Detection of Trypanosoma cruzi and Trypanosoma rangeli infection in triatomine vectors by amplification of the histone H2A/SIRE and the sno-RNA-C11 genes

Trypanosoma rangeli is non pathogenic for humans but of important medical and epidemiological interest because it shares vertebrate hosts, insect vectors, reservoirs and geographic areas with T. cruzi, the etiological agent of Chagas disease. Therefore, in this work, we set up two PCR reactions, TcH2AF/R and TrFR2, to distinguish T. cruzi from T. rangeli in mixed infections of vectors based on amplification of the histone H2A/SIRE and the small nucleolar RNA Cl1 genes, respectively. Both PCRs were able to appropriately detect all T. cruzi or T. rangeli experimentally infected-triatomines, as well as the S35/S36 PCR which amplifies the variable region of minicircle kDNA of T. cruzi. In mixed infections, whereas T. cruzi DNA was amplified in 100% of samples with TcH2AF/R and S35/S36 PCRs, T. rangeli was detected in 71% with TrF/R2 and in 6% with S35/S36. In a group of Rhodnius colombiensis collected from Coyaima (Colombia), T. cruzi was identified in 100% with both PCRs and T. rangeli in 14% with TrF/R2 and 10% with S35/S36 PCR. These results show that TcH2AF/R and TrF/R2 PCRs which are capable of recognizing all T. cruzi and T. rangeli strains and lineages could be useful for diagnosis as well as for epidemiological field studies of T. cruzi and T. rangeli vector infections.

Quinta do Pa��o do Monsul - Uma proposta de classificação

Trabalho de Projecto Mestrado em Patrim��nio, ��rea de especializa����o em Patrim��nio Hist��rico

SleepLab V.2.0: plataforma integrada de teste de algoritmos para classificação n��o supervisionada do sono

Disserta����o para obten����o do Grau de Mestre em Engenharia Biom��dica

Classificação Internacional de Funcionalidade (CIF): Conceitos, Preconceitos e Paradigmas. Contributo de um Construto para o Percurso Real em Meio Natural de Vida.

A autora aborda o historial, g��nese e objectivos da Classificação Internacional de Funcionalidade para crian��as e jovens, implementada pela Organiza����o Mundial de Sa��deem 2007. Sublinha o papel de complementaridadade da CIF relativamente a outras classifica����es como a Classificação Internacional de Doen��as (CID), em que a primeira visa caracterizar as capacidades e dificuldades de crian��as e jovens com defici��ncia, ou seja, caracterizar do ponto de vista funcional cada crian��a e jovem, independentemente do diagn��stico etiol��gico m��dico. Trata-se da mudan��a de paradigma bio-m��dico para a do indiv��duo que apresenta determinadas compet��ncias e dificuldades, em que estas ��ltimas s��o definidas em fun����o do ambiente ��� facilitador ou actuando como barreira. Assim, �� dado enfoque ao ambiente para que este seja modificado transformando os factores que actuam como barreira em facilitadores, que anulem ou atenuem as dificuldades. Sendo a defici��ncia e ou doen��a uma experi��ncia universal, a CIF vem colmatar uma importante lacuna na dificuldade de comunica����o e articula����o entre os diversos actores intervenientes no apoio socio educativo e m��dico destas crian��as,criando uma linguagem acess��vel aos t��cnicos envolvidos ��� educadores, terapeutas e outros profissionais ligados �� inf��ncia, por for��a envolvidos, dando uma maior ��nfase �� interac����o crian��a/meio numa perspectiva hol��stica de bem estar bio-psico-social.

Ankylosing spondylitis: genomic and functional characterization of candidate genes and their repercussion in clinical practice

RESUMO: Introdu����o: A espondilite anquilosante (EA) �� uma doen��a inflamat��ria cr��nica caracterizada pela inflama����o das articula����es sacroil��acas e da coluna. A anquilose progressiva motiva uma deteriora����o gradual da fun����o f��sica e da qualidade de vida. O diagn��stico e o tratamento precoces podem contribuir para um melhor progn��stico. Neste contexto, a identifica����o de biomarcadores, assume-se como sendo muito ��til para a pr��tica cl��nica e representa hoje um grande desafio para a comunidade cient��fica. Objetivos: Este estudo teve como objetivos: 1 - caracterizar a EA em Portugal; 2 - investigar poss��veis associa����es entre genes, MHC e n��o-MHC, com a suscetibilidade e as caracter��sticas fenot��picas da EA; 3 - identificar genes candidatos associados a EA atrav��s da tecnologia de microarray. Material e M��todos: Foram recrutados doentes com EA, de acordo com os crit��rios modificados de Nova Iorque, nas consultas de Reumatologia dos diferentes hospitais participantes. Colecionaram-se dados demogr��ficos, cl��nicos e radiol��gicos e colhidas amostras de sangue perif��rico. Selecionaram-se de forma aleat��ria, doentes HLA-B27 positivos, os quais foram tipados em termos de HLA classe I e II por PCR-rSSOP. Os hapl��tipos HLA estendidos foram estimados pelo algoritmo Expectation Maximization com recurso ao software Arlequin v3.11. As variantes al��licas dos genes IL23R, ERAP1 e ANKH foram estudadas atrav��s de ensaios de discrimina����o al��lica TaqMan. A an��lise de associa����o foi realizada utilizando testes da Cochrane-Armitage e de regress��o linear, tal como implementado pelo PLINK, para vari��veis qualitativas e quantitativas, respetivamente. O estudo de express��o g��nica foi realizado por Illumina HT-12 Whole-Genome Expression BeadChips. Os genes candidatos foram validados usando qPCR-based TaqMan Low Density Arrays (TLDAs). Resultados: Foram inclu��dos 369 doentes (62,3% do sexo masculino, com idade m��dia de 45,4 �� 13,2 anos, dura����o m��dia da doen��a de 11,4 �� 10,5 anos). No momento da avalia����o, 49,9% tinham doen��a axial, 2,4% perif��rica, 40,9% mista e 7,1% entesop��tica. A uve��te anterior aguda (33,6%) foi a manifesta����o extra-articular mais comum. Foram positivos para o HLA-B27, 80,3% dos doentes. Os hapl��tipo A*02/B*27/Cw*02/DRB1*01/DQB1*05 parece conferir suscetibilidade para a EA, e o A*02/B*27/Cw*01/DRB1*08/DQB1*04 parece conferir prote����o em termos de atividade, repercuss��o funcional e radiol��gica da doen��a. Tr��s variantes (2 para IL23R e 1 para ERAP1) mostraram significativa associa����o com a doen��a, confirmando a associa����o destes genes com a EA na popula����o Portuguesa. O mesmo n��o se verificou com as variantes estudadas do ANKH. N��o se verificou associa����o entre as variantes g��nicas n��o-MHC e as manifesta����es cl��nicas da EA. Foi identificado um perfil de express��o g��nica para a EA, tendo sido validados catorze genes - alguns t��m um papel bem documentado em termos de inflama����o, outros no metabolismo da cartilagem e do osso. Conclus��es: Foi estabelecido um perfil demogr��fico e cl��nico dos doentes com EA em Portugal. A identifica����o de variantes g��nicas e de um perfil de express��o contribuem para uma melhor compreens��o da sua fisiopatologia e podem ser ��teis para estabelecer modelos com relev��ncia em termos de diagn��stico, progn��stico e orienta����o terap��utica dos doentes. -----------ABSTRACT: Background: Ankylosing Spondylitis (AS) is a chronic inflammatory disorder characterized by inflammation in the spine and sacroiliac joints leading to progressive joint ankylosis and in progressive deterioration of physical function and quality of life. An early diagnosis and early therapy may contribute to a better prognosis. The identification of biomarkers would be helpful and represents a great challenge for the scientific community. Objectives: The present study had the following aims: 1- to characterize the pattern of AS in Portuguese patients; 2- to investigate MHC and non-MHC gene associations with susceptibility and phenotypic features of AS and; 3- to identify candidate genes associated with AS by means of whole-genome microarray. Material and Methods: AS was defined in accordance to the modified New York criteria and AS cases were recruited from hospital outcares patient clinics. Demographic and clinical data were recorded and blood samples collected. A random group of HLA-B27 positive patients and controls were selected and typed for HLA class I and II by PCR-rSSOP. The extended HLA haplotypes were estimated by Expectation Maximization Algorithm using Arlequin v3.11 software. Genotyping of IL23R, ERAP1 and ANKH allelic variants was carried out with TaqMan allelic discrimination assays. Association analysis was performed using the Cochrane-Armitage and linear regression tests as implemented in PLINK, for dichotomous and quantitative variables, respectively. Gene expression profile was carried out using Illumina HT-12 Whole-Genome Expression BeadChips and candidate genes were validated using qPCR-based TaqMan Low Density Arrays (TLDAs). Results: A total of 369 patients (62.3% male; mean age 45.4��13.2 years; mean disease duration 11.4��10.5 years), were included. Regarding clinical disease pattern, at the time of assessment, 49.9% had axial disease, 2.4% peripheral disease, 40.9% mixed disease and 7.1% isolated enthesopathic disease. Acute anterior uveitis (33.6%) was the most common extra-articular manifestation. 80.3% of AS patients were HLA-B27 positive. The haplotype A*02/B*27/Cw*02/DRB1*01/DQB1*05 seems to confer susceptibility to AS, whereas A*02/B*27/Cw*01/DRB1*08/DQB1*04 seems to provide protection in terms of disease activity, functional and radiological repercussion. Three markers (two for IL23R and one for ERAP1) showed significant single-locus disease associations. Association of these genes with AS in the Portuguese population was confirmed, whereas ANKH markers studied did not show an association with AS. No association was seen between non-MHC genes and clinical manifestations of AS. A gene expression signature for AS was established; among the fourteen validated genes, a number of them have a well-documented inflammatory role or in modulation of cartilage and bone metabolism. Conclusions: A demographic and clinical profile of patients with AS in Portugal was established. Identification of genetic variants of target genes as well as gene expression signatures could provide a better understanding of AS pathophysiology and could be useful to establish models with relevance in terms of susceptibility, prognosis, and potential therapeutic guidance.

Cartografia de zonamento geot��cnico em frentes de escava����o de maci��os rochosos heterog��neos (t��nel do Mar��o): aplica����o da classificação geomec��nica GSI|2013

Este estudo pretende discutir a validade da aplica����o da mais recente vers��o do ���GSI ��� Geological Strength Index��� (Hoek et al., 2013) em maci��os rochosos heterog��neos por aplica����o num projecto de obra subterr��nea em execu����o. O estudo envolveu a caracteriza����o geol��gicageot��cnica nas duas frentes de escava����o da frente ���Poente��� da empreitada de constru����o do T��nel do Mar��o (N de Portugal) tendo sido cartografados 305 metros no conjunto das duas frentes em 74 avan��os consecutivos. Para esta caracteriza����o foram coligidos e uniformizados dados geol��gicos, geot��cnicos e geomec��nicos relativos aos levantamentos realizados a cada avan��o. A t��cnica da amostragem linear foi aplicada para a cartografia das descontinuidades que permitiu estabelecer o grau de compartimenta����o do maci��o rochoso. Al��m disso, procedeu���se a um tratamento estat��stico das descontinuidades, bem como dos par��metros geol��gico���geot��cnicos e geomec��nicos associados. Os zonamentos geot��cnicos e geomec��nicos das sec����es em an��lise foram realizados sempre em estreita liga����o com o conhecimento das caracter��sticas do maci��o in situ. Procurou���se estabelecer termos de compara����o entre a vers��o do GSI|2013 e as vers��es anteriores (GSI|98, GSI|2001) por aplica����o para os avan��os cartografados. Pretende���se que este trabalho contribua para um maior conhecimento da mais recente vers��o do ���Geological Strength Index��� (2013) para a sua aplicabilidade em projectos de geoengenharia.

New mechanisms that regulate the expression of genes implicated in the process of ketogenesis

Disserta����o para obten����o do Grau de Mestre em Biotecnologia

S��ndromes Auto - Inflamat��rias

As s��ndromes auto-inflamat��rias s��o constitu��das por um grupo heterog��neo de patologias isoladas e caracterizadas ao longo dos ��ltimos 15 anos, gra��as aos avan��os marcados no conhecimento do genoma humano e ao desenvolvimento de t��cnicas laboratoriais que permitiram identificar, de forma segura e reprodut��vel, os genes respons��veis pelas v��rias doen��as inclu��das sob esta designa����o comum. Em todas estas doen��as e s��ndromes existem epis��dios recorrentes de febre e inflama����o, localizada ou sist��mica, sem o envolvimento de agentes infecciosos ou quaisquer tipo de mecanismos auto-imunes. Neste trabalho, al��m da elabora����o de uma classificação poss��vel das s��ndromes auto-inflamat��rias, dedicaremos especial aten����o ��s s��ndromes peri��dicas associadas �� criopirina (CAPS), que podem constituir importante causa de diagn��stico diferencial com a forma sist��mica de artrite idiop��tica juvenil (AIJ), com a qual partilham numerosas caracter��sticas cl��nicas comuns.

DETECTION OF VIRULENCE GENES IN ENVIRONMENTAL STRAINS OF Vibrio cholerae FROM ESTUARIES IN NORTHEASTERN BRAZIL

The objectives of this study were to detect the presence of Vibrio cholerae in tropical estuaries (Northeastern Brazil) and to search for virulence factors in the environmental isolates. Water and sediment samples were inoculated onto a vibrio-selective medium (TCBS), and colonies with morphological resemblance to V. cholerae were isolated. The cultures were identified phenotypically using a dichotomous key based on biochemical characteristics. The total DNA extracted was amplified by PCR to detect ompW and by multiplex PCR to detect the virulence genes ctx, tcp, zot and rfbO1. The results of the phenotypic and genotypic identification were compared. Nine strains of V. cholerae were identified phenotypically, five of which were confirmed by detection of the species-specific gene ompW. The dichotomous key was efficient at differentiating environmental strains of V. cholerae. Strains of V. cholerae were found in all four estuaries, but none possessed virulence genes.

Classificação de sons urbanos usando motifs e MFCC

A classificação autom��tica de sons urbanos �� importante para o monitoramento ambiental. Este trabalho apresenta uma nova metodologia para classificar sons urbanos, que se baseia na descoberta de padr��es frequentes (motifs) nos sinais sonoros e utiliza-los como atributos para a classificação. Para extrair os motifs �� utilizado um m��todo de descoberta multi-resolu����o baseada em SAX. Para a classificação s��o usadas ��rvores de decis��o e SVMs. Esta nova metodologia �� comparada com outra bastante utilizada baseada em MFCC. Para a realiza����o de experi��ncias foi utilizado o dataset UrbanSound dispon��vel publicamente. Realizadas as experi��ncias, foi poss��vel concluir que os atributos motif s��o melhores que os MFCC a discriminar sons com timbres semelhantes e que os melhores resultados s��o conseguidos com ambos os tipos de atributos combinados. Neste trabalho foi tamb��m desenvolvida uma aplica����o m��vel para Android que permite utilizar os m��todos de classificação desenvolvidos num contexto de vida real e expandir o dataset.

Classificação dos cr��ditos sobre a insolv��ncia ��� Os cr��ditos subordinados

Disserta����o de Mestrado em Solicitaria

Acelera����o da classificação de documentos com recurso a GPU

Disserta����o para obten����o do Grau de Mestre em Engenharia Inform��tica

Regulation of autoimmune neuroinflammation by stress-responsive genes

Dissertation presented to obtain the Ph.D degree in Biology

AMINOGLYCOSIDE RESISTANCE GENES IN Pseudomonas aeruginosa ISOLATES FROM CUMANA, VENEZUELA

The enzymatic modification of aminoglycosides by aminoglycoside-acetyltransferases (AAC), aminoglycoside-adenyltransferases (AAD), and aminoglycoside-phosphotransferases (APH), is the most common resistance mechanism in P. aeruginosa and these enzymes can be coded on mobile genetic elements that contribute to their dispersion. One hundred and thirty seven P. aeruginosa isolates from the University Hospital, Cumana, Venezuela (HUAPA) were evaluated. Antimicrobial susceptibility was determined by the disk diffusion method and theaac, aadB and aph genes were detected by PCR. Most of the P. aeruginosa isolates (33/137) were identified from the Intensive Care Unit (ICU), mainly from discharges (96/137). The frequency of resistant P. aeruginosaisolates was found to be higher for the aminoglycosides tobramycin and amikacin (30.7 and 29.9%, respectively). Phenotype VI, resistant to these antibiotics, was the most frequent (14/49), followed by phenotype I, resistant to all the aminoglycosides tested (12/49). The aac(6��)-Ib,aphA1 and aadB genes were the most frequently detected, and the simultaneous presence of several resistance genes in the same isolate was demonstrated. Aminoglycoside resistance in isolates ofP. aeruginosa at the HUAPA is partly due to the presence of the aac(6��)-Ib, aphA1 andaadB genes, but the high rates of antimicrobial resistance suggest the existence of several mechanisms acting together. This is the first report of aminoglycoside resistance genes in Venezuela and one of the few in Latin America.

PERFORMANCE OF CONVENTIONAL PCRs BASED ON PRIMERS DIRECTED TO NUCLEAR AND MITOCHONDRIAL GENES FOR THE DETECTION AND IDENTIFICATION OF Leishmania spp.

In visceral leishmaniasis, the detection of the agent is of paramount importance to identify reservoirs of infection. Here, we evaluated the diagnostic attributes of PCRs based on primers directed to cytochrome-B (cytB), cytochrome-oxidase-subunit II (coxII), cytochrome-C (cytC), and the minicircle-kDNA. Although PCRs directed to cytB, coxII, cytC were able to detect different species of Leishmania, and the nucleotide sequence of their amplicons allowed the unequivocal differentiation of species, the analytical and diagnostic sensitivity of these PCRs were much lower than the analytical and diagnostic sensitivity of the kDNA-PCR. Among the 73 seropositive animals, the asymptomatic dogs had spleen and bone marrow samples collected and tested; only two animals were positive by PCRs based on cytB, coxII, and cytC, whereas 18 were positive by the kDNA-PCR. Considering the kDNA-PCR results, six dogs had positive spleen and bone marrow samples, eight dogs had positive bone marrow results but negative results in spleen samples and, in four dogs, the reverse situation occurred. We concluded that PCRs based on cytB, coxII, and cytC can be useful tools to identify Leishmania species when used in combination with automated sequencing. The discordance between the results of the kDNA-PCR in bone marrow and spleen samples may indicate that conventional PCR lacks sensitivity for the detection of infected dogs. Thus, primers based on the kDNA should be preferred for the screening of infected dogs.