Plasmid profile in oral Fusobacterium nucleatum from humans and Cebus apella monkeys

Fusobacterium nucleatum is a strict anaerobe and is indigenous of the human oral cavity. This organism is commonly recovered from different monomicrobial and mixed infections in humans and animals. In this study, the plasmid profile, the plasmid stability and the penicillin-resistance association in oral F. nucleatum isolated from periodontal patients, healthy subjects and Cebus apella monkeys were evaluated. Forty-five F. nucleatum strains from patients, 38 from healthy subjects and seven from C. apella were identified and analyzed. Plasmid extraction was performed in all the isolated strains. These elements were found in 26.7% strains from patients and one strain from C. apella. Strains from healthy subjects did not show any plasmid. Most of strains showed two plasmid bands ranging from 4 to 16 Kb, but digestions with endonucleases showed that they belonged to a single plasmid. The plasmid profile was similar and stable in human and monkey strains. Also, plasmids were classified into three groups according to size. Two strains were positive to beta-lactamase production and no plasmid DNA-hybridization with a beta-lactamase gene probe was observed, suggesting a chromosomal resistance.

Measurement of lead concentration in biological tissues by atomic spectroscopy techniques

Dissertation for the degree of Doctor of Philosophy in Physics

Behavioral and synaptic circuit analysis in models of neuropsychiatric disorders: Dissecting the in vivo role of the postsynaptic density proteins nArgBP2 and Shank3 using genetic engineered mice

Dissertation presented to obtain the Ph.D degree in Biology

Cardiovascular profile after intravenous injection of Africanized bee venom in awake rats

The manifestations caused by Africanized bee stings depend on the sensitivity of the victim and the toxicity of the venom. Previous studies in our laboratory have demonstrated cardiac changes and acute tubular necrosis (ATN) in the kidney of rats inoculated with Africanized bee venom (ABV). The aim of the present study was to evaluate the changes in mean arterial pressure (MAP) and heart rate (HR) over a period of 24 h after intravenous injection of ABV in awake rats. A significant reduction in basal HR as well as in basal MAP occurred immediately after ABV injection in the experimental animals. HR was back to basal level 2 min after ABV injection and remained normal during the time course of the experiment, while MAP returned to basal level 10 min later and remained at this level for the next 5 h. However, MAP presented again a significant reduction by the 7th and 8th h and returned to the basal level by the 24th h. The fall in MAP may contribute to the pathogenesis of ATN observed. The fall in MAP probably is due to several factors, in addition to the cardiac changes already demonstrated, it is possible that the components of the venom themselves or even substances released in the organism play some role in vascular beds.

Staphylococcus aureus ampicillin-resistant from the odontological clinic environment

The aim of this research was to evaluate the prevalence of Sthaphylococcus spp. and S. aureus in the odontological clinic environment (air), their production of beta-lactamase and antibacterial susceptibility to the major antibiotics utilized in medical particle. During 12 months of samples collect were isolated 9775 CFU by MSA medium suggesting a high amount of Staphylococcus spp. in the clinic environment which can appear through aerosols. A total of 3149 colonies (32.2%) were suggestive of pathogenic staphylococci. Gram coloration, catalase test, colony-mallow growing on chromogenic medium, and coagulase test confirmed the identity of 44 (0.45%) S. aureus isolates. Of these, 35 isolates (79.5%) showed production of beta-lactamase by CefinaseTM discs and resistance to ampicillin, erythromycin (7 isolates) and tetracycline (1 isolate) suggesting the existence of multiresistant isolates. The evaluation of the oxacillin MIC by Etest® assays showed susceptibility patterns suggesting the inexistence of the mecA gene in chromosomal DNA. These results point out to the need of a larger knowledge on the contamination means and propagation of this microorganism into the odontological clinic.

Virulence profile of ten Paracoccidioides brasiliensis isolates: association with morphologic and genetic patterns

Ten isolates of Paracoccidioides brasiliensis were examined for differences in virulence in outbred mice intravenously inoculated with the fungus, associated with mycelial morphology, and genetic patterns measured by random amplified polymorphic DNA (RAPD). Virulence was evaluated by viable yeast cell recovery from lungs and demonstration of histopathologic lesions in different organs. The results showed that the isolates presented four virulence degrees: high virulence, intermediate, low and non-virulence. RAPD clustered the isolates studied in two main groups with 56% of genetic similarity. Strains with low virulence, Pb265 or the non-virulent, Pb192, showed glabrous/cerebriform morphology and high genetic similarity (98.7%) when compared to the other isolates studied. The same was observed with Bt79 and Bt83 that shared 96% genetic similarity, cottony colonies and high virulence. The RAPD technique could only discriminate P. brasiliensis isolates according to glabrous/cerebriform or cottony colonies, and also high from low virulence strains. Isolates with intermediate virulence such as Pb18, Pb18B6, Bt32 and Bt56 showed variability in their similarity coefficient suggesting that RAPD was able to detect genetic variability in this fungal specie. Virulence profile of P. brasiliensis demonstrated that both mycelial morphologic extreme phenotypes may be associated with fungal virulence and their in vitro subculture time. Thus, RAPD technique analysis employed in association with virulence, morphologic and immunologic aspects might prove adequate to detect differences between P. brasiliensis isolates.

Análise do Desempenho de Técnicas de Otimização no Problema de Escalonamento

A optimização nas aplicações modernas assume um carácter fortemente interdisciplinar, relacionando-se com a necessidade de integração de diferentes técnicas e paradigmas na resolução de problemas reais complexos. O problema do escalonamento é recorrente no planeamento da produção. Sempre que uma ordem de fabrico é lançada, é necessário determinar que recursos serão utilizados e em que sequência as atividades serão executadas, para otimizar uma dada medida de desempenho. Embora ainda existam empresas a abordar o problema do escalonamento através de simples heurísticas, a proposta de sistemas de escalonamento tem-se evidenciado na literatura. Pretende-se nesta dissertação, a realização da análise de desempenho de Técnicas de Optimização, nomeadamente as meta-heurísticas, na resolução de problemas de optimização complexos – escalonamento de tarefas, particularmente no problema de minimização dos atrasos ponderados, 1||ΣwjTj. Assim sendo, foi desenvolvido um protótipo que serviu de suporte ao estudo computacional, com vista à avaliação do desempenho do Simulated Annealing (SA) e o Discrete Artificial Bee Colony (DABC). A resolução eficiente de um problema requer, em geral, a aplicação de diferentes métodos, e a afinação dos respetivos parâmetros. A afinação dos parâmetros pode permitir uma maior flexibilidade e robustez mas requer uma inicialização cuidadosa. Os parâmetros podem ter uma grande influência na eficiência e eficácia da pesquisa. A sua definição deve resultar de um cuidadoso esforço experimental no sentido da respectiva especificação. Foi usado, no âmbito deste trabalho de mestrado, para suportar a fase de parametrização das meta-heurísticas em análise, o planeamento de experiências de Taguchi. Da análise dos resultados, foi possível concluir que existem vantagem estatisticamente significativa no desempenho do DABC, mas quando analisada a eficiência é possível concluir que há vantagem do SA, que necessita de menos tempo computacional.

Pathogenicity of Entamoeba dispar under xenic and monoxenic cultivation compared to a virulent E. histolytica

Two xenic isolates and cloned cultures of Entamoeba dispar were submitted to monoxenization using Crithidia fasciculata as the associated organism. Growth in monoxenic cultivation and ability of xenic and monoxenic trophozoites to destroy VERO cells and produce lesions in hamster livers were compared to those of a virulent E. histolytica. Parental and cloned E. dispar under monoxenic cultivation showed a remarkable lower growth than the monoxenic E. histolytica and were avirulent in both in vivo and in vitro tests. When xenically cultured, trophozoites of E. dispar showed a moderate lytic activity against VERO cells (1.5 to 41.8% of destruction) but caused severe hepatic lesions in hamsters as those caused by the virulent E. histolytica (29 to 100% in prevalence and 0.86 to 4.00 in lesion degree). Although E. dispar has not been associated with invasive disease in men, the ability of xenic trophozoites to produce prominent tissue damage in experimental conditions has indicated that some strains have a considerable pathogenic potential when in presence of bacteria.

Estado da Arte da Computação Evolutiva Aplicada à Eletrónica

A Computação Evolutiva enquadra-se na área da Inteligência Artificial e é um ramo das ciências da computação que tem vindo a ser aplicado na resolução de problemas em diversas áreas da Engenharia. Este trabalho apresenta o estado da arte da Computação Evolutiva, assim como algumas das suas aplicações no ramo da eletrónica, denominada Eletrónica Evolutiva (ou Hardware Evolutivo), enfatizando a síntese de circuitos digitais combinatórios. Em primeiro lugar apresenta-se a Inteligência Artificial, passando à Computação Evolutiva, nas suas principais vertentes: os Algoritmos Evolutivos baseados no processo da evolução das espécies de Charles Darwin e a Inteligência dos Enxames baseada no comportamento coletivo de alguns animais. No que diz respeito aos Algoritmos Evolutivos, descrevem-se as estratégias evolutivas, a programação genética, a programação evolutiva e com maior ênfase, os Algoritmos Genéticos. Em relação à Inteligência dos Enxames, descreve-se a otimização por colônia de formigas e a otimização por enxame de partículas. Em simultâneo realizou-se também um estudo da Eletrónica Evolutiva, explicando sucintamente algumas das áreas de aplicação, entre elas: a robótica, as FPGA, o roteamento de placas de circuito impresso, a síntese de circuitos digitais e analógicos, as telecomunicações e os controladores. A título de concretizar o estudo efetuado, apresenta-se um caso de estudo da aplicação dos algoritmos genéticos na síntese de circuitos digitais combinatórios, com base na análise e comparação de três referências de autores distintos. Com este estudo foi possível comparar, não só os resultados obtidos por cada um dos autores, mas também a forma como os algoritmos genéticos foram implementados, nomeadamente no que diz respeito aos parâmetros, operadores genéticos utilizados, função de avaliação, implementação em hardware e tipo de codificação do circuito.

Molecular detection of "Candidatus Mycoplasma haemominutum" in a lion (Panthera leo) from a brazilian zoological garden

Although Mycoplasma haemofelis and "Candidatus Mycoplasma haemominutum" infections have been reported in wild cats from United States, their presence among native and captive wild cats in Brazil is still unknown. A 12 year old healthy male lion (Panthera leo) from the Zoological Garden of Curitiba, Brazil was anesthetized for transportation and dental evaluation. A blood sample was obtained for a complete blood cell count (CBC) and PCR analysis. DNA was extracted and fragments of Mycoplasma haemofelis and "Candidatus Mycoplasma haemominutum" 16S ribosomal RNA gene were amplified in PCR assays. CBC results were within reference intervals. A weak band of 192 pb for "Candidatus Mycoplasma haemominutum" was observed, and no band was amplified from Mycoplasma haemofelis reaction. A weak PCR band associated with normal CBC results and without visible parasitemia or clinical signs may suggest a chronic subclinical infection with "Candidatus Mycoplasma haemominutum". The lack of clinical signs may also represent the low pathogenicity of this organism; however, it is noteworthy that immune suppression caused by management and/or corticoids treatment may induce parasitemia and anemia in this animal. This detection suggests further studies in captive wild cats in Brazilian Zoological Gardens.

Cutaneous infection caused by Corynebacterium pseudodiphtheriticum: a microbiological report

We report here a rare case of cutaneous infection due to Corynebacterium pseudodiphtheriticum. The patient presented to the clinical laboratory with a skin ulcer on his left leg. Gram-stained preparation of the purulent secretion revealed the presence of numerous rod-shaped Gram-positive organisms in the absence of any other species. The organism was grown in pure culture on sheep blood agar and was further identified as C. pseudodiphtheriticum using a commercial identification system (API-Coryne, BioMérieux, France). The infection was successfully treated with ciprofloxacin. This case emphasizes the importance of the clinical microbiology laboratory in correctly identifying Gram-positive organisms obtained in pure culture from skin ulcers.

Clinical and Bacteriological Survey of Diabetic Foot Infections in Lisbon

AIMS: An epidemiological survey of diabetic foot infections (DFIs) in Lisbon, stratifying the bacterial profile based on patient demographical data, diabetic foot characteristics (PEDIS classification), ulcer duration and antibiotic therapy. METHODS: A transversal observational multicenter study, with clinical data collection using a structured questionnaire and microbiological products (aspirates, biopsies or swabs collected using the Levine method) of clinically infected foot ulcers of patients with diabetes mellitus (DM). RESULTS: Forty-nine hospitalized and ambulatory patients were enrolled in this study, and 147 microbial isolates were cultured. Staphylococcus was the main genus identified, and methicillin-resistant Staphylococcus aureus (MRSA) was present in 24.5% of total cases. In the clinical samples collected from patients undergoing antibiotic therapy, 93% of the antibiotic regimens were considered inadequate based on the antibiotic susceptibility test results. The average duration of an ulcer with any isolated multi-drug resistant (MDR) organism was 29 days, and previous treatment with fluoroquinolones was statistically associated with multi-drug resistance. CONCLUSIONS: Staphylococcus aureus was the most common cause of DFIs in our area. Prevalence and precocity of MDR organisms, namely MRSA, were high and were probably related to previous indiscriminate antibiotic use. Clinicians should avoid fluoroquinolones and more frequently consider the use of empirical anti-MRSA therapy.

ABCC Subfamily Vacuolar Transporters are Involved in Pb (Lead) Detoxification in Saccharomyces cerevisiae

The present work has as objective to contribute for the elucidation of the mechanism associated with Pb detoxification, using the yeast Saccharomyces cerevisiae as a model organism. The deletion of GTT1 or GTT2 genes, coding for functional glutathione transferases (GST) enzymes in S. cerevisiae, caused an increased susceptibility to high Pb concentrations (500-1000 μmol L(-1)). These results suggest that the formation of glutathione-Pb conjugate (GS-Pb), dependent of GSTs, is important in Pb detoxification. The involvement of ATP-binding cassette (ABC) vacuolar transporters, belonging to class C subfamily (ABCC) in vacuolar compartmentalization of Pb, was evaluated. For this purpose, mutant strains disrupted in YCF1, VMR1, YBT1 or BPT 1 genes were used. All mutants tested, without vacuolar ABCC transporters, presented an increased sensitivity to 500-1000 μmol L(-1) Pb comparative to wild-type strain. Taken together, the obtained results suggest that Pb detoxification, by vacuolar compartmentalization, can occur as a result of the concerted action of GSTs and vacuolar ABCC transporters. Pb is conjugated with glutathione, catalysed by glutathione transferases and followed to the transport of GS-Pb conjugate to the vacuole by ABCC transporters.

Corynebacterium striatum infecting a malignant cutaneous lesion: the emergence of an opportunistic pathogen

We described a case of a 27-year old male patient with skin and soft tissue infection of a neoplastic lesion caused by Corynebacterium striatum, an organism which has been rarely described as a human pathogen. Identification was confirmed by DNA sequencing. Successful treatment with penicillin was achieved. The role of the C. striatum as an emerging opportunistic pathogen is discussed.

Sífilis congénita : caracterização da infecção e avaliação de técnicas laboratoriais para o seu diagnóstico

RESUMO: Um dos maiores problemas da sífilis é a infecção intra-uterina do feto, que pode resultar em morte fetal com aborto espontâneo. Os objectivos desta tese foram comparar os cinco testes serológicos para o diagnóstico de sífilis congénita e optimizar e aplicar a várias amostras clínicas, colhidas de indivíduos com suspeita de infecção por Treponema pallidum, uma técnica de PCR-Multiplex e uma técnica de PCR em tempo real. Na globalidade dos soros estudados obtiveram-se os seguintes resultados: o RPR reactivo em 87/517; TPHA reactivo em 135/517; e EIA reactivo em 127/517. A pesquisa de anticorpos específicos de tipo IgM foi efectuada em 33 soros sendo estudada pelas técnicas de imunofluorescência indirecta e de westernblot. Quanto aos resultados obtidos pelas duas técnicas, o teste FTA-Abs-IgM demonstrou reactividade em 3/33, enquanto que a técnica de westernblot-M, apresentou reactividade em 18/33. Para a avaliação de uma técnica de PCR-TR, foram estudadas 318 amostras de 236 indivíduos classificados, com base em critérios clínicos e laboratoriais, em diferentes estádios de infecção por Treponema pallidum e em indivíduos sem infecção por aquele microrganismo. Relativamente a estas técnicas foi possível observar amplificação de ADN de Treponema pallidum em 133/318 pela técnica de PCR-TR e 90/318 pela técnica de PCR-Multiplex. Mediante os resultados obtidos e de outros estudos efectuados parece poder concluir-se que o teste EIA é o mais indicado para o rastreio da infecção por Treponema pallidum, devendo um resultado reactivo por esta técnica, ser confirmado com a realização de um teste não treponémico (RPR). Relativamente ao diagnóstico de infecção congénita, o teste westernblot para pesquisa de anticorpos específicos de tipo IgM, parece ser o mais apropriado. A técnica PCR-TR, parece ser a mais indicada, pois apresenta uma maior sensibilidade e especificidade que a PCR-Multiplex.----------------- ABSTRACT:The syphilis major problem is the intrauterine infection of the fetus, which may result in fetal death with spontaneous abortion. The objectives of this thesis were to compare the five serological tests for the diagnosis of congenital syphilis and optimize and apply several clinical samples taken from individuals suspected of infection with Treponema pallidum, a PCR-Multiplex and real-time PCR. In all sera studied were obtained the following results: the RPR reactive in 87/517; reactive TPHA in 135/517, and 127/517 in reactive EIA. The search for specific IgM antibodies was performed on 33 sera that were studied by indirect immunofluorescence and westernblot. The results obtained by both techniques, the test FTA-Abs-IgM showed reactivity in 3/33, while the technique westernblot-M, showed reactivity in 18/33. For an RT-PCR evaluation, we studied 318 samples from 236 individuals classified based on clinical and laboratory criteria at different stages of infection by Treponema pallidum and in individuals without infection by that organism. For these techniques it was possible to observe ADN amplification of Treponema pallidum in 133/318 by RT-PCR and 90/318 by PCR-Multiplex. Based on obtained results and in other studies seems that we can conclude that the EIA test is the most suitable for Treponema pallidum screening infection, and a reactive result by this technique, should be confirmed with the realization of a non-treponemal test (RPR). For the congenital infection diagnosis, testing for antibodies westernblot specific IgM appears to be the most appropriate. The RT-PCR seems to be the most suitable, since it has a higher sensitivity and specificity than the PCR-Multiplex.