Hepatite C

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Plano Estratégico para o Turismo do Concelho de Amares

Dissertação apresentada à Universidade Fernando Pessoa como parte dos requisitos para a obtenção do grau de Mestre em Ciências da Comunicação, ramo de Marketing e Publicidade

Neutralization activity in a geographically diverse East London cohort of human immunodeficiency virus type 1-infected patients: clade C infection results in a stronger and broader humoral immune response than clade B infection.

The array of human immunodeficiency virus (HIV) subtypes encountered in East London, an area long associated with migration, is unusually heterogeneous, reflecting the diverse geographical origins of the population. In this study it was shown that viral subtypes or clades infecting a sample of HIV type 1 (HIV-1)-positive individuals in East London reflect the global pandemic. The authors studied the humoral response in 210 treatment-naïve chronically HIV-1-infected (>1 year) adult subjects against a panel of 12 viruses from six different clades. Plasmas from individuals infected with clade C, but also plasmas from clade A, and to a lesser degree clade CRF02_AG and CRF01_AE, were significantly more potent at neutralizing the tested viruses compared with plasmas from individuals infected with clade B. The difference in humoral robustness between clade C- and B-infected patients was confirmed in titration studies with an extended panel of clade B and C viruses. These results support the approach to develop an HIV-1 vaccine that includes clade C or A envelope protein (Env) immunogens for the induction of a potent neutralizing humoral response.

Health status of an elderly population in Sharpeville, South Africa

The objective of this cross-sectional study was a comprehensive nutrition and health assessment to provide a basis for future intervention strategies for an elderly population attending a day-care centre. Socio-demographic, health and 24-hour recall dietary intake questionnaires were administered and anthropometric and biochemical measurements taken. The results indicate that the majority of respondents had an income of between R501 and R1 000 (South African rand) per month and most of them reported an occasional lack of funds to meet basic household needs, confirming the presence of food insecurity. Daily dietary intakes (mean [+ or -] Standard Deviation [SD]) of the women were 5 395 [+ or -] 2 946 kJ energy, 47 [+ or -] 27 g protein, 28 [+ or -] 21 g fat and 196 [+ or -] 123 g carbohydrates compared to 8 641 [+ or -] 3 799 kJ, 86 [+ or -] 48 g, 49 [+ or -] 32 g and 301 [+ or -] 139 g of the men, respectively. The majority (83.6%) of the women were overweight (body mass index [BMI] [greater than or equal to] 25) or obese (BMI [greater than or equal to] 30) whilst 78% had a mid-upper arm circumference (MUAC) of [greater than or equal to] 21.7 cm. Mean intakes of micronutrients were low in comparison to reference standards and serum zinc levels were suboptimal. Obesity, hypertension and raised total serum cholesterol levels indicated an increased risk for coronary heart disease. It can be concluded that a low income, household food insecurity and risk factors associated with malnutrition and non-communicable diseases were prevalent in this elderly population. OPSOMMING Die doelwit van hierdie dwarssnitstudie was ‘n omvattende bepaling van voeding- en gesondheidstatus om as basis te dien vir toekomstige intervensiestrategieë vir ’n groep bejaardes wat ’n dagsentrum besoek. Sosiodemografiese, gesondheid- en 24-uur herroep-dieetinname vraelyste is voltooi en antropometriese en biochemiese metings is geneem. Die resultate het bevestig dat die meerderheid respondente ‘n maandelikse inkomste van tussen R501 en R1 000 (Suid-Afrikaanse rand) gehad het. Die meeste het ‘n geldtekort vir basiese huishoudelike behoeftes gerapporteer wat dui op huishoudelike voedselinsekuriteit. Daaglikse dieetinnames (gemiddeld±standaardafwyking [SA]) van die vroue was onderskeidelik 5 395±2 946 kJ energie, 47±27 g proteïen, 28±21 g vet en 196±123 g koolhidrate in vergelyking met 8 641±3 799 kJ, 86±48 g, 49±32 g en 301±139 g vir die mans. Die meerderheid (83.6%) van die vroue was oorgewig (liggaamsmassa-indeks [LMI] >25) of vetsugtig (LMI > 30) en 78% het ’n middel-bo-armomtrek (MUAC) van > 21.7 cm gehad. Gemiddelde mikronutriëntinnames was laag in vergelyking met die verwysingstandaarde en serumsink was suboptimaal. Vetsug, hipertensie en verhoogde totale serumcholesterolvlakke het op ‘n verhoogde risiko van kardiovaskulêre siekte gedui. Die resultate het dus bewys dat lae inkomste, huishoudelike voedselinsekuriteit en die risikofaktore wat met wanvoeding en leefstylsiektes geassosieer word, teenwoordig was.

Measles virus superinfection immunity and receptor redistribution in persistently infected NT2 cells

A recombinant measles virus (MV) expressing red fluorescent protein (MVDsRed1) was used to produce a persistently infected cell line (piNT2-MVDsRed1) from human neural precursor (NT2) cells. A similar cell line (piNT2-MVeGFP) was generated using a virus that expresses enhanced green fluorescent protein. Intracytoplasmic inclusions containing the viral nucleocapsid protein were evident in all cells and viral glycoproteins were present at the cell surface. Nevertheless, the cells did not release infectious virus nor did they fuse to generate syncytia. Uninfected NT2 cells express the MV receptor CD46 uniformly over their surface, whereas CD46 was present in cell surface aggregates in the piNT2 cells. There was no decrease in the overall amount of CD46 in piNT2 compared to NT2 cells. Cell-to-cell fusion was observed when piNT2 cells were overlaid onto confluent monolayers of MV receptor-positive cells, indicating that the viral glycoproteins were correctly folded and processed. Infectious virus was released from the underlying cells, indicating that persistence was not due to gross mutations in the virus genome. Persistently infected cells were superinfected with MV or canine distemper virus and cytopathic effects were not observed. However, mumps virus could readily infect the cells, indicating that superinfection immunity is not caused by general soluble antiviral factors. As MVeGFP and MVDsRed1 are antigenically indistinguishable but phenotypically distinct it was possible to use them to measure the degree of superinfection immunity in the absence of any cytopathic effect. Only small numbers of non-fusing green fluorescent piNT2-MVDsRed1 cells (1 : 300 000) were identified in which superinfecting MVeGFP entered, replicated and expressed its genes.

Two functionally linked amino acids in the stem 2 region of measles virus haemagglutinin determine infectivity and virulence in the rodent central nervous system.

Rodent brain-adapted measles virus (MV) strains, such as CAM/RB and recombinant MVs based on the Edmonston strain containing the haemagglutinin (H) of CAM/RB, cause acute encephalitis after intracerebral infection of newborn rodents. We have demonstrated that rodent neurovirulence is modulated by two mutations at amino acid positions 195 and 200 in the H protein, one of these positions (200) being a potential glycosylation site. In order to analyse the effects of specific amino acids at these positions, we introduced a range of individual and combined mutations into the open reading frame of the H gene to generate a number of eukaryotic expression plasmids. The functionality of the mutant H proteins was assessed in transfected cells and by generating recombinant viruses. Interestingly, viruses caused acute encephalitis only if the amino acid Ser at position 200 was coupled with Gly at position 195, whereas viruses with single or combined mutations at these positions, including glycosylation at position 200, were attenuated. Neurovirulence was associated with virus spread and induction of neuronal apoptosis, whereas attenuated viruses failed to infect brain cells. Similar results were obtained by using primary brain-cell cultures. Our findings indicate that a structural alteration in the stem 2 region of the H protein at position 195 or 200 interferes with infectivity of rodent neurons, and suggest that the interaction of the viral attachment protein with cellular receptors on neurons is affected.

Measles virus minigenomes encoding two autofluorescent proteins reveal cell-to-cell variation in reporter expression dependent on viral sequences between the transcription units.

Transcription from morbillivirus genomes commences at a single promoter in the 3' non-coding terminus, with the six genes being transcribed sequentially. The 3' and 5' untranslated regions (UTRs) of the genes (mRNA sense), together with the intergenic trinucleotide spacer, comprise the non-coding sequences (NCS) of the virus and contain the conserved gene end and gene start signals, respectively. Bicistronic minigenomes containing transcription units (TUs) encoding autofluorescent reporter proteins separated by measles virus (MV) NCS were used to give a direct estimation of gene expression in single, living cells by assessing the relative amounts of each fluorescent protein in each cell. Initially, five minigenomes containing each of the MV NCS were generated. Assays were developed to determine the amount of each fluorescent protein in cells at both cell population and single-cell levels. This revealed significant variations in gene expression between cells expressing the same NCS-containing minigenome. The minigenome containing the M/F NCS produced significantly lower amounts of fluorescent protein from the second TU (TU2), compared with the other minigenomes. A minigenome with a truncated F 5' UTR had increased expression from TU2. This UTR is 524 nt longer than the other MV 5' UTRs. Insertions into the 5' UTR of the enhanced green fluorescent protein gene in the minigenome containing the N/P NCS showed that specific sequences, rather than just the additional length of F 5' UTR, govern this decreased expression from TU2.

Identification of protease and ADP-ribose 1''-monophosphatase activities associated with transmissible gastroenteritis virus non-structural protein 3.

The replicase polyproteins, pp1a and pp1ab, of porcine Transmissible gastroenteritis virus (TGEV) have been predicted to be cleaved by viral proteases into 16 non-structural proteins (nsp). Here, enzymic activities residing in the amino-proximal region of nsp3, the largest TGEV replicase processing product, were characterized. It was shown, by in vitro translation experiments and protein sequencing, that the papain-like protease 1, PL1pro, but not a mutant derivative containing a substitution of the presumed active-site nucleophile, Cys1093, cleaves the nsp2|nsp3 site at 879Gly|Gly880. By using an antiserum raised against the pp1a/pp1ab residues 526–713, the upstream processing product, nsp2, was identified as an 85 kDa protein in TGEV-infected cells. Furthermore, PL1pro was confirmed to be flanked at its C terminus by a domain (called X) that mediates ADP-ribose 1''-phosphatase activity. Expression and characterization of a range of bacterially expressed forms of this enzyme suggest that the active X domain comprises pp1a/pp1ab residues Asp1320–Ser1486.