Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 4 uppsatser.

Diss. (sammanfattning) Umeå : Sveriges lantbruksuniv., 2003. / Härtill 4 uppsatser.

Thesis (Dr. rer. nat.)--Universität Tübingen, 2004. / Includes bibliographical references. Available in PDF format via the World Wide Web.

No description available.

Bacillus subtilis é uma bactéria gram positiva de solo, não patogênica, não colonizadora de tecidos, naturalmente transformável e formadora de esporos utilizada como modelo de estudo de bactérias gram-positivas. Essas características acarretam em vantagens para a produção de proteases de interesse industrial e para utilização como veículo de antígenos vacinais, porém a falta de vetores induzíveis torna sua utilização como ferramenta biológica pouco explorada. No presente trabalho descrevemos a construção de diferentes vetores capazes de expressar os antígenos subunidade B da toxina termo-lábil (LTB) e subunidade estrutural da fímbria CFA/I (CFAB) de Escherichia coli enterotoxigênica (ETEC) e avaliamos seu potencial vacinal. Foi avaliada a imunogenicidade de linhagens capazes de expressar LTB sob o controle de diferentes promotores: PgsiB (induzido em condições de estresse), PlepA (promotor constitutivo) e Pspac (induzido pela adição de IPTG) e em diferentes locais da célula (ancorada à parede celular ou secretada para o meio externo). Avaliamos ainda a imunogenicidade de linhagens capazes de co-expressar LTB e a listeriolisina O (LLO) de Listeria monocytogenes. O antígeno CFAB foi produzido no citoplasma ou ancorado à parede celular de B. subtilis em condições de estresse e as linhagens bacterianas administradas sozinhas ou conjuntamente com a toxina termo-lábil (LT) como adjuvante de mucosa. Camundongos imunizados com células ou esporos de B. subtilis recombinantes desencadearam respostas de anticorpos sistêmicos e secretados específicos para os antígenos (LTB e CFAB), não alterados pela adição do adjuvante. A expressão de LLO causou a supressão da resposta de anticorpos específicos para o antígeno LTB. Os resultados obtidos demonstram a viabilidade do uso de B. subtilis como veículo vacinal. / Bacillus subtilis is a gram positive, generally regarded as safe and spore forming soil bacteria used as a model for genetic and phisiological studies. This safety status allow its use as host for production of industrial protases and its application as vaccine vehicles, however the lack of epissomal inducible expression systems disable the exploration of this organism as a biotechnologic tool. In this work we describe the construction of epissomal vectors able to express the B subunit of the heat-labile toxin (LTB) and the structural subunit of the CFA/I fimbrae (CFAB) from the enterotoxigenic Escherichia coli (ETEC). We evaluate strains able to express LTB under the control of three promoters: PgsiB (stress inducible), PlepA (constitutive) e Pspac (IPTG inducible) and allowing the expression of LTB secreted or anchored to the cell wall We also evaluate the immunogenicity of strains able to co-express LTB and the listeriolysin O (LLO) from Listeria monocytogenes. CFAB was expressed in the cytoplasm or anchored to the cell wall and administred alone or with the mucosal adjuvant LT. Mice immunized both with cells or spores elicited secreted and systemic specific antibodies responses, which were not altered by the addition of the adjuvant LT. LLO expression suppressed the antibodies responses against LTB. The data shows the ability of B. subtilis to be used as vaccine vehicle.

A transmissão de parasitas da malária por mosquitos vetores é dependente do desenvolvimento bem sucedido das formas infectantes de Plasmodium sp., especialmente os esporozoítas, que são as formas que infectam o hospedeiro vertebrado. A manipulação genética de mosquitos vetores tem sido uma estratégia alternativa na tentativa de controle da malária. Um componente extremamente importante desta estratégia é a escolha de uma molécula efetora capaz de reduzir a transmissão do patógeno. Microplusina é um peptídeo antimicrobiano rico em cisteína, originalmente descrito como um componente antimicrobiano da hemolinfa e dos ovos de carrapato bovino Rhipicephalus (Boophilus) microplus. Testes anteriores utilizando o modelo experimental mosquito Aedes aegypti infectado por Plasmodium gallinaceum mostraram que a microplusina é altamente tóxico para esporozoítas de Plasmodium gallinaceum em concentração relativamente baixa, sem apresentar toxicidade aos mosquitos vetores Aedes aegypti. Nosso objetivo foi analisar a expressão da microplusina e seu efeito na infecção de P. gallinaceum em mosquitos transgênicos. Obtivemos quatro linhagens através da integração de um transgene contendo a região promotora do gene da vitelogenina de Ae. aegypti, peptídeo sinal maltase-like I de Ae. aegypti e a sequência codificadora da microplusina (PMOS [3xP3-EGFP-AeVg Micro]). A atividade anti esporozoítas da microplusina expressa pelos mosquitos transgênicos mostrou diferença significante as linhagens. O desenho de novas moléculas utilizando como molde moléculas efetoras existentes e testadas, possibilitará o aperfeiçoamento da expressão de genes exógenos em mosquitos transgênicos, tornando-os refratários ao parasita. / Transmission of malaria parasites by mosquito vectors is dependent on the successful development of Plasmodium sp. infective forms, particularly the sporozoites, which are the forms that enter the vertebrate host. The genetic manipulation of mosquito vectors has been a strategy for malaria control. An extremely important component of this strategy is the effector molecule of choice which reduces parasite transmission. Microplusin is a cysteine-rich antimicrobial peptide originally described as an hemolymph and eggs antimicrobial component of the cattle tick Boophilus microplus. Previous tests using the experimental model Plasmodium gallinaceum infected Aedes aegypti showed that microplusin is highly toxic to P. gallinaceum sporozoites in relatively low concentration, without showing toxicity to the mosquito vector A. aegypti. Our goal was to analyze transgenic mosquitoes expressing microplusin and its effect on infection of P. gallinaceum. We obtained four lines through the integration of transgene that containing the promoter region of the A. aegypti vitelogenin gene, the maltase-like I signal peptide of A. aegypti and microplusin coding sequence (pMos[3xP3-EGFPAeVg-Micro]). The activity anti sporozoites microplusin expressed by transgenic mosquitoes showed significant differences between strains. The design of effector molecules using information from existing and tested molecules as template will enable the improvement of the expression of foreign genes in transgenic mosquitoes, making them resistant to the parasite.

No description available.

The development of bio-resorbable implant materials is rapidly going on. Sterilization of those materials is inevitable to assure the hygienic requirements for critical medical devices according to the medical device directive (MDD, 93/42/EG). Biopolymer-containing biomaterials are often highly sensitive towards classical sterilization procedures like steam, ethylene oxide treatment or gamma irradiation. Supercritical CO2 (scCO2) treatment is a promising strategy for the terminal sterilization of sensitive biomaterials at low temperature. In combination with low amounts of additives scCO2 treatment effectively inactivates microorganisms including bacterial spores. We established a scCO2 sterilization procedure under addition of 0.25% water, 0.15% hydrogen peroxide and 0.5% acetic anhydride. The procedure was successfully tested for the inactivation of a wide panel of microorganisms including endospores of different bacterial species, vegetative cells of gram positive and negative bacteria including mycobacteria, fungi including yeast, and bacteriophages. For robust testing of the sterilization effect with regard to later application of implant materials sterilization all microorganisms were embedded in alginate/agarose cylinders that were used as Process Challenge Devices (PCD). These PCD served as surrogate models for bioresorbable 3D scaffolds. Furthermore, the impact of scCO2 sterilization on mechanical properties of polysaccharide-based hydrogels and collagen-based scaffolds was analyzed. The procedure was shown to be less compromising on mechanical and rheological properties compared to established low-temperature sterilization methods like gamma irradiation and ethylene oxide exposure as well as conventional steam sterilization. Cytocompatibility of alginate gels and scaffolds from mineralized collagen was compared after sterilization with ethylene oxide, gamma irradiation, steam sterilization and scCO2 treatment. Human mesenchymal stem cell viability and proliferation were not compromised by scCO2 treatment of these materials and scaffolds. We conclude that scCO2 sterilization under addition of water, hydrogen peroxide and acetic anhydride is a very effective, gentle, non-cytotoxic and thus a promising alternative sterilization method especially for biomaterials.

No description available.

Thesis (PhD (Food Science))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Species of Alicyclobacillus are acid-tolerant and heat-resistant bacteria that cause spoilage of heat-treated fruit juices stored at room temperature. During the past decade, Alicyclobacillus spp. have become a major cause of spoilage in pasteurised fruit juices leading to significant economic losses world-wide. Spoilage has been reported in apple, pear, orange, peach, mango and white grape juice, as well as in fruit juice blends, fruit juice containing drinks and tomato products, such as tomato juice and canned tomatoes. Spoilage is characterised by a medicinal smell and guaiacol production. These endospore-formers have been shown to survive pasteurisation conditions of 95 °C for 2 min, grow at temperatures between 25° and 60 °C and a pH range of 2.5 to 6.0. Knowledge of this organism is limited, both locally and internationally and the route of contamination to the final product is not well established. In this study the fruit concentrate processing environment was investigated as a potential source and route of contamination for the final product. Species of Alicyclobacillus were isolated from orchard soil, various stages during processing and from fruit juice and concentrates. The isolates were identified based on morpholological, biochemical and physiological properties. Identification to species level was done by 16S ribosomal RNA gene sequencing and strain differentiation by RAPD-PCR. Results indicate that species of A. acidoterrestris and Alicyclobacillus acidocaldarius were found in orchard soil and throughout the processing environment. This is the first report on the isolation of these species from orchard soil, vinegar flies and the fruit processing environment. The 16 isolates identified as A. acidoterrestris grouped into four clusters based on RAPD-PCR banding patterns, suggesting that they belong to at least four genotypic groups. Isolates from the fruit concentrate, wash water and soil located outside of the fruit processing plant grouped into one cluster. Concluded from these results, A. acidoterrestris found in the wash water and soil outside of the factory could act as a potential reservoir of organisms for the contamination of the final fruit concentrate. Thus good manufacturing practices play an essential role in controlling incidence of spoilage caused by these bacteria. Fruit juices can be treated using ultraviolet (UV-C) light with a wavelength of 254 nm, which has a germicidal effect against micro-organisms. Alicyclobacillus acidoterrestris spores were inoculated into tap water, used wash water from a fruit processing plant and grape juice concentrate. Ultraviolet dosage levels (J L−1) of 0, 61, 122, 183, 244, 305 and 367 were applied using a novel UV-C turbulent flow system. The UV treatment method was shown to reliably achieve in excess of a 4 log10 reduction (99.99%) per 0.5 kJ L-1 of UV-C dosage in all the liquids inoculated with A. acidoterrestris. The applied novel UV technology could serve as an alternative to thermal treatments of fruit juices for the inactivation of Alicyclobacillus spores or in the treatment of contaminated processing wash water. Finally, the thermal inactivation at 95 °C for two strains of A. acidoterrestris isolated from contaminated fruit juice concentrates were investigated in a 0.1% (m/v) peptone buffer solution (pH 7.04) and grape juice (pH 4.02, 15.5 °Brix). The thermal inactivation of A. acidoterrestris spores followed first-order kinetics, suggesting that as the microbial population is exposed to a specific high temperature, the spores inactivated at a constant rate. D-values determined in the buffer solution were calculated to be 1.92 min and 2.29 min, while in grape juice D-values were found to be 2.25 min and 2.58 min for the two strains tested. From this study it is clear that the D-value is dependant on the strain tested, but also on the soluble solids of the solution the cells are suspended in. The results indicated that the spores of A. acidoterrestris isolated from South African fruit juice concentrate may survive after the pasteurisation treatment commonly applied during manufacturing. / AFRIKAANSE OPSOMMING: Spesies van Alicyclobacillus is suur-tolerante en hittebestande bakterieë wat bederf veroorsaak in hitte-behandelde vrugtesappe wat teen kamertemperatuur gestoor word. Gedurende die afgelope dekade het Alicyclobacillus spp. ‘n belangrike oorsaak van bederf in gepasteuriseerde vrugtesappe geword en beduidende ekonomiese verliese wêreldwyd veroorsaak. Bederf is aangeteken in appel-, peer-, lemoen-, perske-, mango- en witdruiwesap, sowel as in vrugtesapversnitte, vrugtesapbevattende drankies en in tamatieprodukte soos tamatiesap en ingemaakte tamaties. Bederf word gekenmerk deur ’n medisinale reuk en guaiacol produksie. Daar is gevind dat hierdie endospoorvormers pasteurisasie teen 95 °C vir 2 min kan oorleef en kan groei by temperature tussen 25° en 60 °C en ‘n pH van 2.5 to 6.0. Plaaslik sowel as internasionaal is kennis van hierdie organisme beperk en die roete van kontaminasie van produkte is nog nie goed vasgestel nie. In hierdie studie is die vrugtekonsentraat-verwerkingsmilieu ondersoek as ‘n moontlike bron en roete van kontaminasie van die finale produk. Spesies van Alicyclobacillus is vanuit vrugteboordgrond, verskeie verwerkingstadia en van vrugtesap en vrugtesapkonsentraat geïsoleer. Die isolate is op grond van morfologiese, biochemiese en fisiologiese eienskappe geïdentifiseer. Identifikasie tot spesiesvlak is deur 16S rDNS sekwensering gedoen en stam differensiasie deur RAPD-PKR. Resultate het aangetoon dat A. acidoterrestris en A. acidocaldarius in vrugteboordgrond sowel as in alle stadia van die verwerkingsmilieu voorkom. Dit is die eerste verslag van die isolering van hierdie spesies uit die Suid-Afrikaanse vrugteverwerkingsmilieu, vrugteboordgrond en asynvlieë. Die 16 isolate, geïdentifiseer as A. acidoterrestris en in vier groepe geplaas op grond van hul RAPD-PKR bandpatrone, dui aan dat hulle aan minstens vier genotipiese groepe behoort. Isolate afkomstig van die vrugtekonsentraat, waswater en die grond buitekant die vrugteverwerkingsaanleg het een groep gevorm. Uit hierdie resultate kan afgelei word dat A. acidoterrestris, wat in die waswater en grond buite die aanleg voorkom, as ‘n moontlike bron van organismes vir die kontaminering van die finale vrugtekonsentraat kan dien. Goeie vervaardigingspraktyke speel dus ‘n noodsaaklike rol in die beheer van bederf veroorsaak deur hierdie bakterieë. Vrugtesappe kan behandel word met ultravioletlig (UV-C) met ‘n golflengte van 254 nm wat ‘n dodende effek op mikro-organismes het. Kraanwater, gebruikte waswater van ‘n vrugtesapvervaardigingsaanleg en druiwesapkonsentraat is met A. acidoterrestris spore geïnokuleer. Ultraviolet toedieningsvlakke (J L−1) van 0, 61, 122, 183, 244, 305 en 367 is aangewend met behulp van ‘n nuwe UV-C drukvloei stelsel. Daar is aangetoon dat die UV-behandelingsmetode ‘n betroubare vermindering (99.99%) van meer as 4 log10 per 0.5 kJ L-1 van ‘n UV-C dosis gee in al die vloeistowwe wat geïnokuleer is met A. acidoterrestris. Die toegepaste nuwe UV-tegnologie kan gebruik word as ‘n alternatief tot die hittebehandeling van vrugtesap vir die deaktivering van Alicyclobacillus spore of in die behandeling van gekontamineerde waswater. Ten slotte is hitte-deaktivering teen 95 °C van twee stamme van A. acidoterrestris, geïsoleer uit gekontamineerde vrugtesapkonsentraat, in ‘n 0.1% (m/v) peptoonbufferoplossing (pH 7.04) en druiwesap (pH 4.02, 15.5 °Brix), ondersoek. Die hitte-deaktivering van A. acidoterrestris spore het eerste-orde kinetika gevolg, wat aandui dat die mikrobe-populasie teen ‘n konstante tempo afsterf, wanneer blootgestel aan ‘n spesifieke hoë temperatuur. Die D-waardes in die bufferoplossing is bereken as 1.92 min en 2.29 min, terwyl daar gevind is dat die D-waardes in druiwesap 2.25 min en 2.58 min is vir die twee betrokke stamme. Vanuit hierdie studie is dit duidelik dat die D-waardes afhang van die betrokke stam, maar ook van die oplosbare vaste stowwe van die oplossing waarin die selle opgelos is. Die resultate dui daarop dat die spore van A. acidoterrestris, wat geïsoleer is uit Suid-Afrikaanse vrugtesapkonsentraat, die pasteurisasiebehandeling wat algemeen tydens vervaardiging toegepas word, kan oorleef. Aangesien die toepassing van strenger hittebehandeling om spore van A. acidoterrestris te deaktiveer onaanvaarbare organoleptiese veranderinge in die produk tot gevolg het, word dit aanbeveel dat die risiko van bederf verminder behoort te word deur die gebruik van goeie vervaardigingspraktyke gedurende vrugteverwerking.