Cilia motility studies in zebrafish embryos

A thesis submitted in fulfilment of the requirements for the degree of Masters in Molecular Genetics and Biomedicine

Frequency of human bocavirus respiratory infections among at-risk patients in São Paulo, Brazil

BACKGROUND AND OBJECTIVES: Human Bocavirus (HBoV) has been described since 2005 as an etiological agent of respiratory virus infections. From 2001 to 2008 we investigated the etiology of HBoV among adults and children in different groups at risk of presenting complications arising from acute respiratory infection, the investigation was carried out in a tertiary hospital health care system in Brazil. METHODS: HBoV DNA was assayed in 598 respiratory samples from community and hospitalized patients by PCR. RESULTS: Of the 598 tested samples, 2.44% (8/328) of children, including five children with heart disease, and 0.4% (1/270) of adult bone-marrow-transplant were HBoV positive. CONCLUSIONS: These data suggested lower HBoV frequency among different at-risk patients and highlights the need to better understand the real role of HBoV among acute respiratory symptomatic patients.

Transplant Glomerulopathy: Clinico-Pathologic Features

Transplant glomerulopathy is a sign of chronic kidney allograft damage. It has a distinct morphology and is associated with poor allograft survival. We aimed to assess the prevalence and clinic-pathologic features of transplant glomerulopathy, as well as determine the functional and histological implications of its severity. We performed a single-centre retrospective observational study during an eight-year period. Kidney allograft biopsies were diagnosed and scored according to the Banff classification, coupled with immunofluorescence studies. The epidemiology, clinical presentation, outcomes (patient and graft survival) and anti-HLA alloantibodies were evaluated. Transplant glomerulopathy was diagnosed in 60 kidney transplant biopsies performed for clinical reasons in 49 patients with ABO compatible renal transplant and a negative T-cell complement dependent cytotoxicity crossmatch at transplantation. The estimated prevalence of transplant glomerulopathy was 7.4% and its cumulative prevalence increased over time. C4d staining in peritubular capillaries (27.6%) was lower than the frequency of anti-HLA antibodies (72.5%), the majority against both classes I and II. Transplant glomerulopathy was associated with both acute (mainly glomerulitis and peritubular capillaritis) and chronic histologic abnormalities. At diagnosis, 30% had mild, 23.3% moderate and 46.7% severe transplant glomerulopathy. The severity of transplant glomerulopathy was associated with the severity of interstitial fibrosis. Other histological features, as well as clinical manifestations and graft survival, were unrelated to transplant glomerulopathy severity.

FREQUENCY, URINALYSIS AND SUSCEPTIBILITY PROFILE OF PATHOGENS CAUSING URINARY TRACT INFECTIONS IN ENUGU STATE, SOUTHEAST NIGERIA

Objective: This study was designed to determine the frequency and causative agent(s) of urinary tract infections (UTIs) in individuals with symptoms of urinary tract infections in Enugu State of Southeast Nigeria, and to determine the antibiotic susceptibility pattern of microbial agents isolated from urine culture.Methods: The study involved 211 individuals (149 females and 62 males) clinically suspected for UTI. Urine samples were collected by the mid-stream ‘clean catch’ method and tested using standard procedures. Antibiotic susceptibility of the isolated pathogens was tested using the Kirby-Bauer technique according to the Clinical and Laboratory Standards Institute (CLSI) guidelines.Results: Microscopy of centrifuged urine samples showed 16 patients had pyuria while 54 had pus cells. Calcium oxalate crystals were found in 14 samples. Urinalysis performed with urine samples showed 17 had protein; seven were nitrite positive and three had moderate to high glucose concentration. Fifty-four urine samples (36.2%) from females and 12 (19.4%) from males showed significant growth upon culture. Gram stain and biochemical tests identified nine different organisms with Escherichia coli as the most common isolated species. Forty three randomly selected strains were further tested for their susceptibility against a panel of antibiotics. Thirty isolates (81.08%) were resistant to four or more antibiotics with the highest resistance shown by E. coli (76.67%). All the Gram- negative isolates were resistant to Ampicilox, Cefuroxime and Amoxicillin.Conclusion: Urinary tract infections were found more in females in the area under study. As found in other studies, E. coli was the most predominant isolate, although other organisms seem to be on the increase.

BLOOD VESSELS IN GANGLIA IN HUMAN ESOPHAGUS MIGHT EXPLAIN THE HIGHER FREQUENCY OF MEGAESOPHAGUS COMPARED WITH MEGACOLON

This study aimed to determine the existence of blood vessels within ganglia of the myenteric plexus of the human esophagus and colon. At necropsy, 15 stillborns, newborns and children up to two years of age, with no gastrointestinal disorders, were examined. Rings of the esophagus and colon were analyzed and then fixed in formalin and processed for paraffin. Histological sections were stained by hematoxylin-eosin, Giemsa and immunohistochemistry for the characterization of endothelial cells, using antibodies for anti-factor VIII and CD31. Blood vessels were identified within the ganglia of the myenteric plexus of the esophagus, and no blood vessels were found in any ganglia of the colon. It was concluded that the ganglia of the myenteric plexus of the esophagus are vascularized, while the ganglia of the colon are avascular. Vascularization within the esophageal ganglia could facilitate the entrance of infectious agents, as well as the development of inflammatory responses (ganglionitis) and denervation, as found in Chagas disease and idiopathic achalasia. This could explain the higher frequency of megaesophagus compared with megacolon.

Information flow analysis using data-dependent logical propositions

Dissertação para obtenção do Grau de Mestre em Engenharia Informática

FREQUENCY OF Candida SPECIES IN A TERTIARY CARE HOSPITAL IN TRIANGULO MINEIRO, MINAS GERAIS STATE, BRAZIL

Infections by Candida species are a high-impact problem in public health due to their wide incidence in hospitalized patients. The goal of this study was to evaluate frequency, susceptibility to antifungals, and genetic polymorphism of Candida species isolated from clinical specimens of hospitalized patients. The Candida isolates included in this study were obtained from blood cultures, abdominal fluids, and central venous catheters (CVC) of hospitalized patients at the Clinical Hospital of the Federal University of Uberlândia during the period of July 2010 - June 2011. Susceptibility tests were conducted by the broth microdilution method. The RAPD-PCR tests used employed initiator oligonucleotides OPA09, OPB11, and OPE06. Of the 63 Candida isolates, 18 (28.5%) were C. albicans, 20 (31.7%) were C. parapsilosis complex species, 14 (22.2%) C. tropicalis, four (6.4%) C. glabrata, four (6.4%) C. krusei, two (3.3%) C. kefyr, and one (1.6%) C. lusitaniae. In vitro resistance to amphotericin B was observed in 12.7% of isolates. In vitroresistance to azoles was not detected, except for C. krusei. The two primers, OPA09 and OPB11, were able to distinguish different species. Isolates of C. albicans and C. parapsilosis complex species presented six and five clusters, respectively, with the OPA09 marker by RAPD-PCR, showing the genetic variability of the isolates of those species. It was concluded that members of the C. parapsilosis complex were the most frequent species found, and most isolates were susceptible to the antifungals amphotericin B, flucozanole, and itraconazole. High genetic polymorphisms were observed for isolates of C. albicans and C. parapsilosis complex species, mainly with the OPA09 marker.

Long-Term and Concentration-Dependent Beneficial Effect of Efavirenz on HDL-Cholesterol in HIV-Infected Patients

AIMS: To investigate the long-term effects of efavirenz on cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein (LDL-C) and triglycerides (TG). METHODS: Thirty-four HIV-infected patients who commenced efavirenz therapy were monitored for 36 months. RESULTS: In patients with baseline HDL-C<40 mg.dL-1 an increase in HDL-C from 31+/-1 mg.dL-1 to 44+/-2 mg.dL-1 (95% confidence interval 5.9, 21.9, P<0.01) was observed and remained throughout the follow-up period. Median efavirenz plasma concentration was 1.98 mg.L-1 and a direct correlation between percentage of HDL-C variation or TC/HDL-C ratio and efavirenz plasma concentrations was found. CONCLUSIONS: There is evidence of a long-term and concentration-dependent beneficial effect of efavirenz on HDL-C in HIV-infected patients.

The mechanism of formate oxidation by metal-dependent formate dehydrogenases

J Biol Inorg Chem (2011) 16:1255–1268 DOI 10.1007/s00775-011-0813-8

Triple Regimen with Rituximab, Plasmapheresis and Intravenous Immunoglobulin in the Treatment of Dialysis Dependent Acute Humoral-Mediated Rejection in Kidney Grafts

Introduction: The clinical importance of humoral-mediated acute rejection has been progressively recognised. Early recognition and treatment with plasmapheresis and intravenous immunoglobulin have recently improved short term prognosis. Case report: In this report we describe the clinical features of three 2nd transplant patients developing severe acute humoral rejection during the first week post-transplant while on anti-thymocyte globulin therapy. Treatment with plasmapheresis/ intravenous immunoglobulin/rituximab resulted in rapid reversal of oliguria,and recovery of renal function within the 1st week of treatment in 2/3 patients. Diagnosis was confirmed by graft biopsies revealing peritubular neutrophiles and C4d deposits. Sequential graft biopsies in all three patients revealed complete histological recovery within two weeks. One patient never recovered renal function, and one patient lost his graft at three months following hemorrhagic shock. After 2 years follow up, the remaining patient maintains a serum creatinine of 1.1mg/dl. Conclusion: The regimen using plasmapheresis plus intravenous immunoglobulin and rituximab was effective in rapidly reversing severe acute humoral rejection.

Differences in Nevirapine Biotransformation as a Factor for its Sex-Dependent Dimorphic Profile of Adverse Drug Reactions

OBJECTIVES: Nevirapine is widely used for the treatment of HIV-1 infection; however, its chronic use has been associated with severe liver and skin toxicity. Women are at increased risk for these toxic events, but the reasons for the sex-related differences are unclear. Disparities in the biotransformation of nevirapine and the generation of toxic metabolites between men and women might be the underlying cause. The present work aimed to explore sex differences in nevirapine biotransformation as a potential factor in nevirapine-induced toxicity. METHODS: All included subjects were adults who had been receiving 400 mg of nevirapine once daily for at least 1 month. Blood samples were collected and the levels of nevirapine and its phase I metabolites were quantified by HPLC. Anthropometric and clinical data, and nevirapine metabolite profiles, were assessed for sex-related differences. RESULTS: A total of 52 patients were included (63% were men). Body weight was lower in women (P = 0.028) and female sex was associated with higher alkaline phosphatase (P = 0.036) and lactate dehydrogenase (P = 0.037) levels. The plasma concentrations of nevirapine (P = 0.030) and the metabolite 3-hydroxy-nevirapine (P = 0.035), as well as the proportions of the metabolites 12-hydroxy-nevirapine (P = 0.037) and 3-hydroxy-nevirapine (P = 0.001), were higher in women, when adjusted for body weight. CONCLUSIONS: There was a sex-dependent variation in nevirapine biotransformation, particularly in the generation of the 12-hydroxy-nevirapine and 3-hydroxy-nevirapine metabolites. These data are consistent with the sex-dependent formation of toxic reactive metabolites, which may contribute to the sex-dependent dimorphic profile of nevirapine toxicity.

A relação entre eficiência e efectividade : aplicação ao internamento por doença cerebrovascular

RESUMO - Contexto: O estudo da relação entre eficiência e efectividade é relevante na investigação dos serviços de saúde, pois pretende-se conhecer os mecanismos que ligam os recursos investidos na prestação aos resultados em saúde experienciados pelos doentes e decorrentes desses mesmos cuidados. Os objectivos definidos para este trabalho consistiram, genericamente, no estudo da efectividade e da eficiência dos cuidados prestados em internamento a uma doença em particular – a doença cerebrovascular – e da relação entre ambas, tanto ao nível do episódio como do hospital. Pretendia-se ainda conhecer a consistência destes resultados, nomeadamente de que forma a relação entre efectividade e eficiência era influenciada pelas características dos hospitais. Metodologia: Foram seguidas duas abordagens: (1) conhecer o impacte dos diferentes níveis de recursos investidos no tratamento (eficiência medida pelos custos) sobre os resultados ao nível da mortalidade; (2) estimar o montante de recursos (medidos pelos custos) associados ao tratamento das complicações potencialmente evitáveis. A análise foi realizada para os episódios de internamento de doença cerebrovascular com alta no período 2005/07. Usou-se a informação da base de dados dos resumos de alta, da contabilidade analítica e da Matriz de Maryland. O estudo considerou medidas de resultados – mortalidade, complicações e custos – e o desempenho foi avaliado a partir da comparação entre os valores observados e esperados. Para o ajustamento pelo risco recorreu-se ao Disease Staging recalibrado aos dados em estudo. Na primeira abordagem usou-se a regressão logística: a mortalidade medida pela efectividade constituiu a variável dependente; a eficiência medida pelos custos, os atributos do hospital e o ano as independentes. O modelo foi também testado em subconjuntos da população. Ao nível do hospital, foram definidos quartis de desempenho na efectividade medida pela mortalidade, analisando-se o comportamento da eficiência medida pelos custos em cada um deles. Estudou-se ainda a análise da correlação entre medidas e os hospitais nos extremos do desempenho. Na segunda abordagem, procedeu-se à comparação entre os custos dos doentes com complicações e sem complicações em doentes de gravidade semelhante. A análise foi realizada para o conjunto de todas as complicações, por tipo de complicação e por hospital. Resultados: Os principais resultados encontrados quanto ao impacte dos diferentes níveis de recursos investidos no tratamento (eficiência medida pelos custos) sobre os resultados ao nível da mortalidade indicaram que, ao nível do episódio, não existia uma relação geral entre eficiência e efectividade, pois o uso de mais recursos traduzia-se quer em melhores quer em piores resultados na mortalidade, quer na ausência de efeito sobre os mesmos, com resultados consistentes ao longo dos anos em estudo. Ao nível do hospital, as conclusões foram consistentes com o encontrado ao nível do episódio – o comportamento dos prestadores na eficiência foi distinto e não acompanhou o que apresentaram na efectividade. Relativamente à estimativa dos custos adicionais decorrentes de complicações dos cuidados, o custo de tratamento dos doentes com complicações foi entre 2,2 a 2,8 vezes o valor dos doentes sem complicações. Estes resultados foram consistentes com a análise por tipo de complicação e por hospital. Foi ainda de assinalar a grande disparidade na frequência de complicações entre hospitais. Conclusão: Os resultados encontrados mostram a importância de uma gestão criteriosa dos recursos actuais e adicionais dedicados à prestação de cuidados de saúde, uma vez que em algumas situações se verifica que o seu acréscimo não se traduz numa melhoria dos resultados em saúde estudados. Sugerem ainda um conjunto de matérias que deverão ser alvo de investigação futura, para conhecer em maior profundidade os mecanismos da relação entre eficiência e efectividade e identificar as circunstâncias em que é possível prosseguir na melhoria quer da eficiência quer da efectividade.

Spectrum and Frequency of GJB2 Mutations in a Cohort of 264 Portuguese Nonsyndromic Sensorineural Hearing Loss Patients

OBJECTIVE: To assess the spectrum and prevalence of mutations in the GJB2 gene in Portuguese nonsyndromic sensorineural hearing loss (NSSHL) patients. DESIGN: Sequencing of the coding region, basal promoter, exon 1, and donor splice site of the GJB2 gene; screening for the presence of the two common GJB6 deletions. STUDY SAMPLE: A cohort of 264 Portuguese NSSHL patients. RESULTS: At least one out of 21 different GJB2 variants was identified in 80 (30.2%) of the 264 patients analysed. Two mutant alleles were found in 53 (20%) of these probands, of which 83% (44/53) harboured at least one c.35delG allele. Twenty-seven (10.2%) of the probands harboured only one mutant allele. Subsequent analysis revealed that the GJB6 deletion del(GJB6-D13S1854) was present in at least 7.4% (2/27) of the patients carrying only one mutant GJB2 allele. Overall, one in five (55/264) of the patients were diagnosed as having DFNB1-related NSSHL, of which the vast majority (53/55) harboured only GJB2 mutations. CONCLUSIONS: This study provides clear demonstration that mutations in the GJB2 gene are an important cause of NSSHL in Portugal, thus representing a valuable indicator as regards therapeutical and rehabilitation options, as well as genetic counseling of these patients and their families.

Gestão de infra-estruturas e avaliação de equipamentos laboratoriais

RESUMO: A confiabilidade e qualidade dos resultados clínicos de um laboratório, estão dependentes da qualidade do equipamento onde estes são processados. A qualidade de um equipamento consiste em apresentar resultados com uma confiabilidade metrológica de acordo com os processos de medição realizados. Desta forma, os equipamentos de medição devem conter características metrológicas de acordo com os requisitos dos processos que estão inseridos, tais como: erro máximo, incerteza, resolução… Todos os equipamentos por muito bons que sejam, apresentam perdas das suas características metrológicas ao longo do tempo, o que se torna importante a determinação periódica das suas calibrações, manutenções e o seu respectivo ajuste. A periodicidade das calibrações, devem ser baseadas, em critérios bem estabelecidos de modo a evitar a ocorrência da não conformidade, devido às medições com os equipamentos fora das suas especificações, garantindo-se assim, a confiabilidade metrológica e o aumento da produtividade devido ao controlo dos processos, evitando-se desta forma qualquer tipo de avaria por parte destes. Os normativos de Qualidade e Certificação estabelecem como requisito a calibração dos equipamentos de medição em intervalos adequados, mas não definem claramente qual o intervalo adequado. O importante é gerir as etapas de calibração e manutenção de acordo com os equipamentos de modo a garantir que os resultados emitidos por estes sejam mantidos em condições confiáveis, dentro do período entre calibrações e manutenções, de modo a que nãoocorra uma condição de falha, e por conseguinte, medições com grande quantidade de erros.------------ ABSTRACT: The reliability and quality of the results of a clinical laboratory, are dependent on the quality of the equipment where they are processed. The quality of a product is to present results with a metrological reliability according to the measuring processes carried out. Thus, measuring equipment must contain metrological characteristics according to the requirements of the processes that are included, such as: maximum error, uncertainty, resolution ... All equipment for very good they are, show losses of its metrological characteristics over time, it becomes important to determine its periodic calibrations, maintenance and its adjustment. The frequency of calibrations should be based on well-established criteria in order to avoid the occurrence of non-compliance due to the measurements with the equipment beyond its specifications, thus ensuring the metrological reliability and increased productivity due to the control of processes, thus avoiding any kind of damage on the part thereof. The normative and Quality Certification as a requirement to establish calibration of measuring equipment at appropriate intervals, but not clearly stipulate the proper range. It is important to manage the steps of calibration and maintenance equipment according to ensure that the results emitted by these conditions are maintained in trusted within the period between calibrations and maintenance, sothat there occurs a fault condition, and therefore, large amount of measurements with errors.

Financial crisis spillovers to the corporate sector: Bank-dependent borrowers versus bond market paticipants

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Finance from the NOVA – School of Business and Economics