Naturally occurring mutations in regulatory proteins among streptococcus pyogenes isolates from distinct human infections

Streptococcus pyogenes (Group A Streptococcus, GAS) is among the most prevalent bacterial pathogens of humans and it is responsible for a wide range of infections, from pharyngitis and impetigo to life-threatening conditions such as necrotizing fasciitis and streptococcal toxic shock syndrome. However, until today, despite many years of research, there is still no consensus regarding which are the genotypic or phenotypic characteristics that confer to an isolate a certain tissue preference or a more invasive potential. The nasopharyngeal mucosa and skin are considered the primary sources of isolates responsible for invasive infections. This suggests a further ability of these isolates to invade and survive in deeper tissues. In the last years, spontaneous mutations occurring in the covRS two-component regulatory system have been considered a possible explanation for the transition from localized to systemic infection. It is estimated that this system controls directly or indirectly the expression of 10-15% of the GAS genome. As a consequence of these mutations, among the distinct patterns of expression of several virulence factors, the downregulation of the extracellular cysteine protease SpeB, has been considered crucial for the switch to a hypervirulent phenotype. The downregulation of SpeB has also been described as a consequence of mutations in a stand-alone transcriptional regulator named RopB. However, contrasting results were reported whether covRS mutations were more prone to occur in certain lineages, namely those more frequently associated with invasive infections or if they occur in a similar proportion among invasive and non-invasive isolates. To address these questions, in the present thesis, we determined the sequence of the covRS and ropB of 191 isolates from invasive infection and pharyngitis and evaluated the production of SpeB, as well of NAD glycohydrolase (NADase) and streptolysin S (SLS), which are two virulence factors supposed to be under the influence of CovRS. Moreover, skin and soft tissue infections (SSTI) are frequently considered the focal points for the development of invasive disease. However, most of the knowledge about GAS skin isolates is from studies that intended to find differences between isolates recovered from invasive and non-invasive infections generically. The majority of these studies use isolates recovered from SSTI but mostly from pharyngitis and compared those together against the isolates recovered from invasive infections, resulting in few data regarding isolates recovered from SSTI. Therefore, we characterized by multiple typing methods a total of 320 isolates from SSTI recovered in Portugal and performed the comparison with invasive isolates recovered during the same period which were previously characterized. All SSTI isolates were also tested for SpeB activity and for those without detectable SpeB activity we determined the sequence of covRS and ropB genes. Overall, we found that isolates with null covS alleles, which are predicted to eliminate the protein function have a significant association with invasive infections comparative with isolates from pharyngitis and SSTI. Additionally, none of these isolates, as expected, had SpeB activity, and, with few exceptions, they showed an increased activity of both NADase and SLS that could explain their potentially higher invasiveness. Even so, this mechanism was found to be uncommon, corresponding to only 10% of invasive isolates, which could be due to an overall fitness cost of these mutations. Moreover, null covS alleles were not more prevalent among isolates from clones frequently associated with invasive infection such as emm1 and emm64 and instead they were distributed throughout diverse genetic backgrounds. The few exceptions regarded the levels of NADase and SLS points to the complexity of the regulatory networks among distinct GAS lineages. Additionally, no null covR alleles were detected in our isolates and ropB null alleles were found in a low fraction of GAS isolates and were not associated with any infection type. Regarding SpeB activity, it was detected in a similar proportion in isolates recovered from the different sources, and therefore its absence was not associated to any type of infection, suggesting that its abrogation cannot by itself explain the higher ability of certain clones to cause invasive disease. Among SSTI isolates, we found that emm89 type isolates were the most prevalent and were significantly associated with these infections when compared with invasive isolates. In contrast, emm1, emm3, and emm64 isolates were associated with invasive infections. Within emm89 isolates, SSTI were only associated to those that lack the hasABC locus, corresponding to a recently emerged acapsular clade (clade 3) that also carries a variant of the ngs-ifs-slo locus. These results suggest that for some unknown reason these isolates may have an increased potential to cause SSTI. As a consequence of known differences in the emm-type between isolates causing these two types of infections, we also found significant associations between the ability to bind to different host proteins. This ability was presumed by inferring the emm-cluster through the emm-type results. The emm-cluster is a recent classification based on the entire sequence of the emm gene (M protein) where each cluster shares binding motifs to host proteins and other structural properties. Therefore, the ability to bind fibrinogen and albumin were significantly associated with invasive isolates, whereas the ability to bind to C4BP and IgG were associated with SSTI isolates. Differences in the presence of superantigen (SAg) genes, SAg profiles and in the distribution of sequence types (ST) determined by Multilocus Sequence Typing (MLST) were also noted. The possible impact of these differences in the ability of the isolates to cause these distinct infections remains to be clarified. Moreover, within each emm type the same MLST defined lineages and SAg profiles could be found in both types of infection, questioning the possibility that these characteristics dictate the tissue tropism of each isolate. In summary, the results described in this thesis indicate that isolates responsible for SSTI are genetically distinct from those recovered from normally sterile sites and while some GAS clones have more capacity to invade deeper tissues, others are more prone to cause SSTI. Moreover, the significant presence of null covS mutations among invasive isolates and the fact that no association was observed regarding the absence of SpeB activity in isolates from different types of infections, suggests that the role of spontaneous mutations impairing the CovRS activity is probably related with the regulation of others virulence factors under its control in addition to SpeB

O rendimento social de inserção: instrumento facilitador da inserção?

Dissertação de Mestrado em Política SocialO Rendimento Social de Inserção é uma medida de política social activa, de combate à pobreza. Este estudo pretende contribuir para uma reflexão sobre a aplicação e resultados da medida. O facto de trabalhar no Instituto de Segurança Social, há 13 anos, permitiu-nos um contacto próximo com indivíduos e famílias que ali acorrem a solicitar RSI. Essa experiência de contacto directo com esta população, tornou-nos particularmente sensíveis e interessadas em compreender melhor as dificuldades e carências com que lidam no seu dia-a-dia e foi esse o ponto de para este estudo, cujo objectivo principal foi o de tentar saber na prática se e em que medida os indivíduos e os agregados conseguem mudar a sua vida e depois autonomizar-se deixando de necessitar de recorrer à prestação em que se constitui RSI: Aqui pretendemos também compreender o enquadramento desta medida de política social activa, de combate à pobreza, contextualizando-a a nível europeu. Procurámos ainda analisar as características da população da amostra recolhida, afim a conhecer mais profundamente a sua inserção social, tendo como critérios a autonomia económica e valorização pessoal. Por fim, identificarmos situações em que o RSI foi factor facilitador da inserção social e situações em que apesar de estarmos perante uma população caracteristicamente semelhante, esta medida foi francamente insuficiente. O RSI como medida activa de combate à pobreza, embora com impacto positivo em grande parte da população abrangida, necessita, pela diversidade de entraves que se levantam, de uma intervenção (no sentido de acompanhamento), mais próxima, abrangente e continuadaThe Social Insertion Income (SII) is an active social policy measure to combat poverty. This study aims to contribute to a reflection on the measure and on implementation and measurement results. Working at the Institute of Social Security during 13 years, enabled us to have a close and near contact experience with the persons and families who ask for SII. That experience of direct contact with this population, made us particularly sensitive and interested in better understanding the difficulties and needs they deal with in daily experience, which was the beginning point for this study, where the main goal was trying no know if and how these people manage to become autonomous, not needing to this income. We investigated the evolution of the measures off Minimum Income, trying to understand the framing of this active social policy measure to combat poverty, in its European context. On a second stage of this work, we tried to analyze the characteristics of the population in the sample, in order to better apprehend their social insertion, being the criteria economic autonomy and personal enhancement. Among the worked cases, we were able to identify situations in which the SII was a facilitating factor of social inclusion as well as situations where, although we are before a characteristically similar population, this measure was clearly insufficient. The SII, as an active measure to combat poverty, produces a positive impact on much of the population covered, nevertheless, it needs to be a much wider and continuous type of intervention, due to the diversity of barriers that may arise

Interaction with smart assistants using alternative and augmentative communication

Smart assistants utilize speech recognition, sensing, artificial intelligence, and networking technologies to enable improved human-machine interaction. However, there remain use cases where smart assistants are not easily usable by humans. For example, voice-activated assistants are not accessible to those that are hearing or speech impaired. Touchscreen based assistants are not usable by those who lack fine motor skills and/or reading ability. This disclosure adds to the modalities by which humans can control and communicate with smart assistants by enabling use of physical objects, facial expressions, gross motor skills, body movements, etc. to provide commands. Collectively, these techniques of control and communication are referred to as alternative and augmentative communication (AAC)

Validation of UVEDAI: An Index for Evaluating the Level of Inflammatory Activity in Uveitis

Introduction Uveitis is the inflammation of the middle layer of the eye, the uvea, and is a major cause of blindness. None of the instruments used in clinical practice are, in themselves, sufficient to evaluate the course of uveitis. Therefore, it is necessary to develop instruments enabling standardized measurement of inflammatory activity. We developed a composite disease activity index for patients with uveitis known as UVEDAI, which considers the overall activity of the eye. The objective of this study was to validate the composite index of ocular inflammation, UVEDAI. Methods A multicenter cross-sectional study involving eight Spanish tertiary hospitals. Sixty-two patients aged ≥ 18 years with acute uveitis were recruited. Participants gave informed consent before participating in the study. A full ophthalmological examination was performed by two ophthalmologists to determine inflammatory activity: one used the UVEDAI score and the other used clinical judgment. The ophthalmologists did not share their findings with each other to avoid introducing bias into the analysis. Construct validity was established by means of factor analysis. The criterion validity of the index was determined using an ordinal multivariate regression model, in which the dependent variable was the degree of uveal inflammation (mild, moderate, or high/severe). Cut-off points were determined for the UVEDAI and for the receiver operating characteristic (ROC) curves. Results Sixty-two patients were included. Total variance with the three components accounted for 80.32% of the construct validity. Each of the three components identified one type of eye involvement. The discriminatory capacity of UVEDAI was 0.867 (95% CI 0.778; 0.955 p < 0.001) for mild versus moderate–high and 0.946 (95% CI 0.879; 1.000 p < 0.001) for high versus mild–moderate. Conclusions The variables included in UVEDAI enable ocular inflammatory activity to be described with a high degree of accuracy. The index may be used to evaluate and classify this activity with considerable discriminatory power.We would like to acknowledge the support of Abbvie: this study was conducted with an unrestricted grant from Abbvie. The Spanish Society of Rheumatology is the sponser and funder of this study and the journal's Rapid Service Fee, and has participated in the study design; in the analysis, and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication. The corresponding author had full access to all study data and had final responsibility for the decision to submit the manuscript for publication

Development of an activity disease score in patients with uveitis (UVEDAI)

To develop a disease activity index for patients with uveitis (UVEDAI) encompassing the relevant domains of disease activity considered important among experts in this field. The steps for designing UVEDAI were: (a) Defining the construct and establishing the domains through a formal judgment of experts, (b) A two-round Delphi study with a panel of 15 experts to determine the relevant items, (c) Selection of items: A logistic regression model was developed that set ocular inflammatory activity as the dependent variable. The construct "uveitis inflammatory activity" was defined as any intraocular inflammation that included external structures (cornea) in addition to uvea. Seven domains and 15 items were identified: best-corrected visual acuity, inflammation of the anterior chamber (anterior chamber cells, hypopyon, the presence of fibrin, active posterior keratic precipitates and iris nodules), intraocular pressure, inflammation of the vitreous cavity (vitreous haze, snowballs and snowbanks), central macular edema, inflammation of the posterior pole (the presence and number of choroidal/retinal lesions, vascular inflammation and papillitis), and global assessment from both (patient and physician). From all the variables studied in the multivariate model, anterior chamber cell grade, vitreous haze, central macular edema, inflammatory vessel sheathing, papillitis, choroidal/retinal lesions and patient evaluation were included in UVEDAI. UVEDAI is an index designed to assess the global ocular inflammatory activity in patients with uveitis. It might prove worthwhile to motorize the activity of this extraarticular manifestation of some rheumatic diseases

Streptococcus pyogenes Causing Skin and Soft Tissue Infections Are Enriched in the Recently Emerged emm89 Clade 3 and Are Not Associated With Abrogation of CovRS

Although skin and soft tissue infections (SSTI) are the most common focal infections associated with invasive disease caused by Streptococcus pyogenes (Lancefield Group A streptococci - GAS), there is scarce information on the characteristics of isolates recovered from SSTI in temperate-climate regions. In this study, 320 GAS isolated from SSTI in Portugal were characterized by multiple typing methods and tested for antimicrobial susceptibility and SpeB activity. The covRS and ropB genes of isolates with no detectable SpeB activity were sequenced. The antimicrobial susceptibility profile was similar to that of previously characterized isolates from invasive infections (iGAS), presenting a decreasing trend in macrolide resistance. However, the clonal composition of SSTI between 2005 and 2009 was significantly different from that of contemporary iGAS. Overall, iGAS were associated with emm1 and emm3, while SSTI were associated with emm89, the dominant emm type among SSTI (19%). Within emm89, SSTI were only significantly associated with isolates lacking the hasABC locus, suggesting that the recently emerged emm89 clade 3 may have an increased potential to cause SSTI. Reflecting these associations between emm type and disease presentation, there were also differences in the distribution of emm clusters, sequence types, and superantigen gene profiles between SSTI and iGAS. According to the predicted ability of each emm cluster to interact with host proteins, iGAS were associated with the ability to bind fibrinogen and albumin, whereas SSTI isolates were associated with the ability to bind C4BP, IgA, and IgG. SpeB activity was absent in 79 isolates (25%), in line with the proportion previously observed among iGAS. Null covS and ropB alleles (predicted to eliminate protein function) were detected in 10 (3%) and 12 (4%) isolates, corresponding to an underrepresentation of mutations impairing CovRS function in SSTI relative to iGAS. Overall, these results indicate that the isolates responsible for SSTI are genetically distinct from those recovered from normally sterile sites, supporting a role for mutations impairing CovRS activity specifically in invasive infection and suggesting that this role relies on a differential regulation of other virulence factors besides SpeB

100 años investigando el mar. El IEO en su centenario (1914-2014).

Se trata de un libro que pretende divulgar a la sociedad las principales investigaciones multidisciplinares llevadas a cabo por el Instituto Español de Oceanografía durante su primer siglo de vida, y dar a conocer la historia del organismo, de su Sede Central y de los nueve centros oceanográficos repartidos por los litorales mediterráneo y atlántico, en la península y archipiélagos.Kongsberg 20

Vehicle environmental ranking methodologies and application to Portugal

Dissertação de Mestrado Integrado em Engenharia do Ambiente apresentada à Faculdade de Ciências e Tecnologia da Universidade de Coimbr

Streptococcus pyogenes como agente de infecção da pele e tecidos moles

Tese de mestrado. Biologia (Microbiologia Aplicada). Universidade de Lisboa, Faculdade de Ciências, 2011Em Portugal, os estudos epidemiológicos em Streptococcus pyogenes englobam sobretudo estirpes responsáveis por infecções do aparelho respiratório superior e por infecções invasivas. No entanto, não há registo de estudos focados em estirpes de S. pyogenes recolhidas de infecções de pele e tecidos moles. Este estudo baseou-se na caracterização de 320 estirpes recolhidas em vários hospitais do país, durante o período de 2003 a 2009, a partir de amostras de pus de lesões cutâneas, como feridas e abcessos. A caracterização das estirpes foi efectuada por diferentes métodos de tipagem, nomeadamente serotipagem T, tipagem emm, determinação dos perfis de toxinas e determinação dos perfis de macro-restrição obtidos por electroforese em campo pulsado (PFGE). Em todos os métodos utilizados verificou-se uma elevada diversidade da população (índice de diversidade de Simpson ≥0.86). As estirpes do tipo emm89 (n=62) foram as mais frequentes, seguidas por estirpes do tipo emm1 (n=55). Quando consideradas em conjunto, estas estirpes correspondem a 36,6% da população total e constituem os dois principais grupos definidos por comparação dos perfis de PFGE. Assim como estes, verificou-se que a maioria dos grupos definidos por PFGE são principalmente constituídos por estirpes do mesmo tipo emm. Relativamente aos perfis de toxinas, observou-se que estirpes com o mesmo tipo emm, normalmente, apresentam um perfil igual ou semelhante. Também se determinou a resistência a alguns antimicrobianos de interesse clínico ou epidemiológico e os respectivos génotipos. No total, observou-se uma resistência aos macrólidos de 10,3%, com uma dominância de estirpes de fenótipo cMLSB em relação ao fénótipo M. A resistência à tetraciclina foi observada em 47 estirpes, das quais 12 eram também resistentes aos macrólidos. Tendo em conta os diferentes tipos emm detectados e o número de estirpes correspondentes, a implementação da vacina experimental 26-valente em todas as faixas etárias poderia prevenir 67,8% das infecções de pele e tecidos moles em Portugal.In Portugal, epidemiological studies of Streptococcus pyogenes concentrate mostly on isolates recovered from pharyngitis and invasive infections. However, there is no record of studies focused on S. pyogenes isolates recovered from skin and soft tissue infections. This work aimed to characterize 320 isolates recovered in several hospitals across the country, from skin lesions, like wounds and abscesses. The typing methods used were T typing, emm typing, exotoxin profiling and pulsed field gel electrophoresis (PFGE) profiling. In all of these, a very diverse population was detected (Simpson’s index of diversity ≥ 0.86). The most prevalent emm type was emm89 (n=62) followed by emm1 (n=55). Together, isolates of these emm types accounted 36.6% of the population and composed the two major clusters defined by comparison of PFGE profiles. Like these clusters, the majority of clusters are composed mostly of isolates with the same emm type. Toxin profiling revealed that isolates with the same emm type, usually share the same or a similar toxin profile. We also determined the resistance to a panel of antibiotics of clinical and epidemiological interest, as well as the associated resistance genotypes. Erythromycin resistance was observed in 33 isolates (10%), of which 11 presented the M phenotype, while 22 were constitutively resistant to clindamycin (cMLSB phenotype). Tetracycline resistance was detected in 47 isolates (14.7%), of which 12 were also resistance to erythromycin. According to the different emm types detected, the implementation in all age groups of the experimental 26-valent M protein-based vaccine could prevent 67.8% of skin and soft tissue infections in Portugal

Can calcium aluminates activate ternesite hydration?

Aluminum hydroxide (AH3) has recently been shown to be able to activate hydration in ternesite, a phase found in some calcium sulfoaluminate (CSA) cements. This study explored the capacity of a number of calcium aluminates (C3A, C12A7, CA and C4A3) to activate ternesite hydraulic reactivity. After laboratory synthesis, the aluminates were blended with ternesite at a ratio of 1:2 and their hydration was monitored with isothermal conduction calorimetry for 7 days at 25 °C. The resulting pastes were analysed with XRD, FTIR and DTA. The presence of ternesite in the pastes altered the aluminate heat flow curves, shortening the induction period and bringing the reaction peak forward, an indication of hastened hydration. Ternesite also altered the reaction products, which included calcium monosulfoaluminate hydrate and strätlingite.This paper has been funded from BIA2016-76466-R project as well as the Regional Government of Madrid Community and European Social Fund (Geomaterials Programme2 S2013/MIT-2914). Authors thank to Ministerio de Economía y Competitividad the young researcher contract given to Myriam Montes. Financial support for Dr. Paula M. Carmona-Quiroga's participation was provided by the Spanish Ministry of the Economy and Competitiveness under project BIA2015-73237-JIN.Peer reviewe