Functional Genomic Screen Identifies Klebsiella pneumoniae Factors Implicated in Blocking Nuclear Factor κB (NF-κB) Signaling

Klebsiella pneumoniae is etiologic agent of community-acquired and nosocomial pneumonia. It has been shown that K. pneumoniae infections are characterized by reduced early inflammatory response. Recently our group have shown that K. pneumoniae dampens the activation of inflammatory responses by antagonizing the activation of the NF-κB canonical pathway. Our results revealed that K. pneumoniae capsule (CPS) was necessary but not sufficient to attenuate inflammation. To identify additional Klebsiella factors required to dampen inflammation, we standardized and applied a high-throughput gain-on-function screen to examine a Klebsiella transposon mutant library. We identified 114 mutants that triggered the activation of NF-κB. Two gene ontology categories accounted for half of the loci identified in the screening, that of metabolism and transport (32% of the mutants), and of enveloperelated genes (17%). Characterization of the mutants revealed that the lack of the enterobactin siderophore was linked to a reduced CPS expression which in turn underlined the NF- κB activation induced by the mutant. The lipopolysaccharide (LPS) O-polysaccharide and the pullulanase (PulA) type 2 secretion system (T2SS) are required for full effectiveness of immune evasion. Importantly, these factors do not play a redundant role. The fact that LPS Opolysaccharide and T2SS mutants-induced responses were dependent on TLR2-TLR4- MyD88 activation suggested that LPS Opolysaccharide and PulA perturbed TLRdependent recognition of K. pneumoniae. Finally, we demonstrate that LPS O-polysaccharide and pulA mutants are attenuated in the pneumonia mouse model. We propose that LPS Opolysaccharide and PulA T2SS could be new targets for designing new antimicrobials. Increasing TLR-governed defence responses might provide also selective alternatives for the management of K. pneumoniae pneumonia.

Klebsiella pneumoniae survives within macrophages by avoiding delivery to lysosomes

Klebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that Klebsiella might be able to persist intracellularly within a vacuolar compartment. This study was designed to investigate the interaction between Klebsiella and macrophages. Engulfment of K. pneumoniae was dependent on host cytoskeleton, cell plasma membrane lipid rafts and the activation of PI 3-kinase (PI3K). Microscopy studies revealed that K. pneumoniae resides within a vacuolar compartment, the Klebsiella containing vacuolae (KCV), which traffics within vacuoles associated with the endocytic pathway. In contrast to UV-killed bacteria, the majority of live bacteria did not colocalize with markers of the lysosomal compartment. Our data suggest that K. pneumoniae triggers a programmed cell death in macrophages displaying features of apoptosis. Our efforts to identify the mechanism(s) whereby K. pneumoniae prevents the fusion of the lysosomes to the KCV uncovered the central role of the PI3K-Akt-Rab14 axis to control the phagosome maturation. Our data revealed that the capsule is dispensable for Klebsiella intracellular survival if bacteria were not opsonized. Furthermore, the environment found by Klebsiella within the KCV triggered the downregulation of the expression of cps. Altogether, this study proves evidence that K. pneumoniae survives killing by macrophages by manipulating phagosome maturation which may contribute to Klebsiella pathogenesis.

Modeling Klebsiella pneumoniae pathogenesis by infection of the wax moth Galleria mellonella

The implementation of infection models that approximate human disease is essential for understanding pathogenesis at the molecular level and for testing new therapies before they are entered into clinical stages. Insects are increasingly being used as surrogate hosts because they share, with mammals, essential aspects of the innate immune response to infections. We examined whether the larva of the wax moth Galleria mellonella could be used as a host model to conceptually approximate Klebsiella pneumoniae-triggered pneumonia. We report that the G. mellonella model is capable of distinguishing between pathogenic and nonpathogenic Klebsiella strains. Moreover, K. pneumoniae infection of G. mellonella models some of the known features of Klebsiella-induced pneumonia, i.e., cell death associated with bacterial replication, avoidance of phagocytosis by phagocytes, and the attenuation of host defense responses, chiefly the production of antimicrobial factors. Similar to the case for the mouse pneumonia model, activation of innate responses improved G. mellonella survival against subsequent Klebsiella challenge. Virulence factors necessary in the mouse pneumonia model were also implicated in the Galleria model. We found that mutants lacking capsule polysaccharide, lipid A decorations, or the outer membrane proteins OmpA and OmpK36 were attenuated in Galleria. All mutants activated G. mellonella defensive responses. The Galleria model also allowed us to monitor Klebsiella gene expression. The expression levels of cps and the loci implicated in lipid A remodeling peaked during the first hours postinfection, in a PhoPQ- and PmrAB-governed process. Taken together, these results support the utility of G. mellonella as a surrogate host for assessing infections with K. pneumoniae.

A qualitative study of community pharmacists’ awareness of and involvement with intermediate care facilities

Rationale, aims and objectives: Intermediate care (IC) describes a range of services targeted at older people, aimed at preventing unnecessary hospitalisation, promoting faster recovery and maximising independence. The introduction of IC has created a new interface between primary and secondary care. Older people are known to be at an increased risk of medication-related problems when transferring between healthcare settings and pharmacists are often not included as part of IC multidisciplinary teams. This study aimed to explore community pharmacists’ (CPs) awareness of IC services and to investigate their views of and attitudes towards the medicines management aspects of such services, including the transfer of medication information.

Method: Semi-structured interviews were conducted, recorded, transcribed verbatim and analysed using a constant comparative approach with CPs practising in the vicinity of IC facilities in Northern Ireland, UK.

Results: Interviews were conducted with 16 CPs. Three themes were identified and named ‘left out of the loop’, ‘chasing things up’ and ‘closing the loop’. CPs felt that they were often ‘left out of the loop’ with regards to both their involvement with local IC services and communication across the healthcare interfaces. As a result, CPs resorted to ‘chasing things up’ as they had to proactively try to obtain information relating to patients’ medications. CPs viewed themselves as ideally placed to facilitate medicines management across the healthcare interfaces (i.e., ‘closing the loop’), but several barriers to potential services were identified.

Conclusion: CPs have limited involvement with IC services. There is a need for improvement of effective communication of patients’ medication information between secondary care, IC and community pharmacy. Increasing CP involvement may contribute to improving continuity of care across such healthcare interfaces, thereby increasing the person-centeredness of service provision.

On the application of optimal control techniques in lossy fieldbuses

The performance of real-time networks is under continuous improvement as a result of several trends in the digital world. However, these tendencies not only cause improvements, but also exacerbates a series of unideal aspects of real-time networks such as communication latency, jitter of the latency and packet drop rate. This Thesis focuses on the communication errors that appear on such realtime networks, from the point-of-view of automatic control. Specifically, it investigates the effects of packet drops in automatic control over fieldbuses, as well as the architectures and optimal techniques for their compensation. Firstly, a new approach to address the problems that rise in virtue of such packet drops, is proposed. This novel approach is based on the simultaneous transmission of several values in a single message. Such messages can be from sensor to controller, in which case they are comprised of several past sensor readings, or from controller to actuator in which case they are comprised of estimates of several future control values. A series of tests reveal the advantages of this approach. The above-explained approach is then expanded as to accommodate the techniques of contemporary optimal control. However, unlike the aforementioned approach, that deliberately does not send certain messages in order to make a more efficient use of network resources; in the second case, the techniques are used to reduce the effects of packet losses. After these two approaches that are based on data aggregation, it is also studied the optimal control in packet dropping fieldbuses, using generalized actuator output functions. This study ends with the development of a new optimal controller, as well as the function, among the generalized functions that dictate the actuator’s behaviour in the absence of a new control message, that leads to the optimal performance. The Thesis also presents a different line of research, related with the output oscillations that take place as a consequence of the use of classic co-design techniques of networked control. The proposed algorithm has the goal of allowing the execution of such classical co-design algorithms without causing an output oscillation that increases the value of the cost function. Such increases may, under certain circumstances, negate the advantages of the application of the classical co-design techniques. A yet another line of research, investigated algorithms, more efficient than contemporary ones, to generate task execution sequences that guarantee that at least a given number of activated jobs will be executed out of every set composed by a predetermined number of contiguous activations. This algorithm may, in the future, be applied to the generation of message transmission patterns in the above-mentioned techniques for the efficient use of network resources. The proposed task generation algorithm is better than its predecessors in the sense that it is capable of scheduling systems that cannot be scheduled by its predecessor algorithms. The Thesis also presents a mechanism that allows to perform multi-path routing in wireless sensor networks, while ensuring that no value will be counted in duplicate. Thereby, this technique improves the performance of wireless sensor networks, rendering them more suitable for control applications. As mentioned before, this Thesis is centered around techniques for the improvement of performance of distributed control systems in which several elements are connected through a fieldbus that may be subject to packet drops. The first three approaches are directly related to this topic, with the first two approaching the problem from an architectural standpoint, whereas the third one does so from more theoretical grounds. The fourth approach ensures that the approaches to this and similar problems that can be found in the literature that try to achieve goals similar to objectives of this Thesis, can do so without causing other problems that may invalidate the solutions in question. Then, the thesis presents an approach to the problem dealt with in it, which is centered in the efficient generation of the transmission patterns that are used in the aforementioned approaches.

More than just a problem with faces: Altered body perception in a group of congenital prosopagnosics

It has been estimated that one out of forty people in the general population suffer from congenital prosopagnosia (CP), a neurodevelopmental disorder characterized by difficulty identifying people by their faces. CP involves impairment in recognising faces, although the perception of non-face stimuli may also be impaired. Given that social interaction does not only depend on face processing, but also the processing of bodies, it is of theoretical importance to ascertain whether CP is also characterised by body perception impairments. Here, we tested eleven CPs and eleven matched control participants on the Body Identity Recognition Task (BIRT), a forced-choice match-to-sample task, using stimuli that require processing of body, not clothing, specific features. Results indicated that the group of CPs was as accurate as controls on the BIRT, which is in line with the lack of body perception complaints by CPs. However the CPs were slower than controls, and when accuracy and response times were combined into inverse efficiency scores (IES), the group of CPs were impaired, suggesting that the CPs could be using more effortful cognitive mechanisms to be as accurate as controls. In conclusion, our findings demonstrate CP may not generally be limited to face processing difficulties, but may also extend to body perception

Análise de sistemas de comunicação óptica com multiplexagem de subportadora e transmissão em banda lateral única

Tese dout., Engenharia Electrónica e Computação, Universidade do Algarve, 2005

Methods to improve creativity and innovation: the effectiveness of creative problem solving

This chapter focuses on the development of organizational creativity, using the CPS methodology, aiming at demonstrating its effectiveness in using the individual and team divergent thinking improvement in identifying organizational problems. A study was undertaken using problem solving teams in seven companies, in which each individual was submitted to a pre-post test in attitudes towards divergent thinking and asked to express the evaluation of the method. All the information reported in the sessions was recorded. The results indicate a change in attitude favourable to divergent thinking, the provision of a professional, efficient method of organizing knowledge in such a way that can help individuals to find original solutions to problems, and an important way to lead teams to creativity and innovation, according with companies different orientations.

Multiuser Detection for the Uplink of Prefix-Assisted DS-CDMA Systems Employing Multiple Transmit and Receive Antennas

In this paper we consider the uplink transmission within a DS-CDMA system employing CP-assisted (Cylic Prefix) block transmission techniques combined with spatial multiplexing techniques that require multiple antennas at both the transmitter and the receiver. We present an efficient frequency-domain receiver structure with iterative MUD (MultiUser Detection). The performance of the proposed receiver can be close to the single user matched filter bound, even for fully loaded systems and/or severely time-dispersive channels.

Taxonomy of fundamental concepts of localization in cyber-physical and sensor networks

Localization is a fundamental task in Cyber-Physical Systems (CPS), where data is tightly coupled with the environment and the location where it is generated. The research literature on localization has reached a critical mass, and several surveys have also emerged. This review paper contributes on the state-of-the-art with the proposal of a new and holistic taxonomy of the fundamental concepts of localization in CPS, based on a comprehensive analysis of previous research works and surveys. The main objective is to pave the way towards a deep understanding of the main localization techniques, and unify their descriptions. Furthermore, this review paper provides a complete overview on the most relevant localization and geolocation techniques. Also, we present the most important metrics for measuring the accuracy of localization approaches, which is meant to be the gap between the real location and its estimate. Finally, we present open issues and research challenges pertaining to localization. We believe that this review paper will represent an important and complete reference of localization techniques in CPS for researchers and practitioners and will provide them with an added value as compared to previous surveys.

Distributed Sensing of Fluid Dynamic Phenomena with the XDense Sensor Grid Network

IEEE International Conference on Cyber Physical Systems, Networks and Applications (CPSNA'15), Hong Kong, China.

Studies of the assembly of the Streptococcus pneumoniae capsular polysaccharide

Dissertation presented to obtain the Ph.D degree in Biology.

Barriers preventing the successful integration of mental health services into Primary Health Care in Nigeria : a mixed methods approach

RESUMO: A Nigéria tem uma população estimada em cerca de 170 milhões de pessoas. O número de profissionais de saúde mental é muito diminuto, contando apenas com 150 psiquiatras o que perfaz aproximadamente um rácio de psiquiatra: população de mais de 1:1 milhão de pessoas. O Plano Nacional de Saúde Mental de 1991 reconheceu esta insuficiência e recomendou a integração dos serviços de saúde mental nos cuidados de saúde primários (CSP). Depois de mais de duas décadas, essa política não foi ainda implementada. Este estudo teve como objetivos mapear a estrutura organizacional dos serviços de saúde mental da Nigéria, e explorar os desafios e barreiras que impedem a integração bem-sucedida dos serviços de saúde mental nos cuidados de saúde primários, isto segundo a perspectiva dos profissionais dos cuidados de saúde primários. Com este objetivo, desenvolveu-se um estudo exploratório sequencial e utilizou-se um modelo misto para a recolha de dados. A aplicação em simultâneo de abordagens qualitativas e quantitativas permitiram compreender os problemas relacionados com a integração dos serviços de saúde mental nos CSP na Nigéria. No estudo qualitativo inicial, foram realizadas entrevistas com listagens abertas a 30 profissionais dos CSP, seguidas de dois grupos focais com profissionais dos CSP de duas zonas governamentais do estado de Oyo de forma a obter uma visão global das perspectivas destes profissionais locais sobre os desafios e barreiras que impedem uma integração bem-sucedida dos serviços de saúde mental nos CSP. Subsequentemente, foram realizadas entrevistas com quatro pessoas-chave, especificamente coordenadores e especialistas em saúde mental. Os resultados do estudo qualitativo foram utilizados para desenvolver um questionário para análise quantitativa das opiniões de uma amostra maior e mais representativa dos profissionais dos CSP do Estado de Oyo, bem como de duas zonas governamentais locais do Estado de Osun. As barreiras mais comummente identificadas a partir deste estudo incluem o estigma e os preconceitos sobre a doença mental, a formação inadequada dos profissionais dos CPS sobre saúde mental, a perceção pela equipa dos CSP de baixa prioridade de ação do Governo, o medo da agressão e violência pela equipa dos CSP, bem como a falta de disponibilidade de fármacos. As recomendações para superar estes desafios incluem a melhoria sustentada dos esforços da advocacia à saúde mental que vise uma maior valorização e apoio governamental, a formação e treino organizados dos profissionais dos cuidados primários, a criação de redes de referência e de apoio com instituições terciárias adjacentes, e o engajamento da comunidade para melhorar o acesso aos serviços e à reabilitação, pelas pessoas com doença mental. Estes resultados fornecem indicações úteis sobre a perceção das barreiras para a integração bem sucedida dos serviços de saúde mental nos CSP, enquanto se recomenda uma abordagem holística e abrangente. Esta informação pode orientar as futuras tentativas de implementação da integração dos serviços de saúde mental nos cuidados primários na Nigéria.------------ABSTRACT: Nigeria has an estimated population of about 170 million people but the number of mental health professionals is very small, with about 150 psychiatrists. This roughly translates to a psychiatrist:population ratio of more than 1:1 million people. The National Mental Health Policy of 1991 recognized this deficiency and recommended the integration of mental health into primary health care (PHC) delivery system. After more than two decades, this policy has yet to be implemented. This study aimed to map out the organizational structure of the mental health systems in Nigeria, and to explore the challenges and barriers preventing the successful integration of mental health into primary health care, from the perspective of the primary health care workers. A mixed methods exploratory sequential study design was employed, which entails the use of sequential timing in the combined methods of data collection. A combination of qualitative and uantitative approaches in sequence, were utilized to understand the problems of mental health services integration into PHC in Nigeria. The initial qualitative phase utilized free listing interviews with 30 PHC workers, followed by two focus group discussions with primary care workers from two Local Government Areas (LGA) of Oyo State to gain useful insight into the local perspectives of PHC workers about the challenges and barriers preventing successful integration of mental health care services into PHC. Subsequently, 4 key informant interviews with PHC co-ordinators and mental health experts were carried out. The findings from the qualitative study were utilized to develop a quantitative study questionnaire to understand the opinions of a larger and more representative sample of PHC staff in two more LGAs of Oyo State, as well as 2 LGAs from Osun State. The common barriers identified from this study include stigma and misconceptions about mental illness, inadequate training of PHC staff about mental health, low government priority, fear of aggression and violence by the PHC staff, as well as non-availability of medications. Recommendations for overcoming these challenges include improved and sustained efforts at mental health advocacy to gain governmental attention and support, organized training and retraining for primary care staff, establishment of referral and supportive networks with neighbouring tertiary facilities and community engagement to improve service utilization and rehabilitation of mentally ill persons. These findings provide useful insight into the barriers to the successful integration of mental health into PHC, while recommending a holistic and comprehensive approach. This information can guide future attempts to implement the integration of mental health into primary care in Nigeria.

Why Have Aggregate Skilled Hours Become So Cyclical Since the Mid-1980's?

This paper documents and discusses a dramatic change in the cyclical behavior of aggregate hours worked by individuals with a college degree (skilled workers) since the mid-1980’s. Using the CPS outgoing rotation data set for the period 1979:1-2003:4, we find that the volatility of aggregate skilled hours relative to the volatility of GDP has nearly tripled since 1984. In contrast, the cyclical properties of unskilled hours have remained essentially unchanged. We evaluate the extent to which a simple supply/demand model for skilled and unskilled labor with capital-skill complementarity in production can help explain this stylized fact. Within this framework, we identify three effects which would lead to an increase in the relative volatility of skilled hours: (i) a reduction in the degree of capital-skill complementarity, (ii) a reduction in the absolute volatility of GDP (and unskilled hours), and (iii) an increase in the level of capital equipment relative to skilled labor. We provide empirical evidence in support of each of these effects. Our conclusion is that these three mechanisms can jointly explain about sixty percent of the observed increase in the relative volatility of skilled labor. The reduction in the degree of capital-skill complementarity contributes the most to this result.

A Type-Preserving Compiler from System F to Typed Assembly Language

L'utilisation des méthodes formelles est de plus en plus courante dans le développement logiciel, et les systèmes de types sont la méthode formelle qui a le plus de succès. L'avancement des méthodes formelles présente de nouveaux défis, ainsi que de nouvelles opportunités. L'un des défis est d'assurer qu'un compilateur préserve la sémantique des programmes, de sorte que les propriétés que l'on garantit à propos de son code source s'appliquent également au code exécutable. Cette thèse présente un compilateur qui traduit un langage fonctionnel d'ordre supérieur avec polymorphisme vers un langage assembleur typé, dont la propriété principale est que la préservation des types est vérifiée de manière automatisée, à l'aide d'annotations de types sur le code du compilateur. Notre compilateur implante les transformations de code essentielles pour un langage fonctionnel d'ordre supérieur, nommément une conversion CPS, une conversion des fermetures et une génération de code. Nous présentons les détails des représentation fortement typées des langages intermédiaires, et les contraintes qu'elles imposent sur l'implantation des transformations de code. Notre objectif est de garantir la préservation des types avec un minimum d'annotations, et sans compromettre les qualités générales de modularité et de lisibilité du code du compilateur. Cet objectif est atteint en grande partie dans le traitement des fonctionnalités de base du langage (les «types simples»), contrairement au traitement du polymorphisme qui demande encore un travail substantiel pour satisfaire la vérification de type.