The Photoreceptor Cell-Specific Nuclear Receptor Gene (PNR ) Accounts for Retinitis Pigmentosa in the Crypto-Jews from Portugal (Marranos), Survivors from the Spanish Inquisition

The last Crypto-Jews (Marranos) are the survivors of Spanish Jews who were persecuted in the late fifteenth century, escaped to Portugal and were forced to convert to save their lives. Isolated groups still exist in mountainous areas such as Belmonte in the Beira-Baixa province of Portugal. We report here the genetic study of a highly consanguineous endogamic population of Crypto-Jews of Belmonte affected with autosomal recessive retinitis pigmentosa (RP). A genome-wide search for homozygosity allowed us to localize the disease gene to chromosome 15q22-q24 (Zmax=2.95 at θ=0 at the D15S131 locus). Interestingly, the photoreceptor cell-specific nuclear receptor (PNR) gene, the expression of which is restricted to the outer nuclear layer of retinal photoreceptor cells, was found to map to the YAC contig encompassing the disease locus. A search for mutations allowed us to ascribe the RP of Crypto-Jews of Belmonte to a homozygous missense mutation in the PNR gene. Preliminary haplotype studies support the view that this mutation is relatively ancient but probably occurred after the population settled in Belmonte.

Expressão em Escherichia coli de antigénios do Cell fusing agent virus (Flaviviridae: Flavivirus) como proteína de fusão

RESUMO: O Cell Fusing Agent Vírus (CFAV), considerado como o primeiro “flavivírus específicos de insectos” (ISF), parece estar exclusivamente adaptado aos seus hospedeiros, não replicando em células de vertebrados. Apesar de ter sido identificado há mais de três décadas (1975), a verdade é que muito pouco se conhece sobre a sua biologia. Dado o seu parentesco filogenético com alguns outros flavivírus encontrados naturalmente em mosquitos de diferentes géneros colhidos em diferentes regiões do globo, este vírus poderá ser usado como modelo para o estudo de ISF. No entanto, necessitam do desenvolvimento de ferramentas básicas, tais como clones moleculares ou baterias de soros contendo anticorpos que reconheçam uma ou mais proteínas codificadas pelo genoma viral, produzidas, por exemplo, a partir de antigénios virais produzidos de forma recombinante. Com este trabalho pretendeu-se a optimização de protocolos que permitiram a expressão e purificação parcial de quatro proteínas [duas proteínas estruturais (C e E) e duas não estruturais (NS3hel e NS5B)] do CFAV em E. coli, todas elas produzidas como proteínas de fusão com “caudas” (tags) de hexahistidina nos seus extremos carboxilo. Para a expansão do CFAV foram utilizadas células Aedes albopictus (C6/36). Após a realização da extracção do RNA viral e a obtenção de cDNA, procedeu-se amplificação, por RT-PCR, das regiões codificantes das proteínas C, E, NS3hel e NS5B, utilizando primers específicos. Os quatro fragmentos de DNA foram independentemente inseridos no vector pJTE1.2/blunt usando E. coli NovaBlue como hospedeira de clonagem e, posteriormente, inseridos em vectores de expressão pET-28b e pET-29b usando E. coli BL21(DE3)pLysS e Rosetta(DE3)pLysS como hospedeiras de expressão. Após da indução, expressão e purificação das proteínas recombinantes C, E, NS3hel e NS5B, foi confirmada a autenticidade destas proteínas produzidas através do método Western Blot com um anticorpo anti-histidina. --------- ABSTRACT: The Cell Fusing Agent virus (CFAV) considered as the first "insect- specific flavivirus" (ISF) and seems to be uniquely adapted to their hosts, not replicating in vertebrate cells. Although it has been known for more than three decades (1975), the truth is very little is known about its biology. Given its close phylogenetic relationship with other flavivirus naturally circulating in various genera of mosquitoes collected from different regions of the globe, this virus could be used as a model for the study of ISF. However, such studies require the development of experimental basic tools, such as molecular clones or serum batteries containing antibodies that recognize one or more proteins encoded by the viral genome, produced, for example, from viral antigens recombinant produced. In this work, we carried out the optimization of protocols that allowed the expression and partial purification of four proteins [two structural proteins (C and E) and two nonstructural proteins (NS3hel and NS5B)] CFAV in E. coli as fusion protein for c-terminal hexahistidine tags. For the expansion of the CFAV we used Aedes albopictus (C6/36) cells. After completion of the viral RNA extraction and cDNA obtained, amplification of the coding regions of the C, E, NS5B and NS3hel proteins was carried out by RT-PCR using specific primers. The four DNA fragments were independently inserted into the vector pJTE1.2/blunt using E. coli NovaBlue as cloning host and then inserted into expression vectors pET-28b and pET-29b using E. coli BL21(DE3)pLysS and Rosetta(DE3)pLysS as expression host. After induction, expression and purification of recombinant C, E, NS3hel and NS5B proteins Western Blot analyses with an anti-histidine antibody confirmed the authenticity of these proteins produced.

THE RELEVANCE OF NUTRITIONAL STATUS AND HISTOPATHOLOGICAL FINDINGS ON THE INFECTIOUS PROCESS OF BALB/C MICE INOCULATED WITH Lacazia loboi

SUMMARY The aim of this study was to evaluate the effects of the protein-calorie malnutrition in BALB/c isogenic mice infected with Lacazia loboi, employing nutritional and histopathological parameters. Four groups were composed: G1: inoculated with restricted diet, G2: not inoculated with restricted diet, G3: inoculated with regular diet, G4: not inoculated with regular diet. Once malnutrition had been imposed, the animals were inoculated intradermally in the footpad and after four months, were sacrificed for the excision of the footpad, liver and spleen. The infection did not exert great influence on the body weight of the mice. The weight of the liver and spleen showed reduction in the undernourished groups when compared to the nourished groups. The macroscopic lesions, viability index and total number of fungi found in the footpads of the infected mice were increased in G3 when compared to G1. Regarding the histopathological analysis of the footpad, a global cellularity increase in the composition of the granuloma was observed in G3 when compared to G1, with large numbers of macrophages and multinucleated giant cells, discrete numbers of lymphocytes were present in G3 and an increase was observed in G1. The results suggest that there is considerable interaction between Jorge Lobo's disease and nutrition.

Spatiotemporal mechanisms for actomyosin ring assembly and contraction in budding yeast cell division

Dissertation presented to obtain the Ph.D degree in Molecular Medicine

Insights into cell wall synthesis and cell division in Staphylococcus aureus

Dissertation presented to obtain the Ph.D degree in Biology

IDENTIFICATION OF CANINE VISCERAL LEISHMANIASIS IN A PREVIOUSLY UNAFFECTED AREA BY CONVENTIONAL DIAGNOSTIC TECHNIQUES AND CELL-BLOCK FIXATION

After the report of a second case of canine visceral leishmaniasis (CVL) in São Bento da Lagoa, Itaipuaçu, in the municipality of Maricá, Rio de Janeiro State, an epidemiological survey was carried out, through active search, totaling 145 dogs. Indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), and rapid chromatographic immunoassay based on dual-path platform (DPP(r)) were used to perform the serological examinations. The parasitological diagnosis of cutaneous fragments was performed by parasitological culture, histopathology, and immunohistochemistry. In the serological assessment, 21 dogs were seropositive by IFA, 17 by ELISA, and 11 by DPP(r), with sensitivity of 66.7%, 66.7% and 50%, and specificity of 87.2%, 90.2% and 94%, respectively for each technique. The immunohistochemistry of bone marrow using the cell-block technique presented the best results, with six positive dogs found, three of which tested negative by the other parasitological techniques. Leishmania sp. was isolated by parasitological culture in three dogs. The detection of autochthonous Leishmania infantum in Itaipuaçu, and the high prevalence of seropositive dogs confirm the circulation of this parasite in the study area and alert for the risk of expansion in the State of Rio de Janeiro.

Dermoscopy in Merkel Cell Carcinoma: a Case Report

Merkel cell carcinoma (MCC) is a rare malignant and primary neuroendocrine carcinoma with several known risk factors. Early diagnosis and aggressive treatment are critical. We report the case of an 82-year old woman with a Merkel cell carcinoma on the face. Clinical and histopathological features are presented. In addition, dermoscopic features and the differential diagnosis of this rare tumor are discussed. Although nodules with atypical dermoscopic vascular pattern and milky-red areas will end up being excised, this report adds more clues to the rarely described dermoscopic morphologic presentation of MCC.