, e as sementes de Penamacor, que estiveram 20 dias em água, encontravam-se totalmente viáveis. Penamacor e Caldeirão apresentaram taxas de germinação de 64,4% e 23,8%, respetivamente, mesmo sem qualquer estratificação. Para Penamacor apenas tratamentos superiores a 46 dias de estratificação mostraram germinações significativamente superiores ao grupo controlo (aproximadamente mais 50%). As plântulas de Monchique apresentaram a menor taxa de mortalidade em viveiro. As sementes de Penamacor, Monchique e Caldeirão apresentam crescimentos significativamente superiores ao Barrocal (mais 15 - 25%), enfatizando estes três locais de proveniência como os mais promissores, dos locais estudados, para viveiristas que utilizem a propagação seminal como método de obtenção de novas plantasN/

Formas urbanas na costa americana do Pacifico

Proponho-me estudar, na minha dissertação, a evolução das formas urbanas, com enfoque particular na temática da dispersão urbana (vulgo "urban sprawl") e da regeneração dos centros, abordando cinco áreas metropolitanas americanas da costa do Pacífico: três no estado da Califórnia, onde vivi alguns anos (Los Angeles, San Diego e San Francisco), e as restantes duas nos estados de Washington e Oregon (Seattle e Portland, respectivamente). O trabalho tem como objectivo fundamental, através do levantamento e análise dos diferentes casos de estudo, a identificação das situações de expansão urbana descontrolada, bem como o estudo de factores que possam ter potenciado o seu aparecimento e crescimento. É também abordada a temática da degeneração dos centros urbanos, onde uma das causas reside precisamente no crescimento sem controlo dos perímetros metropolitanos, retirando recursos e atractividade aos centros, numa espiral negativa de resolução complexa. São também identificados vários modelos de organização urbana, de diferentes origens e densidades, que vão do crescimento orgânico às comunidades planeadas. Também se incluem projectos de renovação urbana que incluem novas formas de urbanização, assim como a reorganização de infraestruturas de transporte como modo de influenciar dinâmicas de crescimento da cidade. É feita uma análise sobre diferentes correntes de opinião, nomeadamente a dos "centralistas", que defendem a densificação dos centros urbanos, e os "descentralistas", que preconizam o oposto, tal como diversas subcorrentes. O trabalho encerra com uma conclusão que terá em linha de conta as actuais tendências demográficas, sociais, económicas, tecnológicas, políticas, energéticas e climáticas.The main theme of this dissertation focuses on the urban forms of the United States Pacific Coast, namely in the metro areas of Seattle, Portland, San Francisco, Los Angeles and San Diego. This work will be subdivided in the following areas: A brief historical background on the cities' organization and urban expansion A timeline on the urban evolution of the cities of Seattle, Portland, San Francisco, Los Angeles and San Diego, focusing on the differences, similarities, causes and consequences of each case, on the following points of view: Urbanization / geography Economical / political Social / Cultural Technological / Innovation Comparative review and analysis of five case studies, focusing the similarities, causes and consequences of urban sprawl Urban forms for the 21st century: current debate tendencies, models, proposals and solutions Centralists vs. Descentralists, or Containment vs. Expansion Centralists: Smart Growth, TOD, New Urbanism Descentralists: Planned communities, Close-gated communities Examples on Centralism and Descentralism Conclusio

Screening for Autism Spectrum Disorders-Validation of the Portuguese Version of the Social Communication Questionnaire

There are no assessment and screening tools for Autism Spectrum Disorders (ASD) validated for the Portuguese population. The Social Communication Questionnaire (SCQ) is an useful screening tool of ASD diagnosis. The main objectives of our study were to produce a Portuguese version of the SCQ (SCQ-PF), study its internal consistency, sensitivity and specificity in order to evaluate its validity as a screening instrument for ASD. We also wanted to study the impact of intellectual disability and verbal impairment and other mental disorders on SCQ-PF psychometric properties. The study included 211 children and adolescents, aged 4-17, divided in three groups: ASD Group (n = 96), Other Mental Disorders Group (OMD) (n = 63) and No Mental Disorders (NMD) Group (n = 52). Parents or other primary caregiver provided information on the SCQ items. The SCQ-PF score was significantly higher in the ASD group than in the other groups (p < 0.001). As to internal consistency, Cronbach's alpha was 87%. ASD subjects were distinguished from subjects without ASD (OMD and NMD Groups) and the area under the curve (AUC) was 0.897 (95% Confidence Interval: 0.852-0.943), for a cutoff of 14, which yielded the highest AUC, with values of sensitivity and specificity 0.76 and 0.93, respectively. These findings show that SCQ- PF with a cutoff of 14 is an acceptable and useful screening tool for ASD in the Portuguese population

Adição de números racionais : uma experiência de ensino no 3º ano

Dissertação de mestrado, Educação (Área de especialização em Didática da Matemática), Universidade de Lisboa, Instituto de Educação, 2015Esta investigação tem como objetivo estudar a aprendizagem por alunos do 3.º ano da adição de números racionais por justaposição retilínea de segmentos de reta, seguindo uma abordagem de cunho exploratório. O quadro concetual tem por base estudos sobre a aprendizagem dos números racionais, nos seus diferentes significados, e o ensino-aprendizagem da adição de frações e de números decimais. Assume-se que (i) a compreensão do conceito de fração é determinante para a aprendizagem dos números racionais e necessária para a compreensão da adição por justaposição retilínea de segmentos de reta; (ii) a aprendizagem da adição de números racionais deve ser iniciada com base em representações pictóricas e contextos tanto quanto possível próximo da realidade dos alunos. Estes princípios informam a elaboração de uma unidade de ensino, com tarefas organizadas numa sequência ordenada de questões que levem a discussões em grande grupo suscetíveis de promover a compreensão e aprendizagem dos temas trabalhados. Esta investigação segue uma abordagem qualitativa e insere-se no paradigma interpretativo. Enquanto investigador e professor desempenho o papel de observador participante. Os participantes do estudo são os alunos de uma turma do 3.º ano de escolaridade. A recolha de dados inclui as produções dos alunos, as informações recolhidas do diário de bordo, as gravações áudio e vídeo das aulas e das três entrevistas. Os resultados mostram que os alunos compreenderam a adição de frações por justaposição retilínea de segmentos de reta tendo como base de aprendizagem as representações pictóricas e a barra numérica. A barra numérica desempenhou um papel muito importante pois revelou ser um modelo de simples utilização e apropriação por parte dos alunos, permitindo estabelecer com facilidade uma relação com um contexto real. Assim, a relação entre representações icónicas e simbólicas pode ser apropriada de forma natural pela criança, permitindo a compreensão gradual das diferentes representações. A adição de números decimais com recurso à reta numérica, pela sua complexidade, mostrou ser um tema onde os alunos apresentam mais erros e dificuldades, pois, ao contrário da barra numérica, revela-se fortemente abstrata e descontextualizada.This research aims to study the learning by grade 3 students of the addition of rational numbers using the number line, following an exploratory approach. The conceptual framework is based on studies on the learning of rational numbers, in their different meanings, and the teaching and learning of the addition of fractions and decimals. It is assumed that (i) the understanding of the concept of fraction is decisive for the learning of rational numbers and necessary for the understanding of addition on number line; (ii) learning the addition of rational numbers should be initiated based on pictorial representations and contexts as much as possible close to reality of the students. These principles inform the development of a teaching unit, with tasks arranged in an ordered sequence of questions that lead to large group discussions that may promote understanding and learning of the topics worked on. This research follows a qualitative approach within the interpretative paradigm. As the researcher and teacher, I assume the role of participant observer. Participants are the students in a grade 3 class. Data collection includes the work produced by the students, the information collected from the logbook, recording audio and video classes and three interviews. The results show that the students understand the addition of fractions based on the pictorial and bar model. The bar model played a very important role because it proved to be of simple use and ownership by the students, allowing to easily establish a relationship with a real context. Thus, the relationship between iconic and symbolic representations may be appropriated in a natural way by the child, allowing the gradual understanding of different representations. The addition of decimal numbers using the number line, because of its complexity, proved to be an area where students make more errors and have difficulties because, unlike bar model, it proves to be highly abstract and decontextualized