mRNA metabolism: nonsense mediated mRNA decay as a tool for gene therapy and the role of human DIS3L2 in transcript degradation

Tese de mestrado em Biologia Humana e Ambiente, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2015

Nutrient deficiencies in apple plants (Pyrus malus L.)

A trial was carried out with one year old 'Ohio Beauty apples (grafted on 'Doucin'), grown on sand cu1ture, receining nutrient solutions lacking the following nutrients at the time: N, P, K, Ca, Mg, S, and B. The main conclusions are as follows: as the adequate and inadequate levels from leaf analysis were, respectively: N -2.22 and 1.53%, P - 0,17 and 0.05%, K - 1.32 and 0.33%; Ca -0.9.4 and 0.52%, Mg - 0.37 and 0.06%; S -0.18 and 0.08%; B -62 and 2k ppm.

Desenvolvimento de Meloidogyne exigua Goeldi, 1887, em raízes de cafeeiros, em três ambientes

Com o objetivo de se obter informações sobre o desenvolvimento de Meloidogyne exigua Goeldi, 1887, em raízes de cafeeiro (Coffea arabica L.) cv. Mundo Novo, em três ambientes, plântulas no estágio do primeiro par de folhas verdadeiras foram transplantadas para copos plásticos com 25O ml de solo. Trinta dias depois, cada muda foi infestada com 600 larvas pré-parasitas e transferidas para as diversas condições de estudo: casa de vegetação, sala de temperatura constante e próximo de plantas adultas num cafezal. As raízes foram coletadas cada dois dias, coloridas e dissecadas, sendo os exemplares de M. exigua classificados de acordo com o estádio de desenvolvimento. Os resultados permitiram concluir que o desenvolvimento de M. exigua foi influenciado pela temperatura, e, que da penetração das larvas às fêmeas com ovos foram necessários, respectivamente, 25,3 dias à temperatura média de 25,8°C, 37,3 dias a 22,1°C e 38 dias a 22,4ºC, para a casa de vegetação, sala de temperatura constante e cafezal. A temperatura base estimada a partir dos resultados obtidos pelo método do coeficiente de variação foi 15°C. Foram necessárias, em média, 6580 unidades de calor, acima de 15°C, para M. exigua completar o seu desenvolvimento.

Viabilidade do tratamento do soro de queijo com digestão anaeróbia

O principal objetivo deste trabalho con sistiu em avaliar a digestão anaeróbia de soro de queijo (68.000mg DQO/l), sem correção de pH, em digestores tipo mistura completa, semi-contínuos em escala laboratorial, mantidos à temperatura de 35° ± 1°C. Foi utilizado como inóculo estéreo bovino, com teor de sólidos totais igual a 8%. O experimento foi desenvolvido num período de 100 dias, onde os primeiros 20 dias foram caracterizados pela aclimatação do inóculo e quatro tempos de retenção (¸) de 20 dias. Foi adicionado em cada reator uma carga de 3,0g DQO/l de reator/dia. No primeiro ¸ a carga foi dividida em 3 partes e adicionada aos digestores em intervalos de 8 horas. No segundo ¸ a carga foi dividida em 2 partes e adicionada aos digestores em intervalos de 12 horas; no terceiro ¸ foi utilizado intervalo de 6 horas para a alimentação com a carga de DQO dividida em 4 porções; o quarto ¸ foi caracterizado pela alimentação da carga total de uma única vez. O acompanhamento do experimento foi feito por análises de acidez, alcalinidade e pH, juntamente com produção de biogás; foi avaliado também a DQO no efluente. Foi constatado que os sistemas de alimentação empregados no presente trabalho, influenciaram no desenvolvimento do processo, sendo que a produção de ácidos graxos voláteis foi acentuada durante os TRH estudados levando a um desequilíbrio populacional e, consequentemente, a uma baixa eficiência de tratamento. Nestas condições, a produção de biogás expressa em 1.biogás/g DQO destruído foi de 0,35 l/g.

Compared nymphal development of Tingis americana (Hemiptera, Tingidae) in two Handroanthus species (Bignoniaceae) and reproductive parameters in seedlings of Handroanthus heptaphyllus

Nymphs and adults of Tingis americana Drake, 1922 were found feeding on leaves of Handroanthus heptaphyllus (Vell.) Mattos and Handroanthus chrysotrichus (Mart. ex A. DC.) Mattos in the Botanic Garden, Porto Alegre, state of Rio Grande do Sul, Brazil. This is the first record of T. americana on these host plants and in the southern Brazil. We aimed to compare the nymphal development on both hosts and to analyze the reproductive parameters on H. heptaphyllus (25 ± 1ºC; 60 ± 10% RH; 16 h photophase). The mean nymphal period (days) was shorter in individuals reared on H. heptaphyllus (12.69 ± 0.076) than on H. chrysotrichus (19.11 ± 0.208) (P < 0.0001), however, nymph viability was similar. On H. heptaphyllus, the mean embryonic period lasted 12.32 ± 0.274 days and the egg viability was 92%. The mean total and daily fecundity were 310.0 ± 19.40 eggs/female and 7.46 ± 0.302 eggs/female/day, respectively. Paired males and females showed similar longevity (P = 0.0691), while unpaired females lived longer than unpaired males (P = 0.0460).

Padronização da técnica de marcação de caramujos Biomphalaria glabrata com radioestroncio

Caramujos Biomphalaria glabrata vetor da esquistossomose mansônica, utilizados em experiências de campo, nas quais se faz necessário sua identificação e/ou localização, devem ser marcados com radioestrôncio 85,89 ou 90. Sua marcação em laboratório deve ser procedida em aquários de vidro sem fundo de areia, temperatura de 26,5 ± 1ºC, pH ajustado ao valor encontrado no "habitat", aeração artificial, alimentação diária com pequenas porções de alface fresca e 25 ml de água desionizada por caramujo. O nível de atividade no caramujo pode ser estimado sabendo-se que para o tempo de marcação de 5 dias e atividades iniciais de 0,036 a 0,362 µCi/ml, a captação global para grupos de 10 caramujos está em torno de 84 ± 2% e que a marcação em água contendo cálcio, praticamente não se altera até concentrações de 50ppm. A sobrevida ao longo de nove semanas não foi influenciada pela presença de radioestrôncio no caramujo.

Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2008.

OBJECTIVE: To provide an update to the original Surviving Sepsis Campaign clinical management guidelines, "Surviving Sepsis Campaign Guidelines for Management of Severe Sepsis and Septic Shock," published in 2004. DESIGN: Modified Delphi method with a consensus conference of 55 international experts, several subsequent meetings of subgroups and key individuals, teleconferences, and electronic-based discussion among subgroups and among the entire committee. This process was conducted independently of any industry funding. METHODS: We used the Grades of Recommendation, Assessment, Development and Evaluation (GRADE) system to guide assessment of quality of evidence from high (A) to very low (D) and to determine the strength of recommendations. A strong recommendation (1) indicates that an intervention's desirable effects clearly outweigh its undesirable effects (risk, burden, cost) or clearly do not. Weak recommendations (2) indicate that the tradeoff between desirable and undesirable effects is less clear. The grade of strong or weak is considered of greater clinical importance than a difference in letter level of quality of evidence. In areas without complete agreement, a formal process of resolution was developed and applied. Recommendations are grouped into those directly targeting severe sepsis, recommendations targeting general care of the critically ill patient that are considered high priority in severe sepsis, and pediatric considerations. RESULTS: Key recommendations, listed by category, include early goal-directed resuscitation of the septic patient during the first 6 hrs after recognition (1C); blood cultures before antibiotic therapy (1C); imaging studies performed promptly to confirm potential source of infection (1C); administration of broad-spectrum antibiotic therapy within 1 hr of diagnosis of septic shock (1B) and severe sepsis without septic shock (1D); reassessment of antibiotic therapy with microbiology and clinical data to narrow coverage, when appropriate (1C); a usual 7-10 days of antibiotic therapy guided by clinical response (1D); source control with attention to the balance of risks and benefits of the chosen method (1C); administration of either crystalloid or colloid fluid resuscitation (1B); fluid challenge to restore mean circulating filling pressure (1C); reduction in rate of fluid administration with rising filing pressures and no improvement in tissue perfusion (1D); vasopressor preference for norepinephrine or dopamine to maintain an initial target of mean arterial pressure > or = 65 mm Hg (1C); dobutamine inotropic therapy when cardiac output remains low despite fluid resuscitation and combined inotropic/vasopressor therapy (1C); stress-dose steroid therapy given only in septic shock after blood pressure is identified to be poorly responsive to fluid and vasopressor therapy (2C); recombinant activated protein C in patients with severe sepsis and clinical assessment of high risk for death (2B except 2C for postoperative patients). In the absence of tissue hypoperfusion, coronary artery disease, or acute hemorrhage, target a hemoglobin of 7-9 g/dL (1B); a low tidal volume (1B) and limitation of inspiratory plateau pressure strategy (1C) for acute lung injury (ALI)/acute respiratory distress syndrome (ARDS); application of at least a minimal amount of positive end-expiratory pressure in acute lung injury (1C); head of bed elevation in mechanically ventilated patients unless contraindicated (1B); avoiding routine use of pulmonary artery catheters in ALI/ARDS (1A); to decrease days of mechanical ventilation and ICU length of stay, a conservative fluid strategy for patients with established ALI/ARDS who are not in shock (1C); protocols for weaning and sedation/analgesia (1B); using either intermittent bolus sedation or continuous infusion sedation with daily interruptions or lightening (1B); avoidance of neuromuscular blockers, if at all possible (1B); institution of glycemic control (1B), targeting a blood glucose < 150 mg/dL after initial stabilization (2C); equivalency of continuous veno-veno hemofiltration or intermittent hemodialysis (2B); prophylaxis for deep vein thrombosis (1A); use of stress ulcer prophylaxis to prevent upper gastrointestinal bleeding using H2 blockers (1A) or proton pump inhibitors (1B); and consideration of limitation of support where appropriate (1D). Recommendations specific to pediatric severe sepsis include greater use of physical examination therapeutic end points (2C); dopamine as the first drug of choice for hypotension (2C); steroids only in children with suspected or proven adrenal insufficiency (2C); and a recommendation against the use of recombinant activated protein C in children (1B). CONCLUSIONS: There was strong agreement among a large cohort of international experts regarding many level 1 recommendations for the best current care of patients with severe sepsis. Evidenced-based recommendations regarding the acute management of sepsis and septic shock are the first step toward improved outcomes for this important group of critically ill patients.

Effects of Laurus nobilis (Lauraceae) on Biomphalaria glabrata (Say, 1818)

Experiments were carried out using aqueous extracts from leaves and flowers of Laurus nobilis on Biomphalaria glabrata. Treatments were performed on blastula stage (± 15 h after first cleavage) and on adult snails (11-18 mm). In both instances they were exposed for 24 h to different concentrations of the extracts on snails (200 to 2500 ppm) and embryos (20 to 300 ppm) at 25 ± 1ºC. The embryos were observed for a period of 20 days after treatment and the snails for 10 days. Results obtained with leaf aqueous extracts have shown a degree of toxicity on embryos starting at a concentration of 125 ppm, the flower extract being effective at 35 ppm. The malformation obtained with the different concentrations falls into the unespecific type category, however some cephalic and shell malformations were found in embryos treated with concentrations over 50 ppm (leaves) and 25 ppm (flowers). The LD90 on adult snails obtained by treatments with flower and leaf extract was observed at concentrations of 340 ppm and 1900 ppm respectively.

Impact of carotid plaque screening on smoking cessation and other cardiovascular risk factors: a randomized controlled trial.

BACKGROUND: Screening of peripheral atherosclerosis is increasingly used, but few trials have examined its clinical impact. We aimed to assess whether carotid plaque screening helps smokers to improve their health behaviors and cardiovascular risk factors. METHODS: We randomly assigned 536 smokers aged 40 to 70 years to carotid plaque ultrasonographic screening (US group) vs no screening (control group) in addition to individual counseling and nicotine replacement therapy for all participants. Smokers with at least 1 plaque received pictures of their plaques with a 7-minute structured explanation. The outcomes included biochemically validated smoking cessation at 12 months (primary outcome) and changes in cardiovascular risk factor levels and Framingham risk score. RESULTS: At baseline, participants (mean age, 51.1 years; 45.0% women) smoked an average of 20 cigarettes per day with a median duration of 32 years. The US group had a high prevalence of carotid plaques (57.9%). At 12 months, smoking cessation rates were high, but did not differ between the US and control groups (24.9% vs 22.1%; P = .45). In the US group, cessation rates did not differ according to the presence or absence of plaques. Control of cardiovascular risk factors (ie, blood pressure and low-density lipoprotein cholesterol and hemoglobin A(1c) levels in diabetic patients) and mean absolute risk change in Framingham risk score did not differ between the groups. The mean absolute risk change in Framingham risk score was +0.6 in the US group vs +0.3 in the control group (P = .56). CONCLUSION: In smokers, carotid plaque screening performed in addition to thorough smoking cessation counseling is not associated with increased rates of smoking cessation or control of cardiovascular risk factors. Trial Registration  clinicaltrials.gov Identifier: NCT00548665.

Congenital heart disease in pregnancies complicated by maternal diabetes mellitus. An international clinical collaboration, literature review, and meta-analysis.

PURPOSE: Investigation of the incidence and distribution of congenital structural cardiac malformations among the offspring of mothers with diabetes type 1 and of the influence of periconceptional glycemic control. METHODS: Multicenter retrospective clinical study, literature review, and meta-analysis. The incidence and pattern of congenital heart disease in the own study population and in the literature on the offspring of type 1 diabetic mothers were compared with the incidence and spectrum of the various cardiovascular defects in the offspring of nondiabetic mothers as registered by EUROCAT Northern Netherlands. Medical records were, in addition, reviewed for HbA(1c) during the 1st trimester. RESULTS: The distribution of congenital heart anomalies in the own diabetic study population was in accordance with the distribution encountered in the literature. This distribution differed considerably from that in the nondiabetic population. Approximately half the cardiovascular defects were conotruncal anomalies. The authors' study demonstrated a remarkable increase in the likelihood of visceral heterotaxia and variants of single ventricle among these patients. As expected, elevated HbA(1c) values during the 1st trimester were associated with offspring fetal cardiovascular defects. CONCLUSION: This study shows an increased likelihood of specific heart anomalies, namely transposition of the great arteries, persistent truncus arteriosus, visceral heterotaxia and single ventricle, among offspring of diabetic mothers. This suggests a profound teratogenic effect at a very early stage in cardiogenesis. The study emphasizes the frequency with which the offspring of diabetes-complicated pregnancies suffer from complex forms of congenital heart disease. Pregnancies with poor 1st-trimester glycemic control are more prone to the presence of fetal heart disease.

Influência da temperatura na biologia de triatomíneos: IX. Rhodnius nasutus Stal, 1859 (Hemiptera, Reduviidae)

Estudou-se a influência da temperatura na biologia de Rhodnius nasutus Stal, 1859 (Hemiptera, Reduviidae), tendo em vista a sua criação em grande escala, em laboratório, para serem utilizados no xenodiagnóstico e fornecer informações que subsidien as ações de controle. Os experimentos realizaram-se em duas câmaras climatizadas, a 25 ± 0,5ºC e 3º ± 1ºC, com umidade relativa de 70 ± 5% e fotoperíodo de 12 horas. Apresentam-se os dados relativos ao ciclo evolutivo, pré-postura, fecundidade, fertilidade, sobrevivência ao jejum e quantidade de sangue ingerido. A duração média do ciclo evolutivo foi de 145,7 dias a 25ºC e de 114,2 dias, a 30ºC. A quantidade de sangue ingerido para completar o ciclo evolutivo foi de 304,3mg e 350,3 mg, a 25º e 30ºC, respectivamente. A sobrevivência ao jejum absoluto foi significativamente maior a 25º do que a 30ºC, ao nível de 1%.

Effect of carbohydrate overfeeding on whole body macronutrient metabolism and expression of lipogenic enzymes in adipose tissue of lean and overweight humans

OBJECTIVE: Lipids stored in adipose tissue can originate from dietary lipids or from de novo lipogenesis (DNL) from carbohydrates. Whether DNL is abnormal in adipose tissue of overweight individuals remains unknown. The present study was undertaken to assess the effect of carbohydrate overfeeding on glucose-induced whole body DNL and adipose tissue lipogenic gene expression in lean and overweight humans. DESIGN: Prospective, cross-over study. SUBJECTS AND METHODS: A total of 11 lean (five male, six female, mean BMI 21.0+/-0.5 kg/m(2)) and eight overweight (four males, four females, mean BMI 30.1+/-0.6 kg/m(2)) volunteers were studied on two occasions. On one occasion, they received an isoenergetic diet containing 50% carbohydrate for 4 days prior to testing; on the other, they received a hyperenergetic diet (175% energy requirements) containing 71% carbohydrates. After each period of 4 days of controlled diet, they were studied over 6 h after having received 3.25 g glucose/kg fat free mass. Whole body glucose oxidation and net DNL were monitored by means of indirect calorimetry. An adipose tissue biopsy was obtained at the end of this 6-h period and the levels of SREBP-1c, acetyl CoA carboxylase, and fatty acid synthase mRNA were measured by real-time PCR. RESULTS: After isocaloric feeding, whole body net DNL amounted to 35+/-9 mg/kg fat free mass/5 h in lean subjects and to 49+/-3 mg/kg fat free mass/5 h in overweight subjects over the 5 h following glucose ingestion. These figures increased (P<0.001) to 156+/-21 mg/kg fat free mass/5 h in lean and 64+/-11 mg/kg fat free mass/5 h (P<0.05 vs lean) in overweight subjects after carbohydrate overfeeding. Whole body DNL after overfeeding was lower (P<0.001) and glycogen synthesis was higher (P<0.001) in overweight than in normal subjects. Adipose tissue SREBP-1c mRNA increased by 25% in overweight and by 43% in lean subjects (P<0.05) after carbohydrate overfeeding, whereas fatty acid synthase mRNA increased by 66 and 84% (P<0.05). CONCLUSION: Whole body net DNL is not increased during carbohydrate overfeeding in overweight individuals. Stimulation of adipose lipogenic enzymes is also not higher in overweight subjects. Carbohydrate overfeeding does not stimulate whole body net DNL nor expression of lipogenic enzymes in adipose tissue to a larger extent in overweight than lean subjects.

Estudo comparado do ciclo evolutivo de triatomíneos a duas temperaturas

We studied the life cicle of several triatominae species: Dipetalogaster maximus (Uhler, 1894); Panstrongylus herreri Wygodzinsky, 1948; Panstrongylus megistus (Burmeister, 1835); Rhodnius ecuadoriensis Lent & Leon, 1958; Rhodnius nasutus Stal, 1859; Rhodnius neglectus Lent, 1954; Rhodnius pictipes Stal, 1872; Rhodnius prolixus Stal, 1859; Rhodnius robustus Larrousse, 1927; Triatoma brasiliensis Neiva, 1911; Triatoma infestans (Klug, 1834); Triatoma maculata (Erichson, 1848); Triatoma matogrossensis Leite & Barbosa, 1953; Triatoma platensis Neiva, 1913; Triatoma protracta (Uhler, 1894); Triatoma sordida (Stal, 1859); Triatoma tibiamaculata (Pinto, 1926) e Triatoma vitticeps (Stal, 1859) (Hemiptera, Reduviidae). The main purpose of the study was to obtain information to improve control measures specially in those peridomiciliar species. The experiments were performed in two climatized chambers, both with an humidity of 70 ± 5% and photoperiod of 12 hours. One was maintained at 25 ± 0,5ºC and the other at 30 ± 1ºC.

T1-weighted MRI as a substitute to CT for refocusing planning in MR-guided focused ultrasound.

Precise focusing is essential for transcranial MRI-guided focused ultrasound (TcMRgFUS) to minimize collateral damage to non-diseased tissues and to achieve temperatures capable of inducing coagulative necrosis at acceptable power deposition levels. CT is usually used for this refocusing but requires a separate study (CT) ahead of the TcMRgFUS procedure. The goal of this study was to determine whether MRI using an appropriate sequence would be a viable alternative to CT for planning ultrasound refocusing in TcMRgFUS. We tested three MRI pulse sequences (3D T1 weighted 3D volume interpolated breath hold examination (VIBE), proton density weighted 3D sampling perfection with applications optimized contrasts using different flip angle evolution and 3D true fast imaging with steady state precision T2-weighted imaging) on patients who have already had a CT scan performed. We made detailed measurements of the calvarial structure based on the MRI data and compared those so-called 'virtual CT' to detailed measurements of the calvarial structure based on the CT data, used as a reference standard. We then loaded both standard and virtual CT in a TcMRgFUS device and compared the calculated phase correction values, as well as the temperature elevation in a phantom. A series of Bland-Altman measurement agreement analyses showed T1 3D VIBE as the optimal MRI sequence, with respect to minimizing the measurement discrepancy between the MRI derived total skull thickness measurement and the CT derived total skull thickness measurement (mean measurement discrepancy: 0.025; 95% CL (-0.22-0.27); p = 0.825). The T1-weighted sequence was also optimal in estimating skull CT density and skull layer thickness. The mean difference between the phase shifts calculated with the standard CT and the virtual CT reconstructed from the T1 dataset was 0.08 ± 1.2 rad on patients and 0.1 ± 0.9 rad on phantom. Compared to the real CT, the MR-based correction showed a 1 °C drop on the maximum temperature elevation in the phantom (7% relative drop). Without any correction, the maximum temperature was down 6 °C (43% relative drop). We have developed an approach that allows for a reconstruction of a virtual CT dataset from MRI to perform phase correction in TcMRgFUS.

Potassium restores vasorelaxation of resistance arterioles in non-hypertensive DOCA/salt fed mice.

BACKGROUND: Potassium-enriched diets exert renal and cardiovascular protective effects, but the underlying mechanisms are largely unknown. METHODS: Using the dorsal skinfold chamber model for intravital microscopy, we examined endothelium-dependent vasorelaxation of precapillary resistance arterioles in response to acetylcholine or the NO donor SNAP in awake mice. Experiments were performed in uni-nephrectomized one renin gene (Ren-1c) C57BL/6 mice (control group) and in mice having received a continuous administration of deoxycorticosterone acetate and a dietary supplementation of 1% sodium chloride for 8weeks (DOCA/salt group). An additional group of DOCA/salt treated animals received a dietary supplement of 0.4% KCl for 3weeks prior to the experiments (DOCA/salt + potassium group). RESULTS: DOCA/salt treatment for 8weeks resulted in hypokalemia, but blood pressure remained unchanged. In DOCA/salt mice, relaxation of resistance arterioles was blunted in response to acetylcholine, and to a lesser extent to SNAP, suggesting endothelial dysfunction. Endothelium-dependent vasorelaxation was restored by the potassium-enriched diet. CONCLUSION: This study is the first to demonstrate a protective effect of potassium on endothelium-dependent vasorelaxation in the absence of confounding anti-hypertensive effects, as observed in most animal models and the clinical situation. We propose that the known cardio- and nephro-protective effects of potassium might - at least in part - be mediated by the salutary effects on endothelium-dependent arteriolar relaxation.