Expression of the human carboxylesterase 2 enzyme (CES2) in mammalian cells

Dissertation to obtain a Master Degree in Biotechnology

A prospective study of cat-scratch disease in Lima-Peru

Cat-Scratch Disease (CSD) is a benign lymphadenitis that may progress to severe or recurrent forms, and it is occasionally associated with morbidity. Between January of 1998 and March of 1999, forty-three suspected CSD patients were assessed in the Hospital Cayetano Heredia and the Instituto de Salud del Niño, in Lima, Peru. Twelve patients had a confirmed diagnosis, 8 of whom were women, and the mean age was 10 years old. The majority (53%) of the cases were encountered in the summer. All patients reported having had contact with cats. Fever, malaise, lymphadenopathy and skin lesions were the most frequent clinical features. Twelve patients had indirect immunofluorescence antibody test titers of between 1/50 and 1/800 for Bartonella henselae and Bartonella clarridgeiae. Two lymph node biopsies were histologically compatible with CSD. No positive blood cultures could be obtained. This is the first Peruvian prospective study able to identify B. henselae and B. clarridgeiae in pediatric patients.

Erythema induratum of Bazin and renal tuberculosis: report of an association

Erythema induratum of Bazin is a disease that usually affects women, in whom erythematous subcutaneous nodules and plaques appear on the posterior part of the lower extremities, some of which ulcerate. In many countries, tuberculosis is still the main etiologic factor. We report a case of a 40-year-old woman who presented a course of protracted and recurrent episodes over five years of cutaneous lesions on her legs. These tend to involute, but new crops appear at irregular intervals. It was painful, erythematous-violaceous nodules, some of which drained a reddish secretion. The histopathologic features of the lesions demonstrated inflammatory infiltration, with predominance of neutrophils in dermis and hypodermis, necrotizing vasculitis in the arterioles and septal fibrosis. There was no granuloma. The Ziehl-Neelsen stain did not revealed acid-fast bacilli, and the culture of biopsy specimen was negative. The tuberculin skin test was strongly positive (17 mm). The chest X-ray was normal. Few months later she presented adynamia and urinary complaints, such as polacyuria and dysuria. It has been done an urynalysis, which demonstrated acid pH urine, sterile pyuria and microscopic hematuria. It was then raised the diagnostic hypothesis of renal tuberculosis. The urine culture for M. tuberculosis was positive in two out of ten samples. The treatment was instituted with rifampin, isoniazid and pyrazinamide, with complete regression. This case illustrates a clear association between erythema induratum and renal tuberculosis, demonstrated by the remission of the cutaneous lesions after the treatment of the renal tuberculosis.

Aborto recorrente das causas na mulher às consequências para o casal

RESUMO: O aborto recorrente (AR) é um evento extremamente traumático com grande impacto na vida dos casais. Apesar de avanços significativos verificados na investigação médica, cerca de 50% dos casos continua sem uma causa identificada. Alguns aspectos como a caracterização inadequada das doentes e das perdas gestacionais, assim como diferentes metodologias utilizadas no seu estudo, têm influenciado a prevalência de alguns dos factores causais e dificultado a compreensão do AR. Da mesma forma, pouco se sabe sobre as diferenças de género na vivência psicológica do aborto recorrente e das suas eventuais repercussões para o relacionamento do casal, centrando-se os poucos estudos existentes preferencialmente na mulher. Por esta razão, o objectivo desta tese foi a caracterização dos factores médicos associados ao AR e das consequências psicológicas desta entidade, contribuindo para promover estratégias clínicas baseadas na evidência específica. Na primeira parte desta tese (capítulos 1 e 2), após uma breve introdução geral e através de uma revisão da literatura, efectua-se uma reflexão sobre o tema, abordando a epidemiologia do aborto recorrente, os factores médicos e os aspectos psicológicos associados. Nos capítulos 3 e 4 descrevemos três estudos efectuados em mulheres portuguesas com aborto recorrente. O primeiro estudo teve por objectivo caracterizar os factores médicos e determinar o padrão da perda recorrente de gravidez, numa coorte de mulheres submetidas a um protocolo de diagnóstico definido. As participantes foram agrupadas de acordo com a paridade (AR primário ou secundário) e a idade gestacional das perdas (embrionárias ou fetais). As anomalias da cavidade uterina, a SAAF e as translocações equilibradas parentais foram os factores mais prevalentes. 15,6% das participantes eram obesas. Em 55,5% dos casos não foi identificado nenhum factor. A história obstétrica materna influenciou significativamente os resultados encontrados: os factores anatómicos e a SAAF foram mais prevalentes em nulíparas e as perdas inexplicadas foram mais frequentes em mulheres com AR secundário. Assim, os nossos dados reforçam os resultados de pesquisas anteriores sobre a importância da obesidade, da síndrome de anticorpos antifosfolípidos e das anomalias uterinas estruturais como factores associados ao AR e mostram que os a paridade é um moderador da importância desses factores. Capítulo 6 94 A ausência de resultados consensuais na literatura sobre a etiologia do AR condiciona a pesquisa sistemática de alguns factores, envolvendo exames dispendiosos, muitas vezes sem que exista evidência que suporte a sua associação com esta entidade. A trombofilia hereditária é uma das condições frequentemente investigadas nestas doentes. O nosso segundo estudo pretende contribuir para clarificar o papel de duas mutações (factor V Leiden e protrombina G20210A) na perda recorrente de gravidez e esclarecer a necessidade do seu rastreio nestas situações. Foi efectuada a pesquisa destes polimorfismos em 100 mulheres com AR inexplicado e num grupo de controlo de multíparas sem história de perdas de gravidez. Na nossa amostra não se verificou uma associação entre perdas embrionárias recorrentes e estas mutações. Nas mulheres com este tipo de perdas, a prevalência do FLV foi inclusive menor do que a verificada nos controlos. Pelo contrário, nas participantes com perdas fetais a prevalência destes polimorfismos foi muito superior à verificada nos controlos, sugerindo uma possível associação entre estas duas entidades. A pequena dimensão deste último subgrupo de mulheres, não nos permitiu contudo tirar conclusões. Uma investigação prospectiva multicêntrica é necessária antes de recomendar a pesquisa da trombofilia hereditária na investigação do AR. Procurámos incluir também nesta tese uma dimensão psicológica e contribuir assim para o conhecimento dos processos relacionais originados pelo AR. No terceiro estudo foram investigadas as diferenças de género na vivência do AR e o seu impacto no relacionamento e sexualidade do casal. Participaram neste estudo 30 casais sem filhos, com pelo menos 3 abortos espontâneos consecutivos. Cada membro do casal respondeu a um conjunto de questionários (Impact of Events Scale, Perinatal Grief Scale, Partnership Questionnaire e Intimate Relationship Scale). Os resultados mostram que as mulheres sofrem mais intensamente do que os homens com o AR, relacionando-se a intensidade do seu sofrimento com a qualidade do relacionamento conjugal. A sexualidade do casal é também afectada pelo stress e pelo sofrimento associados ao AR. Uma avaliação e acompanhamento deste tipo de problemas são imprescindíveis para ajudar estes casais a manterem a qualidade afectiva e sexual da sua relação. Finalmente, no capítulo 5 sumariámos as conclusões de toda a contribuição pessoal para a investigação sobre os factores associados e repercussões para o casal da perda recorrente de gravidez.-------------------ABSTRACT: Recurrent miscarriage (RM), a rare condition, has been described as a traumatic event for couples. Parental chromosomal anomalies, maternal thrombophilic disorders and structural uterine anomalies have been directly associated with RM. However, despite significant advances in medical research, the vast majority of cases remain unexplained. Aspects as the ethnic diversity of the population with different expression of genes, the inappropriate characterization of patients and of pregnancy losses, as well as different methodologies used in their study, have influenced the prevalence of etiological factors and have hampered the understanding of this problem. Similarly, little is known about gender differences in psychological experience of RM and its implications for the relationship of the couple. The first objective of this thesis is the characterization of the medical factors and of the psychological consequences related with RM, in the Portuguese population, helping to promote specific evidence-based clinical strategies. In the first part of this thesis, and after a brief general introduction (Chapter 1), a critical review of literature on the definition, the epidemiology and the dimensions involved, with a special emphasis on associated medical and psychological aspects of recurrent miscarriage, is presented (Chapter 2). In Chapters 3 and 4 we describe three studies carried out in Portuguese couples with RM. The first study aimed to investigate the etiological factors and the pattern of pregnancy loss in a cohort of women with RM. Subjects were divided in groups according to their parity (primary or secondary RM) and time of pregnancy loss (embryonic or fetal). Parental chromosome anomalies, uterine anomalies and antiphospholipid syndrome were the most prevalent medical factors. 15.6% of the women were obese. In the majority of cases (55.5%) no identifiable cause was detected. Parity influenced significantly our results. There was a higher prevalence of anatomic factors and antiphospholipid syndrome in primary RM. On the other hand, unexplained losses were more frequent in secondary RM. Except for the parental chromosomal abnormalities; the frequency of risk factors was similar among women with fetal or embryonic losses. Our data emphasizes the results of previous research on the importance of obesity, antiphospholipid syndrome and structural uterine abnormalities as known risk factors for RM, and shows that parity is an important moderator of the weight of those risk factors. Our second study aims to clarify the role of two mutations (factor V Leiden and prothrombin G20210A) and elucidate the need for their screening in Portuguese women with RM. FVL and PT G20210A analysis was carried out in 100 women with three or more consecutive miscarriages and a control group of 100 parous controls with no history of pregnancy losses. Secondary analysis was made regarding gestational age at miscarriage (embryonic and fetal loss). Overall the prevalence of FVL and PT G20210A was similar in RM women compared with controls. In the RM embryonic subgroup of women, FVL prevalence was inclusively lower than that of controls. Conversely in women with fetal losses both polymorphisms were much more frequent, although statistical significance was not reached due to the small size of this subgroup of patients. These data indicate that inherited maternal thrombophilia is not associated with RM prior to 10 weeks of gestation. Therefore, its screening is not indicated as an initial approach in Portuguese women with RM and a negative personal history of thromboembolic.96 Our second study aims to clarify the role of two mutations (factor V Leiden and prothrombin G20210A) and elucidate the need for their screening in Portuguese women with RM. FVL and PT G20210A analysis was carried out in 100 women with three or more consecutive miscarriages and a control group of 100 parous controls with no history of pregnancy losses. Secondary analysis was made regarding gestational age at miscarriage (embryonic and fetal loss). Overall the prevalence of FVL and PT G20210A was similar in RM women compared with controls. In the RM embryonic subgroup of women, FVL prevalence was inclusively lower than that of controls. Conversely in women with fetal losses both polymorphisms were much more frequent, although statistical significance was not reached due to the small size of this subgroup of patients. These data indicate that inherited maternal thrombophilia is not associated with RM prior to 10 weeks of gestation. Therefore, its screening is not indicated as an initial approach in Portuguese women with RM and a negative personal history of thromboembolic. In our third study, we investigate gender differences in RM experience and its impact on the couple's relationship and sexuality. Each member of 30 couples with RM answered a set of questionnaires, including the Impact of Events Scale (Horowitz et al., 1979), the Perinatal Grief Scale (Toedter et al., 1988), the Partnership Questionnaire (Hahlweg, 1979) and the Intimate Relationship Scale (Hetherington e Soeken, 1990). Results showed that men do grieve, but less intensely than women. Although the quality of the couple‟s relationship seemed not to be adversely affected by RM, both partners described sexual changes after those events. Grief was related to the quality of communication in the couple for women, and to the quality of sex life for men. An understanding of such issues is critical in helping these couples to maintain sexual and affective quality of their relationship. Finally, in Chapter 5, conclusions and clinical implications of all personal contribution to the investigation on associated factors and relational consequences of recurrent miscarriage are presented.

Incidência de "Falsos-Positivos" em Idosos com Fibrilhação Auricular Paroxística Submetidos a Teste de Inclinação

The autonomic nervous system (ANS) plays a role as a modulator in the pathogenesis of paroxysmal atrial fibrillation (PAF). The clinical pattern of vagally mediated PAF has been observed mainly in young patients. Neurocardiogenic responses during orthostatic stress are related to autonomic reflexes in which the vagal influence predominates. AIM: To evaluate the susceptibility of elderly patients with PAF to activation of vasovagal syncope mechanisms. METHODS: We performed passive head-up tilt testing (HUT) in 34 patients (62% women, aged 72 +/- 7 years), with > or = 1 year of clinical history of PAF--19 without structural heart disease, 11 with hypertensive heart disease and 4 with coronary artery disease (who had no previous myocardial infarction, had undergone myocardial revascularization, and had no documented ischemia) (PAF group), and compared the results with those obtained in a group of 34 age-matched patients (53% women, aged 74 +/- 6 years), who underwent HUT due to recurrent syncope (Sc group). In this group, 21 had no documented heart disease and none had a clinical history of AF. There was no diabetes, congestive heart failure or syncope in the PAF group. After a supine resting period, the subjects were tilted at 70 degrees for 20 minutes while in sinus rhythm. No provocative agents were used to complement the HUT. ECG and blood pressure were continuously monitored (Task Force Monitor, CNSystems). The test was considered positive when syncope or presyncope occurred with bradycardia and/or arterial hypotension. Abnormal responses were classified as cardioinhibitory, vasodepressor or mixed. RESULTS: HUT was positive in seven patients of the PAF group--vasodepressor response in five and mixed in two (20.5% of the total; 26.3% of those without heart disease)--and in eight patients (vasodepressor in six and mixed in two) of the Sc group (p=NS). During HUT, three patients of the PAF group had short periods of self-limited PAF (in one, after vasodepressor syncope). There were no differences in gender distribution, age or heart disease. No cardioinhibitory responses or orthostatic hypotension were observed. CONCLUSION: In elderly patients with PAF, a significant number of false positive results during passive HUT may be expected, suggesting increased vasovagal reactions despite aging. This suggests that ANS imbalances may be observed in this population.

Parotidite Recorrente Como Manifestação Inicial de Lúpus Eritematoso Sistémico

Neste artigo apresenta-se o caso clínico de uma doente de 45 anos de idade com tumefacção recorrente das parótidas, que, após quatro anos de evolução, é internada por febre vespertina, sudorese nocturna, toracalgia esquerda com padrão pleurítico, derrame pleural esquerdo, poliartrite e poliartralgia simétricas (padrão inflamatório) dos punhos, articulações interfalângicas proximais das mãos, joelhos e tibiotársicas, acompanhadas de rigidez matinal. Este quadro tinha dois meses de evolução e, após investigação, foi comprovado o diagnóstico de Lúpus Eritematoso Sistémico.

Hemorragia Digestiva Alta Recorrente por Pseudo-Aneurisma da Artéria Gastroduodenal

Os autores apresentam o caso clínico de um homem de 39 anos de idade com antecedentes de pancreatite crónica, que apresenta um quadro de hemorragia digestiva alta recorrente, com repercussão hemodinâmica, endoscopias altas sucessivas sem alterações, e cujos exames imagiológicos revelam presença de mal-formação vascular do tronco celíaco. Na sequência de mais um episódio de hemorragia digestiva alta com franca repercussão hemodinâmica, é internado no Serviço de Medicina,realizando arteriografia selectiva que mostrou a presença de pseudo-aneurisma da artéria gastroduodenal. Após a embolização, verificou-se uma progressiva e mantida melhoria da sua situação clínica. Descrevem os autores um quadro de hemorragia digestiva alta recorrente por malformação arterial associada à pancreatite crónica.

Derrame Pericárdico Recidivante. Um Caso de Angiosarcoma Cardíaco

A case of angiosarcoma of the right atrium in a 59-year-old woman is reported, presenting as a recurrent hematic pericardial effusion. The usefulness of echocardiography in making the diagnosis is emphasized. The tumor was already disseminated at the time of the diagnosis. The patient was on palliative therapy, dying a few months later. The histological examination was done at autopsy. This case highlights the difficulty in diagnosis and the various modalities available for an accurate diagnosis of a rare clinical entity. It has a rapid and aggressive course with a very poor prognosis.

Primary Antiphospholipid Syndrome: Pregnancy Outcome in a Portuguese Population

Introduction:Women with antiphospholipid syndrome(APS) may suffer from recurrent miscarriage, fetal death, fetal growth restriction (FGR), pre-eclampsia, placental abruption, premature delivery and thrombosis. Treatment with aspirin and low molecular weight heparin (LMWH) combined with close maternal-fetal surveillance can change these outcomes. Objective: To assess maternal and perinatal outcome in a cohort of Portuguese women with primary APS. Patients and Methods: A retrospective analysis of 51 women with primary APS followed in our institution (January 1994 to December 2007). Forty one(80.4%) had past pregnancy morbidity and 35.3%(n=18) suffered previous thrombotic events. In their past they had a total of 116 pregnancies of which only 13.79 % resulted in live births. Forty four patients had positive anticardiolipin antibodies and 33 lupus anticoagulant. All women received treatment with low dose aspirin and LMWH. Results: There were a total of 67 gestations (66 single and one multiple). The live birth rate was 85.1%(57/67) with 10 pregnancy failures: seven in the first and second trimesters, one late fetal death and two medical terminations of pregnancy (one APS related). Mean (± SD) birth weight was 2837 ± 812 g and mean gestational age 37 ± 3.3 weeks. There were nine cases of FGR and 13 hypertensive complications(4 HELLP syndromes). 54.4% of the patients delivered by caesarean section. Conclusions: In our cohort, early treatment with aspirin and LMWH combined with close maternal-fetal surveillance was associated with a very high chance of a live newborn.

Antero-Medialisation of the Tibial Tubercle for Patellar Instability

We reviewed 19 patients (24 knees) with patellofemoral instability treated surgically with antero-medialisation of the tibial tubercle and lateral retinacular release. Twenty-two knees had recurrent patellar dislocation and two patellar subluxation. Lateral retinacular release was performed arthroscopically in 15 knees. Average follow-up was 52 (16-86) months. There was one postoperative haemarthrosis and one failed fixation, which needed surgical revision. The average Lysholm score improved from 63.3 to 98 and only one knee had persistent patello-femoral pain postoperatively. The patellar tilt angle improved from 9.4 degrees to 5.5 degrees . There were no redislocations. We find that the surgical technique produces a consistent correction of patellar instability, but long-term studies are needed to confirm whether it can prevent arthritic degeneration.

Norovírus Associated Encephalopathy

clinical presentation is self limited. It is classified into five groups (genogroups I through V). There are numerous reports of neurologic complications, namely afebrile seizures, but only two reports of associated encephalopathy. Case Report: A 12 month old girl with previous history of a pneumonia treated with amoxicillin-clavulanic acid and clarythromycin, presented in our emergency department with strabismus, ataxia for 3 days, later associated with vomiting and diarrhea. On admission she had ataxia and an episode of strabismus, but her later neurologic exam was normal. Laboratory data revealed: 10,9 g/dL hemoglobin, 11.200/μL leukocytes, 29,1% neutrophils and 65,2% lymphocytes, 488.000/μL platelets and negative CRP. The brain MRI showed middle ear, maxillary sinus and ethmoidal opacification, with no other abnormalities. During the first day of admission she had a tonic (?) seizure for 20 minutes. CSF analysis showed 5,6 cells/μL, 100% lymphocytes, 80 mg/dL glucose and 154,1 mg/dL protein. The EEG revealed short duration paroxystic activity located to the vertex. She was treated with acyclovir, ciprofloxacin, cefthriaxone and phenytoin. Her symptoms resolved by the third day of admission. Blood samples were tested for numerous pathogens, including serology for Borrelia, which was positive for IgG but negative for IgM. Fecal sample analysis revealed positive PCR for norovirus, although it was negative in CSF samples. IL-6 was measured in the CSF and was negative (5,8 pg/mL). She had a history of recurrent otitis media and pernieal candidiasis, which led to a detailed immune function study, which showed Immunology tests revealed diminished IgA (< 0,244 g/L) and absent antibody response to vaccinations. Since she was only 13 months old when she was tested, only follow up will determine the relevance of these values. Follow up at two years of age showed no delays and a normal development. Conclusion: Norovirus encephalitis is a rare entity, although gastrointestinal infection with this agent is relatively common. Here we present a case of a probable norovirus associated encephalopathy, although PCR for norovirus was negative in CSF samples and there was no CSF cytokine increase. It was not associated with adverse neurologic outcome and so far her development is normal, unlike the evolution described in previous case reports.

Tradução com adaptação cultural para portugês e determinação da reprodutibilidade do questionnaire on respiratory symptoms in preschool children

RESUMO: Introdução: A asma brônquica é uma entidade frequente em idade pediátrica, apresentando uma grande heterogeneidade clínica e significativa morbilidade quando não controlada. A identificação de crianças sintomáticas pode atrasar ou até mesmo diminuir a ocorrência de algumas alterações estruturais. Reconhece-se a necessidade de questionários sobre sintomas respiratórios em língua portuguesa, devidamente validados, que tenham como população-alvo os grupos etários inferiores a 3 anos. Deste modo, será possível não só um conhecimento mais rigoroso da asma e da sibilância infantil mas também a uniformização de metodologias para o desenvolvimento de estratégias a nível nacional. Objetivos: Tradução com adaptação cultural para português e determinação da reprodutibilidade do Questionnaire on respiratory symptoms in preschool children de Strippoli e colaboradores. Material e métodos: A escolha do questionário obedeceu a vários critérios, entre os quais o grupo etário, o tipo e número de perguntas. O Questionnaire on respiratory symptoms in preschool children de Strippoli e colaboradores é um questionário de autopreenchimento, dirigido a crianças entre os 12 e os 24 meses de idade e destinado a estudos epidemiológicos ao nível da comunidade. Aborda aspetos referentes a sintomas respiratórios (sibilância, tosse crónica, sintomas das vias aéreas superiores), cuidados médicos, terapêutica, características ambientais, história familiar e situação social. Procedemos à sua tradução, com especial atenção para a adaptação do ponto de vista cultural e linguístico, utilizando o método da tradução / retroversão, amplamente utilizado e descrito na literatura internacional. Seguidamente determinámos a reprodutibilidade da versão final em língua portuguesa – Questionário de sintomas respiratórios em idade pré-escolar – utilizando o teste-reteste. Para tal, incluíram-se crianças entre os 12 e os 36 meses de idade recrutadas num Centro de Saúde e em creches de Lisboa. A distribuição dos questionários decorreu em duas fases: na primeira fase foram entregues pessoalmente nos locais de recrutamento e na segunda fase foram enviados por correio para os domicílios das crianças, respeitando-se um intervalo mínimo de 2 semanas entre ambos. Resultados: Na primeira fase foram distribuídos 180 questionários, com uma taxa de reposta de 41% (n=74). Na segunda fase enviaram-se para os respetivos domicílios 70 questionários,obtendo-se uma taxa de resposta de 66% (n=46). Para a análise de reprodutibilidade foram incluídos apenas os questionários preenchidos em ambos os momentos pelo mesmo indivíduo (mãe, pai ou representante legal) (n=41). A idade média das crianças foi, na primeira fase, de 22,5 meses e, na segunda fase, de 23,7 meses, com um predomínio do sexo feminino (F:M =1:0,6). A mediana do tempo decorrido entre os dois momentos de preenchimento dos questionários foi de 26 dias. Obtivemos valores de concordância globalmente bons a muito bons, à semelhança do sucedido no trabalho original. Conclusões: Procedemos à tradução e avaliação da reprodutibilidade do Questionnaire on respiratory symptoms in preschool children. Pretende-se que venha a ser uma ferramenta útil para estudos epidemiológicos e programas de rastreio na comunidade, contribuindo deste modo para uma otimização da abordagem da asma / sibilância infantil a nível nacional. -------------ABSTRACT: Background: Asthma is a very common feature in childhood, with important clinical heterogeneity and morbidity if not properly controlled. Identifying symptomatic children may delay or even reduce several structural changes. The development of questionnaires on respiratory symptoms in Portuguese for children under 3 years old will allow not only a more accurate knowledge of infantile asthma and recurrent wheezing but also the standardization of methodologies to develop nationwide strategies. Objectives: The aim of this study was to translate and adapt to the Portuguese culture and to determine the repeatability of the Questionnaire on respiratory symptoms in preschool children by Strippoli et al. Material and methods: The choice of the questionnaire took in consideration several criteria, among which the target age, the type and the number of questions. The Questionnaire on respiratory symptoms in preschool children by Strippoli et al is a parent-completed questionnaire for assessment of respiratory symptoms in 1 to 2-year-old children, developed for cross-sectional and longitudinal studies. It contains sections on respiratory symptoms (wheezing, chronic cough and upper airways symptoms), healthcare utilization, treatment, environmental exposure, family history and social situation. For the process of translation we used the method of translation and back-translation, with particular concern to cultural and linguistic adaptation. To assess the repeatability of the final Portuguese version - Questionário de sintomas respiratórios em idade pré-escolar - we used the test–retest analyses. The questionnaires were distributed to parents of children between 12 and 36 months old attending nurseries and a Primary Care Center of Lisbon. The distribution took place in two phases: the first questionnaires were delivered in person (phase one) and an identical questionnaire was posted to the families that participated in the first phase, 2 weeks after the first one was returned (phase two). Results: The response rates were 41% (180/74) in the first phase and 66% (70/46) in the second phase. For test–retest analyses, we included the 41 children with the same respondent (mother, father or legal representative) in both occasions. The median age of the children was 22,5 months at the first phase and 23,7 months at the second phase, with a predominance of girls (F:M = 1:0,6). The median time between the fillings of both questionnaires was 26 days. Globally, agreement values were good to excellent, similarly to the original work. Conclusion: In the present study we translated the Questionnaire on respiratory symptoms in preschool children and assessed its repeatability. Overall, we expect it to be a valuable tool for epidemiological studies and community-based screening programs, thus contributing to improve the management of infantile asthma / recurrent wheezing nationwide.

Análise da estrutura e da função da proteína morfogenética RodZ de Bacillus subtilis

Resumo: RodZ é um componente do sistema morfogenético das células bacterianas. É uma proteína transmembranar que localiza em bandas ao longo do eixo longitudinal da célula. Em Bacillus subtilis, RodZ consiste numa porção citoplasmática, RodZn, e em uma parte extra-citoplasmática, RodZc. RodZn contém um domínio em helixturn- helix (HTH), enquanto que RodZc pode ser dividido num domínio coiled-coil e num domínio terminal C, de função desconhecida. Um segmento transmembranar (TM) único separa RodZn de RodZc. A eliminação de rodZ causa alongamento do nucleóide e leva à produção de células polares nucleadas. Aqui, mostramos que RodZn é estruturado, estável e em hélice α. Descobrimos que as substituições Y32A e L33A na suposta hélice de reconhecimento (3) do motivo HTH, bem como as substituições Y49A e F53A, fora do motivo HTH (4), causam divisão assimétrica, mas apenas as últimas levam à deslocalização sub-celular de RodZ. Sugerimos que as hélices 3 e 4 são utilizadas para uma interacção proteína-proteína ou proteína- DNA essencial para divisão celular enquanto que 4 deve contactar um componente do citosqueleto, possivelmente MreB, uma vez que a correcta localização sub-celular de RodZ depende desta proteína. Em todos os mutantes as células polares são anucleadas, pelo que concluímos que o alongamento do nucleóide não é um prérequisito para divisão assimétrica. RodZc é largamente não estruturado mas com conteúdo de folha , sendo estabilizado pelo domínio coiled-coil. Mostramos uma relação homóloga entre RodZc e a bomba de transporte Na+/Ca2+ NCX1 e identificámos dois resíduos no domínio C, G265 e N275, essenciais para a manutenção da forma celular. Estes resíduos fazem parte de um motivo em gancho que pode actuar como um local de interacção com um ligando desconhecido. RodZn e RodZc são monoméricos em solução. Contudo, na membrana, RodZ interage consigo própria num sistema de dois híbridos (Split-Ubiquitin) em levedura, sugerindo que possa formar multímeros in vivo.-----------ABSTRACT: RodZ is a transmembrane component of the bacterial core morphogenic apparatus. RodZ localizes in bands long the longitudinal axis of the cell, and it is though to functionally link the cell wall to the actin cytoskeleton. In Bacillus subtilis, RodZ consists of a cytoplasmic moiety, RodZn, and an extracytoplasmic moiety, RodZc. RodZn contains a predicted helix-turn-helix domain, whereas RodZc is thought to contain a coiled-coil region and a terminal C domain of unknown function. A single transmembrane domain separates RodZn from RodZc. Deletion of rodZ causes elongation of the nucleoid and leads to the production of polar minicells containing DNA. Here, we have studied the structure and function of RodZn and RodZc. We show that RodZn is a stable, folded, -helical domain. We discovered that the Y32A and L33A substitutions within the presumptive recognition helix (3) of the HTH motif, as well as the Y49A and F53A substitutions outside of the HTH motif (in 4) cause asymmetric cell division. However, only the substitutions in 4 cause sub-celular delocalization of RodZ. We suggest that 3 and 4 are used for a protein-protein or protein-DNA interaction important for cell division, whereas 4 is likely to contact a cytoskeletal component, presumably MreB. The polar cells formed by all the mutants are anucleate. We conclude that nucleoid elongation is not a prerequisite for asymmetric division. RodZc appears to be a largely unstructured domain, with some -sheet content, and is stabilized by the coiled-coil region. We show a homology relationship between RodZc and the NCX1 Na+/Ca2+ transporter and we found two residues within the C domain, G265 and N275, that are important for cell shape determination. These residues are predicted to be essential determinants of a claw-like motif, which may act as a binding site for an unknown ligand. Both the isolated RodZn and RodZc proteins are monomeric in solution. However, because full-length RodZ interacts with itself in a split-ubiquitin yeast two-hybrid assay, we suggest that it may dimerize or form higher order multimers in vivo.

Fungal agents in different anatomical sites in Public Health Services in Cuiabá, state of Mato Grosso, Brazil

INTRODUCTION: A contribution to the regional epidemiological profile of the most common fungal agents in Public Health Services in Cuiabá, state of Mato Grosso, including university hospitals and polyclinics. METHODS: Clinical specimens (n = 1,496) from 1,078 patients were collected, submitted to direct mycological exam (potash or stick tape method) and cultured in specific mediums. Dermatophytic and non-dermatophytic agents were identified according to micromorphology (Ridell technique). RESULTS: The majority of the 1,496 specimens were skin (n = 985) and nail exams (n = 472). Of the 800 positive cultures, 246 (30.8%) corresponded to dermatophytes and 336 (42%) to yeasts of the genus Candida, 190 (23.7%) to other yeasts, 27 (3.4%) to non-dermatophytic filamentous fungi and one (0.1%) the agent of subcutaneous mycosis. Lesions considered primary occurred in greater numbers (59.5%) than recurrent lesions (37.4%), with a greater concentration of positivity occurring on the arms and legs. CONCLUSIONS: Comorbidities, allergies and diabetes mellitus were conditions associated with greater positivity in direct mycological exams and cultures. Positive culture was considered a definitive diagnosis of fungal infection and confirmed 47.8% of diagnostic hypotheses.

Os Factores Genéticos na Perda Gestacional

Pregnancy loss is the most common obstetric complication. Multiple factors have been associated with recurrent or sporadic pregnancy loss, and genetic factors, particularly at earlier gestational ages, are the most important ones. The proportion of miscarriages due to chromosomal factors decreases with increasing gestational age. The most common chromosomal abnormalities in early losses are autosomal trisomies, monosomy X and polyploidy. In later losses, aneuploidies are similar to those found in live newborns (trisomies 21,18 and 13, X monosomy and polysomy of sex chromosomes. In cases of recurrent miscarriage the most common cytogenetic changes are trisomies, polyploidy, monosomy X and unbalanced translocations. Identification of the causes of pregnancy loss facilitates the families’ grief and may indicate if there is the risk of repetition, in order to reduce recurrence. The investigation recommended in each case is far from consensual, and the cost/benefit analysis of diagnostic exams is essential. The determination of the karyotype of the products of conception is indicated in cases of fetal loss and recurrent miscarriage, while the parental karyotypes should be performed only in selected cases. Couples with identified genetic conditions should be counseled about reproductive options, including prenatal or pre-implantation diagnosis. Surveillance of a future pregnancy should be multidisciplinary and adjusted in each case. The cytogenetic factors, due to their high prevalence and complexity, have a fundamental, but still not completely clear, role in pregnancy loss.