A clinico-pathological study of 163 untreated cases of chronic hepatitis C

We performed a clinico-pathological study of 163 untreated cases of chronic hepatitis C. Eighty five percent of the patients were clinically asymptomatic and their physical examinations sbowed unremarkable or minimal changes at the time of the liver biopsy Liver function tests tended to present slight abnormalities, involving mild elevations of the activity of the aminotransferases and gamma-glutamil transferase levels. In spite of these mild abnormalities advanced chronic liver disease ivas histologically detected in eighty nine percent of the patients, mainly showing chronic active hepatitis. The most characteristic histological finding ivas an interlobular bile duct damage which correlated with the presence of tymphoid aggregates in the portal tracts and with the development of fibrosis.

Safety evaluation of SPF66 malaria vaccine in Brazil

The frequency and description of side effects secondaiy to the subcutaneous application of SPf66 malaria vaccine and placebo are reported for each dose of application in the participants of the vaccine efficacy trial in Brazil. Side effects evaluated two hours after each application were detected in 8.0%, 30.2% and 8.8%, for the Is', and 3"' dose, respectively, in the SPf66group, and in 7.0%, 8.5% and 2.9% in the placebo group. Local reactions such as mild inflammation, nodule and pain or erythema frequently accompanied by pruritus were the most common reactions detected in both groups (3-8%, 29.1% and 8.5% in the SPf66 group and 4.0%, 7.6% and 2.5% in the placebo group). Among vaccinees, local side effects after the 2nd dose were more frequent in females. Systemic side effects were expressed mainly through general symptoms referred by the participants and were most frequent after the 1st dose in both groups (4.3% in the SPf66 group and 3-0% in the placebo group). Muscle aches and fever were refewred by few participants. No severe adverse reactions were detected for either dose of application or group.

Efeitos da laquea����o proximal dos vasos curtos e da fundoplicatura total sobre a microcircula����o do fundo g��strico : estudo microvascular em cobaias

RESUMO: A opera����o de Nissen, por laparoscopia, �� considerada a cirurgia antirefluxo mais adequada por ser a que melhor replica a fisiologia normal da v��lvula gastresof��gica na maioria dos doentes com sintomas t��picos de doen��a do refluxo gastresof��gico (DRGE). S��o crit��rios t��cnicos o encerramento seguro dos pilares do diafragma e a cria����o de fundoplicatura completa (360 graus), curta (inferior a dois cent��metros), lassa e sem tens��o ��� desiderando para o qual a laquea����o proximal dos vasos curtos g��stricos �� crucial. Realizei a opera����o de Nissen, por laparoscopia, em sessenta mulheres e quarenta homens com DRGE, sem mortalidade operat��ria, no Servi��o de Cirurgia 6 do Hospital dos Capuchos, CHLC, EPE. Os cem doentes apresentavam m��dia et��ria de 46 anos e queixas, com tempo de evolu����o entre 1 e 43 anos, de pirose (90%), regurgita����o (80%), azia (73%), epigastralgias (54%). A endoscopia alta revelou esofagite de grau Savary-Miller 0-I (62%), II (23%), III (8%), IV (7%); h��rnia de deslizamento (71%), h��rnia paraesof��gica (8%), sem h��rnia (21%); a pHmetria de 24h diagnosticou padr��o misto (38%), levantado (20%), deitado (20%), inconclusiva (22%) e a manometria diagnosticou EEI hipot��nico (35%), perist��lise esof��gica normal (88%), hipomotilidade ligeira (5%) e foi omissa (7%). H��rnia hiatal, esofagite grave, inefic��cia do controlo sintom��tico com inibidor da bomba de prot��es e desejo de descontinuidade terap��utica constitu��ram as indica����es para tratamento cir��rgico. Por celioscopia, efetuei laquea����o dos vasos curtos g��stricos (70%), cruroplastia e fundoplicatura total (seda 2/0), curta (dimens��o m��dia 1,5-2 cm), lassa, sem tens��o e sem calibra����o intraoperat��ria do es��fago. A fundoplicatura de Nissen laparosc��pica mostrou-se segura e eficaz no tratamento da DRGE. A sua idoneidade foi ainda comprovada pela normaliza����o da pHmetria de 24 horas e da manometria p��s-operat��rias, com significado estat��stico, num grupo de catorze volunt��rios assintom��ticos. Em catamnese com recuo m��dio 30,7 meses 94% dos indiv��duos persistem assintom��ticos. Interrogando-me acerca das repercuss��es desta opera����o sobre a microcircula����o do fundo g��strico coloquei, como premissa, a possibilidade de na opera����o de Nissen a laquea����o dos vasos curtos poder induzir modifica����o no di��metro arteriolar da parede do fundo g��strico. Para pesquisar a influ��ncia da laquea����o dos vasos curtos g��stricos e da fundoplicatura total sobre o calibre arteriolar da parede do est��mago no c��rdia, no fundo e na regi��o dos vasos curtos g��stricos, idealizei um Projeto de investiga����o experimental em cobaias. O Projeto foi desenvolvido no Centro de Investiga����o do Departamento de Anatomia da FCM-UNL. Para a sua realiza����o obtive autoriza����o da Comiss��o Cient��fica e Pedag��gica da FCM-UNL, requeri a acredita����o como investigador �� Dire����o Geral de Veterin��ria e, por recorrer �� utiliza����o de animais, submeti-o �� Comiss��o de ��tica da FCM-UNL, que o aprovou por unanimidade. Para limitar o n��mero de animais utilizados ao m��nimo necess��rio, calculei, por m��todo estat��stico, a quantidade de cobaias necess��rias. Subdividindo-as num grupo de ensaio (GE), onde realizei a opera����o de Nissen, e num grupo de controlo (GC), onde apenas procedi a tra����o g��strica, defini e apliquei protocolos de anestesia, de cirurgia e de eutan��sia, segundo os princ��pios dos 3R ��� Replacement, Reduction, Refinement da t��cnica de experimenta����o humana de Russell e Burch (1959) ��� uma estrutura ��tica amplamente aceite para a realiza����o de experimenta����o cient��fica humanizada com animais. A utiliza����o das t��cnicas de estudo angiomorfol��gico permitiu-me analisar e descrever a anatomia normal, a vasculariza����o arterial macrosc��pica, a microangioarquitetura, por microscopia eletr��nica de varrimento de moldes de corros��o vascular, e a histologia da parede do est��mago da cobaia. Procedi, tamb��m, �� defini����o dos crit��rios morfol��gicos que considerei suscet��veis de valida����o deste modelo animal para o estudo proposto. Por raz��es acad��micas, foi necess��rio abreviar o Projeto encurtando, em cerca de dois anos, o prazo dispon��vel para conclus��o do estudo. Apreciando-o com o Gabinete de An��lise Epidemiol��gica e Estat��stica do Centro de Investiga����o do CHLC, EPE, optou-se, perante a escassez de elementos ap��s j�� terem sido recrutados 46 animais, por uma amostra, suplementar, de dimens��o de conveni��ncia de oito cobaias (quatro em cada grupo), condicionada pelo limite temporal universit��rio e pelo respeito pela dignidade dos animais. Neste subgrupo procedi, por microscopia eletr��nica de varrimento, �� medi����o dos calibres arteriolares nos moldes vasculares do c��rdia, do fundo e da zona dos vasos curtos g��stricos tanto no GC como no GE efetuando 469 medi����es no primeiro e 461 no ��ltimo. Os dados foram enviados ao Centro de Investiga����o do CHLC, EPE que procedeu �� sua an��lise estat��stica (ANOVA). A referida an��lise revelou que as arter��olas do plexo mucoso e as do plexo submucoso do c��rdia, do fundo e da regi��o dos vasos curtos g��stricos, mostraram aumento de calibre no GE. O aumento foi, estatisticamente, significativo por ser superior a 50% do calibre do GC. Nos vasos curtos, a diferen��a foi mais pequena, mas persistiu sendo, estatisticamente, significativa. Os vasos retos dilataram na base, na sua emerg��ncia do plexo seroso, apenas no fundo g��strico. Na cobaia a opera����o de Nissen ��� fundoplicatura total com laquea����o dos vasos curtos g��stricos ���, provocou vasodilata����o arteriolar do fundo g��strico. Considero que essa vasodilata����o constituiu acomoda����o �� modifica����o introduzida e infiro que o mesmo possa acontecer no ser humano. Admito, assim, que tamb��m ocorra vasodilata����o no ser humano, na sequ��ncia da laquea����o dos vasos curtos g��stricos, pela analogia microvascular entre as duas esp��cies e que essa vasodilata����o corresponda, igualmente, a um mecanismo de adapta����o arteriolar visando, por exemplo, suprir a perda incorrida pela laquea����o. A associa����o experimental entre laquea����o dos vasos curtos g��stricos e realiza����o de fundoplicatura total, que exerce aumento inerente de press��o sobre a JEG, n��o s�� n��o provocou d��fice da microcircula����o do es��fago distal ou do est��mago proximal como desencadeou um mecanismo de vasodilata����o f��ndica que refor��a o conceito de seguran��a da opera����o de Nissen para tratamento da DRGE. -------------- ABSTRACT: The laparoscopic Nissen operation is considered to be the most appropriate antirefluxsurgery because it suitably replicates the standard physiology of the gastroesophageal valve in most patients with typical symptoms of gastroesophageal reflux disease (GERD). The technical criteria includes the safe shutdown of the diafragmatic crura(cruroplasty) and the creation of a complete fundoplication (360 degrees), short (lesser than two inches), floppy and without tension ��� a goal for which the proximal ligation of the gastric short vessels is crucial. The laparoscopic Nissen operation was performed in sixty women and forty men with GERD, without any operative mortality, at the Surgical Department of the Hospital dos Capuchos, CHLC, EPE. The one hundred patients, averaged 46 years old, complained of heartburn (90%), regurgitation (80%) and upper abdominal pain (54 %). The endoscopy process revealed Savary-Miller esophagitis of grade 0-I (62%), II (23%), III (8%), IV (7%), sliding hernia (71%), paraesophageal hernia (8%) or no herniation (21%). The pHmetry/24h diagnosed mixed pattern (38%), raised (20%), lying (20%) or inconclusive (22%). The manometry diagnosed hypotensive LES (35%), normal esophageal peristalsis (88%), mild hypomotility (5%) and was absent (7%). Hiatal hernia, severe esophagitis, ineffective symptomatic control with proton pump inhibitor and request for treatment discontinuation were the signs for surgical action. A laparoscopic ligation of short gastric vessels (70%), cruroplasty and fundoplication (silk 2/0), short (average size 1.5���2 cm) and floppy, without tension and without intraoperative calibration of the esophagus were thus performed. The laparoscopic Nissen fundoplication behaved safe and effective in treating GERD. In a group of 14 asymptomatic volunteers its reputation was confirmed with statistical significance by normalization of postoperative pHmetry/24h and manometry. 94% of the individuals remained asymptomatic up to 30.7 months (average) in the follow-up. Interrogating myself about the impact of this operation on the microcirculation of the gastric fundus I put premised on the possibility of the ligation of the short gastric vessels in the Nissen procedure can induce changes in the arteriolar diameter in the Wall of the gastric fundus. To explore the influence of ligation of the short gastric vessels and the fundoplication at the arteriolar caliber of the cardia, the fundus and the region of the short vessels of the gastric wall, I designed a project of experimental research in guinea pigs with two interdependent components: one veterinary and another technical where I applied angiomorphological studies. The project was developed at the Research Centre of the Department of Anatomy FCMUNL. For its accomplishment I got permission from the Scientific and Pedagogical Committee of the FCM-UNL, I requested for accreditation as a researcher at the General Directorate of Veterinary and, by resorting to the use of animals I submitted it to the Ethics Committee of the FCM-UNL, which approved it unanimously. The guinea pigs were divided into two experimental groups: an experimental group (EG), in which the Nissen procedure was performed and a control group (CG) in which only a gastric traction was done. Protocols of anesthesia, surgery and euthanasia were applied according to the 3Rs ��� Replacement, Reduction, Refinement of the technique of human experimentation of Burch and Russell (1959) ��� a widely accepted ethical framework for conducting scientific experiments using animals humanely. Using histological and angiomorphological techniques, I performed the analysis and the description of the normal, macro and microvascular, anatomy of the guinea pig stomach and I defined the morphological criteria that I considered susceptible for validation of this animal model for the proposed study. By means of scanning electron microscopy I measured the arteriolar calibers of the vascular casts of the cardia, of the fundus and of the short gastric vessels in both CG and EG, making 469 measurements in the former and 461 in the latter. The data were sent to the Research Center of the CHLC which conducted the statistical analysis (ANOVA). The data were sent to the Centre for Research of the CHLC, EPE which proceeded to statistical analysis (ANOVA). This analysis revealed that the arterioles plexus of the mucosal and submucosal plexus of the cardia, fundus and region of the short gastric vessels, showed increased caliber in EG. The increase was statistically significant for being greater than 50% CG gauge. In the short gastric vessels, the difference was smaller, but persisted and statistically significant. Straight vessels were dilated at the base, on its emergence of the plexus serous only in the fundus. In the guinea pig, the Nissen procedure - complete fundoplication with ligation of the short gastric vessels - caused arteriolar vasodilation on the gastric fundus. I believe that this vasodilation constituted some accommodation to the modification introduced and infer that the same might happen in humans. I admit therefore that vasodilation also occurs in humans following the ligation of the short gastric vessels by microvascular analogy between the two species and that this vasodilation corresponds also to na adaptation mechanism arteriolar, for example, to compensate the loss incurred by ligation. The association of experimental ligation of the short gastric vessels with conducting complete fundoplication, which exerts increased pressure on the EGJ, not only did not cause a microcirculation deficit of the distal esophagus or proximal stomach as triggered a mechanism of fundic vasodilation which reinforces the security concept of the Nissen procedure for treatment of GERD.

Retirement behavior of the elderly in Europe: The importance of being healthy

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

Pathogenesis of hepatic septal fibrosis associated with Capillaria hepatica infection of rats

Septal fibrosis is a common form of hepatic fibrosis, but its etiology and pathogenesis are poorly understood. Rats infected with the helminth Capillaria hepatica constitute a good experimental model of such fibrosis. To investigate the pathogenetic contribution of the several parasitic factors involved, the following procedures were performed in rats: a) regarding the role of eggs, these were isolated and injected either into the peritoneal cavity or directly into the liver parenchyma; b) for worms alone, 15-day-old infection was treated with mebendazole, killing the parasites before oviposition started; c) for both eggs and worms, rats at the 30th day of infection were treated with either mebendazole or ivermectin. Eggs only originated focal fibrosis from cicatricial granulomas, but no septal fibrosis. Worms alone induced a mild degree of perifocal septal fibrosis. Systematized septal fibrosis of the liver, similar to that observed in the infected controls, occurred only in the rats treated with mebendazole or ivermectin, with dead worms and immature eggs in their livers. Thus, future search for fibrogenic factors associated with C. hepatica infection in rats should consider lesions with both eggs and worms.

Acute liver failure in children: observations in Vit��ria, Esp��rito Santo State, Brazil

In this communication we report 46 cases of acute liver failure in children diagnosed at the Hospital Infantil Nossa Senhora da Gl��ria in Vit��ria, E Santo. Serology for IgM anti-HAV, IgM anti-HBc, HbsAg, anti-HCV and biochemical tests were performed in all cases in a routine laboratory. The M/F ratio was 1.1:1 and the mean age was 4.7±3.2 years, without gender difference. Anti-HAV IgM+ in 38 (82.6%) cases, anti-HbcIgM+ in two (4.3 %) cases and 6 (13.1%) cases were negative for all viral markers investigated. Anti- HCV+ in one anti-HAV IgM+ case. HbsAg+ in two anti-HbcIgM+ and in two HAVIgM+ cases. Among the six A, B and C negative cases, four (8.6%) did not have the suspected exogenous intoxication. Mortality was 50%, without gender or age differences. These results demonstrate that HAV infection is the main etiology of acute liver failure in children in Brazil, confirming that, although it is a self limited, relatively mild illness, it can cause serious and even fatal disease. The observation of four cases without A, B and C viral markers and no history of exogenous intoxication, agree with the observation of non A-E acute sporadic hepatitis in Northeastern Brazil.

Molecular characterization of a de novo t (11;18) translocation associated with Peter��s anomaly

Disserta����o para obten����o do grau de Mestre em Gen��tica Molecular e Biomedicina

Clinical presentation of parvovirus B19 infection in HIV-infected patients with and without AIDS

Human parvovirus B19 replicates in erythrocyte precursors. Usually, there are no apparent hematological manifestations. However, in individuals with high erythrocyte turnover, as in patients with sickle-cell disease and in the fetus, the infection may lead to severe transient aplasia and hydrops fetalis, respectively. In AIDS patients, persistent infection may result in chronic anemia. By contrast, in HIV-positive patients without AIDS the infection evolves as a mild exanthematous disease. Two clinical descriptions exemplify these forms of presentation. In the first, an AIDS patient presented with bone marrow failure that responded to immunoglobulin. In the second, an HIV-positive patient without AIDS had a morbilliform rash, and needed no treatment. Knowing that an AIDS patient has chronic B19 anemia lessens concern about drug anemia; protects the patient from invasive diagnostic maneuvers; and prevents the patient from disseminating the infection. In AIDS patients with pure red cell aplasia, a search for parvovirus B19 DNA in the serum or in the bone marrow is warranted.

Comparative clinical and ultrasound study of egg-negative and egg-positive individuals from Schistosoma mansoni low morbidity endemic areas, and hospitalized patients with hepatosplenic disease

Two hundred and twenty three subjects from a Schistosoma mansoni low morbidity endemic area and nine hospitalized hepatosplenic patients were submitted to stool test and clinical examination and abdomen ultrasound assessments. According to stool examination and ultrasound results, they were grouped as follows: G1 - 63 Schistosoma mansoni egg-negative individuals; G2 - 141 egg-positive patients and without evidence of periportal fibrosis; G3 - 19 egg-positive patients with periportal echogenicity (3-6mm); and G4 - 9 hepatosplenic patients with periportal echogenicity (> 6mm). Hepatomegaly detected by physical examination of the abdomen evaluated in the midclavicular line was verified in G1, G2 and G3, respectively, in 11.1, 12.1 and 26.3%. In G1, G2 and G3, periportal thickening occurred only in schistosomal patients (8.5%). Mild pathological alterations in patients that cannot yet be detected by clinical examination were detectable in the liver by ultrasound and can be due to fibrosis. The degree of mild periportal fibrosis was diminished in 57.9% of patients 12 months after treatment of schistosomiasis with oxamniquine. At ultrasonography, the mean liver left lobe measurement of G3 was larger than that of G1, and that of G4 larger than that of G1 and G2. The mean size of the spleen of G4 was significantly larger than that of the other three groups, and that of G3 larger than that of G1 and G2.

Significance of bile-duct changes in schistosomiasis

Lesions involving the intra-hepatic biliary ducts in schistosomiasis have been reported in the literature, both in mice and man, but there are no data concerning their quantitative, evolutionary or post curative chemotherapeutic aspects on record. In order to obtain such data an investigation on this subject was attempted. Mice infected with 50 Schistosoma mansoni cercariae were submitted to a liver biopsy at the 9th week post-infection, and treated with 400mg/bw praziquantel immediately afterwards. Infected and non-infected controls were submitted to the same procedures. By 19 weeks from cercarial exposure all surviving animals were sacrificed. The biliary ducts were counted on histological sections and the results were expressed as biliary ducts/portal spaces. This quantitative evaluation was compared with that from normal controls and revealed hyperplasia as the main biliary duct change (p<0.007) in schistosomiasis. Hyperplastic changes underwent only mild partial and not statistically significant regression after specific chemotherapy (p>0.05). Infected and untreated animals presented ductal changes that did not differ from those of the treated group. Measurements of serum bilirrubin (total and direct), and gamma-glutamyl-transpeptidase (gamma-GT) did not reveal significant differences when animals from the several groups were compared. Thus, bile ducts exhibit a proliferative response in relation to neighboring S. mansoni injury to portal areas, but although these lesions are histopathologically impressive, they lack a functional or prognostic significance.

Spore differentiation in relation to the infectious cycle of the enteric pathogen Clostridium difficile

Clostridium difficile is a gram positive, spore former, anaerobic bacterium that is able to cause infection and disease, with symptoms ranging from mild diarrhea to pseudomembranous colitis, toxic megacolon, sepsis and death. In the last decade new strains have emerged that caused outbreaks of increased disease severity and higher recurrence, morbidity and mortality rates, and C. difficile is now considered both a main nosocomial pathogen associated with antibiotic therapy as well as a major concern in the community.(...)

Isolation and encapsulation of a natural colourant using green technologies

The use of natural pigments instead of synthetic colourants is receiving growing interest in the food industry. In this field, cactus pears (Opuntia spp.) have been identified to be a promising betalainic crops covering a wide coloured spectrum. The aim of this work was to develop adequate clean and mild methodologies for the isolation and encapsulation of betacyanins, from cactus pear fruits (Opuntia spp.). Firstly, two different emerging technologies, namely PLE (Pressurized Liquid Extraction) and HPCDAE (High Pressure Carbon Dioxide-Assisted Extraction), were exploited to isolation of betacyanins form cactus pear fruits. Different process conditions were tested for the maximum recovery of betacyanins. Results showed that highest extraction yields were achieved for HPCDAE and mass ratio of pressurized carbon dioxide vs. acidified water was the parameter that most affected the betacyanins extraction. At optimum conditions of HPCDAE, Opuntia spp. extract presented a total betacyanin content of 211 �� 10 mg/100 g whereas extracts obtained using conventional extraction, PLE in static and in dynamic mode presented a total betacyanin content of 85 �� 3, 191 �� 2 and 153 �� 5 mg/100 g, respectively. HPCDAE has proven to be a successful technology to extract betacyanins from Opuntia spp. fruits. Afterward, Supercritical Fluid Technology was exploited to develop lipidic particles of betalain-rich extract. A betacyanin-rich conventional extract was encapsulated by PGSS�� (Particles from Gas Saturated Solutions) technique. Different process conditions were tested in order to model the encapsulation of betacyanins. The pressure had a negative effect on betacyanin encapsulation. Lower pressures leads to an increase in the betacyanin encapsulation. This effect was more pronounced at higher temperatures and lower equilibrium time. At these conditions, Opuntia spp. particles presented 64.4 �� 4.5 mg/100 g and high antioxidant capacity. When compared with the Opuntia spp. dried extract, lipidic particles contributed to a better homogenization of the pink colour after incorporation in ice cream.

Gold nanoparticles for the detection of DNA adducts as biomarkers of exposure to acrylamide

The main objective of this thesis was the development of a gold nanoparticle-based methodology for detection of DNA adducts as biomarkers, to try and overcome existing drawbacks in currently employed techniques. For this objective to be achieved, the experimental work was divided in three components: sample preparation, method of detection and development of a model for exposure to acrylamide. Different techniques were employed and combined for de-complexation and purification of DNA samples (including ultrasonic energy, nuclease digestion and chromatography), resulting in a complete protocol for sample treatment, prior to detection. The detection of alkylated nucleotides using gold nanoparticles was performed by two distinct methodologies: mass spectrometry and colorimetric detection. In mass spectrometry, gold nanoparticles were employed for laser desorption/ionisation instead of the organic matrix. Identification of nucleotides was possible by fingerprint, however no specific mass signals were denoted when using gold nanoparticles to analyse biological samples. An alternate method using the colorimetric properties of gold nanoparticles was employed for detection. This method inspired in the non-cross-linking assay allowed the identification of glycidamide-guanine adducts and DNA adducts generated in vitro. For the development of a model of exposure, two different aquatic organisms were studies: a goldfish and a mussel. Organisms were exposed to waterborne acrylamide, after which mortality was recorded and effect concentrations were estimated. In goldfish, both genotoxicity and metabolic alterations were assessed and revealed dose-effect relationships of acrylamide. Histopathological alterations were verified primarily in pancreatic cells, but also in hepatocytes. Mussels showed higher effect concentrations than goldfish. Biomarkers of oxidative stress, biotransformation and neurotoxicity were analysed after prolonged exposure, showing mild oxidative stress in mussel cells, and induction of enzymes involved in detoxification of oxygen radicals. A qualitative histopathological screening revealed gonadotoxicity in female mussels, which may present some risk to population equilibrium.

First report of an accident with the speckled forest pit viper (Bothriopsis taeniata) in Brazil

The first reported case of an accident with Bothriopsis taeniata in Brazil is described. The victim, a 43-year-old man, was bitten just above his right heel and presented a clinical condition compatible with mild Bothrops poisoning: local edema with hemorrhage at the bite site and pain, although without coagulopathy.

Transcriptional regulation of the mannosyltransferase-encoding gene pigm in inherited glycosylphosphatidylinositol (GPI) deficiency

RESUMO:O glicosilfosfatidilinositol (GPI) �� um complexo glicolip��dico utlizado por dezenas de prote��nas, o qual medeia a sua ancoragem �� superf��cie da c��lula. Prote��nas de superf��cie celular ancoradas a GPI apresentam v��rias fun����es essenciais para a manuten����o celular. A defici��ncia na s��ntese de GPI �� o que caracteriza principalmente a defici��ncia heredit��ria em GPI, um grupo de doen��as autoss��micas raras que resultam de muta����es nos genes PIGA, PIGL, PIGM, PIGV, PIGN, PIGO e PIGT, os quais sao indispens��veis para a bioss��ntese do GPI. Uma muta����o pontual no motivo rico em GC -270 no promotor de PIGM impede a liga����o do factor de transcri����o (FT) Sp1 �� sua sequ��ncia de reconhecimento, impondo a compacta����o da cromatina, associada �� hipoacetila����o de histonas, e consequentemente, impedindo a transcri����o de PIGM. Desta forma, a adi����o da primeira manose ao GPI �� comprometida, a s��ntese de GPI diminui assim como as prote��nas ligadas a GPI �� superficie das c��lulas. Pacientes com Defici��ncia Heredit��ria em GPI-associada a PIGM apresentam trombose e epilesia, e aus��ncia de hem��lise intravascular e anemia, sendo que estas duas ��ltimas caracter��sticas definem a Hemoglobin��ria Parox��stica Nocturna (HPN), uma doen��a rara causada por muta����es no gene PIGA. Embora a muta����o que causa IGD seja constitutiva e esteja presente em todos os tecidos, o grau de defici��ncia em GPI varia entre c��lulas do mesmo tecido e entre c��lulas de tecidos diferentes. Por exemplo nos granul��citos e linf��citos B a defici��ncia em GPI �� muito acentuada mas nos linf��citos T, fibroblastos, plaquetas e eritr��citos �� aproximadamente normal, da�� a aus��ncia de hem��lise intravascular. Os eventos transcricionais que est��o na base da express��o diferencial da ��ncora GPI nas c��lulas hematopoi��ticas s��o desconhecidos e constituem o objectivo geral desta tese. Em primeiro lugar, os resultados demonstraram que os n��veis de PIGM mRNA variam entre c��lulas prim��rias hematopoi��ticas normais. Adicionalmente, a configura����o dos nucleossomas no promotor de PIGM �� mais compacta em c��lulas B do que em c��lulas eritr��ides e tal est�� correlacionado com os n��veis de express��o de PIGM, isto ��, inferior nas c��lulas B. A presen��a de v��rios motivos de liga����o para o FT espec��fico da linhagem megacarioc��tica-eritr��ide GATA-1 no promotor de PIGM sugeriu que GATA-1 desempenha um papel regulador na sua transcri����o. Os resultados mostraram que muito possivelmente GATA-1 desempenha um papel repressor em vez de activador da express��o de PIGM. Resultados preliminares sugerem que KLF1, um factor de transcri����o restritamente eritr��ide, regula a transcri����o de PIGM independentemente do motivo -270GC. Em segundo lugar, a investiga����o do papel dos FTs Sp demonstrou que Sp1 medeia directamente a transcri����o de PIGM em ambas as c��lulas B e eritr��ide. Curiosamente, ao contr��rio do que acontece nas c��lulas B, em que a transcri����o de PIGM requer a liga����o do FT geral Sp1 ao motivo -270GC, nas c��lulas eritr��ides Sp1 regula a transcri����o de PIGM ao ligar-se a montante e n��o ao motivo -270GC. Para al��m disso, demonstrou-se que Sp2 n��o �� um regulador directo da transcri����o de PIGM quer nas c��lulas B quer nas c��lulas eritr��ides. Estes resultados explicam a aus��ncia de hem��lise intravascular nos doentes com IGD associada a PIGM, uma das principais caracter��sticas que define a HPN. Por ��ltimo, resultados preliminares mostraram que a repress��o da transcri����o de PIGM devida �� muta����o patog��nica -270C>G est�� associada com a diminui����o da frequ��ncia de interac����es gen��micas em cis entre PIGM e os seus genes ���vizinhos���, sugerindo adicionalmente que a regula����o de PIGM e desses genes �� partilhada. No seu conjunto, os resultados apresentados nesta tese contribuem para o conhecimento do controlo transcricional de um gene housekeeping, espec��fico-detecido, por meio de FTs gen��ricos e espec��ficos de linhagem.-------------ABSTRACTC: Glycosylphosphatidylinositol (GPI) is a complex glycolipid used by dozens of proteins for cell surface anchoring. GPI-anchored proteins have various functions that are essential for the cellular maintenance. Defective GPI biosynthesis is the hallmark of inherited GPI deficiency (IGD), a group of rare autosomal diseases caused by mutations in PIGA, PIGL, PIGM, PIGV, PIGN, PIGO and PIGT, all genes indispensable for GPI biosynthesis. A point mutation in the -270GC-rich box in the core promoter of PIGM disrupts binding of the transcription factor (TF) Sp1 to it, imposing nucleosome compaction associated with histone hypoacetylation, thus abrogating transcription of PIGM. As a consequence of PIGM transcriptional repression, addition of the first mannose residue onto the GPI core and thus GPI production are impaired; and expression of GPI-anchored proteins on the surface of cells is severely impaired. Patients with PIGM-associated IGD suffer from life-threatening thrombosis and epilepsy but not intravascular haemolysis and anaemia, two defining features of paroxysmal nocturnal haemoglobinuria (PNH), a rare disease caused by somatic mutations in PIGA. Although the disease-causing mutation in IGD is constitutional and present in all tissues, the degree of GPI deficiency is variable and differs between cells of the same and of different tissues. Accordingly, GPI deficiency is severe in granulocytes and B cells but mild in T cells, fibroblasts, platelets and erythrocytes, hence the lack of intravascular haemolysis.The transcriptional events underlying differential expression of GPI in the haematopoietic cells of PIG-M-associated IGD are not known and constitute the general aim of this thesis. Firstly, I found that PIGM mRNA levels are variable amongst normal primary haematopoietic cells. In addition, the nucleosome configuration in the promoter of PIGM is more compacted in B cells than in erythroid cells and this correlated with the levels of PIGM mRNA expression, i.e., lower in B cells. The presence of several binding sites for GATA-1, a mega-erythroid lineage-specific transcription factor (TF), at the PIGM promoter suggested that GATA-1 has a role on PIGM transcription. My results showed that GATA-1 in erythroid cells is most likely a repressor rather than an activator of PIGM expression. Preliminary data suggested that KLF1, an erythroid-specific TF, regulates PIGM transcription but independently of the -270GC motif. Secondly, investigation of the role of the Sp TFs showed that Sp1 directly mediates PIGM transcriptional regulation in both B and erythroid cells. However, unlike in B cells in which active PIGM transcription requires binding of the generic TF Sp1 to the -270GC-rich box, in erythroid cells, Sp1 regulates PIGM transcription by binding upstream of but not to the -270GC-rich motif. Additionally, I showed that Sp2 is not a direct regulator of PIGM transcription in B and erythroid cells. These findings explain lack of intravascular haemolysis in PIGM-associated IGD, a defining feature of PNH. Lastly, preliminary work shows that transcriptional repression of PIG-M by the pathogenic -270C>G mutation is associated with reduced frequency of in cis genomic interactions between PIGM and its neighbouring genes, suggesting a shared regulatory link between these genes and PIGM. Altogether, the results presented in this thesis provide novel insights into tissuespecific transcriptional control of a housekeeping gene by lineage-specific and generic TFs.