Avaliação do efeito antibacteriano de extratos de folhas de batata-doce (Ipomoea batatas L. frente a bactérias de interesse em alimentos e correlação com os compostos fenólicos

RESUMOA batata-doce (Ipomoea batatas L.) é um vegetal muito cultivado nos países tropicais, usado na alimentação humana e na animal, sendo uma boa fonte de energia, sais minerais e vitaminas. Constitui, ainda, fonte de polifenóis antioxidativos, entre eles, antocianinas e ácidos fenólicos. O objetivo deste estudo foi avaliar a atividade antibacteriana de diferentes extratos de folhas frescas de batata-doce, frente a bactérias de interesse alimentar, e correlacioná-la com os teores de polifenóis totais e de antocianinas. Foram avaliados teores de polifenóis totais e de antocianinas, bem como a atividade antibacteriana de extratos alcoólicos, de infusão e de decocção de folhas de dois acessos de batata-doce, em Porto Alegre, RS, frente a três agentes de interesse em alimentos e alimentação, quais sejam, inóculos padronizados de SalmonellaEnteritidis (ATCC 11076), Staphylococcus aureus(ATCC 25923) e Escherichia coli (ATCC 11229). A avaliação da atividade antibacteriana, expressa em intensidade de atividade de inibição bacteriana (IINIB) e em intensidade de atividade de inativação bacteriana (IINAB), foi baseada em testes de diluição em sistema de tubos múltiplos; as antocianinas foram determinadas por meio de pH diferencial e os polifenóis pelo método de Folin & Ciocalteu. Os extratos alcoólicos inibiram e, ou, inativaram todos os inóculos testados, não obstante o Staphylococcus aureus ter sido significativamente mais resistente. Os extratos obtidos por decocção e infusão, além de apresentarem concentrações muito baixas de antocianinas e menores teores de polifenóis do que os seus correspondentes alcoólicos, não apresentaram atividade antibacteriana. A atividade antibacteriana dos extratos testados foi positivamente correlacionada com os compostos fenólicos e, ou, antocianinas.

Essential oils of Origanum vulgare L. subsp glandulosum (Desf.) letswaart from Tunisia: chemical composition and antioxidant activity

BACKGROUND: Characterisation of the essential oils from O. glandulosum collected in three locations of Tunisia, chemical composition and the evaluation of their antioxidant activities were carried out. RESULTS: The essential oils from Origanum vulgare L. subsp. glandulosum (Desf.) letswaart collected from three localities of north Tunisia - Krib, Bargou and Nefza - were obtained in yields of 2.5, 3.0 and 4.6% (v/w), respectively. The essential oils were analysed by GC and GC/MS and assayed for their total phenolics content, by the Folin-Ciocalteu method, and antioxidant effectiveness, using the 2,2-diphenyl-1-picrylhydrazil (DPPH) radical scavenging assay. The main components of these essential oils, from Nefza, Bargou and Krib, were p-cymene (36%, 40% and 46%), thymol (32%, 39% and 18%), gamma-terpinene (24%, 12% and 16%) and carvacrol (2%, 2% and 15%), respectively). The ability to scavenge the DPPH radicals, expressed by IC50, ranged from 59 to 80 mg L-1. The total phenolic content, expressed in gallic acid equivalent (GAE) g kg(-1) dry weight, varied from 9.37 to 17.70 g kg(-1) dw. CONCLUSIONS: A correlation was identified between the total phenolic content of the essential oils and DPPH radical scavenger capacity. The occurrence of a p-cymene chemotype of O. glandulosum in the northern region of Tunisia is demonstrated.

Tipificação de Mycoplasma mycoides subsp. mycoides SC e detecção da sua aderência a células epiteliais pulmonares

Dissertação apresentada para a obtenção do Grau de Mestre em Genética Molecular e Biomedicina, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Genetics of berry colour and anthocyanin content variation in grapevine (Vitis vinifera L.subsp. vinifera)

Dissertation presented in fulfillment of the requirements for the Degree of Doctor of Philosophy in Biology (Molecular Genetics) at the Instituto de Tecnologia Química e Biológica da Universidade Nova de Lisboa

"Multiplex PCR" identification of the atypical and monophasic Salmonella enterica subsp. enterica serotype 1,4,[5],12:i:- in São Paulo State, Brazil: frequency and antibiotic resistance patterns

Salmonella spp. are the etiologic agents of salmonellosis, a worldwide spread zoonoses causing foodborne outbreaks and clinical diseases. By serological identification, Salmonella enterica subsp. enterica serotype 1,4,[5],12:i:- accounted for 8.8% of human and 1.6% of nonhuman Salmonella strains isolated in São Paulo State, during 1991-2000. A total of 28.6% of them amplified a fragment corresponding to H:1,2 (flagellar phase two) through PCR analysis and were further assigned as S. Typhimurium. Antimicrobial resistance was detected in 36.3% of the 369 PCR-negative strains tested, including the multiresistance to ampicillin, chloramphenicol, sulfonamides, tetracycline, and streptomycin.

Salmonella enterica subsp houtenae serogroup O:16 in a HIV positive patient: case report

We described a case of salmonellosis in a 33-year old HIV-infected patient. The patient presented oral and esophageal candidiasis, intense epigastric and retrosternal pain. During the physical examination he was hypochloraemic, acyanotic, hypohydrated, anicteric and afebrile. Admittance laboratorial tests indicated: red cells 3.6 millions/mm³; hemoglobin, 10.1 g/dL; leukocyte count, 3,000/mm³, with 1% of eosinophils, 14% of non-segmented and 53% of segmented neutrophils and 31% of lymphocytes. The blood culture was positive for Salmonella enterica subsp houtenae serogroup O:16. This is probably the first human report of bacteremia due to Salmonella enterica subsp houtenae in Brazil associated to HIV-infected patient.

CD19 LYMPHOCYTE PROLIFERATION INDUCED BY Bifidobacterium animalis subsp. lactis IN C57BL/6 MICE EXPERIMENTALLY INFECTED WITH Toxoplasma gondii

Toxoplasmosis is frequently acquired through the oral route by the ingestion of cysts or oocysts of Toxoplasma gondii. Once ingested, the parasites penetrate the intestinal epithelial cells and rapidly disseminate to all organs in the host. During T. gondii infection, the intestinal microbiota plays an important role in stimulating a protective immune response against the parasite. In this sense the use of probiotics is worthy of note since they are live microorganisms that have beneficial effects on the host through stimulation of the immune response that can be important in the control of T. gondii proliferation and dissemination in the host. In the present study, the action of the probiotic Bifidobacterium animalis subsp. lactis was investigated in C57BL/6 mice infected with oocysts of ME49 strain of T. gondii. The probiotic had an immunomodulatory action, inducing CD19 lymphocyte proliferation and consequently increasing anti-T. gondii antibody level.Bifidobacterium animalis subsp. lactisprovided protection in supplemented mice, compared to the control group. In addition, supplemented animals had milder inflammatory process in the small intestine, indicating that the probiotic protects the intestinal mucosa during infection with T. gondii. It was concluded that the probioticB. animalis subsp. lactis induces humoral immune response capable of providing protection against T. gondii infection.

EVALUATION OF SUBLETHAL EFFECTS OF Ipomoea cairica LINN. EXTRACT ON LIFE HISTORY TRAITS OF DENGUE VECTORS

Plant derived insecticides have considerable potential for mosquito control because these products are safer than conventional insecticides. This study aimed to investigate sublethal activities of Ipomoea carica or railway creeper crude acethonilic extract against life history trait of dengue vectors, Aedes albopictus and Aedes aegypti. The late third instar larvae of Ae. albopictus and Ae. aegypti were exposed to a sublethal dose at LC50 and larvae that survived were further cultured. Overall, Ipomea cairica crude extracts affected the whole life history of both Aedes species. The study demonstrated significantly lower egg production (fecundity) and eggs hatchability (fertility) in Ae. albopictus. The sublethal dose of crude extracts reduced significantly the width of larval head capsule and the wing length of both sexes in both Aedes species. The significance of sublethal effects of I. cairica against Aedes mosquitoes was an additional hallmark to demonstrate further activity of this plant despite its direct toxicity to the larvae. The reduced reproductive capacity as well as morphological and physiological anomalies are some of the effects that make I. cairica a potential candidate to be used as a new plant-based insecticide to control dengue vectors.

Armazenamento de sementes de ingazeira (Inga vera Subsp. affinis (DC.) T.D. Pennington).

O objetivo do trabalho foi estudar a germinação de semente de Inga vera subsp (Mimosaceae).

Producción de ácido Itacónico por Aspergillus terreus y de Ácido Hialurónico por Streptococcus equi subsp. equi en Biorreactores de Tanque Agitado. Production of Itaconic Acid by Aspergillus terreus and of Hyaluronic Acid by Sreptococcus equi subsp. equi in Stirred Tank Bioreactors.

El proyecto aborda el problema general de establecer una metodología para el cambio de escala de la producción de metabolitos en biorreactores de tanque agitado.En el desarrollo de procesos de producción de metabolitos a partir de microorganismos, el cambio de escala es particularmente complejo, dado que los microorganismos experimentan un continuo cambio en sus rutas metabólicas durante el período de producción. Esto hace que en el proceso del cambio de escala, las mayores dificultades se encuentren en el desarrollo del inóculo y problemas ocasionados por modificaciones en las características de transferencia de calor, masa y momento.Dentro de este contexto general se definen dos objetivos específicos. Estos son: el estudio de la producción de ácido itacónico por Aspergillus terreus y el de la producción de ácido hialurónico por Streptococcus equi. subsp. equi, con la finalidad de desarrollar una metodología de trabajo experimental y teórica que permita sistematizar el estudio del factibilidad técnico-económica de plantas de producción, vinculando la investigación del procesos a escala de laboratorio con la producción a mayor escala.La hipótesis de trabajo es que el estudio de la producción de Aspergillus terreus y de Streptococcus equi en un biorreactor de tanque agitado a escala de laboratorio permitirá establecer los parámetros que contribuirán a realizar el cambio de escala de su producción y esto será verificado experimentalmente.Los trabajos se realizarán utilizando un biorreactor a escala de laboratorio especialmente diseñado para este tipo de trabajo. Los resultados experimentales se interpretarán con técnicas estadísticas y matemáticas de diferente complejidad a efectos de establecer los criterios de cambio de escala y luego se realizarán experiencias en un biorreactor piloto con el objeto de verificar la metodología seleccionada.El desarrollo del proyecto permitirá:1.- obtener información técnica útil sobre la producción de ácido itacónico, el que tiene importantes aplicaciones en la industria del plástico. La producción por medio del Aspergillus terreus MJL05 se realizará utilizando glicerol como fuente de carbono, el que constituye el principal subproducto en los procesos de manufactura de biodiesel. De este modo se podrá analizar la factibilidad técnica de una ruta alternativa para emplear este subproducto.2.- obtener información técnica útil sobre la producción de ácido hialurónico, biopolímero de alto valor agregado con importantes aplicaciones en medicina, y contribuir así a realizar el cambio de escala de su producción.

Meiose em Tripsacum australe Cutler e Anderson (T. dactyhides subsp. hispidum Hitchcock)

Depois de uma breve introdução, mostrando a importância que o gênero Tripsacum desempenha hoje nos problemas da origem do milho, fizemos um estudo detalhado da meiose na nova espécie Tripsacum australe, descrita recentemente por CUTLER e ANDERSON (3) e espécie esta encontrada em estado selvagem na América do Sul. Todas as fases da meiose mostraram-se normais e o número cte cromosômios, facilmente determinado nas fases diacinese, metáfase I, metáfase II, é de 18 para a fase haplóide. Esta espécie não difere, quanto ao número de cromosômios, da forma diplóide Tripsacum dactyloide e da espécie Tripsacum floridanum, estudadas por LONGLEY (5). Segundo MANGELSDORF e REEVES (10) as formas de Tripsacum encontradas na América Central têm 72 cromosômios e são consideradas como autotetraplóides. Entretanto, no que se refere à presença de "knobs" nos cromosômios, esta espécie parece diferir da espécie estudada por LONGLEY (5). Tripsacum australe não apresenta "knobs" nas extremidades dos cromosômios e provavelmente também nas outras regiões pois as figuras que puderam ser examinadas não mostraram essa estrutura. Segundo MANGELSDORF e REEVES (10) os "knobs" presentes no milho teriam vindo de Tripsacum, por meio de cruzamento entre estes dois gêneros. Assim sendo, os tipos de milho cultivados próximos ao centro de distribuição das espécies de Tripsacum até então conhecidas, e que é a região da América Central, principalmente o México, deveriam se apresentar bastante contaminados por este gênero e apresentariam mais "knobs" do que aqueles tipos de milho cultivados ionge da referida região. Observações de vários autores (6, 7, 9, 20, 11 e 12) confirmam esta hipótese, inclusive aquelas realizadas por um dos autores deste trabalho (Graner, não publicado) em material sul-americano. Tendo sido encontrada agora esta nova espécie de Tripsacum na América do Sul, aparentemente sem "knobs", torna-se interessante verificar se ela não poderia ter contribuido para o estabelecimento das formas de milho sem "knobs" encontradas na América do Sul. Cruzamentos entre milho e Tripsacum australe foram realizados por um dos autores (Addison), não lendo porém produzido sementes. Outras pesuisas tornam-se então necessárias afim de que se possa tirar conclusões a respeito de tão importante assunto.

Expression of Staphylococcus aureus clumping factor A in Lactococcus lactis subsp. cremoris using a new shuttle vector.

Staphylococcus aureus harbors redundant adhesins mediating tissue colonization and infection. To evaluate their intrinsic role outside of the staphylococcal background, a system was designed to express them in Lactococcus lactis subsp. cremoris 1363. This bacterium is devoid of virulence factors and has a known genetic background. A new Escherichia coli-L. lactis shuttle and expression vector was constructed for this purpose. First, the high-copy-number lactococcal plasmid pIL253 was equipped with the oriColE1 origin, generating pOri253 that could replicate in E. coli. Second, the lactococcal promoters P23 or P59 were inserted at one end of the pOri253 multicloning site. Gene expression was assessed by a luciferase reporter system. The plasmid carrying P23 (named pOri23) expressed luciferase constitutively at a level 10,000 times greater than did the P59-containing plasmid. Transcription was absent in E. coli. The staphylococcal clumping factor A (clfA) gene was cloned into pOri23 and used as a model system. Lactococci carrying pOri23-clfA produced an unaltered and functional 130-kDa ClfA protein attached to their cell walls. This was indicated both by the presence of the protein in Western blots of solubilized cell walls and by the ability of ClfA-positive lactococci to clump in the presence of plasma. ClfA-positive lactococci had clumping titers (titer of 4,112) similar to those of S. aureus Newman in soluble fibrinogen and bound equally well to solid-phase fibrinogen. These experiments provide a new way to study individual staphylococcal pathogenic factors and might complement both classical knockout mutagenesis and modern in vivo expression technology and signature tag mutagenesis.

Biochemical, immunological and toxicological characteristics of the crystal proteins of Bacillus thuringiensis subsp. medellin

Characterization of the insecticidal and hemolytic activity of solubilized crystal proteins of Bacillus thuringiensis (Bt) subsp. medellin (Btmed) was performed and compared to solubilized crystal proteins of isolates 1884 of B. thuringiensis subsp. israelensis (Bti) and isolate PG-14 of B. thuringiensis subsp. morrisoni (Btm). In general, at acid pH values solubilization of the Bt crystalline parasporal inclusions (CPI) was lower than at alkaline pH. The larvicidal activity demonstrated by the CPI of Btmed indicated that optimal solubilization of CPI takes place at a pH value of 11.3, in Bti at pH values from 5.03 to 11.3 and in Btm at pH values from 9.05 to 11.3. Hemolytic activity against sheep red blood cells was mainly found following extraction at pH 11.3 in all Bt strains tested. Polyacrylamide gel electrophoresis under denaturing conditions revealed that optimal solubilization of the CPI in all Bt strains takes place at the alkaline pH values from 9.05 to 11.3. An enriched preparation of Btmed crystals was obtained, solubilized and crystal proteins were separated on a size exclusion column (Sephacryl S-200). Three main protein peaks were observed on the chromatogram. The first peak had two main proteins that migrate between 90 to 100 kDa. These proteins are apparently not common to other Bt strains isolated to date. The second and third peaks obtained from the size exclusion column yielded polypeptides of 68 and 28-30 kDa, respectively. Each peak independently, showed toxicity against 1st instar Culex quinquefasciatus larvae. Interestingly, combinations of the fractions corresponding to the 68 and 30 kDa protein showed an increased toxicity. These results suggest that the 94 kDa protein is an important component of the Btmed toxins with the highest potency to kill mosquito larvae. When crystal proteins of Bti were probed with antisera raised independently against the three main protein fractions of Btmed, the only crystal protein that showed cross reaction was the 28 kDa protein. These data suggest that Btmed could be an alternative bacterium for mosquito control programs in case mosquito larval resistance emerges to Bti toxic proteins.

Cloning, Expression and Toxicity of a Mosquitocidal Toxin Gene of Bacillus thuringiensis subsp. medellin

Bacillus thuringiensis (Bt) subsp. medellin (Btmed) produces parasporal crystalline inclusions which are toxic to mosquito larvae. It has been shown that the inclusions of this bacterium contain mainly proteins of 94, 68 and 28-30 kDa. EcoRI partially digested total DNA of Btmed was cloned by using the Lambda Zap II cloning kit. Recombinant plaques were screened with a mouse policlonal antibody raised against the 94 kDa crystal protein of Btmed. One of the positive plaques was selected, and by in vivo excision, a recombinant pBluescript SK(-) was obtained. The gene encoding the 94 kDa toxin of Btmed DNA was cloned in a 4.4 kb DNA fragment. Btmed DNA was then subcloned as a EcoRI/EcoRI fragment into the shuttle vector pBU4 producing the recombinant plasmid pBTM3 and used to transform by electroporation Bt subsp. israelensis (Bti) crystal negative strain 4Q2-81. Toxicity to mosquito larvae was estimated by using first instar laboratory reared Aedes aegypti, and Culex quinquefasciatus larvae challenged with whole crystals. Toxicity results indicate that the purified inclusions from the recombinant Bti strain were toxic to all mosquito species tested, although the toxicity was not as high as the one produced by the crystal of the Btmed wild type strain. Poliacrylamide gel electrophoresis indicate that the inclusions produced by the recombinant strain Bti (pBTM3) were mainly composed of the 94 kDa protein of Btmed, as it was determined by Western blot