DNA nanoprobes for molecular detection

Dissertação apresentada para obtenção do Grau de Doutor em Engenharia Biológica – especialidade Engenharia Genética, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Random amplified polymorphic DNA (RAPD) analysis of Lutzomyia longipalpis laboratory populations

The phlebotomine sand fly Lutzomyia longipalpis has been incriminated as a vector of American visceral leishmaniasis, caused by Leishmania chagasi. However, some evidence has been accumulated suggesting that it may exist in nature not as a single but as a species complex. Our goal was to compare four laboratory reference populations of L. longipalpis from distinct geographic regions at the molecular level by RAPD-PCR. We screened genomic DNA for polymorphic sites by PCR amplification with decamer single primers of arbitrary nucleotide sequences. One primer distinguished one population (Marajó Island, Pará State, Brazil) from the other three (Lapinha Cave, Minas Gerais State, Brazil; Melgar, Tolima Department, Colombia and Liberia, Guanacaste Province, Costa Rica). The population-specific and the conserved RAPD-PCR amplified fragments were cloned and shown to differ only in number of internal repeats.

Light controlled synthesis of nucleic acids

Dissertação apresentada para obtenção do grau de Doutor em Bioquímica - especialidade Biotecnologia, pela Universidade Nova de Lisboa,Faculdade de Ciências e Tecnologia

A CASE REPORT OF INTRASPINAL PARACOCCIDIOIDOMYCOSIS

The authors report a case of paraplegia caused by a lumbar intraspinal paracoccidioidomycosis (PCM) granuloma. Clinical neurological diagnosis of a compressive spinal cord lesion was confirmed by spinal magnetic resonance imaging (MRI). Patient was submitted to surgery with total excision of the lesion. Histopathological analysis confirmed the diagnosis of PCM. Patient is on sulfamethoxazole/trimethoprim combined with fluconazole and is experiencing positive neurological recovery.

Molecular modelling of ABC transporters: from ATP hydrolysis to substrate transport

Dissertação para a obtenção de grau de doutor em Bioquímica pelo Instituto de Tecnologia Química e Biológica da Universidade Nova de Lisboa

Functional and structural characterization of the RNase II-family of enzymes

Dissertation presented to obtain a Doctoral Degree in Biology by Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa

Molecular characterization of Dengue viruses type 1 and 2 isolated from a concurrent human infection

In 2001, an autochthonous case of dual viremia, resulting from naturally acquired dengue virus DEN-1 and DEN-2 infections was detected during the dengue outbreak that occurred in Barretos, a city with about 105,000 inhabitants in the North region of São Paulo State. Serotype identification was based on virus isolation to C6/36 mosquito cells culture and immunofluorescence assays using type-specific monoclonal antibodies. The double infection was also confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). Comparative analysis of the 240-nucleotide sequences of E/NS1 gene junction region between the genome of DEN-1 and DEN-2 isolates of the corresponding reference Nauru and PR 159S1 strains, respectively, showed some nucleotide differences, mainly silent mutations in the third codon position. Results of maximum likelihood phylogenetic analysis of E/NS1 gene sequences indicated that both genotypes of DEN-1 and DEN-2 viruses recovered from double infection in Barretos belonged to genotypes I and III, respectively.

Molecular typing of dengue virus type 2 in Brazil

Strain typing is a critical tool for molecular epidemiological analysis and can provide important information about the spread of dengue viruses. Here, we performed a molecular characterization of DEN-2 viruses isolated in Brazil during 1990-2000 from geographically and temporally distinct areas in order to investigate the genetic distribution of this serotype circulating in the country. Restriction site-specific polymerase chain reaction (RSS)-PCR presented the same pattern for all 52 Brazilian samples, showing the circulation of just one DEN-2 variant. Phylogenetic analysis using progressive pairwise alignments from 240-nucleotide sequences of the E/NS1 junction in 15 isolates showed that they belong to genotype III (Jamaica genotype).

Tuberculous brain abscess in a patient with AIDS: case report and literature review

Tuberculous brain abscesses in AIDS patients are considered rare with only eight cases reported in the literature. We describe the case of a 34-year-old woman with AIDS and previous toxoplasmic encephalitis who was admitted due to headache and seizures. A brain computed tomography scan disclosed a frontal hypodense lesion with a contrast ring enhancement. Brain abscess was suspected and she underwent a lesion puncture through a trepanation. The material extracted was purulent and the acid-fast smear was markedly positive. Timely medical and surgical approaches allowed a good outcome. Tuberculous abscesses should be considered in the differential diagnosis of focal brain lesions in AIDS patients. Surgical excision or stereotactic aspiration, and antituberculous treatment are the mainstay in the management of these uncommon lesions.

Mapping of a Leishmania major gene/locus that confers pentamidine resistance by deletion and insertion of transposable element

Pentamidine (PEN) is an alternative compound to treat antimony-resistant leishmaniasis patients, which cellular target remains unclear. One approach to the identification of prospective targets is to identify genes able to mediate PEN resistance following overexpression. Starting from a genomic library of transfected parasites bearing a multicopy episomal cosmid vector containing wild-type Leishmania major DNA, we isolated one locus capable to render PEN resistance to wild type cells after DNA transfection. In order to map this Leishmania locus, cosmid insert was deleted by two successive sets of partial digestion with restriction enzymes, followed by transfection into wild type cells, overexpression, induction and functional tests in the presence of PEN. To determine the Leishmania gene related to PEN resistance, nucleotide sequencing experiments were done through insertion of the transposon Mariner element of Drosophila melanogaster (mosK) into the deleted insert to work as primer island. Using general molecular techniques, we described here this method that permits a quickly identification of a functional gene facilitating nucleotide sequence experiments from large DNA fragments. Followed experiments revealed the presence of a P-Glycoprotein gene in this locus which role in Leishmania metabolism has now been analyzed.

Human vaccinia-like virus outbreaks in São Paulo and Goiás States, Brazil: virus detection, isolation and identification

Since October 2001, the Adolfo Lutz Institute has been receiving vesicular fluids and scab specimens of patients from Paraíba Valley region in the São Paulo and Minas Gerais States and from São Patricio Valley, in the Goiás State. Epidemiological data suggested that the outbreaks were caused by Cowpox virus or Vaccinia virus. Most of the patients are dairy milkers that had vesiculo-pustular lesions on the hands, arms, forearms, and some of them, on the face. Virus particles with orthopoxvirus morphology were detected by direct electron microscopy (DEM) in samples of 49 (66.21%) patients of a total of 74 analyzed. Viruses were isolated in Vero cell culture and on chorioallantoic membrane (CAM) of embryonated chicken eggs. Among 21 samples submitted to PCR using primers for hemagglutinin (HA) gene, 19 were positive. Restriction digestion with TaqI resulted in four characteristic Vaccinia virus fragments. HA nucleotide sequences showed 99.9% similarity with Cantagalo virus, described as a strain of Vaccinia virus. The only difference observed was the substitution of one nucleotide in the position 616 leading to change in one amino acid of the protein in the position 206. The phylogenetic analysis showed that the isolates clustered together with Cantagalo virus, other Vaccinia strains and Rabbitpox virus.

Stochastic model of transcription initiation of closely spaced promoters in escherichia coli

Dissertação para obtenção do Grau de Mestre em Engenharia Biomédica

Molecular and seroepidemiologic studies of Enterovirus 71 infection in the State of Pará, Brazil

In many countries, the Enterovirus 71 (EV-71) Picornaviridae family is associated to hand, foot and mouth disease in addition to acute neurological diseases while in Brazil these viruses are more closely associated to the latter group. The aim of this research was to use the first EV-71 isolate of the Northern region of Brazil in molecular and seroepidemiologic studies. Two (2.2%) out of 88 stool samples (44 cases of AFP), collected from January 1998 to December 2000 were positive for EV-71 isolation (73442/PA/99). Nucleotide sequence of the gen that codifies the VP1 protein showed that isolate 73442/PA/99 was similar to the EV-71 strains belonging to genotype B - more closely identified with EV-71 from North America. Neutralization test with 389 sera samples collected from January 1998 to November 2001, from individuals ranging from 0 to 15 years of age living in the city of Belém, State of Pará showed the following results in relation to isolate 73442/PA/99 and prototype BrCr: a total of 207 individuals (53.2%) had neutralization antibodies to both viruses, 167 (42.9%) had no antibodies and 15 showed the presence of neutralizing antibodies to one of the two viruses. Only 20.2% of the children aged 0 to 3 had neutralizing antibodies to EV-71, indicating that these children were more susceptible to the infection. Both the seroprevalence study and VP1 sequencing were important to demonstrate the spread and the molecular pattern of the EV-71 circulating in the Northern Region of Brazil.

Analysis of the genetic polymorphism of Paracoccidioides brasiliensis and Paracoccidioides cerebriformis "Moore" by random amplified polymorphic DNA (RAPD) and 28S ribosomal DNA sequencing: Paracoccidioides cerebriformis revisited

Our purpose was to compare the genetic polymorphism of six samples of P. brasiliensis (113, 339, BAT, T1F1, T3B6, T5LN1), with four samples of P. cerebriformis (735, 741, 750, 361) from the Mycological Laboratory of the Instituto de Medicina Tropical de São Paulo, using Random Amplified Polymorphic DNA Analysis (RAPD). RAPD profiles clearly segregated P. brasiliensis and P. cerebriformis isolates. However, the variation on band patterns among P. cerebriformis isolates was high. Sequencing of the 28S rDNA gene showed nucleotide conservancy among P. cerebriformis isolates, providing basis for taxonomical grouping, and disclosing high divergence to P. brasiliensis supporting that they are in fact two distinct species. Moreover, DNA sequence suggests that P. cerebriformis belongs in fact to the Aspergillus genus.

St. Louis encephalitis vírus: first isolation from a human in São Paulo state, Brasil

This paper reports the isolation of St. Louis encephalitis virus (SLEV) from a febrile human case suspected to be dengue, in São Pedro, São Paulo State. A MAC-ELISA done on the patient's acute and convalescent sera was inconclusive and hemagglutination inhibition test detected IgG antibody for flaviviruses. An indirect immunofluorescent assay done on the C6/36 cell culture inoculated with the acute serum was positive for flaviviruses but negative when tested with dengue monoclonal antibodies. RNA extracted from the infected cell culture supernatant was amplified by RT-PCR in the presence of NS5 universal flavivirus primers and directly sequenced. Results of BLAST search indicated that this sequence shares 93% nucleotide similarity with the sequence of SLEV (strain-MSI.7), confirmed by RT-PCR performed with SLEV specific primers. Since SLEV was identified as the cause of human disease, it is necessary to improve surveillance in order to achieve early detection of this agent in the state of São Paulo and in Brazil. This finding is also an alert to health professionals about the need for more complete clinical and epidemiological investigations of febrile illnesses as in the reported case. SLEV infections can be unrecognized or confused with other ones caused by an arbovirus, such as dengue.