Seleção de genótipos de trigo para rendimento de grãos e qualidade de panificação em ensaios multiambientes

O objetivo deste estudo foi selecionar linhagens de trigo para qualidade de panificação e para rendimento de grãos (RG), em ensaios multiambientes, e identificar ambientes de teste ideais para a seleção. Foram avaliados 39 genótipos de trigo, sendo 18 linhagens e nove cultivares testemunhas, em 2010, e 11 linhagens e quatro cultivares testemunhas, em 2011, no Estado do Paraná. Algumas linhagens e locais não foram repetidos entre os dois anos. O delineamento experimental utilizado foi o de blocos casualizados, com três repetições. A metodologia, em gráfico biplot GGE (Genotype and Genotype-by-Environment), foi utilizada para a avaliação de genótipos e ambientes de teste. Na safra 2010, os locais de Nova Fátima, Pitangueiras e Ventania foram discriminantes (maximizaram a expressão do RG e diferenciação entre genótipos) e representativos do conjunto de ambientes avaliados. Em 2011, Rolândia e Astorga destacaram-se como ambientes ideais para seleção simultânea para RG e qualidade de panificação. As linhagens BIO-08528 e BIO-08228 foram classificadas como genótipos ideais para o RG e a concentração proteica dos grãos (CPG), respectivamente. Na safra 2011, as linhagens BIO-10161 e BIO-10141 foram superiores para RG e para qualidade de panificação, respectivamente. O teste de sedimentação SDS e a CPG correlacionaram-se entre si (r = 0,61**) e foram moderadamente associados com a força de glúten (r = 0,49** e 0,74**), indicando que podem ser utilizados na seleção indireta para qualidade de panificação.

Effects of physical exercise training in DNA damage and repair - could the difference be in hOGG1 Ser326Cys polymorphism?

Acute physical exercise is associated with increased oxygen consumption, which could result in an increased formation of reactive oxygen species (ROS). ROS can react with several organic structures, namely DNA, causing strand breaks and a variety of modified bases in DNA. Physical exercise training seems to decrease the incidence of oxidative stress-associated diseases, and is considered as a key component of a healthy lifestyle. This is a result of exercise-induced adaptation, which has been associated with the possible increase in antioxidant activity and in oxidative damage repair enzymes, leading to an improved physiological function and enhanced resistance to oxidative stress (Radak et al. 2008). Human 8-oxoguanine DNA glycosylase 1 (hOGG1) is involved in the base excision repair (BER) pathway and encodes an enzyme responsible for removing the most common product of oxidative damage in DNA, 8-hydroxyguanine (8-OH-G). The genetic polymorphism of hOGG1 at codon 326 results in a serine (Ser) to cysteine (Cys) amino acid substitution (Ser326Cys). It has been suggested that the carriers of at least one hOGG1Cys variant allele exhibit lower 8-OH-G excision activity than the wild-type (Wilson et al. 2011). The aim of this study was to investigate the possible influence of hOGG1 Ser326Cys polymorphism on DNA damage and repair activity in response to 16 weeks of combined physical exercise training, in thirty healthy Caucasian men. Comet assay was carried out using peripheral blood lymphocytes and enabled the evaluation of DNA damage, both strand breaks and FPG-sensitive sites, and DNA repair activity. Genotypes were determined by PCR-RFLP analysis. The subjects with Ser/Ser genotype were considered as wild-type group (n=20), Ser/Cys and Cys/Cys genotype were analyzed together as mutant group (n=10). Regarding differences between pre and post-training in the wild-type group, the results showed a significant decrease in DNA strand breaks (DNA SBs) (p=0.002) and also in FPG-sensitive sites (p=0.017). No significant differences were observed in weight (p=0.389) and in lipid peroxidation (MDA) (p=0.102). A significant increase in total antioxidant capacity (evaluated by ABTS) was observed (p=0.010). Regarding mutant group, the results showed a significant decrease in DNA SBs (p=0.008) and in weight (p=0.028). No significant differences were observed in FPG-sensitive sites (p=0.916), in ABTS (p=0.074) and in MDA (p=0.086). No significant changes in DNA repair activity were observed in both genotype groups. This preliminary study suggests the possibility of different responses in DNA damage to physical exercise training, considering the hOGG1 Ser326Cys polymorphism.

Associação entre polimorfismo inserção/delecção da enzima conversora da angiotensina na diabetes mellitus tipo 2: estudo da população portuguesa

A Diabetes mellitus tipo 2 (DM2) é uma patologia de etiologia múltipla à qual estão associados vários factores genéticos. A Enzima Conversora da Angiotensina (ECA) tem sido alvo de vários estudos pela sua relação com factores pró-inflamatórios, pró-oxidantes e pró-fibrose, sendo o polimorfismo de Inserção/Delecção o mais estudado. Neste contexto, o objectivo deste estudo é assim verificar a distribuição deste polimorfismo numa amostra de indivíduos de nacionalidade portuguesa e verificar a sua possível associação com a DM2. Para tal, foram analisadas 87 amostras (controlos n =24 e diabéticos n =63) de indivíduos de nacionalidade portuguesa. As amostras foram submetidas a um processo de extracção de ADN, sendo posteriormente amplificadas por Polymerase Chain Reaction e analisadas por eletroforese em gel de agarose a 1%. Observou-se uma prevalência de 8% (n=7) com genótipo I/I, 38% (n=33) com genótipo I/D e 54% (n=47) com genótipo D/D. A amostra em estudo demonstrou assim estar sob o equilíbrio Hardy-Weinberg. Observou-se também uma associação entre níveis mais elevados de glicemia e o genótipo I/I (p=0,019). Na análise da utilização de insulina no controlo dos níveis de glicemia na DM2, observou-se uma maior proporção de indivíduos com genótipo D/D. Este estudo demonstra a importância do investimento da caracterização genética em patologias metabólicas multifactoriais como a DM2.

Two-way MANCOVA: an application to public health

The aim of this work is to use the MANCOVA model to study the influence of the phenotype of an enzyme - Acid phosphatase - and a genetic factor - Haptoglobin genotype - on two dependent variables - Activity of Acid Phosphatase (ACP1) and the Body Mass Index (BMI). Therefore it's used a general linear model, namely a multivariate analysis of covariance (Two-way MANCOVA). The covariate is the age of the subject. This covariate works as control variable for the independent factors, serving to reduce the error term in the model. The main results showed that only the ACP1 phenotype has a significant effect on the activity of ACP1 and the covariate has a significant effect in both dependent variables. The univariate analysis showed that ACP1 phenotype accounts for about 12.5% of the variability in the activity of ACP1. In respect to this covariate it can be seen that accounts for about 4.6% of the variability in the activity of ACP1 and 37.3% in the BMI.

FASL polymorphism is associated with response to bacillus Calmette-Guérin immunotherapy in bladder cancer

Objective Deregulation of FAS/FASL system may lead to immune escape and influence bacillus Calmette-Guérin (BCG) immunotherapy outcome, which is currently the gold standard adjuvant treatment for high-risk non–muscle invasive bladder tumors. Among other events, functional promoter polymorphisms of FAS and FASL genes may alter their transcriptional activity. Therefore, we aim to evaluate the role of FAS and FASL polymorphisms in the context of BCG therapy, envisaging the validation of these biomarkers to predict response. Patients and methods DNA extracted from peripheral blood from 125 patients with bladder cancer treated with BCG therapy was analyzed by Polymerase Chain Reaction—Restriction Fragment Length Polymorphism for FAS-670 A/G and FASL-844 T/C polymorphisms. FASL mRNA expression was analyzed by real-time Polymerase Chain Reaction. Results Carriers of FASL-844 CC genotype present a decreased recurrence-free survival after BCG treatment when compared with FASL-844 T allele carriers (mean 71.5 vs. 97.8 months, P = 0.030) and have an increased risk of BCG treatment failure (Hazard Ratio = 1.922; 95% Confidence Interval: [1.064–3.471]; P = 0.030). Multivariate analysis shows that FASL-844 T/C and therapeutics scheme are independent predictive markers of recurrence after treatment. The evaluation of FASL gene mRNA levels demonstrated that patients carrying FASL-844 CC genotype had higher FASL expression in bladder tumors (P = 0.0027). Higher FASL levels were also associated with an increased risk of recurrence after BCG treatment (Hazard Ratio = 2.833; 95% Confidence Interval: [1.012–7.929]; P = 0.047). FAS-670 A/G polymorphism analysis did not reveal any association with BCG therapy outcome. Conclusions Our results suggest that analysis of FASL-844 T/C, but not FAS-670 A/G polymorphisms, may be used as a predictive marker of response to BCG immunotherapy.

Fatty acids, IL6, and TNFα polymorphisms: an example of nutrigenetics in Crohn's disease

Objectives - The aim of this work was to study the interaction between genetic polymorphisms (single-nucleotide polymorphisms, SNPs) of pro- and anti-inflammatory cytokines and fat intake on the risk of developing Crohn's disease (CD) or modifying disease activity. Methods - Seven SNPs in interleukin 1 (IL1), tumor necrosis factor alpha (TNFalpha), lymphotoxin alpha (LTalpha), and IL6 genes were analyzed in 116 controls and 99 patients with CD. The type of fat intake was evaluated, and the interaction between SNPs and dietary fat in modulating disease activity was analyzed. Results - Individuals who were homozygous for the IL6-174G/C polymorphism had a six-fold higher risk for CD (odds ratio (OR)=6.1; 95% confidence interval (95% CI)=1.9-19.4), whereas the TT genotype on the TNFalpha-857C/T polymorphism was associated with more active disease (OR=10.4; 95% CI=1.1-94.1). A high intake of total, saturated, and monounsaturated fats, as well as a higher ratio of n-6/n-3 polyunsaturated fatty acid (PUFA), was associated with a more active phenotype (P<0.05). Furthermore, there was an interaction between dietary fat intake and SNPs, with a high intake of saturated and monounsaturated fats being associated with active disease, mainly in patients carrying the variant alleles of the 857 TNFalpha polymorphism (OR=6.0, 95% CI=1.4-26.2; OR=5.17; 95% CI=1.4-19.2, respectively) and the 174 IL6 polymorphism (OR=2.95; 95% CI=1.0-9.1; OR=3.21; 95% CI=1.0-10.4, respectively). Finally, low intake of n-3 PUFA and high n-6/n-3 PUFA ratio in patients with the TNFalpha 857 polymorphism were associated with higher disease activity (OR=3.6; 95% CI=1.0-13.0; OR=5.92; 95% CI=1.3-26.5, respectively). Conclusions - These results show that different types of fat may interact with cytokine genotype, modulating disease activity.

Q192R polymorphism of the paraoxonase-1 gene as a risk factor for obesity in Portuguese women

Introduction - Obesity became a major public health problem as a result of its increasing prevalence worldwide. Paraoxonase-1 (PON1) is an esterase able to protect membranes and lipoproteins from oxidative modifications. At the PON1 gene, several polymorphisms in the promoter and coding regions have been identified. The aims of this study were i) to assess PON1 L55M and Q192R polymorphisms as a risk factor for obesity in women; ii) to compare PON1 activity according to the expression of each allele in L55M and Q192R polymorphisms; iii) to compare PON1 activity between obese and normal-weight women. Materials and methods - We studied 75 healthy (35.9±8.2 years) and 81 obese women (34.3±8.2 years). Inclusion criteria for obese subjects were body mass index ≥30 kg/m2 and absence of inflammatory/neoplasic conditions or kidney/hepatic dysfunction. The two PON1 polymorphisms were assessed by real-time PCR with TaqMan probes. PON1 enzymatic activity was assessed by spectrophotometric methods, using paraoxon as a substrate. Results - No significant differences were found for PON1 activity between normal and obese women. Nevertheless, PON1 activity was greater (P<0.01) for the RR genotype (in Q192R polymorphism) and for the LL genotype (in L55M polymorphism). The frequency of allele R of Q192R polymorphism was significantly higher in obese women (P<0.05) and was associated with an increased risk of obesity (odds ratio=2.0 – 95% confidence interval (1.04; 3.87)). Conclusion - 55M and Q192R polymorphisms influence PON1 activity. The allele R of the Q192R polymorphism is associated with an increased risk for development of obesity among Portuguese Caucasian premenopausal women.

Relationship of PON1 192 and 55 gene polymorphisms to calcific valvular aortic stenosis

Introduction and Objectives - Paraoxonases may exert anti-atherogenic action by reducing lipid peroxidation. Previous studies examined associations between polymorphisms in the paraoxonase 1 (PON1) gene and development of coronary artery disease (CAD), with inconsistent results. Given the similarities in clinical and pathophysiological risk factors of CAD and calcific aortic valve stenosis (CAVS), we postulated a link between PON1 alleles and CAVS progression. Methods - We investigated the association between PON1 55 and 192 single nucleotide polymorphisms (SNPs), their enzyme activity, and CAVS progression assessed by aortic valve area and transvalvular peak velocity in 67 consecutive patients with moderate CAVS and 251 healthy controls. Results - PON1 paraoxonase activity was higher in CAVS patients (P<0.001). The PON1 genotype Q192R SNP (P=0.03) and variant allele (R192) (P=0.01) frequencies differed between CAVS patients and controls. Significant association existed between PON1 enzyme activity, phenotypic effects of PON1 192 genotype polymorphisms, and CAVS progression, but not between PON1 55 and high-density lipoprotein (P=0.44) or low-density lipoprotein cholesterol (P=0.12), between 192 genotype and high-density lipoprotein (P=0.24) or low-density lipoprotein cholesterol (P=0.52). Conclusion - The PON1 genotype Q192R SNP has an important effect on CAVS disease progression. This study helps outline a genotype-phenotype relationship for PON1 in this unique population.

5' and 3' UTR thymidylate synthase polymorphisms modulate the risk of colorectal cancer independently of the intake of methyl group donors

Thymidylate synthase, as a rate-limiting step in DNA synthesis, catalyses the conversion of dUMP into dTMP using 5,10-methylenotetrahydrofolate as the methyl donor. Two polymorphisms have been described in this gene: a repeat polymorphism in the 5' promoter enhancer region (3R versus 2R) and a 6 bp deletion in the 3' unstranslated region. Both of these may affect protein levels. The present case control study was aimed at investigating the influence of these two polymorphisms on the development of colorectal cancer (CRC), as well as their potential interaction with folate, vitamin B6 and vitamin B12 intake. A total of 196 cases and 200 controls, matched for age and sex distribution, were included in the study. No association was found between CRC and the 28 bp repeat polymorphism, but it was observed that individuals with the 6 bp/del and del/del genotypes had a significantly lower risk of developing the disease (OR=0.47; 95% CI 0.30-0.72). A combined genotype (2R/2R; 6 bp/del+del/del) was also found, which was associated with an even lower risk of developing of the disease (OR=0.42; 95% CI 0.26-0.69). No significant interaction between these polymorphisms and vitamin intake was observed. These results indicate for the first time that the 6 bp/del allele might be a protective factor in the development of CRC, independent of the intake of methyl group donors.

The D1822V APC polymorphism interacts with fat, calcium, and fiber intakes in modulating the risk of colorectal cancer in Portuguese persons

Background - Both genetic and environmental factors affect the risk of colorectal cancer (CRC). Objective - We aimed to examine the interaction between the D1822V polymorphism of the APC gene and dietary intake in persons with CRC. Design - Persons with CRC (n = 196) and 200 healthy volunteers, matched for age and sex in a case-control study, were evaluated with respect to nutritional status and lifestyle factors and for the D1822V polymorphism. Results - No significant differences were observed in energy and macronutrient intakes. Cases had significantly (P < 0.05) lower intakes of carotenes, vitamins C and E, folate, and calcium than did controls. Fiber intake was significantly (P = 0.004) lower in cases than in controls, whereas alcohol consumption was associated with a 2-fold risk of CRC. In addition, cases were significantly (P = 0.001) more likely than were controls to be sedentary. The homozygous variant for the APC gene (VV) was found in 4.6% of cases and in 3.5% of controls. Examination of the potential interactions between diet and genotype found that a high cholesterol intake was associated with a greater risk of colorectal cancer only in noncarriers (DD) of the D1822V APC allele (odds ratio: 1.66; 95% CI: 1.00, 2.76). In contrast, high fiber and calcium intakes were more markedly associated with a lower risk of CRC in patients carrying the polymorphic allele (DV/VV) (odds ratio: 0.50; 95% CI: 0.27, 0.94 for fiber; odds ratio: 0.51; 95% CI: 0.28, 0.93 for calcium) than in those without that allele. Conclusion - These results suggest a significant interaction between the D1822V polymorphism and the dietary intakes of cholesterol, calcium, and fiber for CRC risk.

Glucose-6-Phosphate dehydrogenase deficiency in children from 0 to 14 years hospitalized at the Pediatric Hospital David Bernardino, Luanda, Angola

The Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymatic defect in the world. The most common clinical manifestations are acute hemolytic anemia associated with drugs, infections, neonatal jaundice and hemolytic non-spherocytic chronic anemia. The main aim of this study was to determine the frequency of major genetic variants of G6PD leading to enzyme deficiency in children from 0 to 14 years at a Pediatric Hospital in Luanda, Angola. A cross-sectional and descriptive analytical study covered a total of 194 children aged from 0 to 14 years, of both genders and hospitalized at the Pediatric Hospital David Bernardino, Luanda between November and December, 2011. The G202A, A376G and C563T mutations of the G6PD gene were determined by real-time PCR with Taqman probes. The disabled A-/A- genotype was detected in 10 girls (10.9%). Among the boys, 21 (20.6%) presented the genotype A-. Considering all the samples, the A- variant was observed in 22.4% of cases. The Mediterranean mutation was not detected in the Angolan sample. Furthermore, no association was found between genotype and anemia, nutritional state and mucosa color. A significant association, however, was observed with jaundice. Based on the results obtained, there is a clear need to identify those with the disabled genotype in the Angolan population in order to avoid cases of drug-induced anemia, particularly in the treatment of malaria, so prevalent in Angola.

Citologia da cúpula vaginal com suspeita de lesão intraepitelial de grau indeterminado: estudo de caso

No presente artigo é relatado um caso de uma paciente de 42 anos, diagnosticada em 2011 com Adenocarcinoma in situ e displasia grave do epitélio de revestimento pavimentoso, que foi tratada por traquelectomia. Em Novembro de 2013, a paciente realizou uma citologia da cúpula vaginal, onde se observaram achados citológicos compatíveis com lesão intraepitelial de baixo grau mas também a presença de células que favorecem o diagnóstico de lesão intraepitelial de alto grau. Não sendo possível classificar a lesão intraepitelial como sendo claramente baixo ou alto grau, atribuiu-se a interpretação de lesão intraepitelial de grau indeterminado. Para confirmação e esclarecimento do diagnóstico foi efetuada biopsia com resultado de displasia grave do epitélio de revestimento pavimentoso vaginal sem evidência de invasão do estroma. Por fim foi indicada a pesquisa e tipificação de vírus do papiloma humano, com resultado positivo para o tipo 16. Diagnósticos citológicos de lesão intraepitelial de grau indeterminado apresentam um follow-up histológico estatisticamente diferente das lesões intraepiteliais de alto e baixo grau, e estão na sua maioria associadas a infeção por vírus do papiloma humano de alto risco. Os achados citológicos do presente estudo apoiam a necessidade de se estabelecer esta lesão como categoria de diagnóstico no Sistema de Bethesda, com um follow-up definido.

Dengue type 2 outbreak in the south of the State of Bahia, Brazil: laboratorial and epidemiological studies

During March 1994 cases of a exanthematic acute disease were reported in the municipalities of Itagemirim, Eunápolis and Belmonte, state of Bahia. Dengue fever was confirmed by serology (MAC-ELISA) and by dengue virus type 2 isolation, genotype Jamaica. Signs and symptoms of classic dengue fever were observed with a high percentual of rash (73.8%) and pruritus (50.5%). Major haemorrhagic manifestations were unfrequent and only bleeding gum was reported. Dengue virus activity spreaded rapidly to important tourism counties like Porto Seguro, Ilhéus, Santa Cruz de Cabrália, Prado, Alcobaça and others, representing a risk for the spreading of dengue virus into the country and abroad.

Anemia de células falciformes na idade pediátrica : contribuição para o estudo das suas principais complicações e indicadores de prognóstico

RESMO: Introdução: A anemia de células falciformes doença hereditária, com repercussão multi-orgânica, tem grande variabilidade na sua expressão clínica. Daí o interesse do estudo de indicadores de prognóstico. A investigação realizada foi precedida de um resumo histórico incidindo sobre a compreensão de aspectos fundamentais da doença ao longo dos tempos. Na primeira parte do estudo e após revisão bibliográfica, foram referidos dados da fisiopatologia como base para os estudos que integram a presente dissertação. Abordou-se o estado da arte relativamente às complicações, aos indicadores de prognóstico e à terapêutica utilizada. Objectivos: Constituíram objectivos deste estudo realizado numa amostra populacional representativa: identificar as lesões a nível dos sistemas cardio-respiratório e nervoso central, avaliando-se as respectivas repercussões; avaliar a presença de indicadores de prognóstico entre as variáveis seleccionadas; estudar a eficácia e toxicidade da HU nos doentes com as formas graves da ACF. Para a prossecução destes objectivos foram delineados para além do estudo global três estudos específicos: Estudo 1- repercussão no sistema cardio-respiratório; Estudo 2- repercussão no sistema nervoso central; Estudo 3- terapêutica com hidroxiureia. Doentes e métodos: Procedeu-se a um estudo prospectivo e multi-institucional durante um período de três anos tendo-se seleccionado para a amostra, e de acordo com critérios pré-definidos, 30 doentes com ACF na fase estável da doença, com idades compreendidas entre os sete e os 18 anos, todos de origem africana à excepção de um caucasiano. O diagnóstico baseou-se em técnicas de electroforese e estudo molecular que definiu o genotipo da doença e a presença da delecção da -talassémia assim como os haplotipos da amostra populacional. Foram utilizadas diferentes metodologias para avaliar a existência de lesão pulmonar e cerebral. Através do estudo estatístico foram seleccionadas diversas variáveis como hipotéticos indicadores de prognóstico. Estudo 1. Para determinar a existência de lesão a nível pulmonar usaram-se duas metodologias diferentes, a avaliação da função pulmonar com estudo da saturação da Hb em O2 no sangue arterial e a tomografia computadorizada de alta resolução. Estudou-se também a possível disfunção cardíaca como repercussão da lesão pulmonar, através do ecocardiograma, e os indicadores de prognóstico com significado estatístico para a lesão encontrada. Estudo 2. O desenho deste estudo foi sobreponível ao anterior, mas com metodologia adequada para o SNC. Procedeu-se ao estudo das lesões cerebrais por meio de exames imagiológicos, (RMN-CE e DTC) e de testes psicológicos. Correlacionaram-se as três metodologias utilizadas e a importância de cada uma para a decisão de atitudes terapêuticas preventivas. Estudo 3. Consistiu num estudo aberto prospectivo não controlado com nove crianças e adolescentes com formas graves de ACF, com o objectivo de avaliar a eficácia da terapêutica com hidroxiureia, durante um período de 24 meses. Todos os doentes completaram no mínimo 15 meses de terapêutica, com uma dose final média de 194 mg/K/dia. Resultados globais: Durante o período anterior à investigação caracterizou-se a amostra populacional estudada quanto ao fenotipo genético, clínico e hematológico de acordo com os critérios utilizados por outros investigadores. Verificou-se: predomínio do haplotipo Bantu na forma homozigótica em 53% dos doentes; número total de EVO ≥3/ano em 87,5% dos doentes; crises de sequestração em 18,75%; dactilites no primeiro ano de vida em 31,2%; quadro de sépsis grave apenas num doente; crises de hiper-hemólise em 50%; e STA em 59,38% dos doentes. Quanto ao fenotipo hematológico evidenciaram-se como factores de risco reticulocitose (13,1x103/l) e hiperbilirrubinémia (2,5 mg/dl) e como factores de bom prognóstico a presença de delecção de um gene da -talassémia em 46,9% dos doentes e valor médio de Hb 8,1 g/dl. Resultados dos estudos parcelares: Estudo 1. Deste estudo infere-se que a DPR ligeira foi diagnosticada em 70% dos doentes, uma vez que as alterações da difusão não foram estatisticamente significativas, o estudo dos gases no sangue não evidenciaram resultados anormais e a TCAR evidenciou alterações em 43,3% dos doentes. Apenas num doente se verificou doença pulmonar obstrutiva relacionada com maior número da STA.O estudo da disfunção cardíaca encontrada em 86,7% dos doentes não reflecte a repercussão da DPR a nível cardíaco, podendo estar associada às alterações fisiopatológicas da própria anemia crónica. Encontraram-se indicadores de prognóstico hematológicos e clínicos. Entre os primeiros, valores de Hb ≥8,5 g/dl e de HbF ≥13% foram considerados indicadores de bom prognóstico para a lesão pulmonar. Em relação aos parâmetros clínicos, as STA não foram consideradas indicadoras de prognóstico para a DPR ao contrário do que se verificou com o número de EVO. Pela análise dos parâmetros genéticos e socio-económicos provou-se a ausência de relação estatisticamente significativa com lesão pulmonar. Estudo 2. Pela RMN-CE foram diagnosticados ES em 33,3% com uma localização preferencial na substância branca profunda em 26,6% dos doentes. Relativamente aos parâmetros hematológicos seleccionados, o valor médio da HbF 8,6% constituíu um indicador de bom prognóstico para o aparecimento de ES, enquanto o valor médio de leucócitos 12.39x103/μl foi considerado um indicador de mau prognóstico. No estudo do DTC apenas um doente apresentou aumento da velocidade do fluxo cerebral na ACM igual a 196 cm/segundos, associado a vasculopatia grave. Os testes psicológicos alterados em 80% dos doentes mostraram ser o método mais sensível para detectar alterações do neurodesenvolvimento, mas sem correlação com os ES em 10% dos doentes. Realça-se a baixa percentagem de DTC patológicos encontrados neste estudo em relação ao número elevado de ES e de testes psicológicos alterados, não se verificando concordância entre os três exames. Dos indicadores de prognóstico estudados a -talassémia foi considerada um factor de protecção para o coeficiente de inteligência da escala de Wechsler. Em relação a parâmetros clínicos estudados os doentes com maior número de EVO, tem em média valores inferiores nos testes psicológicos. Estudo 3. Neste estudo verificou-se que o valor médio da HbF aumentou significativamente de 7,0±4% para 13,7±5,3% (p=0,028) ao fim de 15 meses de terapêutica com hidroxiureia. Clinicamente todos os doentes responderam significativamente com uma redução de 80% no número de EVO, 69% no número de internamentos, 76% no número de dias de hospitalização e 67% no número de transfusões. Deste modo comprovou-se não só a eficácia desta terapêutica neste grupo pediátrico como também a falta de efeitos secundários significativos. Considera-se a necessidade de estudos mais prolongados e em grande séries, para com segurança se usar a HU antes que a lesão orgânica se estabeleça, portanto logo nos primeiros anos de vida. Conclusão: Na amostra populacional estudada foram evidenciadas lesões pulmonares e cerebrais na grande maioria dos doentes que condicionaram a sua qualidade de vida. Foram identificados indicadores de prognóstico que poderão eventualmente ditar medidas terapêuticas precoces com o objectivo de diminuir a morbilidade e a mortalidade neste grupo etário. Demonstrou-se que a terapêutica com a HU foi eficaz e bem tolerada----------ABSTRACT: Background: Sickle cell anemia (SCA), a hereditary disease characterized by pain and lifetime multi-organic lesion, is a challenge for all that work with carriers of this disease. The clinical expression variability of SCA is a constant reality and a problem to be solved in the current world of investigation, for which the knowledge of prognostic indicators responsible for the different aspects of clinical evolution diversity wiil be an added value. The study is preceded by a historical summary of the most important factors in the evolution of SCA, which are in themselves, an incentive for future research. In the first part of the study, after an extensive bibliographical revision, physiopathology data is referred to in general and specifically regarding the target organs, that constituted the base for the studies presented in the dissertation. The state of the art for the complications to be studied, the choice of prognostic indicators and the therapeutics application, were approached for the renewed interest in the theme. Aims: In regard to the investigation, the objective was to study the lesions in the most affected organs of a chosen pediatric group, to investigate prognostic indicators for lung and cerebral lesions and to evaluate the protective effect of hydroxyurea in children with severe outcomes. Patients and methods: A prospective and multi-institutional study was carried out during a three-year period, February 1998 to March 2001, with children and adolescents followed up at a Immunohematology Outpatient Clinic of Dona Estefânia's Hospital, Lisbon. Based in predefined criteria, 30 children with SCA were selected in a stable phase of the disease, aged from seven to 18 years old, all of whom were of African origin with exception of one who was Caucasian. The diagnosis was based on electrophoresis techniques and molecular study that allowed to define the genotype, the presence of deletional alpha-thalassemia as well as haplotypes in the population. Different methodologies were used to evaluate the existence of lung and cerebral lesion. Statistical study of the different variables selected the prognostic indicators. In Study 1, to determine the existence of lung lesion two different methodologies were used: pulmonar function study with arterial blood gases determination; and high resolution computerized tomography. Heart dysfunction as a repercussion of lung lesion was also studied through echocardiography, and prognostic indicators were statistically significant for lesions found. The design of Study 2 was similar to Study 1, but with the appropriate methodology for CNS. After neurological examination, which was normal in all patients (control group), cerebral lesions were studied with imagiologic exams (MRN-CE and TCD) and psychological tests. These three methodologies were correlated and the importance of each one in the decision of the therapeutic profilactic attitudes. Study 3 consisted of a controlled prospective open study in children with severe forms of SCA, with the aim of the evaluating therapeutic effectiveness of hydroxyurea, during a period of 24 months. Results: In the global overall study preceding the Studies 1,2 and 3, there were a prevalence of haplotype Bantu (53%) and other risk factors, namely the number of VOC (87,5%), sequestration crisis (18,75%), dactilytis in first year of life(31,2%), hyperhemolysis crisis (50%) and ATC in more than half of the patients (59,38%). This group of bad prognostic indicators, associated with the population of the lower class according to the Graffar scale, demonstrates the importance of primary health care services, information provided to the children and their relatives, as well as the interest in prophylactic therapeutics, specific screening and prenatal diagnosis. Study 1. It was evident from this study that slight RPD was diagnosed in 70% of the patients, because alterations of the diffusion had no statistical significance and arterial blood gases determinations were normal. Only one patient had restrictive lung disease related with numerous ACS. However ACS was not considered a prognostic indicator for RPD, contrary to the number of EVO. HRTC revealed discreet fibrotic lines that could be related with slight RPD, but the lack of correlation of these two exams (33%) supports the value of lung function tests for precocious diagnosis of RPD. Heart dysfunction was found in 86,7% of patients, does not reflect the repercussion of RPD, but with the physiopathology of chronic anemia. Hematologic and clinical prognostic indicators were found. Good prognostic indicators for the non-evolution of RPD with average Hb values of ≥ 8,5 g/dl and average HbF values of ≥13%, respectively. The genetic and social-economic factors had no statistical significance; nevertheless, they were more prevalent among Bantu haplotype (53,3%) in patients with RPD. Study 2. RMN-CE detected SI in 33,3% of the patients, with preferential location in deep white substance in 26,6% and in front lobe in 20%. This distribution can be related to structural aspects of the brain and with the high sensibility of this organ to hypoxia. From the hematological parameters selected, average HbF value 8,6% and average leucocyte count 12.39x103/μl were prognostic indicators with different meaning to SI. The increase in the total bilirubin related to hyperhemolysis clinically explains the genesis of SI In the TCD study, only one patient had increased cerebral flow speed >196 cm/sec in CMA, which corresponded to serious vasculopathy in AngioMR. This patient never present previously neurological symptoms and had several hyperhemolysis crisis and VOC as risk factors. Low percentage of pathological TCD in this study, in relation to the high number of SI and altered tests, although without correlation among the three exams, is probably attributed to factors related to the methodology, aspects of cerebral physiopathology or perhaps a sign of good prognostic if the duration of study had not been so short. TCD should be used as a screening method in the age groups with higher risk of AVC and should never be considered separately in prophylactic therapeutics indication. Psychological tests were the most sensitive method to detect neurodevelopment impairment; in 80% of patients the neuropsychologics tests were altered, but without correlation with SI (10%). Since SI can become evident during the first two years of life and develop with time, the first psychological tests should be carried out between 3 and 5 years of age to timely be referred to special education and stimulation programs. Prognostic indicators to psychological tests were also found: alpha-thalassemia was found to be a protection factor of the IQ, just as other hematologic factors (hematocrit, MGCV and erythrocytes count). In relation to clinical parameters, although without statistical significance, patients with larger number of VOC had average lower scores versus the average in tests, except in TP. Results from different studies were conclusive as to the type of lesion found and the importance of prognostic indicators. Study 3. All the patients completed a minimum of 15 months therapeutic treatment with the final average daily dose of 19±4 mg/kg/day. The average value of the fetal hemoglobin increased significantly from 7,0±3,9% to 13,7±5,3% (p=0.028). The HbF average values increased from 6% to 15% after 15 months of therapeutic treatment. Clinically there was a reduction of 80% in the number of VOE , 69% in the number of hospitalization, 76% in the number of days of hospitalization and 67% in the number of transfusions. Once again the effectiveness of this treatment in this pediatric group, as well as the lack of any significant secondary effects, was evident. The study confirms the need for further detailed research in order to safely effect the appropriate treatment prior to the development of organic lesions, which ideally should be in the first year of life. Conclusions: These results allow us to clarify the importance of either pulmonary lesions or either nervous central system impairment among patients, children and adolescents, with sickle cell anemia. These lesions were demonstrated in most of the patients studied compromising their quality of life and the mortality. The treatment with HU is proved to be effective and having low toxicity.

Aplicação de sistemas moleculares na detecção rápida de MDR-TB e XDR-TB

Mycobacterium tuberculosis é o agente etiológico da tuberculose em humanos e segundo as estimativas da Organização Mundial da Saúde, um terço da população mundial está infectada com esta bactéria, calculando-se que no ano de 2008 aproximadamente 9,4 milhões de pessoas contraíram tuberculose activa. Associada a esta tendência, encontra-se o aumento alarmante da incidência de tuberculose resistente aos antibióticos, mais propriamente da tuberculose multirresistente e extensivamente resistente. Nesta Dissertação estudaram-se três sistemas de detecção molecular (INNO-LiPA Rif. TB, Innogenetics, Ghent, Bélgica, MTBDRplus e MTBDRsl, GenoType, GmbH, Nehren, Alemanha), que permitem detectar o complexo M. tuberculosis e as mutações mais comuns associadas à resistência aos antibióticos de primeira e segunda linha. Para o efeito, na primeira parte do trabalho os três sistemas em estudo foram testados em 21 isolados clínicos pertencentes à colecção do Laboratório de Micobactérias do Instituto de Higiene e Medicina Tropical (IHMT, UNL), com o propósito de avaliar a sua capacidade para identificar o complexo M. tuberculosis e detectar mutações ligadas à resistência aos fármacos de primeira e segunda linha. Na segunda parte do trabalho, os sistemas INNO-LiPA Rif. TB e MTBDRplus foram testados em 33 amostras respiratórias com baciloscopia positiva, com o propósito de aferir a “performance” destes sistemas para a detecção directa de tuberculose multirresistente em amostras respiratórias. Na primeira parte do trabalho os três sistemas em estudo apresentaram elevada sensibilidade na identificação do complexo M. tuberculosis em culturas, bem como na detecção de mutações ligadas à resistência aos antibióticos de primeira linha, com excepção do etambutol. No que diz respeito à detecção da resistência aos antibióticos de segunda linha, não foi possível calcular os valores de sensibilidade e especificidade. Na segunda parte do trabalho, o INNO-LiPA Rif. TB demonstrou ser o sistema mais robusto para a análise directa de amostras respiratórias com baciloscopia positiva, para um diagnóstico precoce de tuberculose e detecção de resistência à rifampicina. O MTBDRplus não se mostrou uma alternativa viável ao INNO-LiPA Rif. TB, pois apresentou baixa sensibilidade para a identificação do complexo M. tuberculosis e vários problemas no passo de amplificação. O MTBDRsl não foi testado, por não ter sido detectada nenhuma amostra multirresistente.