Exploration génétique de l'hypothyroïdie congénitale par dysgénésie thyroïdienne

L'hypothyroïdie congénitale par dysgénésie thyroïdienne (HCDT, ectopie dans plus de 80 %) a une prévalence de 1 cas sur 4000 naissances vivantes. L’HCDT est la conséquence d'une défaillance de la thyroïde embryonnaire à se différencier, à se maintenir ou à migrer vers sa localisation anatomique (partie antérieure du cou), qui aboutit à une absence totale de la thyroïde (athyréose) ou à une ectopie thyroïdienne (linguale ou sublinguale). Les HCDT sont principalement non-syndromiques (soit 98% des cas sont non-familiale), ont un taux de discordance de 92% chez les jumeaux monozygotes, et ont une prédominance féminine et ethnique (i.e., Caucasienne). La majorité des cas d’HCDT n’a pas de cause connue, mais est associée à un déficit sévère en hormones thyroïdiennes (hypothyroïdie). Des mutations germinales dans les facteurs de transcription liés à la thyroïde (NKX2.1, FOXE1, PAX8, NKX2.5) ont été identifiées dans seulement 3% des patients atteints d’HCDT sporadiques et l’analyse de liaisons exclue ces gènes dans les rares familles multiplex avec HCDT. Nous supposons que le manque de transmission familiale claire d’HCDT peut résulter de la nécessité d’au moins deux « hits » génétiques différents dans des gènes importants pour le développement thyroïdien. Pour répondre au mieux nos questions de recherche, nous avons utilisé deux approches différentes: 1) une approche gène candidat, FOXE1, seul gène impliqué dans l’ectopie dans le modèle murin et 2) une approche en utilisant les techniques de séquençage de nouvelle génération (NGS) afin de trouver des variants génétiques pouvant expliquer cette pathologie au sein d’une cohorte de patients avec HCDT. Pour la première approche, une étude cas-contrôles a été réalisée sur le promoteur de FOXE1. Il a récemment été découvert qu’une région du promoteur de FOXE1 est différentiellement méthylée au niveau de deux dinucléotides CpG consécutifs, définissant une zone cruciale de contrôle de l’expression de FOXE1. L’analyse d’association basée sur les haplotypes a révélé qu’un haplotype (Hap1: ACCCCCCdel1C) est associé avec le HCDT chez les Caucasiens (p = 5x10-03). Une réduction significative de l’activité luciférase est observée pour Hap1 (réduction de 68%, p<0.001) comparé au promoteur WT de FOXE1. Une réduction de 50% de l’expression de FOXE1 dans une lignée de cellules thyroïdienne humaine est suffisante pour réduire significativement la migration cellulaire (réduction de 55%, p<0.05). Un autre haplotype (Hap2: ACCCCCCC) est observé moins fréquemment chez les Afro-Américain comparés aux Caucasiens (p = 1.7x10-03) et Hap2 diminue l’activité luciférase (réduction de 26%, p<0.001). Deux haplotypes distincts sont trouvés fréquemment dans les contrôles Africains (Black-African descents). Le premier haplotype (Hap3: GTCCCAAC) est fréquent (30.2%) chez les contrôles Afro-Américains comparés aux contrôles Caucasiens (6.3%; p = 2.59 x 10-9) tandis que le second haplotype (Hap4: GTCCGCAC) est trouvé exclusivement chez les contrôles Afro-Américains (9.4%) et est absent chez les contrôles Caucasiens (P = 2.59 x 10-6). Pour la deuxième approche, le séquençage de l’exome de l’ADN leucocytaire entre les jumeaux MZ discordants n’a révélé aucune différence. D'où l'intérêt du projet de séquençage de l’ADN et l’ARN de thyroïdes ectopiques et orthotopiques dans lesquelles de l'expression monoallélique aléatoire dans a été observée, ce qui pourrait expliquer comment une mutation monoallélique peut avoir des conséquences pathogéniques. Finalement, le séquençage de l’exome d’une cohorte de 36 cas atteints d’HCDT a permis d’identifier de nouveaux variants probablement pathogéniques dans les gènes récurrents RYR3, SSPO, IKBKE et TNXB. Ces quatre gènes sont impliqués dans l’adhésion focale (jouant un rôle dans la migration cellulaire), suggérant un rôle direct dans les défauts de migration de la thyroïde. Les essais de migration montrent une forte diminution (au moins 60% à 5h) de la migration des cellules thyroïdiennes infectées par shRNA comparés au shCtrl dans 2 de ces gènes. Des zebrafish KO (-/- et +/-) pour ces nouveaux gènes seront réalisés afin d’évaluer leur impact sur l’embryologie de la thyroïde.

Insuficiência ovárica prematura

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

The effect of chaperone compounds on glucocerebrosidase stability and activity in fibroblasts from Gaucher Disease patients with different genotypes

Dissertação de mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2014

The Outcome of Infantile Onset Pompe Disease in South of Iran

Background: Infantile Onset Pompe Disease (IOPD) is a rare autosomal recessive neuromuscular disorder. It is associated with cardiomegaly, hypotonia, paresis, and death in the first year of life. Since 2006, following the use of Alglucosidase alfa as Enzyme Replacement Therapy (ERT), the patients’ survival is improved to a noticeable extent. Objectives: The purpose of this study is to examine the outcome of IOPD patients in South of Iran and the degree of responsiveness to ERT. Patients and Methods: All patients who were diagnosed with IOPD on the bases of clinical symptoms, and enzyme assay on dried blood spot, were included in the study; and were followed up regarding cardiac function, locomotor activity, and cognition. Results: Six patients with IOPD were identified. All these six patients suffered from Hypertrophic Cardiomyopathy (HCM). Four (67%) of them also had generalized hypotonia. Three patients expired during the first weeks due to severe respiratory infection. One of them also got involved with Acute Cardiopulmonary Failure while receiving the fifth dose of ERT; and expired. However, the remaining two patients had a significant improvement after the maximum of 117 weeks of following up both cardiac and locomotor findings. These two patients were the same patients who showed cardiac symptoms from the beginning but did not have generalized hypotonia. Conclusions: Although ERT has a significant effect on enhancing the survival of IOPD patients, it should be associated with meticulous heart-respiratory cares during the first months of treatment and preventing infection especially nosocomial infections.

EVERSUN: A phase 2 trial of alternating sunitinib and everolimus as first-line therapy for advanced renal cell carcinoma

Background We hypothesised that alternating inhibitors of the vascular endothelial growth factor receptor (VEGFR) and mammalian target of rapamycin pathways would delay the development of resistance in advanced renal cell carcinoma (aRCC). Patients and methods A single-arm, two-stage, multicentre, phase 2 trial to determine the activity, feasibility, and safety of 12-week cycles of sunitinib 50 mg daily 4 weeks on / 2 weeks off, alternating with everolimus 10 mg daily for 5 weeks on / 1 week off, until disease progression or prohibitive toxicity in favourable or intermediate-risk aRCC. The primary end point was proportion alive and progression-free at 6 months (PFS6m). The secondary end points were feasibility, tumour response, overall survival (OS), and adverse events (AEs). The correlative objective was to assess biomarkers and correlate with clinical outcome. Results We recruited 55 eligible participants from September 2010 to August 2012. Demographics: mean age 61, 71% male, favourable risk 16%, intermediate risk 84%. Cycle 2 commenced within 14 weeks for 80% of participants; 64% received ≥22 weeks of alternating therapy; 78% received ≥22 weeks of any treatment. PFS6m was 29/55 (53%; 95% confidence interval [CI] 40% to 66%). Tumour response rate was 7/55 (13%; 95% CI 4% to 22%, all partial responses). After median follow-up of 20 months, 47 of 55 (86%) had progressed with a median progression-free survival of 8 months (95% CI 5–10), and 30 of 55 (55%) had died with a median OS of 17 months (95% CI 12–undefined). AEs were consistent with those expected for each single agent. No convincing prognostic biomarkers were identified. Conclusions The EVERSUN regimen was feasible and safe, but its activity did not meet pre-specified values to warrant further research. This supports the current approach of continuing anti-VEGF therapy until progression or prohibitive toxicity before changing treatment.

Part I: Enzyme replacement versus neurotrophic factor viral vector-mediated gene therapy of Parkinson’s disease, Part II: The effects of orally dosed tolcapone and entacapone on dopamine metabolism in the dorsal striatum and nucleus accumbens in rats

Part I: Parkinson’s disease is a slowly progressive neurodegenerative disorder in which particularly the dopaminergic neurons of the substantia nigra pars compacta degenerate and die. Current conventional treatment is based on restraining symptoms but it has no effect on the progression of the disease. Gene therapy research has focused on the possibility of restoring the lost brain function by at least two means: substitution of critical enzymes needed for the synthesis of dopamine and slowing down the progression of the disease by supporting the functions of the remaining nigral dopaminergic neurons by neurotrophic factors. The striatal levels of enzymes such as tyrosine hydroxylase, dopadecarboxylase and GTP-CH1 are decreased as the disease progresses. By replacing one or all of the enzymes, dopamine levels in the striatum may be restored to normal and behavioral impairments caused by the disease may be ameliorated especially in the later stages of the disease. The neurotrophic factors glial cell derived neurotrophic factor (GDNF) and neurturin have shown to protect and restore functions of dopaminergic cell somas and terminals as well as improve behavior in animal lesion models. This therapy may be best suited at the early stages of the disease when there are more dopaminergic neurons for neurotrophic factors to reach. Viral vector-mediated gene transfer provides a tool to deliver proteins with complex structures into specific brain locations and provides long-term protein over-expression. Part II: The aim of our study was to investigate the effects of two orally dosed COMT inhibitors entacapone (10 and 30 mg/kg) and tolcapone (10 and 30 mg/kg) with a subsequent administration of a peripheral dopadecarboxylase inhibitor carbidopa (30 mg/kg) and L- dopa (30 mg/kg) on dopamine and its metabolite levels in the dorsal striatum and nucleus accumbens of freely moving rats using dual-probe in vivo microdialysis. Earlier similarly designed studies have only been conducted in the dorsal striatum. We also confirmed the result of earlier ex vivo studies regarding the effects of intraperitoneally dosed tolcapone (30 mg/kg) and entacapone (30 mg/kg) on striatal and hepatic COMT activity. The results obtained from the dorsal striatum were generally in line with earlier studies, where tolcapone tended to increase dopamine and DOPAC levels and decrease HVA levels. Entacapone tended to keep striatal dopamine and HVA levels elevated longer than in controls and also tended to elevate the levels of DOPAC. Surprisingly in the nucleus accumbens, dopamine levels after either dose of entacapone or tolcapone were not elevated. Accumbal DOPAC levels, especially in the tolcapone 30 mg/kg group, were elevated nearly to the same extent as measured in the dorsal striatum. Entacapone 10 mg/kg elevated accumbal HVA levels more than the dose of 30 mg/kg and the effect was more pronounced in the nucleus accumbens than in the dorsal striatum. This suggests that entacapone 30 mg/kg has minor central effects. Also our ex vivo study results obtained from the dorsal striatum suggest that entacapone 30 mg/kg has minor and transient central effects, even though central HVA levels were not suppressed below those of the control group in either brain area in the microdialysis study. Both entacapone and tolcapone suppressed hepatic COMT activity more than striatal COMT activity. Tolcapone was more effective than entacapone in the dorsal striatum. The differences between dopamine and its metabolite levels in the dorsal striatum and nucleus accumbens may be due to different properties of the two brain areas.

Revascularization versus medical therapy for renal-artery stenosis

Percutaneous revascularization of the renal arteries improves patency in atherosclerotic renovascular disease, yet evidence of a clinical benefit is limited.

A high-value, low-cost bubble continuous positive airway pressure system for low-resource settings:technical assessment and initial case reports

Acute respiratory infections are the leading cause of global child mortality. In the developing world, nasal oxygen therapy is often the only treatment option for babies who are suffering from respiratory distress. Without the added pressure of bubble Continuous Positive Airway Pressure (bCPAP) which helps maintain alveoli open, babies struggle to breathe and can suffer serious complications, and frequently death. A stand-alone bCPAP device can cost $6,000, too expensive for most developing world hospitals. Here, we describe the design and technical evaluation of a new, rugged bCPAP system that can be made in small volume for a cost-of-goods of approximately $350. Moreover, because of its simple design--consumer-grade pumps, medical tubing, and regulators--it requires only the simple replacement of a <$1 diaphragm approximately every 2 years for maintenance. The low-cost bCPAP device delivers pressure and flow equivalent to those of a reference bCPAP system used in the developed world. We describe the initial clinical cases of a child with bronchiolitis and a neonate with respiratory distress who were treated successfully with the new bCPAP device.

Gestão integrada da doença renal crónica em Portugal : o panorama atual e o papel do profissional de nutrição na obtenção de melhores resultados em saúde

RESUMO - A doença renal crónica (DRC) resulta da perda de função renal, sendo necessário a terapêutica de substituição, no estádio terminal. Em Portugal está atualmente em vigor o modelo de Gestão Integrada da Doença, que tem inerente o cumprimento de objetivos e metas pelas unidades de diálise. Uma alimentação adequada é um pilar fundamental ao sucesso do tratamento desta doença, o que torna o profissional de nutrição indispensável. Este trabalho pretendeu avaliar o cumprimento das metas e objetivos estabelecidos no modelo referido, e relacionar os resultados obtidos com a existência de contacto entre o profissional de nutrição e os pacientes. Para a persecução dos objetivos, foram analisadas duas bases de dados disponibilizadas pela Direção Geral da Saúde: a base de dados da Plataforma de Gestão Integrada da Doença Renal Crónica em 2012 e a do Questionário de Avaliação da Satisfação dos Doentes em Hemodiálise em 2013. Verificou-se uma melhoria contínua ao longo dos anos do cumprimento das metas e objetivos preconizados em Portugal para o tratamento da DRC, com um cumprimento da maioria no ano de 2012. No entanto, os parâmetros ferritina e albumina sérica ficaram aquém da recomendação. Observou-se um nível elevado de satisfação do paciente quanto ao trabalho do profissional de nutrição, apesar de ser frequente a inexistência de contacto entre ambas as partes. Os resultados obtidos demonstram também que o profissional de nutrição tem um papel importante para a obtenção de melhores resultados de saúde nos pacientes em tratamento por hemodiálise, pelo que se sugere um acompanhamento da totalidade deste tipo de população por este profissional.

Ultrastructural and proliferative features of the ventral lobe of the prostate in non-obese diabetic mice (NOD) following androgen and estrogen replacement associated to insulin therapy

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Importance of early and continuous use of protein restriction on the progression of adriamycin nephropathy

Three experimental protocols were carried out with the aim of evaluating the role of protein restriction on the progression of the established adriamycin-induced nephropathy, and whether the protective effect of the diet persists after the diet is discontinued. The effect of a low protein diet (LPD) was studied for 6 weeks in protocol 1, 16 weeks in protocol 2 and for 28 weeks in protocol 3. In protocol 3, one group (LL) received LPD and another (NN) was given a normal protein diet (NPD). A third group (LN) received LPD for 16 weeks and then NPD for 12 weeks and a fourth group (NL) was fed NPD for 16 weeks and then LPD for 12 weeks. In protocol I the tubulo- interstitial index (TILl) of rats on LPD (Md = 2, P25 = 0.0; P75 = 3.5) after six weeks, was smaller than that of the animals on NPD (Md = 6.0; P25 = 3.0; P75 = 8.0; p < 0.05). In protocol 2, the group taking LPD presented an area of interstitial fibrosis (IF) (Md= 0.5%, P25 0.2%; P75 = 1.9%) smaller than that of the NPD group (Md = 6.8%; P25 = 5.2%; P75 = 7.1%; P < 0.05). No significant difference in the area of glomerulosclerosis (GSA) was observed between the animals on LPD (Md = 0.0%; P25 = 0.0%, P75 = 0.0%) and NPD (Md = 0.37%; P25 = 02% P75 = 1.25%; p > 0.05). In protocol 3, the group LL showed GSA (Md = 1.3%; P25 0.6%, P75 = 2.5%) and IF (Md = 3.60/0; P25 = 1.6%; P75 = 5.9%) smaller that those of LN (GSA Md = 10.1%; P25 = 6.6%; P75 = 14.8%; IF; Md = 17.3%; P25 = 14.1%; P75 = 24,5%), NL (GSA: Md = 9.1%; P25 = 5,8%; P75 = 11.7%; IF; Md = 25.0%; P25 = 20.4%; P75 = 30%), and NN (GSA: Md = 6. 75%; P25 = 4.9%; P75 = 11.7%; IF: Md = 20.9%; P25 = 16.2%; P75 = 32.4%). In conclusion, in order to be effective, LPD must be introduced early and maintained for a long period of tune.