Mediterranean spotted fever and identification of new agents of rickettsioses in Portugal : epidemiological determinants, host and microbial features in portuguese patients

Resumo: a febre botonosa, tamb��m conhecida por febre escaro-nodular (FEN) �� uma doen��a end��mica nos Pa��ses da bacia do Mediterr��neo, ��frica, M��dio Oriente, ��ndia e Paquist��o. O agente etiol��gico respons��vel por esta patologia �� a bact��ria Rickettsia conorii. Contudo, em alguns pa��ses, como Portugal e It��lia, esta patologia �� causada por duas estirpes diferentes: R conorii Malish e R conorii Israeli spotted fever strain. O principal vector e reservat��rio �� o ixod��deo Rhipicephalus sanguineus. Mesmo com uma elevada taxa de subnotifica����o detectada no nosso Pa��s, a taxa incid��ncia da FEN �� de 8.4/105 habitantes (1989-2005), uma das mais altas quando comparada coom a de outros pa��ses da bacia do Mediterr��neo. De todos os distritos portugueses, Bragan��a e Beja s��o aqueles que apresentam as taxas de incid��ncia mais elevadas, 56,8/105 habitantes e 47,4 / 105 habitantes respectivamente. Em Portugal, as altera����es clim��ticas verificadas na ��ltima d��cada, nomeadamente a subida das temperaturas m��dias anuais, parecem ter influenciado o ciclo de vida do vector e a sua din��mica sazonal, permitindo ao R. sanguineus completar mais de um ciclo de vida por ano. Este facto, e a possibilidade deste vector se manter activo noutros meses do ano, nomeadamente nos meses de inverno, tem influenciado consequentemente o padr��o de distribui����o anual dos casos de FEN. A febre escaro-nodular caracteriza-se clinicamente como uma doen��a exantem��tica, com um processo de vasculite generalizado. Apesar de na generalidade ser considerada uma doen��a benigna (quando tratada atempadamente e com terap��utica adequada e espec��fica)e de estarem descritos casos graves em cerca de 5-6% dos doentes, em Portugal essa percentagem aumentou e consequentemente levou a um aumento de casos fatais. Este facto tornou-se mais evidente em 1997, no Hospital Distrital de Beja e no Hospital Garcia de Orta, onde a taxa de letalidade atingiu os 32% e 18% respectivamente.Para al��m dos factores de co-morbilidade encontrados nos doentes mais graves, como diabetes mellitus, ou o atraso na institui����o da terap��utica espec��fica, foi colocada de que a estirpe R. conorii Israel spotted fever strain pudesse ser mais virulenta ou ent��o estivesse associada a diferentes manifesta����es cl��nicas que dificultassem o diagn��stico cl��nico e a institui����o atempada da terap��utica. Houve ainda a necessidade de avaliar alguns par��metros imunol��gicos dos doentes e tentar identificar que factores, nomeadamente que citoquinas, poderiam estar envolvidos na resposta a uma infec����o por R.conorii.Face a estas quest��es foi avaliada e comparada a epidemiologia, manifesta����es cl��nicas e laboratoriais de 140 doentes (71 infectados com R. conorii Malish e 69 infectados com R. conorii Israel spotted fever strain). Concluiu-se que existe uma sobreposi����o de manifesta����es clinicas entre os dois grupos de doentes, mas que a percentagem da escara de inocula����o �� significativamente inferior em doentes infectados com R. conorii Israel spotted fever strain. Dos resultados mais importantes encontrados neste estudo concluiu-se que a estirpe R. conorii Malish e �� demonstrado, pela primeira vez, estatisticamente que o alcoolismo �� um factor de risco para a morte de doentes com FEN. Associadas a factores de um mau progn��sitco da doen��a, est��o as manifesta����es gastrointestinais, que poder��o ser ou n��o reflexo de altera����es do sistema nervoso central, e ainda a altera����o de par��metros laboratoriais como a presen��a de hiperbilirubinemia e aumento dos valores da ureia.A maior parte dos estudos realizados sobre os mecanismos da resposta imunit��ria �� infec����o por R. conorii e as interac����es hospedeiro - agente etiol��gico t��m sido elucidados com base em modelos animais. Poucos estudos t��m sido efectuados em doentes e nenhum estudo pr��vio tinha sido realizado no sentido de avaliar localmente (escara/pele) quais os mediadores ou outras mol��culas envolvidas na resposta imunit��ria ��s rickettsioses. Foi avaliado o n��vel de express��o g��nica de RNA mensageiro (RNAm)de diferentes citoquinas em amostras de pele de doentes com FEN pela t��cnica de PCR em tempo real.Os resultados deste estudo mostraram que, quando comparado com o grupo controlo, os 23 doentes analisados apresentavam n��veis estatisticamente significativos, mais elevados de express��o g��nica de interfer��o (IFN-��, Tumor necrosis factor (TFN-��, interleucina 10 (IL-10, RANTES (regulated by activation, normal T-cell-expressed and secreted chemokine)e indolamina 2-3 desoxigenase (IDO),uma enzima envolvida no controlo e limita����o do crescimento intracelular das rickettsias, atrav��s da degrada����o do triptofano. Seis dos 23 doentes apresentaram ainda niveis de express��o elevados de ��xido n��trico indut��vel (iNOS)que actua como microbicida. Encontrou-se uma correla����o positiva entre a express��o de RNAm de TNF-��, ��, iNOS e IDO e os casos menos graves de FEN sugerindo um tipo de resposta imunit��ria tipo Th1, i.e. com papel protector na resposta �� infec����o.Verificou-se tamb��m que os valores de express��o gen��tica do RNAm de IL-10, estavam inversamente correlacionados com a express��o do RNAm de TNF-�� e IFN-��. Os casos menos graves de FEN parecem assim envolver um balan��o entre a resposta pr��-inflamat��ria e anti-inflamat��ria. J�� os n��veis de express��o g��nica do RNAm de IL-10 estavam inversamente correlacionados com a express��o RNAm de TNF-�� e IFN-��. Os casos menos graves de FEN parecem assim envolver um balan��o entre uma resposta pr��-inflamat��ria e anti-inflamat��ria. J�� os n��veis de express��o RNAm da quimoquina RANTES foram estatisticamente mais elevados em doentes graves.Nesta disserta����o �� ainda descrita uma nova rickettsiose presente em Portugal, causada pela bact��ria R. sibirica mongolitimonae, que foi identificada laboratorialmente por isolamento do agente, e por detec����o do DNA em biopsia de pele. A presen��a deste agente foi ainda corroborada pela detec����o em paralelo do mesmo agente no ixod��deos como R. africae like e em pulgas como R. felis e R.typhi alertam para a possibilidade de exist��ncia de outras rickettsioses que possam estar diagnosticadas em Portugal. Abstract: Mediterranean spotted fever (MSF), a tick-borne disease caused by Rickettsia conorii, is widley distributed in the Old World, being endemic in the southern Europe, Africa, Middle East, India and Pakistan. In Portugal two strains cause disease: R.conorii Malish and R.conorii Israeli spotted fever.Rhipicephalus sanguineus, the brown dog tick, is considered the main vector and reservoir. MSF is characterized by seasonality, and most of cases are encountered in late spring and summer, peaking in July and August. However, CEVDI/INSA laboratory has observed that the incidence of MSF cases has changed during winter season.The increasing annual averages of air temperatures and warmer and drier winters might have influenced the dynamics of the life cycle and activity of R. sanguineus, and indirectley the number MSF cases during the so called MSF off-season.In the period of 1989-2005, the incidence rate of MSF was 8.4/105 inhabitants, one of the highest rates compared with other endemic countries. In the Portugal during the same period, the highest incidence rates were reported in the districts of Bragan��a, with 56.8/105 inhabitants, and Beja, with 47.4/105 inhabitants. Severe cases of MSF are reported in 6% of the patients, but it seems that this pattern of disease in Portugal has been changing.This factor became more evident in 1997, with a reported case fatality rate of 32% and 18% in patients with MSF admited at Beja and Garcia Orta Hospitals, respectively. Although it was found that diabetes mellitus and delay in therapy have been implicated as a risk factor for death, the hypothesis was considered, that the new ISF strain isolated from Portugueses patients in the same year (1997)causes different or atypical clinical conorii Malish strain. The local (skin biopsies) immune response to R. conorii infection was also evaluated.A prospective study was performed to characterized epidemiological, clinical, laboratory features and determined risk factors for a fatal outcome. One hundred forty patients (51% patients were infected with Rickettsia conorii Malish stain and 49% with Israeli spotted fever strain)with diagnosis documented with identification of the causative rickettsial strain were admitted to 13 Portugueses Hospitals during 1994-2006.Comparison of the clinical manifestations of MSF caused by Malish and ISF strains revealed tremendous overlap that would not permit clinical recognition of the strain envolved, but an eschar was observed in a significantly higher percentage of patients with Malish than ISF strain.A fatal outcome was significantly more likely for patients with ISF strain infection meaning that ISF strain was more virulent than Malish strain, and also alcoholism was a host risk factor for a fatal outcome.The pathophysiology of a fatal outcome involved significantly greater incidence of petechial rash, gastrointestinal symptoms, confusion/obtundation, dehydration, tachypnea, hepatomegaly, leukocytosis, coagulopathy, azotemia, hyperbilirubinemia, and elevated hepatic enzymes and creatine kinase. Multivariate analysis revealed that acute renal failure and hyperbilirubinemia were most strong associated with a fatal oucome of infections with both strains.The immune response to R. conorii infection determined with both strains. The immune response to R. conorii infection determined by the expression levels of inflammatory and immune mediators in skin biopsies collected from untreated patients with Mediterranean spotted fever reveal that intralesional expression of mRNA of TNF-��, IFN-��, IL-10, RANTES, and indoleamine-2, 3-dioxygenase (IDO)an enzyme involved in limiting rickettsial growth by tryptophan degradation, were elevated in skin of MSF patients compared to controls. Six patients had elevated levels of inducible nitric oxide synthase (NOS2, a source microbicidal nitric oxide.Positive correlations among TNF-��, IFN-��, NOS2,IDO and mild-to-moderate disease suggested that type 1 polarization plays a protective role. Significantly high levels of intralesional IL-10 were inversely correlated with IFN-�� and TNF-��. The chemokine RANTES was significantly higher in patients with several MSF. It seems that MSF patients with mild-to-moderate disease have a strong and balanced intralesional pro-inflammatory and anti-inflammatory response, while severe disease is associated with higher chemokine expression.Whether these findings are simply a correlate of mild and severe disease or contribute to anti-rickettsial immunity and pathogenesis remains to be determined.In this dissertation is also described a new rickettsiois present in Portugal caused by R.sibirica mongolitimonae strain, identified based on agent isolation and DNA detection by PCR technique in a skin biopsy.The presence of this agent corroborated by its detection also in Rhipicephalus pusillus tick. Also, pathogenic tick and flea-borne rickettsial agents such as R. africae strain detected in Rhipicephalus bursa tick, and R.felis and R.typhi detected in different fleas species raise the alert for the possible existence of other rickettsioses in Portugal that might be underdiagnosed.

Cochineal, a precious source of red: cochineal dyes characterization by high performance liquid chromatography with diode array detection and principal component analysis

Presented at Faculdade de Ci��ncias e Tecnologias, Universidade de Lisboa, to obtain the Master Degree in Conservation and Restoration of Textiles

Experimental design methodologies for the identification of Michaelis-Menten type kinetics

The main objective of this work was to investigate the application of experimental design techniques for the identification of Michaelis-Menten kinetic parameters. More specifically, this study attempts to elucidate the relative advantages/disadvantages of employing complex experimental design techniques in relation to equidistant sampling when applied to different reactor operation modes. All studies were supported by simulation data of a generic enzymatic process that obeys to the Michaelis-Menten kinetic equation. Different aspects were investigated, such as the influence of the reactor operation mode (batch, fed-batch with pulse wise feeding and fed-batch with continuous feeding) and the experimental design optimality criteria on the effectiveness of kinetic parameters identification. The following experimental design optimality criteria were investigated: 1) minimization of the sum of the diagonal of the Fisher information matrix (FIM) inverse (A-criterion), 2) maximization of the determinant of the FIM (D-criterion), 3) maximization of the smallest eigenvalue of the FIM (E-criterion) and 4) minimization of the quotient between the largest and the smallest eigenvalue (modified E-criterion). The comparison and assessment of the different methodologies was made on the basis of the Cram��r-Rao lower bounds (CRLB) error in respect to the parameters vmax and Km of the Michaelis-Menten kinetic equation. In what concerns the reactor operation mode, it was concluded that fed-batch (pulses) is better than batch operation for parameter identification. When the former operation mode is adopted, the vmax CRLB error is lowered by 18.6 % while the Km CRLB error is lowered by 26.4 % when compared to the batch operation mode. Regarding the optimality criteria, the best method was the A-criterion, with an average vmax CRLB of 6.34 % and 5.27 %, for batch and fed-batch (pulses), respectively, while presenting a Km���s CRLB of 25.1 % and 18.1 %, for batch and fed-batch (pulses), respectively. As a general conclusion of the present study, it can be stated that experimental design is justified if the starting parameters CRLB errors are inferior to 19.5 % (vmax) and 45% (Km), for batch processes, and inferior to 42 % and to 50% for fed-batch (pulses) process. Otherwise equidistant sampling is a more rational decision. This conclusion clearly supports that, for fed-batch operation, the use of experimental design is likely to largely improve the identification of Michaelis-Menten kinetic parameters.

Diversity in Teams: Source of Conflict or Source of Creativity? Investigating Hidden Mental Models

The workforce in organizations today is becoming increasingly diverse. Consequently the role of diversity management is heavily discussed with respect to the question how diversity influences the productivity of a group. Empirical studies show that on one hand there is a potential for increasing productivity but on the other hand it might be as well that conflicts arise due to the heterogeneity of the group. Usually according empirical studies are based on interviews, questionnaires and/or observations. These methods imply that answers are highly selective and filtered. In order to make the invisible visible, to have access to mental models of team members the paper will present an empirical study on the self-understanding of groups based on an innovative research method, called ���mind-scripting���.

Characterization and identification of printed objects

A study about the physical appearance of pre-photographic, photomechanical, photographic and digital positive reflective prints was made, relating the obtained images with the history, materials and technology used to create them. The studied samples are from the Image Permanence Institute (IPI) study collection. The digital images were obtained using a digital SLR on a copystand and a compound light microscope, with different lighting angles (0��, 45��and 90��) and magnifications from overall views on the copystand down to a 20x objective lens on the microscope. Most of these images were originally created by IPI for www.digitalsamplebook.org, a web tool for teaching print identification, and will be used on the www.graphicsatlas.org website, along with textual information on identification, technology and history information about these reproduction processes.

In-situ X-ray diffraction studies during growth of Ni-Ti shape memory alloy films and their complementary ex-situ characterization

Shape Memory Alloy (SMA) Ni-Ti films have attracted much interest as functional and smart materials due to their unique properties. However, there are still important issues unresolved like formation of film texture and its control as well as substrate effects. Thus, the main challenge is not only the control of the microstructure, including stoichiometry and precipitates, but also the identification and control of the preferential orientation since it is a crucial factor in determining the shape memory behaviour. The aim of this PhD thesis is to study the optimisation of the deposition conditions of films of Ni-Ti in order to obtain the material fully crystallized at the end of the deposition, and to establish a clear relationship between the substrates and texture development. In order to achieve this objective, a two-magnetron sputter deposition chamber has been used allowing to heat and to apply a bias voltage to the substrate. It can be mounted into the six-circle diffractometer of the Rossendorf Beamline (ROBL) at the European Synchrotron Radiation Facility (ESRF), Grenoble, France, enabling an in-situ characterization by X-ray diffraction(XRD) of the films during their growth and annealing. The in-situ studies enable us to identify the different steps of the structural evolution during deposition with a set of parameters as well as to evaluate the effect of changing parameters on the structural characteristics of the deposited film. Besides the in-situ studies, other complementary ex-situ characterization techniques such as XRD at a laboratory source, Rutherford backscattering spectroscopy(RBS), Auger electron spectroscopy (AES), cross-sectional transmission electron microscopy (X-TEM), scanning electron microscopy (SEM), and electrical resistivity (ER) measurements during temperature cycling have been used for a fine structural characterization. In this study, mainly naturally and thermally oxidized Si(100) substrates, TiN buffer layers with different thicknesses (i.e. the TiN topmost layer crystallographic orientation is thickness dependent) and MgO(100) single crystals were used as substrates. The chosen experimental procedure led to a controlled composition and preferential orientation of the films. The type of substrate plays an important role for the texture of the sputtered Ni-Ti films and according to the ER results, the distinct crystallographic orientations of the Ni-Ti films influence their phase transformation characteristics.

Photographic source for the history of portuguese-speaking Africa, 1870-1914

History in Africa, n.18, p��g.67-82

Radio frequency identification: o futuro da gest��o de stocks na grande distribui����o

Disserta����o apresentada como requisito parcial para obten����o do grau de Mestre em Estat��stica e Gest��o de Informa����o

Avalia����o de software em open source para a gest��o da Biblioteca UNL no Campus de Caparica

O presente trabalho parte da caracteriza����o da Biblioteca UNL para desenvolver um estudo de avalia����o dos principais Sistemas Integrados de Gest��o de Bibliotecas (SIGB) em open source. Os SIGB s��o utilizados na automatiza����o dos processos de trabalho das bibliotecas, atrav��s de diversos m��dulos funcionais e op����es de configura����o, e permitem a interoperabilidade atrav��s de normas e protocolos. O open source define-se como abordagem para o desenvolvimento e distribui����o de software baseada no livre acesso ao c��digo fonte, estando assim em oposi����o ao conceito de software propriet��rio. A Biblioteca UNL �� caracterizada atrav��s da sua organiza����o, dos seus espa��os e recursos, dos seus processos de gest��o documental e da infraestrutura tecnol��gica subjacente. O open source �� abordado a partir da sua contextualiza����o hist��rica, da organiza����o dos seus projectos, das vantagens e inconvenientes para as institui����es e do desenvolvimento e adop����o de SIGB open source.

Identification of mating type genes in the bipolar basidiomycetous yeast rhodosporidium toruloides: first insight into the MAT Locus structure of the sporidiobolales

Eukaryotic Cell, Vol.7, N��6

Neutral C60 effusive source for atomic collisions with fullerene

Review of scientific instruments, Vol.72, N��9

Functional domains of Bacillus subtilis transcription factor AraR and identification of aminoacids important for nucleoprotein complex assembly and effector-binding.

Journal of Bacteriology (Apr 2006) 3024-3036

Mineralogia da frac����o argilosa da Forma����o de Vale ��lvaro (Bragan��a, NE Portugal)

The studied materials were sampled from several conglomerate and carbonate sandstone units, overlapped for 23 meters. This formation represents a debris flow dominated alluvial fan alternating with quiet sedimentary conditions. These deposits of probably Paleogene age were placed upon mafic and ultramafic rocks that are the exclusive source of sediments. Optical and SEM identification, microanalysis and XRD studies (with decomposition procedures) of clay fractions obtained after high-speed centrifugation were performed in order to characterise the clay minerals content. The results of the analytical program allowed the establishment of the following remarks: a) Fe-rich montmorillonite dominance over paligorskite, chlorite, chlorite-smectite mixed-layers, serpentine and talc; b) smectites in the 12.4 - 15 A range, expanding to about 17 A after EG treatment; c) serpentine and talc as secondary minerals in the interior of altered clasts; d) chlorite and clorite smectite mixed-layer compositions in the borders of the clasts and in the cement. The composition of sediments results from coarse clasts eroded from mafic and ultramafic rocks and clayey material. Clasts show evidences of post-depositional weathering (coatings of chlorite and smectite). Clayey material has the contributions of i) inherired chlorite, smectite and chlorite-smectite mixed-layers; ii ) authigenic crystallisation of Fe-montmorillonite (due to availability of Fe in the crystallising solutions following previous weathering events); iii) authigenic paligorskite associated to a carbonate cement.

Freshwater arsenic detoxification through selenium-enriched food supplements. A proteomic approach

Disserta����o apresentada para a obten����o do grau de doutor em Bioqu��mica pela Universidade Nova de Lisboa, Faculdade de Ci��ncias e Tecnologia

Sigla WEBGIS portal: an open source Web-based GIS application for the portuguese bluetongue Entomologiacal surveillance program.

Disserta����o apresentada como requisito parcial para obten����o do grau de Mestre em Ci��ncia e Sistemas de Informa����o Geogr��fica